# Patent

## Title
Combinatorial antibody libraries and uses thereof

## Abstract

Methods for making a combinatorial antibody library from human germline segments are provided. Also provided are libraries of nucleic acid molecules compiled from germline segments encoding VL chains and libraries of nucleic acid molecules encoding VH chains, and resulting antibody libraries. The libraries are provided as addressable libraries. Methods for screening antibody libraries against a target protein antigen, and the identified or selected antibodies are provided.

## Background
RELATED APPLICATIONS
This application is a U.S. National Stage Application under 35 U.S.C. §371 of International Patent Application No. PCT/US09/63299, filed Nov. 4, 2009, which claims the benefit of priority to U.S. Provisional Application No. 61/198,764, and to U.S. Provisional Application No. 61/211,204, filed Mar. 25, 2009, the entire contents of which are each incorporated by reference.
This application also is related to International PCT Application No. PCT/US09/63303, entitled “Anti-DLL4 Antibodies and Uses Thereof,” filed Nov. 4, 2009, which claims priority to U.S. Provisional Application No. 61/198,764 and to U.S. Provisional Application No. 61/211,204. This application also is related to U.S. Provisional Application No. 61,280,618, entitled “Methods for Affinity-Maturation-Based Antibody Optimization,” filed Nov. 4, 2009. Where permitted, the subject matter of each of the above-noted related applications is incorporated by reference in its entirety.


INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY
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FIELD OF THE INVENTION
Methods for making a combinatorial antibody library from human germline segments are provided. Also provided are libraries of nucleic acid molecules compiled from germline segments encoding VL chains and libraries of nucleic acid molecules encoding VH chains, and resulting antibody libraries. The libraries are provided as addressable libraries. Methods for screening antibody libraries against a target protein antigen, and the identified or selected antibodies are provided.
BACKGROUND
Numerous therapeutic and diagnostic monoclonal antibodies (MAbs) are used in the clinical setting to treat and diagnose human diseases, for example, cancer and autoimmune diseases. For example, exemplary therapeutic antibodies include Rituxan (Rituximab), Herceptin (Trastuzumab), Avastin (Bevacizumab) and Remicade (Infliximab). In designing antibody therapeutics, it is desirable to create antibodies, for example, antibodies that modulate a functional activity of a target, and/or improved antibodies such as antibodies with higher specificity and/or affinity and/or and antibodies that are more bioavailable, or stable or soluble in particular cellular or tissue environments.
Available techniques for generating antibody therapeutics are limited. Current methods include using antibody libraries to select variant proteins with desired properties in vitro. The libraries are generated to contain mutational diversity by targeted and non-targeted methods (e.g., Marks et al., J. Mol. Biol. (1991) 222, 581-597; Winters et al. (1994) Annu Rev. Immunol. 12:433-55; Rosok et al. (1996) J. Biol. Chem., 271:22611-22618; Kim et al. (2005) Mol. Cells 20:17-29; Mondon et al. (2008) Frontiers in Bioscience, 13:1117-1129; Benhar et al. (2007) Expert Opin. Biol. Ther., 7:763-779; and Knappik et al. (2000) J. Mol. Biol., 296:57-86) . Each of these antibody libraries has its limitations. Accordingly, it is among the objects herein is to provide methods for making antibody libraries, and antibodies produced by the methods.
SUMMARY
Provided herein are human combinatorial antibody libraries generated by rearrangement of human germline segments. Included among the combinatorial antibody libraries provided herein are libraries containing a plurality of antibodies, whereby each member antibody in the library contains a variable light (VL) chain and a variable heavy (VH) chain or a sufficient portion thereof to form an antigen binding site. Each VL chain of the antibodies in the library are encoded by a nucleic acid molecule that contains Vκ and a Jκ human germline segment or degenerate codons thereof, or a Vλ and a Jλ human germline segment or degenerate codons thereof, whereby the segments are linked in-frame. Each VH chain of the antibodies in the library are encoded by a nucleic acid molecule that contains a human VH and a human JH germline segment and any sequence of nucleotides between the VH and a JH germline segments, whereby the segments are linked in-frame. The human combinatorial antibody libraries contain at least about or 50 or 100 more different members. Each member in the library contains an antigen binding site and is a functional and productive antibody.
In such an example of a human combinatorial antibody library, the VH germline segment, the sequence of nucleotides between the VH and JH and the JH germline segment of the nucleic acid molecule encoding a VH chain are linked such that the VH segment is 5′ to the sequence of nucleotides between the VH and JH which is 5′ to the JH segment; and the Vκ and Jκ or Vλand Jλ germline segments of the nucleic acid molecule encoding a VL chain are linked such that the Vκ segment is 5′ to the Jκ segment or the Vλ segment is 5′ to the Jλ segment The sequence of nucleotides between the VH and JH germline segments is at or is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides in length. In some examples, the sequence of nucleotides between the VH and JH germline segments encodes a peptide mimetic.
Also provided herein is a human combinatorial antibody library containing a plurality of antibodies, whereby each member antibody contains a modified variable light (VL) chain and/or a modified variable heavy chain (VH) chain or a sufficient portion thereof to form an antigen binding site. The VL chain in each library is encoded by a nucleic acid molecule that contains a Vκ and a Jκ human germline segment or degenerate codons thereof, or a Vλ and a Jλ human germline segment or degenerate codons thereof, whereby the segments are linked in-frame. Each VH chain of antibodies in the library are encoded by a nucleic acid molecule that contains a VH, DH and a JH human germline segment or degenerate codons thereof, whereby the segments are linked in-frame. The resultant protein of the VL chain and the VH chain are modified by amino acid replacement or insertion of amino acids into a CDR. The CDR can be any one or more up to all of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or a CDRL3, for example, a CDRH3. The amino acids that can be inserted or replaced correspond to a peptide mimetic.
In all of the examples above, the peptide mimetic can be a TPO, EPO, G-CSF, IL-5, human brain natriuretic peptide (hBNP-32), exendin 4, GLP-1, GLP-2, glucagon, PACAP-38, CD209L, TNF, VEGF, MMP inhibitor, or CTLA-4 peptide mimetic. In particular of a peptide mimietic is a mimetic that mimics Epo activiation of its receptor. The peptide mimetic further can include a flanking sequence at the carboxy and/or N-terminal end, such as an amino acid or amino acids. For example, the flanking sequence can include glycine or a proline. Exemplary of peptide mimetics are any set forth in any of SEQ ID NOS: 891, and 987-1014.
In the libraries provided above, the nucleotides between the VH and JH germline segments is a human germline DH segment or degenerate codons thereof. Hence, a human combinatorial antibody library provided herein includes a library containing a plurality of antibodies, each member antibody contains a variable light (VL) chain and a variable heavy (VH) chain or a sufficient portion thereof to form an antigen binding site. Each VL chain of the antibodies in the library are encoded by a nucleic acid molecule that contains a Vκ and a Jκ human germline segment or degenerate codons thereof, or a Vλ and a Jλ human germline segment or degenerate codons thereof, whereby the segments are linked in-frame. Each VH chain of the antibodies in the library are encoded by nucleic acid molecule that contains a VH, DH and a JH germline segment, whereby the segments are linked in-frame. The human combinatorial antibody library contains at least about or 50 or 100 more different members. In such examples of a human combinatorial antibody library, the VH, DH and JH segments of the nucleic acid molecule encoding a VH chain are linked such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment; and the Vκ and Jκ or Vλ and Jλ germline segments of the nucleic acid molecule encoding a VL chain are linked such that the Vκ segment is 5′ to the Jκ segment or the Vλ segment is 5′ to the Jλ segment.
In all of the human combinatorial antibody libraries provided herein , the libraries can be provided as addressable libraries. In such addressable libraries, the antibody within each address is the same antibody and is different from the antibodies at all other addresses. For example, the addressable antibodies in the library are arranged in a spatial array. The spatial array can be a multiwall plate, wherein each individual locus of the plate corresponds to a different antibody member. The antibody members can be immobilized to the surface of the wells of the plate or can be present in solution. In another example, the addressable antibodies are attached to a solid support. In such an example, the solid support can be a filter, chip, slide, bead or cellulose, and the different antibody members are immobilized to the surface thereof. In some examples, the solid support is a biacore chip. In any of the addressable libraries provided herein, the members can be identifiably labeled. For example, the label can be colored, chromogenic, luminescent, chemical, fluorescent or electronic.
The human combinatorial libraries provided herein include members whereby a plurality of nucleic acid molecules encode a VH chain and a plurality of nucleic acid molecules encode a VL chain such that the plurality of nucleic acid molecules encoding the VH chain and the VL chain are sufficient to generate at least about or 50 or 100 different antibodies. Hence, in the libraries provided herein, in the nucleic acid molecules encoding a VH chain all or a subset of germline VH segments are linked with all or a subset of DH segments which are linked with all or a subset of germline JH segments to generate the plurality of nucleic acid molecules encoding a VH chain; and in the nucleic acid molecules encoding a VL chain all or a subset of germline Vλ segments are linked with all or a subset of germline Jλ segments, or all or a subset of germline Vλ segments are linked to all or a subset of germline Jλ segments to generate a plurality of nucleic acid molecules encoding a VL chain.
In the libraries provided herein, the VH chains in the members in the library are encoded by rearranged nucleic acid sequences combined by joining a human VH germline segment, a DH germline segment and a JH germline segment. The human VH germline segment is an IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6 or IGHV7 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS: 10-238. In examples of the combinatorial antibody libraries provided herein, the VH gene segment can have one nucleiotide added or removed at the V-D joint of the nucleic acid molecule encoding the VH chain to maintain the reading frame of the VH chain. For example, a nucleotide is inserted at the 3′ end of a VH nucleotide sequence to add a nucleotide between the VH and DH. The nucleotide can be any nucleotide, in particular, the nucleotide is a guanine (G). The DH segment is an IGHD1, IGHD2, IGHD3, IGHD4, IGHD5, IGHD6, or IGHD7 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS:239-272. In the examples of the combinatorial antibody libraries provided herein, the DH gene segment can have one nucleotide inserted or deleted at the V-D joint and/or the D-J joint of the nucleic acid encoding the VH chain to maintain the reading frame of the VH chain. The nucleotide insertion or deletion is chosen to maximize the hydrophilicity of the DH. For example, a nucleotide from the 5′ end of a DH is deleted and/or a nucleotide from the 3′ end of a DH is deleted. In another example, a nucleotide is inserted at the 3′ end of a DH sequence to add a nucleotide between the DH and JH. The added nucleotide can be any nucleotide, in particular a guanine (G). The JH germline segment is an IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5 or IGHJ6 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS:273-285. In examples of the human combinatorial antibody library provided herein, the J H gene segment can have one or more nucleotides inserted or deleted at the D-J joint of the nucleic acid sequence encoding a VH chain to maintain the reading frame of the VH chain. For example, the nucleotide insertion or deletion is chosen to maintain the reading frame of the JH. In one examples, a nucleotide from the 5′ end of the the JH is deleted. In another example, a nucleotide from the 3′ end of the JH is deleted.
In the libraries provided herein, the VL chains in the members in the library are encoded by rearranged nucleic acid sequences encoding a kappa light chain combined by joining a human Vκ germline segment and a Jκ germline segment or are encoded by rearranged nucleic acid sequences encoding a lambda light chain combined by joining a human Vλ germline segment and a Jλ germline segment. The human Vκ is an IGKV1, IGKV2, IGKV3, IGKV4, IGKV5 and IGKV6, and genes or alleles thereof, for example, any set forth in any of SEQ ID NOS: 286-355 and 868. In examples of the combinatorial antibody libraries provided herein, the Vκ gene segment can have one or more nucleiotides inserted or deleted at the V-J joint of the nucleic acid molecule encoding the VL chain to maintain the reading frame of the VL chain. For example, a nucleotide is deleted at the 3′ end of a Vκ. In another example, a nucleotide is inserted at the 3′ end of a of a Vκ nucleotide sequence to add a nucleotide between the Vκ and Jκ. The nucleotide can be any nucleotide, in particular, the nucleotide is a guanine (G). The human Jκ is an IGKJ1, IGKJ2, IGKJ3, IGKJ4 or IGKJ5 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS: 356-364. In examples of the combinatorial antibody libraries provided herein, the Jκ gene segment has one or more nucleotides inserted or deleted at the V-J joint of the nucleic acid sequence encoding a VL chain to maintain the reading frame of the VL chain. The nucleotide insertion or deletion is chosen to maintain the reading frame of the Jκ. In some examples, a nucletode from the 5′ end of the Jκ is deleted. The human Vλ is an IGLV1, IGLV2, IGLV3, IGLV4, IGLV5, IGLV6, IGLV7, IGLV8, IGLV9, IGLV10 or IGLV11 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS:365-441. In examples of the combinatorial antibody libraries provided herein, the Vλ has one or more nucleotides inserted or deleted at the V-J joint of the nucleic acid sequence encoding a VL to maintain the reading frame of the VL chain. For example, a nucleotide from the 3′ end of a Vλ nucleotide sequence is deleted. In another example, a nucleotide is inserted at the 3′ end of a Vλ nucleotide sequence to add a nucleotide between the Vλ and Jλ. The nucleotide can be any nucleotide, in particular a guanine (G). The human Jλ is an IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6 or IGLJ7 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS:442-451. In examples of the combinatorial antibody library provided herein, the Jλ has one nucleotide inserted or deleted at the V-J joint of the nucleic acid sequence encoding a VL chain to maintain the reading frame of the VL chain. The nucleotide insertion or deletion can be chosen to maintain the reading frame of the Jλ. For example, a nucleotide from the 5′ end of the Jλ is deleted.
The human combinatorial antibody libraries contain a plurality of members each encoded by a plurality of nucleic acid molecules encoding a VH chain and a plurality of nucleic acid molecules encoding a VL chain. The plurality of nucleic acid molecules can correspond to all combinations or permutations of rearranged germline segments or a subset thereof. Generally, the libraries provided herein include libraries containing at or about 50, 102, 103, 104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 105, 106, 107, 108, 109 or more different members. For example, libraries provided herein include those that contain 103, 2×103, 3×103, 4×103, 5×103, 6×103, 4×103, 7×103, 8×103, 9×103, 104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104 or more different members.
For example, the plurality of nucleic acid molecules encoding a VH chain are generated from a subset of germline segments selected based on sequence similarities or differences, gene family, length, composition, CDR length or composition, species, functionality, specificity, group or subgroup. In one example, the plurality of nucleic acid molecules encoding a VH chain are generated from a subset of germline segments selected based on CDR and the CDR is CDR3. In another example, the plurality of nucleic acid molecules encoding a VH chain are selected based on gene family, whereby one germline segment from each of a VH, DH, and/or JH gene family is selected or one germline segment from a subset of a VH, DH, and/or JH gene family is selected. In such an example, the VH gene family is selected from among a IGHV1-18, IGHV1-2, IGHV1-24, IGHV1-3, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV1-8, IGHV2-26, IGHV2-5, IGHV2-5, IGHV2-70, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-35, IGHV3-38, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-7, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3-9, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-4, IGHV4-59, IGHV4-61, IGHV5-51, IGHV6-1, and IGHV7-81; the DH gene family is selected from among a IGHD1-1, IGHD1-14, IGHD1-20, IGHD1-26, IGHD1-7, IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-11, IGHD4-17, IGHD4-23, IGHD4-4, IGHD5-12, IGHD5-18, IGHD5-24, IGHD5-5, IGHD6-13, IGHD6-19, IGHD6-25, IGHD6-6 and IGHD7-27; and the JH gene family is selected from among a IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5 and IGHJ6.
For example, the plurality of nucleic acid molecules encoding a VL chain are generated from a subset of germline segments selected based on sequence similarities or differences, gene family, length, composition, CDR length or composition, species, functionality, specificity, group, subgroup. In one example, the plurality of nucleic acid molecules encoding a VL chain are generated from a subset of germline segments selected based on CDR and the CDR is CDR3. In another example, the plurality of nucleic acid molecules encoding a VH chain are selected based on gene family, whereby one germline segment from each of a Vκ and/or Jκ or Vλ and/or Jλ gene family is selected or one germline segment from a subset of a Vκ and/or Jκ or Vλ and/or Jλ gene family is selected. In such an example, the Vκ gene family is selected from among a IGKV1-12, IGKV1-12, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1-NL1, IGKV1/OR2, IGKV1D-12, IGKV1D-13, IGKV1D-16, IGKV1D-17, IGKV1D-33, IGKV1-D-37, IGKV1-D-39, IGKV1D-42, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-30, IGKV2-40, IGKV2D-24, IGKV2D-26, IGKV2D-28, IGKV2D-29, IGKV2-D-30, IGKV2D-40, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3-7, IGKV3-NL1, IGV3-NL2, IGKV3-NL3, IGKV3-NL4, IGKV3-NL5, IGKV3/OR2-268, IGKV3D-11, IGKV3D-15, IGKV3D-20, iGKV3D-7, IGKV4-1, IGKV5-2, IGKV6-21, IGKV6D-21, IGKV6D-41, and IGKV1-39; the Jκ gene family is selected from among a IGKJ1, IGKJ2, IGKJ3, IGKJ4 and IGKJ5; the Vλ gene family is selected from among a IGLV1-36, IGLV1-40, IGLV1-41, IGLV1-44, IGLV1-47, IGLV1-50, IGLV1-51, IGLV10-54, IGLV11-55, IGLV2-11, IGLV2-14, IGLV2-18, IGLV2-23, IGLV2-33, IGLV2-8, IGLV3-1, IGLV3-10, IGLV3-12, IGLV3-16, IGLV3-19, IGLV3-21, IGLV3-22, IGLV3-25, IGLV3-27, IGLV3-32, IGLV3-9, IGLV4-3, IGLV4-60, IGLV4-69, IGLV5-37, IGLV5-39, IGLV5-45, IGLV5-8, IGLV5-52, IGLV6-57, IGLV7-43, IGLV7-46, IGLV8-61, IGLV8-61and IGLV9-49; and the Jλ gene family is selected from among a IGLJ1, IGLJ2, IGLJ4, IGLJ5, IGLJ6 and IGLJ7.
In any of the combinatorial antibody libraries provided herein, each antibody member in the library is productive and functional. Hence, in some examples, member antibodies in the library contains a VH chain and/or a VL chain that is encoded by a nucleic acid molecule that is modified to remove stop codons and/or restriction enzyme sites.
In any of the combinatorial antibody libraries provided herein, the VH chain is encoded by a nucleic acid molecule having a sequence of nucleotides set forth in any of SEQ ID NOS: 1059-1410, or a subset thereof and the VL chain is encoded by a nucleic acid molecule having a sequence of nucleotides set forth in any of SEQ ID NOS: 1411-1422, 1424-1439 and 1441-1471, or a subset thereof. The antibody libraries provided herein include libraries containing members whereby the VH chain has a sequence of amino acids set forth in any of SEQ ID NOS: 1475-1826 or a subset thereof and the VL chain has a sequence of amino acids set forth in any of SEQ ID NOS: 1827-1838, 1840-1855 and 1857-1888 or a subset thereof.
The human combinatorial antibody libraries provided herein include those having members that are full length antibodies or are fragments or portions thereof of antibodies, whereby the fragment or portion of the antibody is sufficient to form an antigen binding site. Thus, any of the the combinatorial antibody libraries provided herein can further contain all of a portion of a constant region, such that the portion of a constant region is sufficient to permit association of a heavy and light chain. Included among fragments or portions of antibody members in the libraries provided herein are a Fab, Fab′, F(ab′)2, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments Fab fragments, Fd fragments, scFv fragments, or a scFab fragments. For example, combinatorial antibody libraries provided herein are Fab libraries, whereby antibody members of the library are Fabs.
Provided herein is a library of nucleic acid molecules containing a plurality of addressable nucleic acid molecules encoding a variable light (VL) chain. In such libraries, each VL chain is encoded by a nucleic acid molecule containing a Vκ and a Jκ human germline segment or a Vλ and a Jλ human germline segment linked in-frame, whereby the nucleic acid molecule within each address is the same and is different from the nucleic acid molecules at all other addresses. Each nucleic acid member are formed from combination of germline segments such that the Vκ and Jκ or Vλ and Jλ germline segments of the nucleic acid molecule encoding a VL chain are linked such that the Vκ segment is 5′ to the Jλ segment or the Vλ segment is 5′ to the Jλ segment. The library includes a plurality of nucleic acid members that can include all permutations of all combinations of germline segments. In some examples, the plurality of nucleic acid members includes a subset of all germline segments such that a subset of germline Vκ segments are linked with all or a subset of germline Jκ segments, or all or a subset of germline Vλ segments are linked to all or a subset of germline Jλ segments to generate a plurality of nucleic acid molecules encoding a VL chain.
In the VL nucleic acid libraries provided herein, nucleic acid molecules encoded a VL chain are generated by by rearranged nucleic acid sequences combined by joining a human Vλ germline segment and a Jκ germline segment. The Vκ is an IGKV1, IGKV2, IGKV3, IGKV4, IGKV5 or IGKV6, and genes and alleles thereof, for example any set forth in any of SEQ ID NOS:286-355 and 868. Included among the Vκ germline segment contained in nucleic acid members in the libraries provided herein are any where the Vκ has one or more nucleotides inserted or deleted a the V-J joint in the nucleic acid molecule encoding a VL to maintain the reading frame of the VL chain. For example, one or more nucleotides at the 3′ end of the Vκ nucleotide sequence is deleted. In other examples, one or more nucleotides is inserted at the 3′ end of a Vκ nucleotide sequence to add a nucleotide between the Vκ and Jκ germline segments. The nucleotide can be any nucleotide, and in particular is a guanine (G). The Jκ germline segment is an IGKJ1, IGKJ2, IGKJ3, IGKJ4 and IGKJ5 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS: 356-364. Included among the Jκ germline segment contained in nucleic acid members in the libraries provided herein are any where the Jκ has one or more nucleotides inserted or deleted at the V-J joint to maintain the reading frame of the VL chain. The insertion or deletion is typically chosen to maintain the reading frame of the Jκ. For example, one or more nucleotides from the 5′ end of the Jκ is deleted.
In some examples, the VL nucleic acid libraries provided herein, nucleic acid molecules encoded a VL chain are generated by by rearranged nucleic acid sequences combined by joining a human Vλ germline segment and a Jλ germline segment. The Vλ germline segment is an IGLV1, IGLV2, IGLV3, IGLV4, IGLV5, IGLV6, IGLV7, IGLV8, IGLV9, IGLV10 and IGLV11 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS: 365-441. Included among the Vλ germline segment contained in nucleic acid members in the libraries provided herein are any where the Vλ has one or more nucleotides inserted or deleted at the V-J joint of the nucleic acid molecule encoding a VL to maintain the reading frame of the VL chain. For example, one or more nucleotides is from the 3′ end of a Vλ nucleotide sequence is deleted. In another example, one or more nucleotides is inserted at the 3′ end of a Vλ nucleotide sequence to add a nucleotide between the Vλ and Jλ. The nucleotide can be any nucleotide, in particular a guanine (G). The Jλ germline segment is an IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6 and IGLJ7 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS: 442-451. Included among Jλ germline segments contained in nucleic acid members in the libraries provided herein are any where the Jλ has one or more nucleotides inserted or deleted at the V-J join of the nucleic acid molecule encoding a VL to maintain the reading frame of the VL chain. The insertion or deletion is typically chosed to maintain the reading frame of the Jλ. For example, a nucleotide from the 5′ end of the Jλ is deleted.
Any of the plurality of nucleic acid molecules encoding a VL chain in the libraries provided herein can be generated from a subset of germline segments selected based on sequence similarities or differences, gene family, length, composition, CDR length or composition, species, functionality, specificity, group, subgroup. For example, the plurality of nucleic acid molecules encoding a VL chain are generated from a subset of germline segments selected based on CDR and the CDR is CDR3. In another example, the plurality of nucleic acid molecules encoding a VL chain are selected based on gene family, whereby one germline segment from each of a Vκ and/or Jκ or Vλ and/or Jλ gene family is selected or one germline segment from a subset of a Vκ and/or Jκ or Vλ and/or Jλ gene family is selected. In such an example, a Vκ germline segment can include any one or more germline segments from a IGKV1-12, IGKV1-12, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1-NL1, IGKV1/OR2, IGKV1D-12, IGKV1D-13, IGKV1D-16, IGKV1-D-17, IGKV1D-33, IGKV1D-37, IGKV1D-39, IGKV1D-42, IGKV1D-43, IGKV1D-8, IGKV1-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-30, IGKV2-40, IGKV2D-24, IGKV2D-26, IGKV2D-28, IGKV2D-29, IGKV2-D-30, IGKV2D-40, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3-7, IGKV3-NL1, IGV3-NL2, IGKV3-NL3, IGKV3-NL4, IGKV3-NL5, IGKV3/OR2-268, IGKV3D-11, IGKV3D-15, IGKV3D-20, IGKV3D-7, IGKV4-1, IGKV5-2, IGKV6-21, IGKV6D-21, IGKV6D-41, or IGKV1-39 gene family; a Jκ germline segments can include any one or more germline segments from a IGKJ1, IGKJ2, IGKJ3, IGKJ4 and IGKJ5 gene family; a Vλ germline segment can include any one or more germline segments from a IGLV1-36, IGLV1-40, IGLV1-41, IGLV1-44,IGLV1-47, IGLV1-50, IGLV1-51, IGLV10-54, IGLV11-55, IGLV2-11, IGLV2-14, IGLV2-18, IGLV2-23, IGLV2-33, IGLV2-8, IGLV3-1, IGLV3-10, IGLV3-12, IGLV3-16, IGLV3-19, IGLV3-21, IGLV3-22, IGLV3-25, IGLV3-27, IGLV3-32, IGLV3-9, IGLV4-3, IGLV4-60, IGLV4-69, IGLV5-37, IGLV5-39, IGLV5-45, IGLV5-8, IGLV5-52, IGLV6-57, IGLV7-43, IGLV7-46, IGLV8-61, IGLV8-61 and IGLV9-49 gene family; and/or a Jλ germline segment can include any one or more germline segments from a IGLJ1, IGLJ2, IGLJ4, IGLJ5, IGLJ6 and IGLJ7 gene family.
In all of the nucleic acid libraries encoding a VL chain provided herein, the nucleic acid molecule encoding a VL chain can be modified to remove stop codons and/or restriction enzyme sites. Exemplary of nucleic acid molecules in the libraries provided herein include any of SEQ ID NOS:1411-1422, 1424-1439 and 1441-1471, or a subset thereof.
Provided herein is a library of nucleic acid molecules containing a plurality of addressable nucleic acid molecules encoding a variable light (VH) chain. In such libraries, each VH chain is encoded by a nucleic acid molecule containing a a VH, a DH and a JH human germline segment linked in-frame, whereby the nucleic acid molecule within each address is the same and is different from the nucleic acid molecules at all other addresses. Each nucleic acid member is formed from combination of germline segments such that the VH, a DH and a JH human germline segment of the nucleic acid molecule encoding a VH chain are linked such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment. The library includes a plurality of nucleic acid members that can include all permutations of all combinations of germline segments. In some examples, the plurality of nucleic acid members includes a subset of all germline segments such that a subset of germline VH segment are linked with all or a subset of germline DH segments which are linked with all or a subset of germline JH segments to generate the plurality of nucleic acid molecules encoding a VH chain.
In the VH nucleic acid libraries provided herein, nucleic acid molecules encoded a VH chain are generated by by rearranged nucleic acid sequences combined by joining a human VH, DH and JH germline segments. The VH is an IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6 and IGHV7 and genes and alleles thereof, for example any set forth in any of SEQ ID NOS: 10-238. Included among the VH germline segment contained in nucleic acid members in the libraries provided herein are any where the VH has one or more nucleotides added or removed at the V-D joint in the nucleic acid molecule encoding a VH to maintain the reading frame of the VH chain. For example, one or more nucleotides at the 3′ end of the VH nucleotide sequence is deleted. In other examples, one or more nucleotides is inserted or added at the 3′ end of a VH nucleotide sequence to add a nucleotide between the VH and DH germline segments. The nucleotide can be any nucleotide, and in particular is a guanine (G). The DH germline segment is an IGHD1, IGHD2, IGHD3, IGHD4, IGHD5, IGHD6, and IGHD7 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS: 239-272. Included among the DH germline segment contained in nucleic acid members in the libraries provided herein are any where the DH has one or more nucleotides inserted or deleted at the V-D and/or the D-J joint to maintain the reading frame of the VH chain. The nucleotide insertion or deletion can be any nucleotide, but typically is chosen to maximize the hydrophilicity of the DH. For example, one or more nucleotides from the 5′ end of the DH is deleted. In other examples, a nucleotide from the 3′ end of a DH is deleted. In further examples, a nucleotide is inserted at the 3′ end of a DH sequence to add a nucleotide been the DH and JH. The nucleotide can be any nucleotide, but typically is a guanine (G). The germline segment is an IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6 and genes and alleles thereof, for example, any set forth in any of SEQ ID NOS:273-285. Included among the JH germline segment contained in nucleic acid members in the libraries provided herein are any where the JH has one or more nucleotides inserted or deleted at the D-J joint to maintain the reading frame of the VH chain. Typically, the nucleotide insertion or deletion is chosen to maintain the reading frame of the JH. For example, one or more nucleotides from the 5′ end of the JH is deleted. In another example, one or more nucleotides from the 3′ end of the JH is deleted.
Any of the plurality of nucleic acid molecules encoding a VH chain in the libraries provided herein can be generated from a subset of germline segments selected based on sequence similarities or differences, gene family, length, composition, CDR length or composition, species, functionality, specificity, group, subgroup. For example, the plurality of nucleic acid molecules encoding a VH chain are generated from a subset of germline segments selected based on CDR and the CDR is CDR3. In another example, the plurality of nucleic acid molecules encoding a VH chain are selected based on gene family, whereby one germline segment from each of a VH, DH, and/or JH gene family is selected or one germline segment from a subset of a VH, DH, and/or JH gene family is selected. In such an example, a VH germline segment can include any one or more germline segments from a IGHV1-18, IGHV1-2, IGHV1-24, IGHV1-3, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV1-8, IGHV2-26, IGHV2-5, IGHV2-70, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16,IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-35, IGHV3-38, IGHV3-43, IGHV3-48, IGHV3-49,IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-7, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3-9, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-4, IGHV4-59, IGHV4-61, IGHV5-51, IGHV6-1 and IGHV7-81 gene family; a the DH germline segment can include any one ore more germline segments from a IGHD1-1, IGHD1-14, IGHD1-20, IGHD1-26, IGHD1-7, IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-11, IGHD4-17, IGHD4-23, IGHD4-4, IGHD5-12, IGHD5-18, IGHD5-24, IGHD5-5, IGHD6-13, IGHD6-19, IGHD6-25, IGHD6-6, and IGHD7-27 gene family; and/or a JH germline segment can include any one or more germline segments from a IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5 and IGHJ6 gene family.
In all of the nucleic acid libraries encoding a VH chain provided herein, the nucleic acid molecule encoding a VH chain can be modified to remove stop codons and/or restriction enzyme sites. Exemplary of nucleic acid molecules in the libraries provided herein include any of SEQ ID NOS: 1059-1410, or a subset thereof.
Also provided herein are libraries of addressable vectors containing any of the above nucleic acid molecules encoding a variable light (VL) chain or a variable heavy (VH) chain. Also provided herein are addressable cells, whereby each cell in the library contains any of the above different vectors.
Also provided herein are a library of nucleic acid molecules containing a plurality of addressable nucleic acid molecules encoding a variable light (VL) chain and a plurality of addressable nucleic acid molecules encoding a variable heavy (VH) chain (i.e. paired nucleic acid libraries). In such libraries, each VL chain is encoded by a nucleic acid molecule that contains a Vκ and a Jκ human germline segments or Vλ and Jλ germline segments linked in-frame and each VH chain is encoded by a nucleic acid molecule that contains a VH, a DH and a JH human germline segment. The resulting nucleic acid members in the the nucleic acid molecule encoding the VL chain can be any provided herein and the nucleic acid molecule encoding the VH chain can be any provided herein. In such addressable libraries, each locus contains a nucleic acid molecule encoding a VH chain and a nucleic acid molecule encoding a VL chain, such that the combination of VH nucleic acid molecules and VL nucleic acid molecules within each address is different from the combination of nucleic acid molecules at all other addresses, i.e. the pairs of nucleic acid libraries at each locus are different.
Provided herein is a method of generating human combinatorial antibody libraries. The method includes the steps of a) combining a VH, a DH and a JH human germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VH chain or a portion thereof and b) combining a Vκ and a Jκ human germline segment or portion thereof, or a Vλ and a Jλ germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VL chain or a portion thereof. In the method provided herein each of the portions of the VH, DH, JH, Vκ, Jκ, Vλ or Jλ in step a) and b) are sufficient to produce an antibody or portion thereof containing a VH or VL or portion thereof that forms a sufficient antigen binding site. In the methods, steps a) and b) are repeated a plurality of times to generate sequences of a plurality of different nucleic acid molecules. The nucleic acid molecules are synthesized to produce two libraries, whereby the first library contains nucleic acid molecules encoding a VH chain or portion thereof and the the second library contains nucleic acid molecules encoding a VL chain or a portion thereof. In the method, a nucleic acid molecule from the first library and from the second library are introduced into a cell (e.g. together such as by co-transformation). The step of introducing nucleic acids into cells is repeated a plurality of times with different pairs of nucleic acid molecules from the first library and the second library resulting in that contain nucleic acid molecules encoding a VH chain and a nucleic acid molecule encoding a VL chain such that the nucleic acid molecules encode a different combination of VH and VL chains from every other cell. The cells are grown to express the antibodies or portions thereof in each cell, thereby producing a plurality of antibodies or portion thereof. The plurality of produced antibody or portion thereof contains a VH and a VL or a sufficient portion thereof to form an antigen binding site and the antibodies or portions thereof are different from those at every other antibody or portions thereof in the library.
The human combinatorial library produced by the method provided herein can be provided as an addressable library. In such methods, each of the various steps can be performed in an addressed format so that throughout the steps of the method the identity of the germline segments, the recombined nucleic acid sequence and/or produced antibody or portion thereof are known by their address. For example, the synthesized nucleic acid sequences are individually addressed, thereby generating a first addressed nucleic acid library and a second addressed nucleic acid library. The nucleic acid molecules can be introduced into addressed cells, whereby each locus contains a cell that contains nucleic acid molecules encoding a different combination of a VH and a VL from every other cell in the addressed library of cells. Upon expression of the antibodies, addressed antibodies are produced whereby each locus contains an antibody containing a VH chain and a VL chain or a portion thereof sufficient to form an antigen binding site. The antibodies or portions thereof at each locus is the same and is different from those at each and every other locus. Hence, the identify of the antibody or portion thereof is known by its address.
In the method of generating a human combinatorial antibody library provided herein, in step a) the VH, DH and JH germline segments or portions thereof of the nucleic acid molecule encoding a VH chain are combined such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment; and in step b) the Vκ and Jκ or Vλ and Jλ germline segments or portions thereof of the nucleic acid molecule encoding a VL chain are linked such that the Vκ segment is 5′ to the Jκ segment or the Vλ segment is 5′ to the Jλ segment. Steps a) and/or b) can be performed manually or can be performed in silico, such as by a computer or computer system capable of or programmed to execute computer-readable instructions based on an algorithm for performing a method of combining human germline segments.
In the method provided herein for generating a human combinatorial antibody library, step a) includes the steps of selecting a VH, a DH and a JH germline segment or portion thereof, generating a V-D joint by modifying the germline sequence of the VH and/or DH germline segments by insertion or deletion of one or more nucleotides in order to maximize the hydrophilicity of the DH germline segment, generating a D-J joint by modifying the germline sequence of the DH and/or JH germline segments by insertion or deletion of one or more nucleotides to maintain the reading frame of the JH, and combining the resulting VH, DH, and JH, to generate a sequence of a nucleic acid molecule encoding a VH chain. In such a method, the V-D joint can be generated by deletion of one or more, for example one, nucleotide from the 5′ end of the DH germline segment. In another example, the V-D joint can be generated by deleting one or more nucleotides from the 3′ end of the VH germline segment. In a further example, the V-D joint can be generated by inserting one or more nucleotides at the 5′ end of the DH germline segment. For example, the inserted or added nucleotide or nucleotides can be any nucleotide, and in particular is a guanine (G). Further, the D-J joint can be generated by deletion of one or more nucleotides from the 5′ end of the JH. In another example, the D-J joint is generated by inserting a nucleotide from the 3′ end of the DH germline segment. The inserted or added nucleotide or nucleotides can be any nucleotide, and in particular is a guanine (G).
In the method provided herein for generating a human combinatorial antibody library, step b) includes the steps of selecting Vκ and Jκ or Vλ and Jλ germline segment or portion thereof, generating a V-J joint by modifying the germline sequence of the Vκ or Jκ by insertion or deletion of one or more nucleotides to maintain the reading frame of the Jκ, or by modifying the germline sequence of the Vλ or Jλ by insertion or deletion of one or more nucleotides to maintain the reading frame of the J,λ , and combining the resulting Vκ and Jκ or Vλ and Jλ to generate a sequence of a nucleic acid molecule encoding a VL chain. In such a method, the V-J joint can be generated by deletion of one or more, for example one, nucleotide from the 5′ end of the of the Jκ or Jλ germline segment. In another example, the V-J joint can be generated by deleting one or more nucleotides from the 3′ end of the Vλ or Vλ germline segment. In a further example, the V-J joint can be generated by inserting one or more nucleotides at the 5′ end of the Jκ or Jλ, germline segment. For example, the inserted or added nucleotide or nucleotides can be any nucleotide, and in particular is a guanine (G).
In the methods of generating a combinatorial library provided herein, steps a) and b) are repeated a plurality of times. Repeating step a) a plurality of times includes selecting N1 (i.e. a first number) of different VH germline segments, selecting N2 (i.e. a second number) of different DH germline segments and selecting N3 (a third number) of different JH sequences. The N1, N2 and N3 numbers can be the same or different, and can include all respective germline segments or a subset thereof. Generally, the N1, N2 and N3 are a number of germline segments that can be all or a subset of VH, DH or JH germline segments, respectively. In the method of repeating step a) a plurality of times, all possible combination of VH, DH and JH combinations are made to generate N1×N2×N3 different nucleic acid sequences encoding a VH chain.
For example, in the method of generating a human combinatorial antibody library provided herein, a VH germline segment (including N1 different VH germline segments) can be selected from all or a subset of an IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6 or IGHV7 and genes and alleles thereof, for example, a VH germline segment set forth in any of SEQ ID NOS: 10-238. A DH germline segment can be selected from all or a subset of an IGHD1, IGHD2, IGHD3, IGHD4, IGHD5, IGHD6, or IGHD7 and genes and alleles thereof, for example, a DH germline segment set forth in any of SEQ ID NOS: 239-272. A JH germline segment can be selected from all or a subset of IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, or IGHJ6 and genes and alleles thereof, for example, a JH germline segment set forth in any of SEQ ID NOS: 273-285.
In any of the above examples, the method can include in step a) selecting a subset of germline segments based on sequence similarities or differences, gene family, length, composition, CDR length or composition, species, functionality, specificity, group or subgroup. For example, the subset of germline segments can be selected based on gene family. In the methods, germline segments can be selected such that one germline segment from each of a a VH, DH, and/or JH gene family is selected or one germline segment from a subset of a VH, DH, and/or JH gene family is selected. VH gene families include, but are not limited to, a IGHV1-18, IGHV1-2, IGHV1-24, IGHV1-3, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV1-8, IGHV2-26, IGHV2-5, IGHV2-70, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-35, IGHV3-38, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-7, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3-9, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-4, IGHV4-59, IGHV4-61, IGHV5-51, IGHV6-1 and IGHV7-81 gene families including genes and alleles thereof. DH gene families include, but are not limited to, a IGHD1-1, IGHD1-14, IGHD1-20, IGHD1-26, IGHD1-7, IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-11, IGHD4-17, IGHD4-23, IGHD4-4, IGHD5-12, IGHD5-18, IGHD5-24, IGHD5-5, IGHD6-13, IGHD6-19, IGHD6-25, IGHD6-6, and IGHD7-27, gene families including genes and alleles thereof. The JH gene families include, but are not limited to, a IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5 and IGHJ6 gene families including genes and alleles thereof.
In the methods of generating a combinatorial library provided herein, steps a) and b) are repeated a plurality of times. Repeating step b) a plurality of times includes selecting N1 (i.e. a first number) of different Vλ germline segments and selecting N2 (i.e. a second number) different Jλ germline segments or selecting N3 (a third number) of different Vκ germline segments and selecting N4 (i.e. a fourth number) of different Jκ germline segments. The N1, N2, N3 and N4 numbers can be the same or different, and can include all respective germline segments or a subset thereof. Generally, the N1, N2, N3 and N4 are a number of germline segments that can be all or a subset of Vλ, Jλ, Vκ, Jκ germline segments, respectively. In the method of repeating step b) a plurality of times, all possible combination of Vλ, Jλ, Vκ, Jκ combinations are made to generate N1×N2 or N3×N4 different nucleic acid sequences encoding a VL chain.
For example, in the method of generating a human combinatorial antibody library provided herein, a Vλ germline segment (including N1 different Vλ germline segments) can be selected from all or a subset of an IGLV1, IGLV2, IGLV3, IGLV4, IGLV5, IGLV6, IGLV7, IGLV8, IGLV9, IGLV10 and IGLV11 and genes and alleles thereof, for example, a Vλ germline segment set forth in any of SEQ ID NOS: 365-441. A Jλ germline segment can be selected from all or a subset of an IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6 and IGLJ7 and genes and alleles thereof, for example, a Jλ germline segment set forth in any of SEQ ID NOS: 442-451. A Vκ germline segment can be selected from all or a subset of IGKV1, IGKV2, IGKV3, IGKV4, IGKV5 and IGKV6, and genes and alleles thereof, for example, a Vκ germline segment set forth in any of SEQ ID NOS: 286-355 and 868. A Jκ germline segment can be selected from all or a subset of a IGKJ1, IGKJ2, IGKJ3, IGKJ4 and IGKJ5 and genes and alleles thereof, for example, a Jκ germline segment set forth in any of SEQ ID NOS: 356-364.
In any of the above examples, the method can include in step b) selecting a subset of germline segments based on sequence similarities or differences, gene family, length, composition, CDR length or composition, species, functionality, specificity, group or subgroup. For example, the subset of germline segments can be selected based on gene family. In the methods, germline segments can be selected such that one germline segment from each of a Vκ and/or Jκ or Vλ and/or Jλ gene family is selected or one germline segment from a subset of a Vκ and/or Jκ or Vλ and/or Jλ gene family is selected. Vκ gene families include, but are not limited to, a IGKV1-12, IGKV1-12, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1-NL1, IGKV1/OR2, IGKV1D-12, IGKV1D-13, IGKV1D-16, IGKV1-D-17, IGKV1D-33, IGKV1D-37, IGKV1D-39, IGKV1D-42, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-30, IGKV2-40, IGKV2D-24, IGKV2D-26, IGKV2D-28, IGKV2D-29, IGKV2-D-30, IGKV2D-40, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3-7, IGKV3-NL1, IGV3-NL2, IGKV3-NL3, IGKV3-NL4, IGKV3-NL5, IGKV3/OR2-268, IGKV3D-11, IGKV3D-15, IGKV3D-20, IGKV3D-7, IGKV4-1, IGKV5-2, IGKV6-21, IGKV6D-21, IGKV6D-41, and IGKV1-39 gene families including genes and alleles thereof. Vλ gene families include, but are not limited to, a IGLV1-36, IGLV1-40, IGLV1-41, IGLV1-44, IGLV1-47, IGLV1-50, IGLV1-50, IGLV1-51, IGLV10-54, IGLV11-55, IGLV2-11, IGLV2-14, IGLV2-18, IGLV2-23, IGLV2-33, IGLV2-8, IGLV3-1, IGLV3-10, IGLV3-12, IGLV3-16, IGLV3-19, IGLV3-21, IGLV3-22, IGLV3-25, IGLV3-27, IGLV3-32, IGLV3-9, IGLV4-3, IGLV4-60, IGLV4-69, IGLV5-37, IGLV5-39, IGLV5-45, IGLV5-8, IGLV5-52, IGLV6-57, IGLV7-43, IGLV7-43, IGLV7-46, IGLV8-61, IGLV8-61 and IGLV9-49 gene families including genes and alleles thereof. The Jλ gene families include, but are not limited to, a IGLJ1, IGLJ2, IGLJ4, IGLJ5, IGLJ6 and IGLJ7 gene families including genes and alleles thereof.
In any of the methods above of generating a human combinatorial antibody library, the germline segments can be included in a user-created database, for example, to provide convenient access to such sequences. In practicing the method, the sequences of the JH, Jκ, and Jλ germline segment in the database are set forth in their correct reading frame (e.g., such as is set forth in Table 13).
The methods provided herein can further include a step after steps a) and/or b) of modifying the nucleic acid sequences encoding a VH chain or a portion thereof and/or modifying the nucleic acid sequences encoding a VL chain or a portion thereof. For example, the nucleic acid sequences can be modified by removing any internal stop codons. Generally, modification of stop codon(s) is made by making only one or two nucleotide changes to the nucleic acid sequences. The codon triplet for a stop codon can be changed to any other codon triplet encoding an amino acid. For example, the stop codon TAA can be modified to be TAT, the stop codon TAG can be modified to be TAT and the stop codon TGA can be modified to be TCA. In another example, the nucleic acid sequences can be modified by removing any internal restriction sites. The nucleotides recognized by a restriction enzyme can be modified to any other nucleotide sequence so long as the sequence is not recognized by a restriction enzyme of interest, i.e. one used in subsequent cloning steps. Generally, only one or two nucleotides changes are made. Typically, modification of restriction sites are made to maximize codon usage in E. coli. 
In the methods of generating a human combinatorial antibody library provided herein, the plurality of nucleic acid sequences encoding a VH chain include, but are not limited to, any set forth in any of SEQ ID NOS: 1059-1410, or a subset thereof. The plurality of nucleic acid sequences encoding a VL chain include, but are not limited to, any set forth in any of SEQ ID NOS: 1411-1422, 1424-1439 and 1441-1471, or a subset thereof.
In any of the methods provided herein of generating a human combinatorial antibody library, the plurality of nucleic acid sequences encoding a VH chain and/or the plurality of nucleic acid sequences encoding a VL chain can be ranked. For example, the sequences can be ranked based on sequence similarity (e.g. performed by sequence alignment or other methods known in the art and described herein). The sequence similarity between and among the plurality of different nucleic acid molecules encoding a VH chain and/or the sequence similarity between and among the plurality of different nucleic acid molecules encoding a VL chain can be determined. A subset of nucleic acid sequences encoding a VH chain and/or a VL chain can be selected (e.g. for synthesis and subsequent expression) such that the selected sequences include those that are the most similar or are the most different from other selected sequences.
In the method provided herein for generating a combinatorial antibody library, the synthesized nucleic acid sequences in the addressed libraries are contained in vectors. Hence, a vector from a first vector library containing nucleic acid sequences encoding a VH chain and a vector from a second vector library containing nucleic acid sequences encoding a VL chain are introduced into addressed cells for generation and production of antibody members of the library. The vector can further contain all or a portion of a constant region sufficient to permit association of heavy and light chains. For example, the vector can contain a CH1 and CL such that the resulting encoded antibody is a Fab. In the methods provided herein, the cells include prokaryotic or eukaryotic cells. For example, cells include E. coli cells. In the methods of generating a combinatorial antibody library provided herein, the addressed cells can be arranged in a spatial array. The spatial array includes, for example, a multi-well plate such that each individual locus of the plate corresponds to a cell that contains nucleic acid molecules encoding a different combination of a VH and a VL compared to every other cell in the addressed library of cells.
The antibody or portions thereof that are expressed in the method provided herein include a full length antibody or a fragment or portion thereof sufficient to form an antigen binding site. For example, the expressed antibody is a Fab. The methods provided herein, further can include a step of purifying the antibodies or portions thereof. The antibodies or portion thereof in the libraries include those that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure. Hence, the addressable library includes antibody members that are purified such that the purified antibodies or portions thereof are addressed, and the antibody within each address is the same antibody and is different from all other antibodies at all other addresses in the library.
Also provided herein, is an addressable combinatorial antibody library produced by the method provide herein of generating a combinatorial antibody library. Such an antibody library includes members each containing a VH chain having a sequence of amino acids selected from among any of SEQ ID NOS: 1475-836 and a VL chain having a sequence of amino acids selected from among any of SEQ ID NOS: 1827-1838, 1840-1855 and 1857-1888.
Provided herein is a method of screening a human combinatorial antibody library for binding or activity against a target protein to identify antibodies or portions thereof that bind to a target protein and/or modulate an activity of a target protein. In such methods, a human combinatorial antibody library is provided. The library includes any human combinatorial antibody library provided herein or any human combinatorial antibody library produced by the methods provided herein. In such methods, the an antibody or portion thereof in the library is contacted with a target protein and binding of the antibody or portion thereof with the target protein and/or modulation of a functional activity by an antibody or portion thereof in the library is assessed. Antibodies or portions thereof that bind to the target protein and/or modulate an activity of the target protein are identified, whereby the identified antibody or portion thereof is designated as a “HIT.” In such methods, for example, the human combinatorial antibody library is an addressable library and contacting is performed in an addressable array, such that the identity of the “Hit” is known by its address. For example, screening is performed in a spatial array, such as a microwell plate.
In the methods of screening provided herein, the target protein is a membrane-bound protein, a cell surface receptor (CSR) or a CSR ligand. The membrane-bound protein or CSR includes, but is not limited to, a cytokine receptor, a receptor kinase, a receptor phosphatase, a receptor involved in cell-cell interactions or a cellular adhesion molecule. For example, the target protein includes, but is not limited to, VEGFR-1, VEGFR-2VEGFR-3, a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-b3, IGF-R1, C-Met, TNF-R1, TNF-R2, BTLA, HVEM, LT-13R, CD20, CD3, CD25, NOTCH, DLL4, G-CSF-R, GM-CSF-R, EPO-R, a cadherin, an integrin, CD52 and CD44, a VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-α, LIGHT, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO.
In some examples, the binding of the antibody or portion thereof to the target protein is assessed. In other examples, modulation of a functional activity of the target protein by an antibody or portion thereof is assessed. The functional activity includes, but is not limited to, cellular proliferation, lymphoma apoptosis, chemotaxis, cancer cell invasion, matrigel, endothelial proliferation, tube formation and signal transduction.
In the methods of screening provided herein, binding can be assessed on cells and/or a functional activity can be assessed in a cell-based activity. In such examples, the cells express the target protein, typically on their surface as a membrane-bound or extracellular receptor, ligand or adhesion protein. For example, the cells can be transiently or stably expressed with a nucleic acid molecule encoding the target protein.
Provided herein is a method of screening that includes after identifying a “Hit”, for example, in a previous iteration of the method of screening as provided above, a second library combinatorial antibody library is provided. In such examples, the second combinatorial antibody library is based on the identified “Hit.” For example, the second library is generated by selecting VH, DH and JH human germline segments that are related by sequence similarity to the germline segments of the identified HIT(s), and combining all possible VH, DH and JH human germline segments or portions thereof in frame to generate a plurality of sequences of nucleic acid molecules each encoding a different VH chain or a portion thereof and selecting Vκ and a Jκ or Vλ and a Jλ human germline segments that are related by sequence similarity to the germline segments of the identified HIT(s), and combining all possible Vκ and a Jκ human germline segments or portions thereof, or all possible Vλ and a Jλ germline segment or portions thereof in frame to generate a plurality of sequences of nucleic acid molecule each encoding a different VL chain or a portion thereof. The portions of the VH, DH, JH, Vκ, Jκ, Vλ or Jλ are sufficient to produce an antibody containing a VH or VL or portion thereof that is sufficient to bind to an antigen. Upon combination of the germline segments, the nucleic acid molecules are synthesized to produce a first library that contains nucleic acid molecules encoding a VH chain or a portion thereof and a second library that contains nucleic acid molecules encoding a VL chain or a portion thereof. A nucleic acid molecule from the first library and from the second library is introduced into a cell and the steps are repeated a plurality of times to produce a library of cells such that each cell contains nucleic acid molecules encoding a different combination of VH and VL from every other cell in the library of cells. The cells are grown to express the antibodies in each cell, thereby producing a plurality of antibodies or portion thereof such that each produced antibody or portion thereof in the library contains a different combination of a VH and a VL chain or a sufficient portion thereof to form an antigen binding site from all other antibodies or portions thereof in the library. The antibodies are further purified to generate a second human combinatorial antibody library. In such examples, the second human combinatorial antibody library is generated such that the sequence similarity between and among related germline segments is or is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
In another example, a second combinatorial antibody library is provided that is generated by selecting a plurality of nucleic acid molecules encoding a VH chain that contain a VH, a DH or a JH human germline segment that is the same as contained in the nucleic acid molecule encoding the identified HIT and selecting a plurality of nucleic acid molecules encoding a VL chain that contains a Vκ and a Jκ or a Vλ and a Jλ human germline segment that is the same as contained in the nucleic acid molecule encoding the identified HIT. A nucleic acid molecule from the plurality of nucleic acid molecules encoding a VL and a nucleic acid molecule from the plurality of nucleic acid molecules encoding a VH are introduced into a cells and the cells are grown. This is repeated a plurality of times to produce a library of cells, whereby each cells contains nucleic acid molecules encoding a different combination of a VH and a VL chain from every other cell in the library. Upon expression of the antibodies in each cell, a plurality of antibodies or portions thereof is produced. Each antibody or portion thereof in the library contains a different combination of a VH and a VL chain or a sufficient portion thereof to form an antigen binding site from all other antibodies or portions thereof in the library. The antibodies or portions thereof are purified to generate a second combinatorial antibody library.
In additional examples, a second combinatorial library is provided based on the identified HIT(s) that is generated by introducing amino acid mutations into the “HIT” to generate a plurality of antibodies or portions thereof. Each antibody member or portion thereof in the second combinatorial antibody library differs from the identified “HIT” by one or more amino acid mutations in its primary sequence. In such examples, the amino acid mutations are in the complementarity determining regions (CDRs) of the identified HIT.
In each of the methods above, the second human combinatorial library is contacted with a target protein and binding of the antibody or portion thereof with the target protein and/or modulation of a functional activity by an antibody or portion thereof in the library is assessed. Antibodies or portions thereof that bind to the target protein and/or modulate an activity of the target protein are identified, whereby the identified antibody or portion thereof is designated as a further “HIT.” In some examples, the second combinatorial antibody library is an addressable library such that the purified antibodies or portions thereof are addressed and each purified antibody within each address is the same antibody and is different from the antibodies at all other addresses. In such examples, contacting is performed in an addressable array, such that the identity of the “Hit” is known by its address. For example, screening is performed in a spatial array, such as a microwell plate.
In any of the examples of screening provided above, the method is repeated iteratively until a further “HIT” is identified having an optimized binding affinity for a target protein and/or having an activity that is optimized against a target protein compared to previous “HITS” in earlier iterations of the method.
Provided herein is an anti-DLL4 antibody that contains a VH chain encoded by a sequence of nucleotides compiled from a VH, DH and JH germline segment and a VL chain encoded by a sequence of nucleotides compiled from a Vκ and Jκ or Vλ and Jλ germline segments. The VH germline segment is an IGHV1, and IGHV5 or an IGHV6 or genes and alleles thereof; the DH germline segment is an IGHD6, and IGHD5 or an IGHD3 or genes and alleles thereof; and the JH germline segment is an IGHJ1 or an IGHJ4 or genes and alleles thereof. The Vκ germline segment is a IGKV3 and the JK is a IGKJ1 or genes and alleles thereof; the Vλ germline segment is a IGLV8 or an IGLV5 and the Jλ germline segment is a IGLJ1 or a IGLJ4 or genes and alleles thereof. The anti-DLL4 antibodies provided herein bind DLL4 and/or modulates an activity of DLL4.
In some examples, the VH germline segment is an IGHV1-46*01, IGHV1-46*02 or an IGHV1-46*03 or an IGHV6-1*01 or an IGHV6-1*02. The DH germline segment is an IGHD6-6*01, IGHD5-18*01, IGHD3*01 or IGHD3-3*02. The JH germline segment is an IGHJ1*01, IGHJ4*01, IGHJ4*02 or IGHJ4*03. The Vκ germline segment is an IGKV3-11*01 or IGKV3-11*02. The JK germline segment is a IGKJ1*01. The Vλ germline segment is a IGLV8-61*01, IGLV8-61*02, IGLV8-61*03 or IGLV5-48*01. The Jλ germline segment that is a IGLJ1*01 or IGLJ4*01. For example, The anti-DLL4 antibody provided herein is an antibody or portion thereof containing a a VH chain encoded by a sequence of nucleotides compiled from an IGHV1-46*01, IGHD6-6*01 and IGHJ1*01 and a VL chain encoded by a sequence of nucleotides compiled from IGKV3-11*01 and IGKJ1*01 germline segments; a VH encoded by a sequence of nucleotides compiled from IGHV5-51*03, IGHD5-18*01 and IGHJ4*01 germline segments and a VL chain encoded by a sequence of nucleotides compiled from an IGLV8-61*01 and IGLJ1*01 germline segments; or a VH chain encoded by a sequence of nucleotides compiled from an IGHV6-1*01, and IGHD3-3*01 and an IGHJ4*01 germline segments and a VL chain encoded by a sequence of nucleotides compiled from an IGLV5-48*01 and a IGLJ4*01 germline segments. Anti-DLL4 antibodies provided herein include, but are not limited to, antibodies containing a VH chain having a sequence of amino acids set forth in SEQ ID NO: 1513 and a VL chain having a sequence of amino acids set forth in SEQ ID NO:1850; a VH chain having a sequence of amino acids set forth in SEQ ID NO: 1803 and a VL chain having a sequence of amino acids set forth in SEQ ID NO:1881; or a VH chain having a sequence of amino acids set forth in SEQ ID NO: 1812 and a VL chain having a sequence of amino acids set forth in SEQ ID NO:1884. For example, exemplary of an anti-DLL4 antibody provided herein is an antibody containing a VH chain having a sequence of amino acids set forth in SEQ ID NO: 1513 and a VL chain having a sequence of amino acids set forth in SEQ ID NO:1850 Antibodies that include portions of any of the above antibodies that form a sufficient antigen binding site that bind to anti-DLL4 and/or modulate an activity of DLL4 also are provided. Also provided are any antibodies that contain conservative amino acid changes in their sequence compared to any of the antibodies provided herein.
Provided herein is an anti-EpoR antibody containing a VH chain encoded by a sequence of nucleotides compiled from a VH, DH and JH germline segment and a VL chain encoded by a sequence of nucleotides compiled from a Vκ and Jκ germline segments. The VH germline segment is an IGHV1 or genes and alleles thereof. The DH germline segment is an IGHD6 or an IGHD3 or genes and alleles thereof. The JH germline segment is an IGHJ1 or genes and alleles thereof. The Vκ germline segment is an IGKV4. The Jκ is an IGKJ1. The anti-EpoR antibodies provided herein bind EpoR and/or modulate an activity of EpoR.
In some examples, the VH germline segment is an IGHV1-46*01, IGHV1-46*02 or an IGHV1-46*03. The the DH germline segment is an IGHD6-6*01, IGHD3-10*01 or IGHD3-10*02. The JH germline segment is an IGHJ1*01, IGHJ4*01, IGHJ4*02 or IGHJ4*03. The Vκ germline segment is an IGKV4-1*01. the Jκ germline segment that is a IGKJ1*01. For example, the anti-EpoR antibody provided herein contains a VH chain encoded by a sequence of nucleotides compiled from an IGHV1-46*01, IGHD3-10*01 and IGHJ4*01 and a VL chain encoded by a sequence of nucleotides compiled from IGKV4-1*01 and IGKJ1*01 germline segments or contains a VH chain encoded by a sequence of nucleotides compiled from IGHV1-46*01, IGHD6-6*01 and IGHJ1*01 germline segments and a VL chain encoded by a sequence of nucleotides compiled from an IGKV4-1*01 and IGKJ1*01 germline segments. Anti-EpoR antibodies provided herein include, but are not limited to, an antibody containing a VH chain having a sequence of amino acids set forth in SEQ ID NO: 1509 and a VL chain having a sequence of amino acids set forth in SEQ ID NO:1838; or a VH chain having a sequence of amino acids set forth in SEQ ID NO: 1513 and a VL chain having a sequence of amino acids set forth in SEQ ID NO:1838. Antibodies that include portions of any of the above antibodies that form a sufficient antigen binding site that bind to anti-EpoR and/or modulate an activity of EpoR also are provided. Also provided are any antibodies that contain conservative amino acid changes in their sequence compared to any of the antibodies provided herein.
Provided herein is an anti-ErbB2 antibody containing a VH chain encoded by a sequence of nucleotides compiled from a VH, DH and JH germline segment and a VL chain encoded by a sequence of nucleotides compiled from a Vκ and Jκ germline segments. The VH germline segment is an IGHV4 or an IGHV1 or genes and alleles thereof. the DH germline segment is an IGHD6 or an IGHD1 or genes and alleles thereof. The JH germline segment is an IGHJ1 or an IGHJ2 or genes and alleles thereof. The Vλ germline segment is a IGKV3 or IGKV4. The Jκ is an IGKJ1 or genes and alleles thereof The anti-ErbB2 antibodies provided herein bind ErbB2 and/or modulate an activity of ErbB2.
In some examples, the VH germline segment is an the VH germline segment is an IGHV1-46*01, IGHV1-46*02 or an IGHV1-46*03 or an IGHV4-31*01, IGHV4-31*02, IGHV4-31*03, IGHV4-31*04, IGHV4-31*05, IGHV4-31*06, IGHV4-31*07, IGHV4-31*08, IGHV4-31*09, IGHV4-31*10. The DH germline segment is an IGHD6-6*01 or IGHD1-26*01. The JH germline segment is an IGHJ1*01 or an IGHJ2*01. The Vκ germline segment is an IGHV3-20*01, IGHV3-20*02 or IGKV4-1*01. The Jκ germline segment that is a IGKJ1*01. For example, the anti-ErbB2 antibody provided herein a VH chain encoded by a sequence of nucleotides compiled from an IGHV4-31*02, IGHD1-26*01 and IGHJ2*01 and a VL chain encoded by a sequence of nucleotides compiled from IGKV3-20*01 and IGKJ1*01 germline segments or contains a VH encoded by a sequence of nucleotides compiled from IGHV1-46*01, IGHD6-6*01 and IGHJ1*01 germline segments and a VL chain encoded by a sequence. Anti-ErbB2 antibodies provided herein include, but are not limited to, an antibody containing a VH chain having a sequence of amino acids set forth in SEQ ID NO: 1760 and a VL chain having a sequence of amino acids set forth in SEQ ID NO:1833; or a VH chain having a sequence of amino acids set forth in SEQ ID NO: 1513 and a VL chain having a seqene of amino acids set forth in SEQ ID NO:1838. Antibodies that include portions of any of the above antibodies that form a sufficient antigen binding site that bind to anti-ErbB2 and/or modulate an activity of ErbB2 also are provided. Also provided are any antibodies that contain conservative amino acid changes in their sequence compared to any of the antibodies provided herein.
Any of the antibodies provided herein can further contain a constant region or a portion of a constant region sufficient to permit association of a heavy and light chain. For example, antibodies provided herein include Fab antibodies. In addition, any of the antibodies provided herein include antibodies having a binding affinity that is or is about 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M or 10−12M or lower. For example, any of the antibodies provided herein have a binding affinity that is or is about 1×10−9M, 2×10−9M, 3×10−9M, 4×10−9M, 5×10−9M, 6×10−9M, 7×10−9M, 8×10−9M, 9×10−9M, 1×10−10M, 2×10−10M, 3×10−10M, 4×10−10M, 5×10−10M, 6×10−10M, 7×10−10M, 8×10−10M, 9×10−10M or less.
Provided herein are methods of treatment using any of the antibodies provided herein, including any of the antibodies identified in the screening method provided herein. Such antibodies can be used to treat diseases or disorders associated with expression and/or activity of the target protein. In one example, provided herein are methods of treatment or uses of treating or formulating a medicament with any of the anti-DLL4 antibodies provided herein for treating a disease or disorder associated with expression and/or activity of DLL4. In another example, provided herein are methods of treatment or uses of treating or formulation of a medicament with any of the anti-EpoR antibodies provided herein for treating a disease or disorder associated with expression and/or activity of EpoR. In an additional example, provided herein are methods of treatment using anti-ErbB2 antibodies provided herein for treating a disease or disorder associated with expression and/or activity of ErbB2. Provided herein is a computer system or a computer readable medium that contains computer-readable instructions executable by a computer device for performing a method of combining human germline segments. The method of combinaing human germline segments includes these steps of (a) accessing a user-created in silico database of all available human antibody germline segments (VH, DH, JH, Vκ, Jκ, Vλ and Jλ); (b) applying an algorithm to generate a collection of every possible recombined full length nucleic acid sequence encoding a heavy chain (5′-VH-DH-JH-3′); (c) applying an algorithm to generate a collection of every possible recombined full length nucleic acid sequence encoding a kappa light chains (5′-Vκ-Jκ-3′) and/or every possible recombined full length nucleic acid sequence encoding a lambda light chains (5′Vλ-Jλ-3′); (d) applying an algorithm to modify nucleotides at the V-D and/or D-J joints of the nucleic acid sequences of (b) and at the V-J joints of the nucleic acid sequences of (c) so that the resulting nucleic acids sequences are in frame; (e) modifying the nucleic acid sequences of (d) to remove any inadvertently generated stop codons; (f) assigning each recombined nucleic acid sequence to a unique locus of an addressable format; and (g) generating an output file that identifies the address of each recombined nucleic acid sequences. The method executed by the computer system or computer readable medium can further include (h) after step (e) adding nucleotides at the 5′ and 3′ termini of the recombined nucleic acid sequences containing a sequence recognized by a restriction enzyme; and (i) modifying the recombined nucleic acid sequence by nucleotide replacement to remove internal nucleotides that are recognized by a restriction enzyme. The method can further modify the recombined nucleic acid sequences to optimize codon usage for bacterial expression. In some examples, the method executed by the computer system or computer readable medium can include before step (f) selecting recombined nucleic acid sequence(s) from the library of recombined nucleic acid sequences based on sequence similarities or differences and assigning only the selected sequences to a locus in an addressable format in step f).
Provided herein is a method that includes execution of computer-readable instructions for performing a method of combining human germline segments by a computer device, whereby the method includes the steps of (a) accessing a user-created in silico database of all available human antibody germline segments (VH, DH, JH, Vκ, Jκ, Vλ and Jλ); (b) applying an algorithm to generate a collection of every possible recombined full length nucleic acid sequence encoding a heavy chain (5′-VH-DH-JH-3′); (c) applying an algorithm to generate a collection of every possible recombined full length nucleic acid sequence encoding a kappa light chains (5′-Vκ-Jκ-3′) and/or every possible recombined full length nucleic acid sequence encoding a lambda light chains (5′-Vλ-Jλ-3′); (d) applying an algorithm to modify nucleotides at the V-D and/or D-J joints of the nucleic acid sequences of (b) and at the V-J joints of the nucleic acid sequences of (c) so that the resulting nucleic acids sequences are in frame; (e) modifying the nucleic acid sequences of (d) to remove any inadvertently generated stop codons; (f) assigning each recombined nucleic acid sequence to a unique locus of an addressable format; and (g) generating an output file that identifies the address of each recombined nucleic acid sequences. The method further includes DNA synthesis of the recombined nucleic acid sequences encoding a heavy chain, encoding a kappa light chain, and/or encoding a lambda light chain, or DNA synthesis of a subset of recombined nucleic acid sequences encoding a heavy chain, encoding a kappa light chain, and/or encoding a lambda light chain.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Schematic Illustration of the Method for Generating Antibody Diversity
FIG. 1 illustrates how antibody diversity is generated, including combinatorial diversity, pairing diversity, and junctional diversity.
FIG. 2: Schematic Illustration of Iterative Methods of Antibody Discovery
FIG. 2a is a depiction of the methods of 1) combining heavy and light germ line segments to generate nucleic acid sequences encoding a variable heavy and light chain; 2) synthesizing the nucleic acid molecules; 3) generating a plurality of antibodies by expression and association of the heavy and light chains; purifying the antibodies (e.g. using Piccolo and FLPC purification) and screening the antibodies for an activity (e.g. binding or a functional activity). FIG. 2b is a depiction of iterative methods to identify additional related antibodies involving repeating the method based on identified “HITS” to identify HITs that are optimized or improved for the activity.
FIG. 3: Plasmid A Vector Map
FIG. 3 is an illustrative vector map of plasmid A, provided and described in detail herein.
FIG. 4: Plasmid D Vector Map
FIG. 4 is an illustrative vector map of plasmid D, provided and described in detail herein.
FIG. 5: Plasmid C Vector Map
FIG. 5 is an illustrative vector map of plasmid C, provided and described in detail herein.
FIG. 6: Plasmid E Vector Map
FIG. 6 is an illustrative vector map of plasmid E, provided and described in detail herein.
FIG. 7: Schematic Illustration of DNA Sequence Compilation Software Modules
FIG. 7 illustrates the 10 modules that are contained within the DNA Sequence Compilation Software. As illustrated, there are four layers, including the GUI (Graphical User Interface), the GUI Controls, the Compilation rules and the NCBI tools. In addition, the associations between the modules are illustrated.
FIG. 8: Algorithm for Heavy and Light Chain Combinations
FIG. 8 is a schematic diagram of the algorithm for generating recombined nucleic acid sequences from germline segments encoding a variable heavy chain or a variable light chain.
FIG. 9: Algorithm for Sequence Compilation
FIG. 9 is a flow chart for modifying recombined nucleic acid sequences encoding a functional variable heavy chain or a variable light chain.
FIG. 10: Algorithm for Ranking Sequences
FIG. 10 is a flow chart for ranking recombined variable heavy and light chains based on diversity score and cluster information.
FIG. 11: Schematic Illustration of Sequence Database File Format
FIG. 11 illustrates the format of the sequence database file. Illustrated are exemplary sequences, including VH1-18 (SEQ ID NO:10), VH1-2 (SEQ ID NO:13), IGHD1-1*01 (SEQ ID NO:239), IGHJ1*01 (SEQ ID NO:273), AI (SEQ ID NO:330), A10 (SEQ ID NO:354), IGKJ1*01 (SEQ ID NO:356), V1-11 (SEQ ID NO:365), V1-13 (SEQ ID NO:366), IGLJ1*01 (SEQ ID NO:442), IGLJ2*01 (SEQ ID NO:443), and Mfe I restriction site (SEQ ID NO:1900).
FIG. 12: DNA Sequence Compilation Software Initial Startup Screen
FIG. 12 depicts the initial startup screen for the DNA Sequence Compilation Software. Illustrated are 5′ End sequence CCATGGCA (SEQ ID NO:1901), 5′ End sequence CCATGGCG, (SEQ ID NO:1902), 3′ End sequence CTAGC (SEQ ID NO:1903) and 3′ End sequence GTACT (SEQ ID NO:1904).
FIG. 13: DNA Sequence Compilation Software Splash Screen
FIG. 13 depicts the splash screen that appears upon application startup.
FIG. 14: DNA Sequence Compilation Software 96-well Plate Screen
FIG. 14 depicts a model 96-well plate screen containing compiled sequences. The sequence of gnl|Fabrus|V3-4_IGLJ7*01 (SEQ ID NO:1906) is illustrated in the sequence information box.
FIG. 15: DNA Sequence Compilation Software Light Chain Manual Compilation Screen
FIG. 15 depicts a model manual compilation screen, which allows the user to compile either a kappa or lambda light chain. Selected in the VL sequence box is V1-11 (SEQ ID NO:365). Selected in the JL sequence box is IGLJ1*01 (SEQ ID NO:442). The full length sequence (SEQ ID NO:1914) is depicted in the Dna sequence box.
FIG. 16: DNA Sequence Compilation Software Heavy Chain Manual Compilation Screen
FIG. 16 depicts a model manual compilation screen, which allows the user to compile a heavy chain. Selected in the VH sequence box is VH1-18 (SEQ ID NO:10). Selected in the DH sequence box is IGHD1-1*01 (SEQ ID NO:239). Selected in the JH sequence box is IGHJ1*01 (SEQ ID NO:373). The full length sequence (SEQ ID NO:1915) is depicted in the Dna sequence box.
FIG. 17: DNA Sequence Compilation Software Light Chain Auto Compilation Screen
FIG. 17 depicts a model light chain auto compilation screen. Illustrated are exemplary sequences of variable light chains set forth in SEQ ID NOS:1880-1881, 1883, 1905-1913 and 1916-1940.
FIG. 18: Light Chain BLAST Grid
FIG. 18 depicts a model BLAST grid for a light chain sequence. Illustrated are exemplary sequences of variable light chains set forth in SEQ ID NOS:1880-1881, 1883, 1905-1913, 1916-1939.
FIG. 19: DNA Sequence Compilation Software Heavy Chain Auto Compilation Screen
FIG. 19 depicts a model heavy chain auto compilation screen. Illustrated are exemplary sequences of variable heavy chains set forth in SEQ ID NOS:1556, 1760, 1769, 1821, 1941-1973.
FIG. 20: Heavy Chain BLAST Grid
FIG. 20 depicts a model of the BLAST grid for a heavy chain sequence. Illustrated are exemplary sequences of variable heavy chains set forth in SEQ ID NOS:1530, 1539, 1974-1995.



DETAILED DESCRIPTION
Outline


    
    
        A. Definitions
        B. Overview
        
            1. Methods of Generating Addressable Combinatorial Antibody Collections
            2. The Resulting Libraries
            3. Applications of the libraries
        
        
        C. Antibodies
        
            1. Antibody Polypeptides
            2. Antibody structural and functional domains
            3. Antibody Sequence and Specificity
        
        
        D. Methods of Generating Members of the Combinatorial Antibody Library
        
            1. Methods for Producing Functional Recombined Germline Variable Region
            
                a. Variable Gene Segments
                
                    i. Germline Segments
                    ii. Modified Germline Segments
                
                
                b. Choosing Germline Segments or Modified Segments Thereof
                c. Sequence Compilation
                d. Further Sequence Modification of Recombined Nucleic Acid Molecules
                
                    i. Codon Usage
                    ii. Adding or Removing Restriction Enzyme Sites
                    iii. Linkers
                    iv. Tags or detectable moieties
                    v. Mutational Diversity
                    vi. Directed Peptides
                
                
                e. Generating Variable Heavy and Light Chain Sequences and Nucleic Acid Molecules
                
                    i. Storage and Collection
                    ii. Determining Sequence Diversity of Collected Nucleic Acid Sequences
                    iii. Generating Nucleic Acid Molecules
                     a) Synthesis
                     b) Recombinant Techniques
                
                
                f. Expressing and Producing Antibodies or Portions or Fragments Thereof Resulting Members in the Library
            
            
            2. Automation
            
                a. User-Created Database
                b. Sequence Compilation
                c. Automation of protein expression and purification
            
            
        
        
        E. Libraries
        
            1. VH nucleic Acid Libraries and Vector Libraries Thereof
            2. VL nucleic acid Libraries and Vector Libraries Thereof
            3. Paired Nucleic Acid Libraries or Vector Libraries Thereof
            4. Antibody Libraries
            5. Addressable Formats
            
                a. Multiwell Plate
                b. Solid Support
            
            
            6. Other Display Methods
            
                a. Cell Display
                b. Phage Display
                c. mRNA Display and Ribosome Display
                d. DNA display
            
            
        
        
        F. Methods of Production of Antibodies
        
            1. Vectors
            2. Cells Expression Systems
            
                a. Prokaryotic Expression
                b. Yeast
                c. Insects
                d. Mammalian Cells
                e. Plants
            
            
            3. Purification
        
        
        G. Application and Uses of the libraries
        
            1. Binding Assays
            2. Functional Activity
            
                a. Differentiation
                b. Alternation of Gene Expression
                c. Cytotoxicity Activity
            
            
            3. Targets
            
                Membrane-bound proteins, receptors and ligands thereof
                
                    i. Notch and Notch ligands
                     a) Notch Proteins
                     b) DLL4
                    ii. ErbB family
                     a) Epidermal Growth Factor Receptor (EGFR)
                     b) Human Epidermal Growth Factor Receptor 2
                
                
                (HER2/neu)
                
                    iii. IGF-R1 (Insulin-like Growth Factor 1 Receptor)
                    iv. c-Met
                    v. CD20-B lymphocyte antigen
                    vii. Erythropoietin Receptor (Epo-R)
                    viii. Cadherins
                     a) P-Cadherin (P-cad/CDH3)
                    ix. CD44
                
                
            
            
            4. Iterative Screening
            5. Directed Evolution
            
                a. Random Mutagenesis
                
                    i. Saturation Mutagenesis
                    ii. Error Prone PCR
                    iii. Cell lines
                    iv. DNA shuffling/antibody chain shuffling
                    v. CDR walking
                    vi. Framework Stabilization
                
                
            
            
            6. Epitope Mapping
            7. In Vivo Assays of Identified Hits
            8. Articles of Manufacture/Kits
            9. Formulations/Administrations and uses of antibodies and polypeptides
        
        
        H. Examples
    
    


A. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
As used herein, “combinatorial library” refers to collections of compounds formed by reacting different combinations of interchangeable chemical “building blocks” to produce a collection of compounds based on permutations of the building blocks. For an antibody combinatorial library, the building blocks are the component V, D and J regions (or modified forms thereof) from which antibodies are formed. For purposes herein, the terms “library” or “collection” are used interchangeably.
As used herein, a combinatorial antibody library is a collection of antibodies (or portions thereof, such as Fabs), where the antibodies are encoded by nucleic acid molecules produced by the combination of V, D and J gene segments, particularly human V, D and J germline segments. The combinatorial libraries herein typically contain at least 50 different antibody (or antibody portions or fragment) members, typically at or about 50, 100, 500, 103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×103, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 106, 107, 108, 109, 1010, or more different members. The resulting libraries or collections of antibodies or portions thereof, can be screened for binding to a target protein or modulation of a functional activity.
As used herein, a human combinatorial antibody library is a collection of antibodies or portions thereof, whereby each member contains a VL and VH chains or a sufficient portion thereof to form an antigen binding site encoded by nucleic acid containing human germline segments produced as described herein.
As used herein, germline gene segments refer to immunoglobulin (Ig) variable (V), diversity (D) and junction (J) or constant (C) genes from the germline that encode immunoglobulin heavy or light (kappa and lambda) chains. There are multiple V, D, J and C gene segments in the germline, but gene rearrangement results in only one segment of each occurring in each functional rearranged gene. For example, a functionally rearranged heavy chain contains one V, one D and one J and a functionally rearranged light chain gene contains one V and one J. Hence, these gene segments are carried in the germ cells but cannot be transcribed and translated into heavy and light chains until they are arranged into functional genes. During B-cell differentiation in the bone marrow, these gene segments are randomly shuffled by a dynamic genetic system capable of generating more than 1010 specificities. For purposes herein, the gene segments are rearranged in vitro by combination or compilation of the individual germline segments.
Reference to a variable germline segment herein refers to V, D and J groups, subgroups, genes or alleles thereof. Gene segment sequences are accessible from known database (e.g., National Center for Biotechnology Information (NCBI), the international ImMunoGeneTics information system® (IMGT), the Kabat database and the Tomlinson's VBase database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin et al., Bioinformatics Tools for Antibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH (2007), pp. 104-107). Tables 3-5 list exemplary human variable germline segments. Sequences of exemplary VH, DH, JH, Vκ, Jκ, Vλ and or Jλ, germline segments are set forth in SEQ ID NOS: 10-451 and 868. For purposes herein, a germline segment includes modified sequences thereof, that are modified in accord with the rules of sequence compilation provided herein to permit practice of the method. For example, germline gene segments include those that contain one amino acid deletion or insertion at the 5′ or 3′ end compared to any of the sequences of nucleotides set forth in SEQ ID NOS:10-451, 868.
As used herein, modified form with reference to a germline segment refers to a sequence of nucleotides that is substantially the same as the sequence of nucleotides of a human germline segment (e.g. a VH, DH, JH, Vκ, Jκ, Vλ and Jλ) except that the sequence of nucleotides contains one or a few nucleotide differences, for example, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 nucleotide differences compared to the corresponding sequence in a germline segment sequence. In some instances, modified germline sequences are modified in accord with the rules herein to remove stop codons, restriction enzyme sites, or to add or delete nucleotides to maintain reading frames.
As used herein, inverted sequence with reference to nucleotides of a germline segment means that the gene segment has a sequence of nucleotides that is the reverse complement of a reference sequence of nucleotides. For purposes herein, the reference sequence of nucleotides is a germline segment, typically a DH germline segment.
As used herein, “compilation,” “compile,” “combine,” “combination,” “rearrange,” “rearrangement,” or other similar terms or grammatical variations thereof refers to the process by which germline segments are ordered or assembled into nucleic acid sequences representing genes. For example, variable heavy chain germline segments are assembled such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment, thereby resulting in a nucleic acid sequence encoding a VH chain. Variable light chain germline segments are assembled such that the VL segment is 5′ to the JL segment, thereby resulting in a nucleic acid sequence encoding a VL chain. A constant gene segment or segments also can be assembled onto the 3′ end of a nucleic acid encoding a VH or VL chain.
As used herein, “linked,” or “linkage” or other grammatical variations thereof with reference to germline segments refers to the joining of germline segments. Linkage can be direct or indirect. Germline segments can be linked directly without additional nucleotides between segments, or additional nucleotides can be added to render the entire segment in-frame, or nucleotides can be deleted to render the resulting segment in-frame. It is understood that the choice of linker nucleotides is made such that the resulting nucleic acid molecule is in-frame and encodes a functional and productive antibody.
As used herein, “in-frame” or “linked in-frame” with reference to linkage of human germline segments means that there are insertions and/or deletions in the nucleotide germline segments at the joined junctions to render the resulting nucleic acid molecule in-frame with the 5′ start codon (ATG), thereby producing a “productive” or functional full-length polypeptide. The choice of nucleotides inserted or deleted from germline segments, particularly at joints joining various VD, DJ and VJ segments, is in accord with the rules provided in the method herein for V(D)J joint generation. For example, germline segments are assembled such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment. At the junction joining the VH and the DH and at the junction joining the DH and JH segments, nucleotides can be inserted or deleted from the individual VH, DH or JH segments, such that the resulting nucleic acid molecule containing the joined VDJ segments are in-frame with the 5′ start codon (ATG).
As used herein, a “functional antibody” or “productive antibody” with reference to a nucleic acid encoding an antibody or portion thereof refers to an antibody or portion thereof, such as Fab, that is encoded by the nucleic acid molecule produced by the methods as described herein. In a functional or productive antibody, the V(D)J germline segments are compiled (i.e. rearranged) such that the encoded antibody or portion thereof is not truncated and/or the amino acid sequence is not out of frame. This means that the nucleic acid molecule does not contain internal stop codons that result in the protein translation machinery terminating protein assembly prematurely.
As used herein, a portion of an antibody includes sufficient amino acids to form an antigen binding site.
As used herein, a reading frame refers to a contiguous and non-overlapping set of three-nucleotide codons in DNA or RNA. Because three codons encode one amino acid, there exist three possible reading frames for given nucleotide sequence, reading frames 1, 2 or 3. For example, the sequence ACTGGTCA will be ACT GGT CA for reading frame 1, A CTG GTC A for reading frame 2 and AC TGG TCA for reading frame 3. Generally for practice of the method described herein, nucleic acid sequences are combined so that the V sequence has reading frame 1.
As used herein, a stop codon is used to refer to a three-nucleotide sequence that signals a halt in protein synthesis during translation, or any sequence encoding that sequence (e.g. a DNA sequence encoding an RNA stop codon sequence), including the amber stop codon (UAG or TAG)), the ochre stop codon (UAA or TAA)) and the opal stop codon (UGA or TGA)). It is not necessary that the stop codon signal termination of translation in every cell or in every organism. For example, in suppressor strain host cells, such as amber suppressor strains and partial amber suppressor strains, translation proceeds through one or more stop codon (e.g. the amber stop codon for an amber suppressor strain), at least some of the time.
As used herein, reference to a variable heavy (VH) chain or a variable light (VL) chain (also termed VH domain or VL domain) refers to the polypeptide chains that make up the variable domain of an antibody. For purposes herein, heavy chain germline segments are designated as VH, DH and JH, and compilation thereof results in a nucleic acid encoding a VH chain. Light chain germline segments are designated as VL or JL, and include kappa and lambda light chains (Vκ and Jκ; Vλ and Jλ) and compilation thereof results in a nucleic acid encoding a VL chain. It is understood that a light chain chain is either a kappa or lambda light chain, but does not include a kappa/lambda combination by virtue of compilation of a Vκ and Jλ.
As used herein, a “degenerate codon” refers to three-nucleotide codons that specifies the same amino acid as a codon in a parent nucleotide sequence. One of skill in the art is familiar with degeneracy of the genetic code and can identify degenerate codons.
As used herein, a “group” with reference to a germline segment refers to a core coding region from an immunoglobulin, i.e. a variable (V) gene, diversity (D) gene, joining (J) gene or constant (C) gene encoding a heavy or light chain. Exemplary of germline segment groups include VH, DH, JH, Vκ, Jκ, Vλ and Jλ.
As used herein, a “subgroup” with reference to a germline segment refers to a set of sequences that are defined by nucleotide sequence similarity or identity. Generally, a subgroup is a set of genes that belong to the same group [V, D, J or C], in a given species, and that share at least 75% identity at the nucleotide level. Subgroups are classified based on IMGT nomenclature (imgt.cines.fr; see e.g., Lefranc et al. (2008) Briefings in Bioinformatics, 9:263-275). Generally, a subgroup represent a multigene family.
As used herein, an allele of a gene refer to germline sequences that have sequence polymorphism due to one or more nucleotide differences in the coding region compared to a reference gene sequence (e.g. substitutions, insertions or deletions). Thus, IG sequences that belong to the same subgroup can be highly similar in their coding sequence, but nonetheless exhibit high polymorphism. Subgroup alleles are classified based on IMGT nomenclature with an asterisk(*) followed by a two figure number. Exemplary allelic subgroup germline segments for VH, DH, JH, Vκ, Jκ, Vλ and Jλ are set forth in Tables 3-5.
As used herein, a “family” with reference to a germline segment refers to sets of germline segment sequences that are defined by amino acid sequence similarity or identity. Generally, a germline family includes all alleles of a gene.
As used herein, a “segment designated DH ” refers to any sequence of nucleotides of at least or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides in length. The sequence of nucleotides is sufficient to code for part of the CDR3 region of the VH chain.
As used herein, reference to a V, D or J gene segment “derived from a germline segment” refers to the corresponding nucleotides in a VH or VL nucleic acid sequence, that by recombination events, derived from a V, D or J germline gene.
As used herein, reference to a V region, D region or J region in an antibody or portion or fragment thereof refers to amino acids encoded by nucleotides that, by recombination events, derive from a corresponding V, D or J germline segment gene.
As used herein, “diversity” with respect to members in a collection refers to the number of unique members in a collection. Hence, diversity refers to the number of different amino acid sequences or nucleic acid sequences, respectively, among the analogous polypeptide members of that collection. For example, a collection of polynucleotides having a diversity of 104 contains 104 different nucleic acid sequences among the analogous polynucleotide members. In one example, the provided collections of polynucleotides and/or polypeptides have diversities of at least at or about 102, 103, 104, 105, 106, 107, 108, 109, 1010 or more.
As used herein, “a diversity ratio” refers to a ratio of the number of different members in the library over the number of total members of the library. Thus, a library with a larger diversity ratio than another library contains more different members per total members, and thus more diversity per total members. The provided libraries include libraries having high diversity ratios, such as diversity ratios approaching 1, such as, for example, at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.
As used herein, “sequence diversity” refers to a representation of nucleic acid sequence similarity and is determined using sequence alignments, diversity scores, and/or sequence clustering. Any two sequences can be aligned by laying the sequences side-by-side and analyzing differences within nucleotides at every position along the length of the sequences. Sequence alignment can be assessed in silico using Basic Local Alignment Search Tool (BLAST), an NCBI tool for comparing nucleic acid and/or protein sequences. The use of BLAST for sequence alignment is well known to one of skill in the art. The Blast search algorithm compares two sequences and calculates the statistical significance of each match (a Blast score). Sequences that are most similar to each other will have a high Blast score, whereas sequences that are most varied will have a low Blast score.
As used herein, antibody refers to immunoglobulins and immunoglobulin portions, whether natural or partially or wholly synthetic, such as recombinantly, produced, including any portion thereof containing at least a portion of the variable region of the immunoglobulin molecule that is sufficient to form an antigen binding site. Hence, an antibody or portion thereof includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding site. For example, an antibody refers to an antibody that contains two heavy chains (which can be denoted H and H′) and two light chains (which can be denoted L and L′), where each heavy chain can be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen binding site (e.g. heavy chains include, but are not limited to, VH, chains VH-CH1 chains and VH-CH1-CH2-CH3 chains), and each light chain can be a full-length light chain or a portion thereof sufficient to form an antigen binding site (e.g. light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H′) pairs with one light chain (L and L′, respectively). Typically, antibodies minimally include all or at least a portion of the variable heavy (VH ) chain and/or the variable light (VL) chain. The antibody also can include all or a portion of the constant region.
For purposes herein, the term antibody includes full-length antibodies and portions thereof including antibody fragments, such as, but not limited to, Fab, Fab′, F(ab′)2, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments Fab fragments, Fd fragments and scFv fragments. Other known fragments include, but are not limited to, scFab fragments (Hust et al., BMC Biotechnology (2007), 7:14). Antibodies include members of any immunoglobulin class, including IgG, IgM, IgA, IgD and IgE.
As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g. VH -CH1-CH2-CH3 or VH -CH1-CH2-CH3- CH4) and two full-length light chains (VL-CL) and hinge regions, such as human antibodies produced by antibody secreting B cells and antibodies with the same domains that are produced synthetically.
As used herein, antibody fragment or antibody portion refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of the antibody sufficient to form an antigen binding site (e.g. one or more CDRs) and thus retains the a binding specificity and/or an activity of the full-length antibody; antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically, e.g. recombinantly produced derivatives. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment can include multiple chains linked together, such as by disulfide bridges and/or by peptide linkers. An antibody fragment generally contains at least about 50 amino acids and typically at least 200 amino acids.
As used herein, an Fv antibody fragment is composed of one variable heavy domain (VH) and one variable light (VL) domain linked by noncovalent interactions.
As used herein, a dsFv refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-VL pair.
As used herein, an Fd fragment is a fragment of an antibody containing a variable domain (VH) and one constant region domain (CH1) of an antibody heavy chain.
As used herein, “Fab fragment” is an antibody fragment that contains the portion of the full-length antibody that results from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure that is produced synthetically, e.g. recombinantly. A Fab fragment contains a light chain (containing a VL and CL portion) and another chain containing a variable domain of a heavy chain (VH) and one constant region domain portion of the heavy chain (CH1); it can be recombinantly produced.
As used herein, a F(ab′)2 fragment is an antibody fragment that results from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a synthetically, e.g. recombinantly, produced antibody having the same structure. The F(ab')2 fragment contains two Fab fragments but where each heavy chain portion contains an additional few amino acids, including cysteine residues that form disulfide linkages joining the two fragments; it can be recombinantly produced.
A Fab′ fragment is a fragment containing one half (one heavy chain and one light chain) of the F(ab′)2 fragment.
As used herein, an Fd′ fragment is a fragment of an antibody containing one heavy chain portion of a F(ab′)2 fragment.
As used herein, an Fv′ fragment is a fragment containing only the VH and VL domains of an antibody molecule.
As used herein, a scFv fragment refers to an antibody fragment that contains a variable light chain (VL) and variable heavy chain (VH), covalently connected by a polypeptide linker in any order. The linker is of a length such that the two variable domains are bridged without substantial interference. Exemplary linkers are (Gly-Ser)n residues with some Glu or Lys residues dispersed throughout to increase solubility.
As used herein, diabodies are dimeric scFv; diabodies typically have shorter peptide linkers than scFvs, and they preferentially dimerize.
As used herein, hsFv refers to antibody fragments in which the constant domains normally present in a Fab fragment have been substituted with a heterodimeric coiled-coil domain (see, e.g., Arndt et al. (2001) J Mol Biol. 7:312:221-228).
As used herein, a polypeptide domain is a part of a polypeptide (a sequence of three or more, generally 5 or 7 or more amino acids) that is a structurally and/or functionally distinguishable or definable. Exemplary of a polypeptide domain is a part of the polypeptide that can form an independently folded structure within a polypeptide made up of one or more structural motifs (e.g. combinations of alpha helices and/or beta strands connected by loop regions) and/or that is recognized by a particular functional activity, such as enzymatic activity or antigen binding. A polypeptide can have one, typically more than one, distinct domains. For example, the polypeptide can have one or more structural domains and one or more functional domains. A single polypeptide domain can be distinguished based on structure and function. A domain can encompass a contiguous linear sequence of amino acids. Alternatively, a domain can encompass a plurality of non-contiguous amino acid portions, which are non-contiguous along the linear sequence of amino acids of the polypeptide. Typically, a polypeptide contains a plurality of domains. For example, each heavy chain and each light chain of an antibody molecule contains a plurality of immunoglobulin (Ig) domains, each about 110 amino acids in length.
As used herein, an Ig domain is a domain, recognized as such by those in the art, that is distinguished by a structure, called the Immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each containing anti-parallel beta strands of amino acids connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond. Individual immunoglobulin domains within an antibody chain further can be distinguished based on function. For example, a light chain contains one variable region domain (VL) and one constant region domain (CL), while a heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each VL, CL, VH, and CH domain is an example of an immunoglobulin domain.
As used herein, a “variable domain” with reference to an antibody is a specific Ig domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies. Each light chain and each heavy chain has one variable region domain (VL, and, VH). The variable domains provide antigen specificity, and thus are responsible for antigen recognition. Each variable region contains CDRs that are part of the antigen binding site domain and framework regions (FRs).
As used herein, “hypervariable region,” “HV,” “complementarity-determining region” and “CDR” and “antibody CDR” are used interchangeably to refer to one of a plurality of portions within each variable region that together form an antigen binding site of an antibody. Each variable region domain contains three CDRs, named CDR1, CDR2, and CDR3. The three CDRs are non-contiguous along the linear amino acid sequence, but are proximate in the folded polypeptide. The CDRs are located within the loops that join the parallel strands of the beta sheets of the variable domain.
As used herein, framework regions (FRs) are the domains within the antibody variable region domains that are located within the beta sheets; the FR regions are comparatively more conserved, in terms of their amino acid sequences, than the hypervariable regions.
As used herein, a constant region domain is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved among antibodies than the variable region domain. Each light chain has a single light chain constant region (CL) domain and each heavy chain contains one or more heavy chain constant region (CH) domains, which include, CH1, CH2, CH3 and CH4. Full-length IgA, IgD and IgG isotypes contain CH1, CH2 CH3 and a hinge region, while IgE and IgM contain CH1, CH2 CH3 and CH4. CH1 and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigen and rotation of the antibody arms. Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g. through interactions with various cells, biomolecules and tissues.
As used herein, humanized antibodies refer to antibodies that are modified to include “human” sequences of amino acids so that administration to a human does not provoke an immune response. Methods for preparation of such antibodies are known. For example, the antibody in which the amino acid composition of the non-variable regions can be based on human antibodies. Computer programs have been designed to identify such regions.
As used herein, “antigen-binding site” refers to the interface formed by one or more complementary determining regions (CDRs; also called hypervariable region). Each antigen binding site contains three CDRs from the heavy chain variable region and three CDRs from the light chain variable region. An antibody molecule has two antigen combining sites, each containing portions of a heavy chain variable region and portions of a light chain variable region. The antigen combining sites can contain other portions of the variable region domains in addition to the CDRs.
As used herein, reference to an “antibody or portion thereof that is sufficient to form an antigen binding site” means that the antibody or portion thereof contains at least 1 or 2, typically 3, 4, 5 or all 6 CDRs of the VH and VL sufficient to retain at least a portion of the binding specificity of the corresponding full-length antibody containing all 6 CDRs. Generally, a sufficient antigen binding site at least requires CDR3 of the heavy chain (CDRH3). It typically further requires the CDR3 of the light chain (CDRL3). As described herein, one of skill in the art knows and can identify the CDRs based on kabat or Chothia numbering (see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). For example, based on Kabat numbering, CDR-LI corresponds to residues L24-L34; CDR-L2 corresponds to residues L50-L56; CDR-L3 corresponds to residues L89-L97; CDR-H1 corresponds to residues H31-H35, 35a or 35b depending on the length; CDR-H2 corresponds to residues HSO-H65; and CDR-H3 corresponds to residues H95-H 102.
As used herein, a “peptide mimetic” is a peptide that mimics the activity of a polypeptide. For example, an erythropoietin (EPO) peptide mimetic is a peptide that mimics the activity of Epo, such as for binding and activation of the EPO receptor.
As used herein, an optimized antibody refers to an antibody, or portion thereof, that has an improved binding affinity for a target protein and/or an improved functional activity compared to a reference antibody. Typically, the antibody is optimized by virtue of one or more amino acid modifications (amino acid deletion, replacement or insertion) compared to a parent antibody not containing the one or more amino acid modifications. Generally, an activity or binding affinity is increased by at or about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more compared to an activity or binding affininity of the parent antibody (e.g. germline Hit not containing the modification(s)).
As used herein, corresponding with reference to corresponding residues, for example “amino acid residues corresponding to”, referes to residues compared among or between two polypeptides that are related sequences (e.g. allelic variants, genes of the same family, species variants). One of skill in the art can readily identify residues that correspond between or among polypeptides. For example, by aligning the sequence of regions encoded by germline segments, one of skill in the art can identify corresponding residues, using conserved and identical amino acids as guides. One of skill in the art can manually align a sequence or can use any of the numerous alignment programs available (for example, BLAST). Hence, an amino acid residues or positions that correspond to each other are those residues that are determined to correspond to one another based on sequence and/or structural alignments with a specified reference polypeptide.
As used herein, a consensus sequence is a sequence containing residues that are the most frequently occurring residues at each position when a plurality of related sequences (e.g. allelic variants, genes of the same family, species variants) are aligned. Hence a consensus sequence represents the residues that are the most abundant in the alignment at each position. For purposes herein, for example, germline sequences, or portions thereof, can be aligned to generate a consensus germline sequence.
As used herein, a locus in a library refers to a location or position, that can contain a member or members of library. The position does not have to be a physical position. For example, if the collection is provided as an array on a solid support, the support contains loci that can or do present members of the array.
As used herein, an address refers to a unique identifier for each locus in a collection whereby an addressed member (e.g. an antibody) can be identified. An addressed moiety is one that can be identified by virtue of its locus or location. Addressing can be effected by position on a surface, such as a well of a microplate. For example, an address for a protein in a microwell plate that is F9 means that the protein is located in row F, column 9 of the microwell plate. Addressing also can be effected by other identifiers, such as a tag encoded with a bar code or other symbology, a chemical tag, an electronic, such RF tag, a color-coded tag or other such identifier.
As used herein, an array refers to a collection of elements, such as antibodies, containing three or more members.
As used herein, a “spatial array” is an array where members are separated or occupy a distinct space in an array. Hence, spatial arrays are a type of addressable array. Examples of spatial arrays include microtiter plates where each well of a plate is an address in the array. Spacial arrays include any arrangement wherein a plurality of different molecules, e.g., polypeptides, are held, presented, positioned, situated, or supported. Arrays can include microtiter plates, such as 48-well, 96-well, 144-well, 192-well, 240-well, 288-well, 336-well, 384-well, 432-well, 480-well, 576-well, 672-well, 768-well, 864-well, 960-well, 1056-well, 1152-well, 1248-well, 1344-well, 1440-well, or 1536-well plates, tubes, slides, chips, flasks, or any other suitable laboratory apparatus. Furthermore, arrays can also include a plurality of sub-arrays. A plurality of sub-arrays encompasses an array where more than one arrangement is used to position the polypeptides. For example, multiple 96-well plates can constitute a plurality of sub-arrays and a single array.
As used herein, an addressable library is a collection of molecules such as nucleic acid molecules or protein agents, such as antibodies, in which each member of the collection is identifiable by virtue of its address.
As used herein, an addressable array is one in which the members of the array are identifiable by their address, the position in a spatial array, such as a well of a microtiter plate, or on a solid phase support, or by virtue of an identifiable or detectable label, such as by color, fluorescence, electronic signal (i.e. RF, microwave or other frequency that does not substantially alter the interaction of the molecules of interest), bar code or other symbology, chemical or other such label. Hence, in general the members of the array are located at identifiable loci on the surface of a solid phase or directly or indirectly linked to or otherwise associated with the identifiable label, such as affixed to a microsphere or other particulate support (herein referred to as beads) and suspended in solution or spread out on a surface.
As used herein, “an addressable combinatorial antibody library” refers to a collection of antibodies in which member antibodies are identifiable and all antibodies with the same identifier, such as position in a spatial array or on a solid support, or a chemical or RF tag, bind to the same antigen, and generally are substantially the same in amino acid sequence. For purposes herein, reference to an “addressable arrayed combinatorial antibody library” means that the antibody members are addressed in an array.
As used herein, a support (also referred to as a matrix support, a matrix, an insoluble support or solid support) refers to any solid or semisolid or insoluble support to which a molecule of interest, typically a biological molecule, organic molecule or biospecific ligand is linked or contacted. Such materials include any materials that are used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, such as, but are not limited to: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications. The matrix herein can be particulate or can be in the form of a continuous surface, such as a microtiter dish or well, a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, or other such materials. When particulate, typically the particles have at least one dimension in the 5-10 mm range or smaller. Such particles, referred collectively herein as “beads”, are often, but not necessarily, spherical. Such reference, however, does not constrain the geometry of the matrix, which can be any shape, including random shapes, needles, fibers, and elongated. Roughly spherical “beads”, particularly microspheres that can be used in the liquid phase, also are contemplated. The “beads” can include additional components, such as magnetic or paramagnetic particles (see, e.g., Dynabeads® (Dynal, Oslo, Norway)) for separation using magnets, as long as the additional components do not interfere with the methods and analyses herein.
As used herein, matrix or support particles refers to matrix materials that are in the form of discrete particles. The particles have any shape and dimensions, but typically have at least one dimension that is 100 mm or less, 50 mm or less, 10 mm or less, 1 mm or less, 100 μm or less, 50 μm or less and typically have a size that is 100 mm3 or less, 50 mm3 or less, 10 mm3 or less, and 1 mm3 or less, 100 μmm3 or less and can be on the order of cubic microns. Such particles are collectively called “beads.”
As used herein, in silico refers to research and experiments performed using a computer. In silico methods include, but are not limited to, molecular modeling studies, biomolecular docking experiments, and virtual representations of molecular structures and/or processes, such as molecular interactions. For purposes herein, the antibody members of a library can be designed using a computer program that selects component V, D and J germline segments from among those input into the computer and joins them in-frame to output a list of nucleic acid molecules for synthesis. Thus, the recombination of the components of the antibodies in the collections or libraries provided herein, can be performed in silico by combining the nucleotide sequences of each building block in accord with software that contains rules for doing so. The process can be performed manually without a computer, but the computer provides the convenience of speed.
As used herein, a database refers to a collection of data items. For purposes herein, reference to a database is typically with reference to antibody databases, which provide a collection of sequence and structure information for antibody genes and sequences. Exemplary antibody databases include, but are not limited to, IMGT®, the international ImMunoGeneTics information system (imgt.cines.fr; see e.g., Lefranc et al. (2008) Briefings in Bioinformatics, 9:263-275), National Center for Biotechnology Information (NCBI), the Kabat database and the Tomlinson's VBase database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin et al., Bioinformatics Tools for Antibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH (2007), pp. 104-107). A database also can be created by a user to include any desired sequences. The database can be created such that the sequences are inputted in a desired format (e.g., in a particular reading frame; lacking stop codons; lacking signal sequences). The database also can be created to include sequences in addition to antibody sequences.
As used herein, “a computer-based system” refers to the hardware, software, and data storage media and methods used to recombine germline segments. The minimum hardware of the computer-based systems provided herein include a central processing unit (CPU), input mean, output means and data storage means. A skilled artisan can select a suitable computer-based systems for use in the methods and systems provided herein.
As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to array image data. The choice of the data storage structure can generally be based on the media and platforms chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the array image information on computer readable medium. The image information can be represented in a word processing text file, formatted in commercially-available software such as MICROSOFT Word®, graphics files or represented in the form of an ASCII file, stored in a database application, such as DB2®, Sybase® and Oracle®. A skilled artisan can adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the information or instructions as described herein.
As used herein, “screening” refers to identification or selection of an antibody or portion thereof from a collection or libriary of antibodies and/or portions thereof, based on determination of the activity or property of an antibody or portion thereof. Screening can be performed in any of a variety of ways, including, for example, by assays assessing direct binding (e.g. binding affinity) of the antibody to a target protein or by functional assays assessing modulation of an activity of a target protein.
As used herein, activity towards a target protein refers to binding specificity and/or modulation of a functional activity of a target protein, or other measurements that reflects the activity of an antibody or portion thereof towards a target protein.
As used herein the term assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the binding of an antibody or portion thereof with a target protein and/or modulation of an activity of a target protein by an antibody or portion thereof, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the binding or activity. Assessment can be direct or indirect. For example, binding can be determined by directly labeling of an antibody or portion thereof with a detectable label and/or by using a secondary antibody that itself is labeled. In addition, functional activities can be determined using any of a variety of assays known to one of skill in the art, for example, proliferation, cytotoxicity and others as described herein, and comparing the activity of the target protein in the presence versus the absence of an antibody or portion thereof.
As used herein, a “target protein” refers to candidate proteins or peptides that are specifically recognized by an antibody or portion thereof and/or whose activity is modulated by an antibody or protion thereof. A target protein includes any peptide or protein that contains an epitope for antibody recognition. Target proteins include proteins involved in the etiology of a disease or disorder by virtue of expression or activity. Exemplary target proteins are described herein.
As used herein, “Hit” refers to an antibody or portion thereof identified, recognized or selected as having an activity in a screening assay.
As used herein, “iterative” with respect to screening means that the screening is repeated a plurality of times, such as 2, 3, 4, 5 or more times, until a “Hit” is identified whose activity is optimized or improved compared to prior iterations.
As used herein, “high-throughput” refers to a large-scale method or process that permits manipulation of large numbers of molecules or compounds, generally tens to hundred to thousands of compounds. For example, methods of purification and screening can be rendered high-throughput. High-throughput methods can be performed manually. Generally, however, high-throughput methods involve automation, robotics or software.
As used herein, “structure/activity relationship (SAR)” refers to the relationship between structure and function of a molecule. For purposes herein, structure is with reference to sequence, for example, a sequence of nucleotides encoding an antibody. By virtue of addressing library members, the identify of each antibody by its sequence is known based on its address. Hence, structure is known and can be correlated to a particular activity. Hence, SAR can be used to assess the affects of changes in structure on an activity.
As used herein, “functional activity” refer to activities of a polypeptide (e.g. target protein) or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity (ability to bind to or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, the ability to specifically bind to a receptor or ligand for the polypeptide and signaling and downstream effector functions. For purposes herein, modulation (i.e. activation or inhibition) of a functional activity of a polypeptide by an antibody or portion thereof in the libraries herein means that a functional activity of the polypeptide is changed or altered in the presence of the antibody compared to the absence of the antibody or portion thereof.
As used herein, “modulate” or “modulation” and other various grammatical forms thereof with reference to the effect of an antibody or portion thereof on the functional activity of a target protein refers to increased activity such as induction or potentiation of activity, as well as inhibition of one or more activities of the target protein. Hence, modulation can include an increase in the activity (i.e., up-regulation or agonist activity) a decrease in activity (i.e., down-regulation or inhibition) or any other alteration in an activity (such as a change in periodicity, frequency, duration, kinetics or other parameter) . Modulation can be context dependent and typically modulation is compared to a designated state, for example, the wildtype protein, the protein in a constitutive state, or the protein as expressed in a designated cell type or condition. The functional activity of a target protein by an antibody or portion thereof can be modulated by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the activity of the target protein in the abasence of the antibody or portion thereof.
As used herein, “agonist” refers to an antibody or portion thereof that modulates signal transduction or other functional activity of a receptor by potentiating, inducing or otherwise enhancing the signal transduction activity or other functional activity of a receptor. Agonists can modulate signal transduction or other functional activity when used alone or can alter signal transduction or other functional activity in the presence of the natural ligand of the receptor or other receptor stimulator to enhance signaling by the receptor compared to the ligand alone.
As used herein, “antagonist” refers to an antibody or portion thereof that modulates signal transduction or other functional activity of a receptor by blocking or decreasing the signal transduction activity or other functional activity of a receptor
As used herein, a label is a detectable marker that can be attached or linked directly or indirectly to a molecule or associated therewith. The detection method can be any method known in the art.
As used herein, binding activity refer to characteristics of a molecule, e.g. a polypeptide, relating to whether or not, and how, it binds one or more binding partners. Binding activities include ability to bind the binding partner(s), the affinity with which it binds to the binding partner (e.g. high affinity), the avidity with which it binds to the binding partner, the strength of the bond with the binding partner and specificity for binding with the binding partner.
As used herein, “affinity” or “binding affinity” refers to the strength with which an antibody molecule or portion thereof binds to an epitope on a target protein or antigen. Affinity is often measured by equilibrium association constant (KA) or equilibrium dissociation constant (KD). Low-affinity antibody-antigen interaction is weak, and the molecules tend to dissociate rapidly, while high affinity antibody-antigen binding is strong and the molecules remain bound for a longer amount of time. A high antibody affinity means that the antibody specifically binds to a target protein with an equilibrium association constant (KA) of greater than or equal to about 106M−1, greater than or equal to about 107M−1, greater than or equal to about 108M−1, or greater than or equal to about 109M−1, 1010M−1, 1011M−1 or 1012M−1. Antibodies also can be characterized by an equilibrium dissociation constant (KD), for example, 10−4M, 10−8M, 10−8M, 10−7M, 10−8M, 10−10M, 10−11M or 10−12M or lower. Generally, antibodies having a nanomolar or sub-nanomolar dissociaton constant are deemed to be high affinity antibodies. Such affinities can be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity data can be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. Scl., 51:660 (1949).
As used herein, “epitope” refers to the localized region on the surface of an antigen or protein that is recognized by an antibody. Peptide epitopes include those that are continuous epitopes or discontinuous epitopes. An epitope is generally determined by the three dimensional structure of a protein as opposed to the linear amino acid sequence.
As used herein, “epitope mapping” is the process of identification of the molecular determinants for antibody-antigen recognition.
As used herein, Basic Local Alignment Search Tool (BLAST) is a search algorithm developed by Altschul et al. (1990) to separately search protein or DNA databases, for example, based on sequence identity. For example, blastn is a program that compares a nucleotide query sequence against a nucleotide sequence database (e.g. GenBank). BlastP is a program that compares an amino acid query sequence against a protein sequence database.
As used herein, a BLAST bit score is a value calculated from the number of gaps and substitutions associated with each aligned sequence. The higher the score, the more significant the alignment.
As used herein, a human protein is one encoded by a nucleic acid molecule, such as DNA, present in the genome of a human, including all allelic variants and conservative variations thereof. A variant or modification of a protein is a human protein if the modification is based on the wildtype or prominent sequence of a human protein.
As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides. The residues are those 20 a-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.
As used herein, non-naturally occurring amino acids refer to amino acids that are not genetically encoded. For example, a non-natural amino acid is an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids. Exemplary non-natural amino acids are known to those of skill in the art.
As used herein, nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long.
As used herein, a peptide refers to a polypeptide that is from 2 to 40 amino acids in length. As used herein, the amino acids which occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.
As used herein, an “amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids and amino acid analogs (i.e., amino acids wherein the α-carbon has a side chain).
As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), and adopted 37 C.F.R. '§§1.821-1.822, abbreviations for amino acids residues are shown in Table 1:







TABLE 1







Table of Correspondence








SYMBOL










1-Letter
3-Letter
AMINO ACID





Y
Tyr
Tyrosine


G
Gly
Glycine


F
Phe
Phenylalanine


M
Met
Methionine


A
Ala
Alanine


S
Ser
Serine


I
Ile
Isoleucine


L
Leu
Leucine


T
Thr
Threonine


V
Val
Valine


P
Pro
Proline


K
Lys
Lysine


H
His
Histidine


Q
Gln
Glutamine


E
Glu
Glutamic acid


Z
Glx
Glu and/or Gln


W
Trp
Tryptophan


R
Arg
Arginine


D
Asp
Aspartic acid


N
Asn
Asparagine


B
Asx
Asn and/or Asp


C
Cys
Cysteine


X
Xaa
Unknown or other









It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH. The abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726). Each naturally occurring L-amino acid is identified by the standard three letter code (or single letter code) or the standard three letter code (or single letter code) with the prefix “L-”; the prefix “D-” indicates that the stereoisomeric form of the amino acid is D.
As used herein, an isokinetic mixture is one in which the molar ratios of amino acids has been adjusted based on their reported reaction rates (see, e.g., Ostresh et al., (1994) Biopolymers 34:1681).
As used herein, modification is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.
As used herein, suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p. 224). Such substitutions can be made in accordance with those set forth in TABLE 2 as follows:










TABLE 2







Original residue
Exemplary conservative substitution









Ala (A)
Gly; Ser



Arg (R)
Lys



Asn (N)
Gln; His



Cys (C)
Ser



Gln (Q)
Asn



Glu (E)
Asp



Gly (G)
Ala; Pro



His (H)
Asn; Gln



Ile (I)
Leu; Val



Leu (L)
Ile; Val



Lys (K)
Arg; Gln; Glu



Met (M)
Leu; Tyr; Ile



Phe (F)
Met; Leu; Tyr



Ser (S)
Thr



Thr (T)
Ser



Trp (W)
Tyr



Tyr (Y)
Trp; Phe



Val (V)
Ile; Leu










Other substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions.

As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
As used herein, a DNA segment is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5′ to 3′ direction , encodes the sequence of amino acids of the specified polypeptide.
As used herein, the term “nucleic acid” refers to single-stranded and/or double-stranded polynucleotides such as deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) as well as analogs or derivatives of either RNA or DNA. Also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acid can refer to polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term also includes, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.
As used herein, “nucleic acid molecule encoding” refers to a nucleic acid molecule which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein or peptide. The nucleic acid molecule includes both the full length nucleic acid sequences as well as non-full length sequences derived from the full length mature polypeptide, such as for example a full length polypeptide lacking a precursor sequence. For purposes herein, a nucleic acid sequence also includes the degenerate codons of the native sequence or sequences which can be introduced to provide codon preference in a specific host.
As used herein, the term “polynucleotide” refers to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), and a DNA or RNA derivative containing, for example, a nucleotide analog or a “backbone” bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phophorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid). The term “oligonucleotide” also is used herein essentially synonymously with “polynucleotide,” although those in the art recognize that oligonucleotides, for example, PCR primers, generally are less than about fifty to one hundred nucleotides in length.
Polynucleotides can include nucleotide analogs, including, for example, mass modified nucleotides, which allow for mass differentiation of polynucleotides; nucleotides containing a detectable label such as a fluorescent, radioactive, luminescent or chemiluminescent label, which allow for detection of a polynucleotide; or nucleotides containing a reactive group such as biotin or a thiol group, which facilitates immobilization of a polynucleotide to a solid support. A polynucleotide also can contain one or more backbone bonds that are selectively cleavable, for example, chemically, enzymatically or photolytically. For example, a polynucleotide can include one or more deoxyribonucleotides, followed by one or more ribonucleotides, which can be followed by one or more deoxyribonucleotides, such a sequence being cleavable at the ribonucleotide sequence by base hydrolysis. A polynucleotide also can contain one or more bonds that are relatively resistant to cleavage, for example, a chimeric oligonucleotide primer, which can include nucleotides linked by peptide nucleic acid bonds and at least one nucleotide at the 3′ end, which is linked by a phosphodiester bond or other suitable bond, and is capable of being extended by a polymerase. Peptide nucleic acid sequences can be prepared using well-known methods (see, for example, Weiler et al. Nucleic acids Res. 25: 2792-2799 (1997)).
As used herein, “similarity” between two proteins or nucleic acids refers to the relatedness between the sequence of amino acids of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity and/or homology of sequences of residues and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. “Identity” refers to the extent to which the amino acid or nucleotide sequences are invariant. Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and/or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions).
“Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exists a number of methods to measure identity between two polynucleotide or polypeptides, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).
As used herein, homologous (with respect to nucleic acid and/or amino acid sequences) means about greater than or equal to 25% sequence homology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence homology; the precise percentage can be specified if necessary. For purposes herein the terms “homology” and “identity” are often used interchangeably, unless otherwise indicated.
In general, for determination of the percentage homology or identity, sequences are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073). By sequence homology, the number of conserved amino acids is determined by standard alignment algorithms programs, and can be used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.
Whether any two molecules have nucleotide sequences or amino acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” or “homologous” can be determined using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
Therefore, as used herein, the term “identity” or “homology” represents a comparison between a test and a reference polypeptide or polynucleotide. As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference nucleic acid or amino acid sequence of the polypeptide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) of the amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of a polypeptide or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often by manual alignment without relying on software.
As used herein, a polypeptide containing a specified percentage of amino acids set forth in a reference polypeptide refers to the proportion of contiguous identical amino acids shared between a polypeptide and a reference polypeptide. For example, an isoform that contains 70% of the amino acids set forth in a reference polypeptide having a sequence of amino acids set forth in SEQ ID NO:XX, which recites 147 amino acids, means that the reference polypeptide contains at least 103 contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:XX.
As used herein, an aligned sequence refers to the use of homology (similarity and/or identity) to align corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.
As used herein, “primer” refers to a nucleic acid molecule that can act as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and a polymerization agent , such as DNA polymerase, RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. It will be appreciated that a certain nucleic acid molecules can serve as a “probe” and as a “primer.” A primer, however, has a 3′ hydroxyl group for extension. A primer can be used in a variety of methods, including, for example, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′ RACE, in situ PCR, ligation-mediated PCR and other amplification protocols.
As used herein, “primer” pair refers to a set of primers that includes a 5′ (upstream) primer that hybridizes with the 5′ end of a sequence to be amplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizes with the complement of the 3′ end of the sequence to be amplified.
As used herein, “specifically hybridizes” refers to annealing, by complementary base-pairing, of a nucleic acid molecule (e.g. an oligonucleotide) to a target nucleic acid molecule. Those of skill in the art are familiar with in vitro and in vivo parameters that affect specific hybridization, such as length and composition of the particular molecule. Parameters particularly relevant to in vitro hybridization further include annealing and washing temperature, buffer composition and salt concentration. Exemplary washing conditions for removing non-specifically bound nucleic acid molecules at high stringency are 0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1% SDS, 50° C. Equivalent stringency conditions are known in the art. The skilled person can readily adjust these parameters to achieve specific hybridization of a nucleic acid molecule to a target nucleic acid molecule appropriate for a particular application.
As used herein, substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.
As used herein, it also is understood that the terms “substantially identical” or “similar” varies with the context as understood by those skilled in the relevant art.
As used herein, an allelic variant or allelic variation references any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and can result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequence. The term “allelic variant” also is used herein to denote a protein encoded by an allelic variant of a gene. Typically the reference form of the gene encodes a wildtype form and/or predominant form of a polypeptide from a population or single reference member of a species. Typically, allelic variants, which include variants between and among species typically have at least 80%, 90% or greater amino acid identity with a wildtype and/or predominant form from the same species; the degree of identity depends upon the gene and whether comparison is interspecies or intraspecies. Generally, intraspecies allelic variants have at least about 80%, 85%, 90% or 95% identity or greater with a wildtype and/or predominant form, including 96%, 97%, 98%, 99% or greater identity with a wildtype and/or predominant form of a polypeptide. Reference to an allelic variant herein generally refers to variations n proteins among members of the same species.
As used herein, “allele,” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide or several nucleotides, and can include substitutions, deletions and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation.
As used herein, species variants refer to variants in polypeptides among different species, including different mammalian species, such as mouse and human.
As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.
As used herein, the term promoter means a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding region of genes.
As used herein, isolated or purified polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification does not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
The term substantially free of cellular material includes preparations of proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the term substantially free of cellular material includes preparations of protease proteins having less that about 30% (by dry weight) of non-protease proteins (also referred to herein as a contaminating protein), generally less than about 20% of non-protease proteins or 10% of non-protease proteins or less that about 5% of non-protease proteins. When the protease protein or active portion thereof is recombinantly produced, it also is substantially free of culture medium, i.e., culture medium represents less than about or at 20%, 10% or 5% of the volume of the protease protein preparation.
As used herein, the term substantially free of chemical precursors or other chemicals includes preparations of protease proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. The term includes preparations of protease proteins having less than about 30% (by dry weight) 20%, 10%, 5% or less of chemical precursors or non-protease chemicals or components.
As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.
As used herein, production by recombinant means by using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.
As used herein, vector (or plasmid) refers to discrete elements that are used to introduce a heterologous nucleic acid into cells for either expression or replication thereof. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
As used herein, an expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
As used herein, vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.
As used herein, operably or operatively linked when referring to DNA segments means that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
As used herein, biological sample refers to any sample obtained from a living or viral source and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants. Also included are soil and water samples and other environmental samples, viruses, bacteria, fungi, algae, protozoa and components thereof. Hence bacterial and viral and other contamination of food products and environments can be assessed. The methods herein are practiced using biological samples and in some embodiments, such as for profiling, also can be used for testing any sample.
As used herein, macromolecule refers to any molecule having a molecular weight from the hundreds up to the millions. Macromolecules include peptides, proteins, nucleotides, nucleic acids, and other such molecules that are generally synthesized by biological organisms, but can be prepared synthetically or using recombinant molecular biology methods.
As used herein, the term “biopolymer” is a biological molecule, including macromolecules, composed of two or more monomeric subunits, or derivatives thereof, which are linked by a bond or a macromolecule. A biopolymer can be, for example, a polynucleotide, a polypeptide, a carbohydrate, or a lipid, or derivatives or combinations thereof, for example, a nucleic acid molecule containing a peptide nucleic acid portion or a glycoprotein, respectively. Biopolymers include, but are not limited to, nucleic acids, proteins, polysaccharides, lipids and other macromolecules. Nucleic acids include DNA, RNA, and fragments thereof. Nucleic acids can be derived from genomic DNA, RNA, mitochondrial nucleic acid, chloroplast nucleic acid and other organelles with separate genetic material.
As used herein, a biomolecule is any compound found in nature, or derivatives thereof. Biomolecules include, but are not limited to: oligonucleotides, oligonucleosides, proteins, peptides, amino acids, peptide nucleic acids (PNAs), oligosaccharides and monosaccharides.
As used herein, a biological particle refers to a virus, such as a viral vector or viral capsid with or without packaged nucleic acid, phage, including a phage vector or phage capsid, with or without encapsulated nucleic acid, a single cell, including eukaryotic and prokaryotic cells or fragments thereof, a liposome or micellar agent or other packaging particle, and other such biological materials.
As used herein, a composition refers to any mixture. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a combination refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof.
As used herein, kit refers to a packaged combination, optionally including instructions and/or reagents for their use.
As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
As used herein, antigenic means that a polypeptide induce an immune response. Highly antigenic polypeptides are those that reproducibly and predictably induce an immune response.
As used herein, a pharmaceutical effect or therapeutic effect refers to an effect observed upon administration of an agent intended for treatment of a disease or disorder or for amelioration of the symptoms thereof.
As used herein, “disease or disorder” refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms. Diseases and disorders of interest herein are those involving a specific target protein including those mediated by a target protein and those in which a target protein plays a role in the etiology or pathology. Exemplary target proteins and associated diseases and disorders are described elsewhere herein.
As used herein, “treating” a subject with a disease or condition means that the subject's symptoms are partially or totally alleviated, or remain static following treatment. Hence treatment encompasses prophylaxis, therapy and/or cure. Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease. Treatment also encompasses any pharmaceutical use of a modified interferon and compositions provided herein.
As used herein, a therapeutic agent, therapeutic regimen, radioprotectant, or chemotherapeutic mean conventional drugs and drug therapies, including vaccines, which are known to those skilled in the art. Radiotherapeutic agents are well known in the art.
As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease or other indication, are ameliorated or otherwise beneficially altered.
As used herein therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound which results in a therapeutic effect following administration to a subject.
As used herein, the term “subject” refers to an animal, including a mammal, such as a human being.
As used herein, a patient refers to a human subject.
As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.
As used herein, prevention or prophylaxis refers to methods in which the risk of developing disease or condition is reduced.
As used herein, an effective amount is the quantity of a therapeutic agent necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.
As used herein, administration refers to any method in which an antibody or protion thereof is contacted with its target protein. Administration can be effected in vivo or ex vivo or in vitro. For example, for ex vivo administration a body fluid, such as blood, is removed from a subject and contacted outside the body with the antibody or portion thereof. For in vivo administration, the antibody or portion thereof can be introduced into the body, such as by local, topical, systemic and/or other route of introduction. In vitro administration encompasses methods, such as cell culture methods.
As used herein, unit dose form refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.
As used herein, a single dosage formulation refers to a formulation for direct administration.
As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass compiled germline antibodies or antibodies obtained therefrom contained in articles of packaging.
As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
As used herein, animal includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs and other animals. Non-human animals exclude humans as the contemplated animal. The germline segments, and resulting antibodies, provided herein are from any source, animal, plant, prokaryotic and fungal. Most germline segments, and resulting antibodies, are of animal origin, including mammalian origin.
As used herein, a control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a sample plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control.
As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a compound, comprising “an extracellular domain” includes compounds with one or a plurality of extracellular domains.
As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 bases” means “about 5 bases” and also “5 bases.”
As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.
As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).
B. Overview
Provided are methods for generating combinatorial libraries (i.e. collections) of functional antibodies, and the resulting libraries. The collections or libraries provided are addressable, where antibodies within each address have the same sequence, are known a priori, and are different from the antibodies at each other address in the collection. The collections can be provided as physical arrays or the members can be otherwise identified so that they can be sorted. The arrayed collections of antibodies can represent the complete repertoire of combined germline portions, a selected portion thereof, or collections of modified forms thereof. The members of the libraries are individually designed and addressed. Because of this, the libraries are highly diverse, permitting creation of libraries with far fewer members than other libraries, but having higher diversity. The libraries provided herein contain as few as 102 members and typically contain about or 108, 104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 108 and more unique members, including about or 106, 107, 108 , 108 and more unique members.
The collections of antibodies are addressable, such as in arrays or other addressable format, such that each member is identifiable and each locus has antibodies that are the same or that have the same binding specificity at each locus. The locus can be a physical locus or can be otherwise identifiable and sortable, such as an RF tag, attachment to label with a bar code, attachment to a chemical tag, in manners described for chemical libraries.
In contrast, other antibody libraries are produced such that they contain mixtures of antibodies at a locus, or contain unidentified members of libraries. Exemplary of such libraries are those described in any of the following: European Patent Application Nos. EP0368684 and EP89311731; International Published Patent Application Nos. WO92/001047, WO 02/38756, WO 97/08320, WO 2005/023993, WO 07/137616 and WO 2007/054816; U.S. Pat. Nos. 6,593,081 and 6,989,250; United States Published Patent Application No. US 2002/0102613, US 2003/153038, US 2003/0022240, US 2005/0119455, US 2005/0079574 and US 2006/0234302; and Orlandi et al. (1989) Proc Natl. Acad. Sci. U.S.A., 86:3833-3837; Ward et al. (1989) Nature, 341:544-546; Huse et al. (1989) Science, 246:1275-1281; Burton et al. (1991) Proc. Natl. Acad. Sci., U.S.A., 88:10134-10137; Marks et al. (1991) J Mol Biol, 222:581-591; Hoogenboom et al. (1991) J Mol Biol, 227:381-388; Nissim et al. (1994) EMBO J, 13:692-698; Barbas et al. (1992) Proc. Natl. Acad. Sci., U.S.A., 89:4457-4461; Akamatsu et al. (1993) J. Immunol., 151:4651-1659; Griffiths et al. (1994) EMBO J, 13:3245-3260; Fellouse (2004) PNAS, 101:12467-12472; Persson et al. (2006) J. Mol. Biol. 357:607-620; Knappik et al. (2000) J. Mol. Biol. 296:57-86; Rothe et al. (2008) J. Mol. Biol. 376:1182-1200; Mondon et al. (2008) Frontiers in Bioscience, 13:1117-1129; and Behar, I (2007) Expert Opin. Biol. Ther., 7:763-779.
Although many of these libraries contain large numbers of members (e.g. 108-1010 members), there is no mechanism to ensure that all members are functional, nor to maximize diversity nor to represent the complete repertoire of germline sequences or a selected portion thereof. Thus, the composition and diversity of the library is not optimal. For example, many existing libraries are developed by PCR amplification of germline sequences. PCR amplification introduces errors into the resulting amplified products. In addition, in some methods hybrid primers are used to facilitate recombination of individual V(D)J segments. This can result in recombination events that are “out-of-frame” resulting in non-functional members. Also, in practicing such methods, members either are pooled (such as in a tube or via phage display) and screened together for binding to a target substrate or are introduced into host cells as mixtures and then colonies are individually picked and grown. Upon identification of a positive interaction or other selected events, any “Hits” must be further characterized in order to be identified.
The combinatorial addressable libraries of antibodies provided herein do not share these problems. Each member of the collection is addressed, such that each member occupies a unique locus, for example, a spatial array or other array or other identifiable address (e.g., presentation in well-plates; being bound to a support or chip, bar-coded, color-coded, RF-tag labeled support or other such addressable format). Displaying members on an address is facilitated because each member is individually generated, and thus the sequence of each member is known. Display of the members can be achieved on any desired format, which permits screening the members not only for binding but also for function. The “Hits” can be quickly identified coincident with the screening results. Hence, structure/activity relationships (SAR) between members of the collections can be performed to identify similarities in characteristics or sequences between and among identified “Hits”. Pharmacokinetics and dose-responses also are available on screening or immediately following “Hit” identification. Further optimization of “Hits” can be performed such as by mutagenesis and iterative screening. Accordingly, the methods provided herein for generating addressable combinatorial antibody collections and the resulting collections offer a robust alternative to identification of antibodies with desired specificities and/or activities, for example, for use as therapeutic antibodies.
1. Methods of Generating Addressable Combinatorial Antibody Collections
In one example of the methods provided herein, variable heavy (VH) and variable light (VL) chain members of the libraries are generated, recombinantly or synthetically by DNA synthesis, from known germline antibody sequences or modified sequences thereof. Thus, the members can represent the entire repertoire of the naïve germline, and are not restricted based on selection against “self” proteins. Combinatorial diversity in the collection exists from recombination (e.g. such as performed in silico by computer software as described herein) of individual V, D and J segments that make up the variable heavy chain and of individual V (Vκ, or Vλ) and J (Jκ, or Jλ) segments that make up the variable light chains (see FIG. 1). The sequences can be joined together by random recombination of all combinations of V(D)J segments generating maximal combinatorial diversity. Alternatively, the V(D)J segments can be recombined using rational or semi-rational approaches such that a specific sequence or subset of sequences are used in generating the members of the library. For example, germline segments can be selected based on sequence similarities or differences or based on shared characteristics (e.g., a V region family, CDR3 length or composition or other biochemical attribute).
In the methods herein, the combinatorial diversity of the resulting members is optimized for functioning sequences that encode a full length polypeptide. Although all combinations of V(D)J segments can be recombined, the joints in the compiled sequences between the different V(D)J sequences are selected so that the resulting sequences are in-frame. Each functioning member occupies an address (e.g. a position in a well or chip) of a collection. In vivo, however, junctional diversity exists upon V(D)J recombination such that nucleotides are often inserted at junction regions, which can result in new amino acids at the junctions. Hence, in some example of the methods herein, the resulting in-frame members can be subjected to mutagenesis, for example, to introduce diversity at the junction regions (e.g., junctional diversity). In such examples, each locus can contain a pool of antibodies with the same V(D)J segments, but differing from each other by one or more mutations (e.g. insertions, deletions or replacements of amino acids).
In addition to generating naïve antibody libraries, the methods provided herein can be used to generate directed antibody libraries, whereby the resulting members are optimized against known targets. For example, the starting sequences of individual V(D)J segments of heavy and light chains can be generated to contain a known binding peptide against a target. The goal of such a library format is to generate a platform that allows the discovery of agonist or antagonist antibodies that mimic therapeutic targets, for example, growth factors, cytokines, hormones or other cellular activators.
Generally, the members of the collections provided herein contain all or a portion of a variable light (VL) and variable heavy (VH) chain, so long as the resulting antibody is sufficient to form an antigen binding site. Hence, in addition to combinatorial diversity, diversity in the collections provided herein is achieved by pairing diversity by combining heavy and light chains (FIG. 1). Thus, the individually recombined VL and VH chains as discussed above can themselves be paired in varying combinations to generate a scalable collection of all possible combinations of VL and VH chains, or a subset thereof. For example, a library can be generated where all members have the same VH chains, but are paired with all or a subset of the individually recombined VL members. The heavy and light chain members can be paired by direct or indirect linkage. The resulting pairs of heavy and light chains can be presented in any desired library format, such as a complete antibody, Fab forms, single chain (sc) Fv forms, or any multimeric form thereof.
2. The Resulting Libraries
Provided herein are libraries of nucleic acid molecules encoding VL chains and libraries of nucleic acid molecules encoding VH chains. Also provided herein are combinatorial antibody libraries that are paired antibody libraries containing at a minimum all or a portion of a VL and VH chains, such that each resulting member in the library is sufficient to form an antigen binding site. The libraries can be naïve libraries representing all or a portion of all possible germline antibodies, or can be modified forms thereof. The resulting members of the paired antibody collections include, but are not limited to, Fab, single chain (sc) Fv, disulfide-stabilized Fv and multimeric formats such as minibodies, bis-scFv, diabodies, triabodies and tetrabodies. The libraries provided herein differ from existing antibody collections because each individual member of the collection is known and, in the case of the antibody libraries, each member is “productive” or “functional” because the encoding nucleic acid molecules lack stop codons that can otherwise truncate the resulting protein before a full length polypeptide can be produced. Typically, all libraries provided herein are in an addressable format, such that the identity of each member of the library is known based on its locus or “address”. Exemplary of antibody collections provided herein are combinatorial Fab libraries, such as addressable combinatorial Fab libraries. Any of the above libraries can include 102, 103, 104 or 105, or more different members.
3. Applications of the Libraries
The resulting libraries can be used for any application or purpose as desired. Because of their diversity, specificity and effector functions, antibodies are attractive candidates for protein-based therapeutics. Thus, the libraries can be used in methods of screening for various activities to identify antibodies with unique functions, such as for use as therapeutic antibodies. For example, the antibody libraries provided herein can be used in screening assays based on function or binding against unknown or known targets. In particular, it is contemplated herein that the resulting libraries can be used in functional assays, such as cell based assays, to discover new MAbs (e.g., Fabs) against selected targets. Hence, the libraries provided herein offer advantages over existing libraries because they permit identification of antibodies that perhaps are low affinity binders, but functionally are ideal therapeutic antibody candidates. Accordingly, both agonist and antagonist antibodies can be easily discovered.
The resulting identified “Hits” can be further optimized against a desired target by iterative screening methods of antibody discovery (FIG. 2). For example, antibody “Hits” identified from binding or activity-based screening assays, can be used to generate further libraries containing V(D)J germline segments related (e.g., by sequence identity or similarity) to the V(D)J germline segments in the identified Hit(s). By virtue of the fact that the collections are arrayed, such that the identity of each individual member in the collection is known, iterative approaches can be used for the rapid expansion of “Hits” to identify antibody “Hits” with improved therapeutic applications. In addition, antibody “Hits” can be used as a scaffold for mutagenesis to generate modified heavy and light chains, and for successive generations of libraries of nucleic acid molecules encoding modified VL chains, libraries of nucleic acid molecules encoding modified VH chains and antibody libraries minimally containing modified VL and VH chains.
Finally, antibody “Hits” identified from the libraries herein and/or further optimized by iterative screening and/or other mutagenesis methods, can be used in a variety of in vitro and in vivo applications by virtue of the specificity for one or more target proteins. For example, the antibodies can be used in diagnostic methods. In another example, the antibodies can be used in methods of treatment and other uses for treating a disease or disorder which is associated with expression or activation of a particular target protein, and for which the antibody can modulate.
The following sections describe exemplary components of the methods and libraries, the methods of generating combinatorial antibody libraries, including arrayed libraries, the resulting libraries and applications of the libraries.
C. Antibodies
Provided herein are methods of generating libraries of addressable combinatorial antibodies, and the resulting libraries and antibodies. The antibodies in the libraries minimally include all or a portion of a variable heavy chain (VH) and/or a variable light (VL) chain so long as the antibody contains a sufficient antibody binding site. For example, the VH and VL chains of the antibodies provided herein typically include one or more, generally two or more, and up to all of the three CDRs making up the antigen binding site. In some examples, the antibodies can be generated to contain a synthetic CDR, whereby a peptide against a known target is grafted into the CDR regions of the variable region to effect directed binding and activation of the target (see e.g., Frederickson et al. (2006) PNAS 103: 14307-14312).
Optionally, the antibodies can include all or a portion of the constant heavy chain (e.g. one or more CH domains such as CH1, CH2, CH3 and CH4 and/or a constant heavy chain (CL)). Hence, the antibodies included in the libraries herein include those that are full-length antibodies, and also include fragments or portions thereof including, for example, Fab, Fab′, F(ab′)2, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments Fab fragments, Fd fragments, scFv fragments, and scFab fragments. For example, antibodies in the libraries provided herein include Fabs.
A description of the structure, sequence and function of antibodies are known to one of skill in the art and one of skill in the art is familiar with the mechanisms that give rise to diversity in the germline. It is contemplated herein that libraries of combinatorial antibodies can be made by recombination of germline DNA sequences that mimic the process of germline recombination during B cell differentiation. Such recombination can be performed in silico (e.g., by a computer) as described herein or can be performed manually using molecular biology techniques. Recombined sequences can be individually generated, such as by DNA synthesis or by recombinant DNA techniques, to generate all permutations of variable heavy and light chain sequences. The antibodies can be expressed in any desired form, and in some instances, pairing of variable and constant regions can be achieved. The result is that the libraries of combinatorial antibodies provided herein can represent the entire naïve antibody repertoire or a subset thereof.
1. Antibody Polypeptides
Antibodies are produced naturally by B cells in membrane-bound and secreted forms. Antibodies specifically recognize and bind antigen epitopes through cognate interactions. Antibody binding to cognate antigens can initiate multiple effector functions, which cause neutralization and clearance of toxins, pathogens and other infectious agents. Diversity in antibody specificity arises naturally due to recombination events during B cell development. Through these events, various combinations of multiple antibody V, D and J gene segments, which encode variable regions of antibody molecules, are joined with constant region genes to generate a natural antibody repertoire with large numbers of diverse antibodies. A human antibody repertoire contains more than 1010 different antigen specificities and thus theoretically can specifically recognize any foreign antigen. Antibodies include such naturally produced antibodies, as well as synthetically, i.e. recombinantly, produced antibodies, such as antibody fragments.
In folded antibody polypeptides, binding specificity is conferred by antigen binding site domains, which contain portions of heavy and/or light chain variable region domains. Other domains on the antibody molecule serve effector functions by participating in events such as signal transduction and interaction with other cells, polypeptides and biomolecules. These effector functions cause neutralization and/or clearance of the infecting agent recognized by the antibody.
2. Antibody Structural and Functional Domains
A full-length antibody contains four polypeptide chains, two identical heavy (H) chains (each usually containing about 440 amino acids) and two identical light (L) chains (each containing about 220 amino acids). The light chains exist in two distinct forms called kappa (κ) and lambda (λ). Each chain is organized into a series of domains organized as immunoglobulin (Ig) domains, including variable (V) and constant (C) region domains. Light chains have two domains, corresponding to the C region (CL) and the V region (VL). Heavy chains have four domains, the V region (VH) and three or four domains in the C region (CH1, CH2 , CH3 and CH4), and, in some cases, hinge region. The four chains (two heavy and two light) are held together by a combination of covalent and non-covalent (disulfide) bonds.
Antibodies include those that are full-lengths and those that are fragments thereof, namely Fab, Fab′, F(ab′)2, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments. The fragments include those that are in single-chain or dimeric form. The Fv fragment, which contains only the VH and VL domain, is the smallest immunoglobulin fragment that retains the whole antigen-binding site (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). Stabilization of Fv are achieved by direct linkage of the VH and VL chains, such as for example, by linkage with peptides (to generate single-chain Fvs (scFv)), disulfide bridges or knob-into-hole mutations. Fab fragments, in contrast, are stable because of the presence of the CH1 and CL domains that hold together the variable chains. Fd antibodies, which contain only the VH domain, lack a complete antigen-binding site and can be insoluble.
3. Antibody Sequence and Specificity
The variable region of the heavy and light chains are encoded by multiple germline gene segments separated by non-coding regions, or introns, and often are present on different chromosomes. For example, the genes for the immunoglobulin heavy chain region contains approximately 65 variable (VH) genes, 27 Diversity (DH) genes, and 6 Joining (JH) genes. The kappa (κ) and lambda (λ) light chains are also each encoded by a similar number of VL and JL gene segments, but do not include any D gene segments. Exemplary VH, DH, JH and VL (Vκ or Vλ) and JL (Jκ or Jλ) germline gene segments are set forth in Tables 3-5.
During B cell differentiation germline DNA is rearranged whereby one DH and one JH gene segment of the heavy chain locus are recombined, which is followed by the joining of one VH gene segment forming a rearranged VDJ gene that encodes a VH chain. The rearrangement occurs only on a single heavy chain allele by the process of allelic exclusion. Allelic exclusion is regulated by in-frame or “productive” recombination of the VDJ segments, which occurs in only about one-third of VDJ recombinations of the variable heavy chain. When such productive recombination events first occur in a cell, this results in production of a p heavy chain that gets expressed on the surface of a pre-B cell and transmits a signal to shut off further heavy chain recombination, thereby preventing expression of the allelic heavy chain locus. The surface-expressed p heavy chain also acts to activate the kappa (κ) locus for rearrangement. The lambda (λ) locus is only activated for rearrangement if the κ recombination is unproductive on both loci. The light chain rearrangement events are similar to heavy chain, except that only the VL and JL segments are recombined. Before primary transcription of each, the corresponding constant chain gene is added. Subsequent transcription and RNA splicing leads to mRNA that is translated into an intact light chain or heavy chain.
The variable regions of antibodies confer antigen binding and specificity due to recombination events of individual germline V, D and J segments, whereby the resulting recombined nucleic acid sequences encoding the variable region domains differ among antibodies and confer antigen-specificity to a particular antibody. The variation, however, is limited to three complementarity determining regions (CDR1, CDR2, and CDR3) found within the N-terminal domain of the heavy (H) and (L) chain variable regions. The CDRs are interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDRs has been precisely defined (see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Each VH and VL is typically composed of three CDRs and four FRs arranged from the amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Sequence variability among VL and VH domains is generally limited to the CDRs, which are the regions that form the antigen binding site. For example, for the heavy chain, generally, VH genes encode the N-terminal three framework regions, the first two complete CDRs and the first part of the third CDR; the DH gene encodes the central portion of the third CDR, and the JH gene encodes the last part of the third CDR and the fourth framework region. For the light chain, the VL genes encode the first CDR and second CDR. The third CDR (CDRL3) is formed by the joining of the VL and JL gene segments. Hence, CDRs 1 and 2 are exclusively encoded by germline V gene segment sequences. The VH and VL chain CDR3s form the center of the Ag-binding site, with CDRs 1 and 2 form the outside boundaries; the FRs support the scaffold by orienting the H and L CDRs. On average, an antigen binding site typically requires that at least four of the CDRs make contact with the antigen's epitope, with CDR3 of both the heavy and light chain being the most variable and contributing the most specificity to antigen binding (see e.g., Janis Kuby, Immunology, Third Edition, New York, W.H. Freeman and Company, 1998, pp. 115-118). CDRH3, which includes all of the D gene segment, is the most diverse component of the Ab-binding site, and typically plays a critical role in defining the specificity of the Ab. In addition to sequence variation, there is variation in the length of the CDRs between the heavy and light chains (see Table 26 in Example 12).
The constant regions, on the other hand, are encoded by sequences that are more conserved among antibodies. These domains confer functional properties to antibodies, for example, the ability to interact with cells of the immune system and serum proteins in order to cause clearance of infectious agents. Different classes of antibodies, for example IgM, IgD, IgG, IgE and IgA, have different constant regions, allowing them to serve distinct effector functions.
These natural recombination events of V, D, and J, can provide nearly 2×107 different antibodies with both high affinity and specificity. Additional diversity is introduced by nucleotide insertions and deletions in the joining segments and also by somatic hypermutation of V regions. The result is that there are approximately 1010 antibodies present in an individual with differing antigen specificities.
The methods provided herein take advantage of the mechanisms responsible for generating diversity between and among germline antibodies, thereby permitting generation of collections of antibodies that can be tested for varied functional or other properties.
D. Methods of Generating Members of the Combinatorial Antibody Library
Provided herein are methods of producing combinatorial antibody libraries and the resulting libraries. Typically, each member in the library contains a variable heavy chain and a variable light chain, or portions thereof sufficient to form an antigen binding site. In the methods provided herein, each antibody member of the library is generated by mimicking natural recombination events by combining known V(D)J gene segment sequences (e.g. from publicly available databases of germline sequences), or modified forms thereof, in various permutations, in-frame, to generate a plurality of nucleic acid sequences encoding functional VH and VL chains. For example, in the steps of the methods nucleic acid molecules encoding the variable heavy (VH) chain are generated by recombining individual V, D and J segments. Nucleic acid molecules encoding the variable light (VL) chain are generated by recombining individual V (Vκ or Vλ) and J (Jκ, or Jλ) segments that make up the variable light chains. The segments can be germline segments, or degenerate sequences thereof. In such examples, the resulting antibodies produced by the method are naïve antibodies. It is contemplated herein, however, that the method can be performed using any modified form of a known germline segment, for example, to introduce further diversity into the library. For example, the method can be performed using reverse complement (i.e. inverted) sequences of DH germline segments. The process of recombining germline segments in-frame can be performed manually using molecular biology techniques or in silico (e.g. using a computer programmed to perform an algorithm).
In the methods, the recombination is effected so that each gene segment is in-frame, such that resulting recombined nucleic acid molecules encodes a functional VH or VL polypeptide. Also, in the methods, each nucleic acid molecule is individually generated and synthesized. In the methods, resulting members of the library are produced by co-expression of nucleic acid molecules encoding the recombined variable region genes together, such that when expressed, a combinatorial antibody member is generated minimally containing a VH and VL chain, or portions thereof. In some examples of the methods, the nucleic acid molecule encoding the VH and VL chain can be expressed as a single nucleic acid molecule, whereby the genes encoding the heavy and light chain are joined by a linker. In another example of the methods, the nucleic acid molecules encoding the VH and VL chain can be separately provided for expression together. Thus, upon expression from the recombined nucleic acid molecules, each different member of the library represents a germline encoded antibody, whereby diversity is achieved by combinatorial diversity of V(D)J segments and pairing diversity of heavy and light chains. In the method, additional diversity can be introduced into the library using any of a number of approaches known in the art, including but not limited to, random mutagenesis, semi-rational or rational mutagenesis.
One or more or all steps of the method can be performed in an addressable format, such that the identity of each member in the process is known by its location at an addressed locus. Hence, provided herein are addressable libraries of germline recombined nucleic acid sequences encoding VH chains, addressable libraries of germline recombined nucleic acid sequences encoding VL chains, and addressable libraries formed by combinations of nucleic acids molecules encoding VL chains and nucleic acid molecules encoding VH chains at each locus. Also provided are addressable cells, each cell at a locus containing different combinations of a recombined nucleic acid molecule encoding a VL and a recombined nucleic acid encoding a VH. Resulting antibody libraries also can be addressable. Such addressable antibody libraries permit rapid identification of “Hits” and assessment of structure/activity relationships between and among “Hits.” The resulting libraries of antibodies can be screened for a variety of activities, such as but not limited to binding, proliferation, cytotoxicity and low affinity leads against difficult antigens, such as self antigens, ion channels, G-protein coupled receptors, novel epitopes, non-protein antigens and the discovery of agonist antibodies.
1. Methods for Producing Functional Recombined Germline Variable Region Genes
Provided herein is a method for generating nucleic acid molecules generated by recombination of germline segments or modified forms thereof, each nucleic acid molecule encoding a different and functional variable region of the heavy or light chains. Variable gene segments include VH, DH, JH, Vκ, Jκ, Vλand Jλ. Germline segments can be selected from but not limited to human, mouse, rat, sheep, pig, goat horse, rabbit or dog germline segments. Exemplary germline segments are of human origin.
a. Variable Gene Segments
i. Germline Segments
In practicing the methods herein, germline segment sequences are obtained from any source that provides antibody germlines gene segments. These include any databases or published literature that sets forth sequences of germline gene segments. Exemplary antibody germline sources include but are not limited to databases at the National Center for Biotechnology Information (NCBI), the international ImMunoGeneTics information system® (IMGT), the Kabat database and the Tomlinson's VBase database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin et al., Bioinformatics Tools for Antibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH (2007), pp. 104-107). If desired, nucleic acid sequences for non-human germline segments also can be obtained from published literature or publicly available databases. For example, an exemplary mouse germline databases is ABG database available at ibt.unam.mx/vir/v_mice.html. The Sequence Listing provided herein provides sequences of exemplary human germline segment sequences collected from the IMGT database and other public database (see e.g., SEQ ID NOS:10-451 and 868).
For example, exemplary human Heavy Chain Germline Segments (SEQ ID NO. 10-285) are listed in Table 3. Exemplary human Light Chain Kappa Germline Segments (SEQ ID NO.286-364 and SEQ ID NO. 868) are listed in Table 4. Exemplary human Light Chain Lambda Germline Segments (SEQ ID NO. 365-451) are listed in Table 5. Germline segments are listed using IMGT gene names and definitions previously approved by the Human Genome Organization (HUGO) nomenclature committee. The segments are named using IMGT nomenclature, whereby the first three letters indicate the locus (IGH, IGK or IGL), the fourth letter represents the gene (e.g., V for V-gene, D for D-gene, J for J-gene), the fifth position indicates the number of the subgroup, followed by a hyphen indicating the gene number classification. For alleles, the IMGT name is followed by an asterisk and a two figure number.
Tables 6-8 list alternative nomenclature for Human Heavy Chain V Genes, Human Light Chain Kappa V Genes, and Human Light Chain Lambda V Genes, respectively (see e.g. Lefranc, M.-P. Exp Clin Immunogenet, 18:100-116 (2001), Zachau, H. G. Immunologist, 4:49-54 (1996), Lefranc, M.-P. Exp Clin Immunogenet, 18:161-174 (2000), Kawasaki et al, Genome Res, 7:250-261 (1997), Lefranc, M.-P. Exp Clin Immunogenet, 18:242-254 (2001). Any desired naming convention can be used to identify antibody germline segments. One of skill in the art can identify a nucleic acid sequence using any desired naming convention. For purposes herein when describing recombined nucleic acid sequences (see e.g., Table 22), VH germline segments are named using IMGT nomenclature without any allele identified. Table 6 lists the IMGT nomenclature and corresponding IMGT nomenclature with the respective allele. VK germline segments are named using Zachau nomenclature. Table 7 lists the Zachau nomenclature and the corresponding IMGT nomenclature. VL germline segments are identified using Kawasaki nomenclature. Table 8 lists the Kawasaki nomenclature and the corresponding IMGT nomenclature. DH, JH, JK and JL germline segments are named using IMGT nomenclature.







TABLE 3







Human Heavy Chain Germline Segments








SEQ ID



NO.












V Segments


10
IGHV1-18*01


11
IGHV1-18*02


12
IGHV1-2*01


13
IGHV1-2*02


14
IGHV1-2*03


15
IGHV1-2*04


16
IGHV1-24*01


17
IGHV1-3*01


18
IGHV1-3*02


19
IGHV1-45*01


20
IGHV1-45*02


21
IGHV1-45*03


22
IGHV1-46*01


23
IGHV1-46*02


24
IGHV1-46*03


25
IGHV1-58*01


26
IGHV1-58*02


27
IGHV1-69*01


28
IGHV1-69*02


29
IGHV1-69*03


30
IGHV1-69*04


31
IGHV1-69*05


32
IGHV1-69*06


33
IGHV1-69*07


34
IGHV1-69*08


35
IGHV1-69*09


36
IGHV1-69*10


37
IGHV1-69*11


38
IGHV1-69*12


39
IGHV1-69*13


40
IGHV1-8*01


41
IGHV1-c*01


42
IGHV1-f*01


43
IGHV1-f*02


44
IGHV2-26*01


45
IGHV2-5*01


46
IGHV2-5*02


47
IGHV2-5*03


48
IGHV2-5*04


49
IGHV2-5*05


50
IGHV2-5*06


51
IGHV2-5*07


52
IGHV2-5*08


53
IGHV2-5*09


54
IGHV2-5*10


55
IGHV2-70*01


56
IGHV2-70*02


57
IGHV2-70*03


58
IGHV2-70*04


59
IGHV2-70*05


60
IGHV2-70*06


61
IGHV2-70*07


62
IGHV2-70*08


63
IGHV2-70*09


64
IGHV2-70*10


65
IGHV2-70*11


66
IGHV2-70*12


67
IGHV2-70*13


68
IGHV3-11*01


69
IGHV3-11*03


70
IGHV3-13*01


71
IGHV3-13*02


72
IGHV3-13*02


73
IGHV3-15*01


74
IGHV3-15*02


75
IGHV3-15*03


76
IGHV3-15*04


77
IGHV3-15*05


78
IGHV3-15*06


79
IGHV3-15*07


80
IGHV3-15*08


81
IGHV3-16*01


82
IGHV3-16*02


83
IGHV3-20*01


84
IGHV3-21*01


85
IGHV3-21*02


86
IGHV3-23*01


87
IGHV3-23*02


88
IGHV3-23*03


89
IGHV3-23*04


90
IGHV3-23*05


91
IGHV3-30*01


92
IGHV3-30*02


93
IGHV3-30*03


94
IGHV3-30*04


95
IGHV3-30*05


96
IGHV3-30*06


97
IGHV3-30*07


98
IGHV3-30*08


99
IGHV3-30*09


100
IGHV3-30*10


101
IGHV3-30*11


102
IGHV3-30*12


103
IGHV3-30*13


104
IGHV3-30*14


105
IGHV3-30*15


106
IGHV3-30*16


107
IGHV3-30*17


108
IGHV3-30*18


109
IGHV3-30*19


110
IGHV3-30-3*01


111
IGHV3-30-3*02


112
IGHV3-33*01


113
IGHV3-33*02


114
IGHV3-33*03


115
IGHV3-33*04


116
IGHV3-33*05


117
IGHV3-35*01


118
IGHV3-38*01


119
IGHV3-38*02


120
IGHV3-43*01


121
IGHV3-43*02


122
IGHV3-48*01


123
IGHV3-48*02


124
IGHV3-48*03


125
IGHV3-49*01


126
IGHV3-49*02


127
IGHV3-49*03


128
IGHV3-49*04


129
IGHV3-49*05


130
IGHV3-53*01


131
IGHV3-53*02


132
IGHV3-53*03


133
IGHV3-64*01


134
IGHV3-64*02


135
IGHV3-64*03


136
IGHV3-64*04


137
IGHV3-64*05


138
IGHV3-66*01


139
IGHV3-66*02


140
IGHV3-66*03


141
IGHV3-66*04


142
IGHV3-7*01


143
IGHV3-7*02


144
IGHV3-72*01


145
IGHV3-72*02


146
IGHV3-73*01


147
IGHV3-73*02


148
IGHV3-74*01


149
IGHV3-74*02


150
IGHV3-74*03


151
IGHV3-9*01


152
IGHV3-d*0153


153
IGHV4-28*01


154
IGHV4-28*02


155
IGHV4-28*03


156
IGHV4-28*04


157
IGHV4-28*05


158
IGHV4-30-2*01


159
IGHV4-30-2*02


160
IGHV4-30-2*03


161
IGHV4-30-2*04


162
IGHV4-30-4*01


163
IGHV4-30-4*02


164
IGHV4-30-4*03


165
IGHV4-30-4*04


166
IGHV4-30-4*05


167
IGHV4-30-4*06


168
IGHV4-31*01


169
IGHV4-31*02


170
IGHV4-31*03


171
IGHV4-31*04


172
IGHV4-31*05


173
IGHV4-31*06


174
IGHV4-31*07


175
IGHV4-31*08


176
IGHV4-31*09


177
IGHV4-31*10


178
IGHV4-34*01


179
IGHV4-34*02


180
IGHV4-34*03


181
IGHV4-34*04


182
IGHV4-34*05


183
IGHV4-34*06


184
IGHV4-34*07


185
IGHV4-34*08


186
IGHV4-34*09


187
IGHV4-34*10


188
IGHV4-34*11


189
IGHV4-34*12


190
IGHV4-34*13


191
IGHV4-39*01


192
IGHV4-39*02


193
IGHV4-39*03


194
IGHV4-39*04


195
IGHV4-39*05


196
IGHV4-39*06


197
IGHV4-39*07



V, D, or J



Segments


198
IGHV4-4*01


199
IGHV4-4*02


200
IGHV4-4*03


201
IGHV4-4*04


202
IGHV4-4*05


203
IGHV4-4*06


204
IGHV4-4*07


205
IGHV4-59*01


206
IGHV4-59*02


207
IGHV4-59*03


208
IGHV4-59*04


209
IGHV4-59*05


210
IGHV4-59*06


211
IGHV4-59*07


212
IGHV4-59*08


213
IGHV4-59*09


214
IGHV4-59*10


215
IGHV4-61*01


216
IGHV4-61*02


217
IGHV4-61*03


218
IGHV4-61*04


219
IGHV4-61*05


220
IGHV4-61*06


221
IGHV4-61*07


222
IGHV4-61*08


223
IGHV4-b*01


224
IGHV4-b*02


225
IGHV5-51*01


226
IGHV5-51*02


227
IGHV5-51*03


228
IGHV5-51*04


229
IGHV5-51*05


230
IGHV5-a*01


231
IGHV5-a*03


232
IGHV5-a*04


233
IGHV6-1*01


234
IGHV6-1*02


235
IGHV7-4-1*01


236
IGHV7-4-1*02


237
IGHV7-4-1*03


238
IGHV7-81*01



D Segments


239
IGHD1-1*01


240
IGHD1-14*01


241
IGHD1-20*01


242
IGHD1-26*01


243
IGHD1-7*01


244
IGHD2-15*01


245
IGHD2-2*01


246
IGHD2-2*02


247
IGHD2-2*03


248
IGHD2-21*01


249
IGHD2-21*02


250
IGHD2-8*01


251
IGHD2-8*02


252
IGHD3-10*01


253
IGHD3-10*02


254
IGHD3-16*01


255
IGHD3-16*02


256
IGHD3-22*01


257
IGHD3-3*01


258
IGHD3-3*02


259
IGHD3-9*01


260
IGHD4-11*01


261
IGHD4-17*01


262
IGHD4-23*01


263
IGHD4-4*01


264
IGHD5-12*01


265
IGHD5-18*01


266
IGHD5-24*01


267
IGHD5-5*01


268
IGHD6-13*01


269
IGHD6-19*01


270
IGHD6-25*01


271
IGHD6-6*01


272
IGHD7-27*01



J Segments


273
IGHJ1*01


274
IGHJ2*01


275
IGHJ3*01


276
IGHJ3*02


277
IGHJ4*01


278
IGHJ4*02


279
IGHJ4*03


280
IGHJ5*01


281
IGHJ5*02


282
IGHJ6*01


283
IGHJ6*02


284
IGHJ6*03


285
IGHJ6*04
















TABLE 4







Human Light Chain Kappa Germline Segments








SEQ ID



NO.






V Segments


286
IGKV1-12*01


287
IGKV1-12*02


288
IGKV1-13*02


289
IGKV1-16*01


290
IGKV1-17*01


291
IGKV1-17*02


292
IGKV1-27*01


293
IGKV1-33*01


294
IGKV1-37*01


295
IGKV1-39*01


296
IGKV1-5*01


297
IGKV1-5*02


298
IGKV1-5*03


299
IGKV1-6*01


300
IGKV1-8*01


301
IGKV1-9*01


302
IGKV1-NL1*01


303
IGKV1/OR2-0*01


304
IGKV1/OR2-108*01


305
IGKV1D-12*01


306
IGKV1D-12*02


307
IGKV1D-13*01


308
IGKV1D-16*01


309
IGKV1D-16*02


310
IGKV1D-17*01


311
IGKV1D-33*01


312
IGKV1D-37*01


313
IGKV1D-39*01


314
IGKV1D-42*01


315
IGKV1D-43*01


316
IGKV1D-8*01


317
IGKV2-24*01


318
IGKV2-28*01


319
IGKV2-29*02


320
IGKV2-29*03


321
IGKV2-30*01


322
IGKV2-40*01


323
IGKV2-40*02


324
IGKV2D-24*01


325
IGKV2D-26*01


326
IGKV2D-26*02


327
IGKV2D-28*01


328
IGKV2D-29*01


329
IGKV2D-29*02


330
IGKV2D-30*01


331
IGKV2D-40*01


332
IGKV3-11*01


333
IGKV3-11*02


334
IGKV3-15*01


335
IGKV3-20*01


336
IGKV3-20*02


337
IGKV3-7*01


338
IGKV3-7*02


339
IGKV3-7*03


340
IGKV3-NL1*01


341
IGKV3-NL2*01



V or J Segments


342
IGKV3-NL3*01


343
IGKV3-NL4*01


344
IGKV3-NL5*01


345
IGKV3/OR2-268*01


346
IGKV3/OR2-268*02


347
IGKV3D-11*01


348
IGKV3D-15*01


349
IGKV3D-20*01


350
IGKV3D-7*01


351
IGKV4-1*01


352
IGKV5-2*01


353
IGKV6-21*01


354
IGKV6D-21*01


355
IGKV6D-41*01


868
IGKV1-39*02



J Segments


356
IGKJ1*01


357
IGKJ2*01


358
IGKJ2*02


359
IGKJ2*03


360
IGKJ2*04


361
IGKJ3*01


362
IGKJ4*01


363
IGKJ4*02


364
IGKJ5*01
















TABLE 5







Human Light Chain Lambda Germline Segments








SEQ ID



NO.






V Segments


365
IGLV1-36*01


366
IGLV1-40*01


367
IGLV1-40*02


368
IGLV1-40*03


369
IGLV1-41*01


370
IGLV1-44*01


371
IGLV1-47*01


372
IGLV1-47*02


373
IGLV1-50*01


374
IGLV1-51*01


375
IGLV1-51*02


376
IGLV10-54*01


377
IGLV10-54*02


378
IGLV10-54*03


379
IGLV11-55*01


380
IGLV2-11*01


381
IGLV2-11*02


382
IGLV2-11*03


383
IGLV2-14*01


384
IGLV2-14*02


385
IGLV2-14*03


386
IGLV2-14*04


387
IGLV2-18*01


388
IGLV2-18*02


389
IGLV2-18*03


390
IGLV2-18*04


391
IGLV2-23*01


392
IGLV2-23*02


393
IGLV2-23*03


394
IGLV2-33*01


395
IGLV2-33*02


396
IGLV2-33*03


397
IGLV2-8*01


398
IGLV2-8*02


399
IGLV2-8*03


400
IGLV3-1*01


401
IGLV3-10*01


402
IGLV3-10*02


403
IGLV3-12*01


404
IGLV3-12*02


405
IGLV3-16*01


406
IGLV3-19*01


407
IGLV3-21*01


408
IGLV3-21*02


409
IGLV3-21*03


410
IGLV3-22*01


411
IGLV3-25*01


412
IGLV3-25*02


413
IGLV3-25*03


414
IGLV3-27*01


415
IGLV3-32*01


416
IGLV3-9*01


417
IGLV3-9*02


418
IGLV4-3*01


419
IGLV4-60*01


420
IGLV4-60*02


421
IGLV4-60*03


422
IGLV4-69*01


423
IGLV4-69*02


424
IGLV5-37*01



V or J Segments


425
IGLV5-39*01


426
IGLV5-39*02


427
IGLV5-45*01


428
IGLV5-45*02


429
IGLV5-45*03


430
IGLV5-48*01


431
IGLV5-52*01


432
IGLV6-57*01


433
IGLV7-43*01


434
IGLV7-46*01


435
IGLV7-46*02


436
IGLV8-61*01


437
IGLV8-61*02


438
IGLV8-61*03


439
IGLV9-49*01


440
IGLV9-49*02


441
IGLV9-49*03



J Segments


442
IGLJ1*01


443
IGLJ2*01


444
IGLJ3*01


445
IGLJ3*02


446
IGLJ4*01


447
IGLJ5*01


448
IGLJ5*02


449
IGLJ6*01


450
IGLJ7*01


451
IGLJ7*02
















TABLE 6







Human Heavy Chain V Genes












IMGT Nomenclature
SEQ ID



IMGT Nomenclature
with alleles
No.















VH1-18
IGHV1-18*01
10



VH1-2
IGHV1-2*02
13



VH1-24
IGHV1-24*01
16



VH1-3
IGHV1-3*02
18



VH1-45
IGHV1-45*02
20



VH1-46
IGHV1-46*01
22



VH1-58
IGHV1-58*02
26



VH1-69
IGHV1-69*06
32



VH1-8
IGHV1-8*01
40



VH2-26
IGHV2-26*01
44



VH2-5
IGHV2-5*01
45



VH2-70
IGHV2-70*13
67



VH3-11
IGHV3-11*01
68



VH3-13
IGHV3-13*01
70



VH3-15
IGHV3-15*01
73



VH3-16
IGHV3-16*02
82



VH3-20
IGHV3-20*01
83



VH3-21
IGHV3-21*01
84



VH3-23
IGHV3-23*01
86



VH3-30
IGHV3-30*03
93



VH3-33
IGHV3-33*01
112



VH3-35
IGHV3-35*01
117



VH3-38
IGHV3-38*02
119



VH3-43
IGHV3-43*01
120



VH3-48
IGHV3-48*02
123



VH3-49
IGHV3-49*03
127



VH3-53
IGHV3-53*01
130



VH3-64
IGHV3-64*02
134



VH3-66
IGHV3-66*03
140



VH3-7
IGHV3-7*01
142



VH3-72
IGHV3-72*01
144



VH3-73
IGHV3-73*02
147



VH3-74
IGHV3-74*01
148



VH3-9
IGHV3-9*01
151



VH4-28
IGHV4-28*01
153



VH4-31
IGHV4-31*02
169



VH4-34
IGHV4-34*01
178



VH4-39
IGHV4-39*01
191



VH4-4
IGHV4-4*07
204



VH4-59
IGHV4-59*01
205



VH4-61
IGHV4-61*08
222



VH5-51
IGHV5-51*03
227



VH6-1
IGHV6-1*01
233



VH7-81
IGHV7-81*01
238

















TABLE 7







Human Light Chain Kappa V Genes











Zachau
IMGT
SEQ ID



Nomencalture
Nomenclature
No.







A1
IGKV2D-30*01
330



A10
IGKV6D-21*01
354



A11
IGKV3D-20*01
349



A14
IGKV6D-41*01
355



A17
IGKV2-30*01
321



A18b
IGKV2-29*02
319



A19
IGKV2-28*01
318



A2
IGKV2D-29*01
328



A20
IGKV1-27*01
292



A23
IGKV2-24*01
317



A26
IGKV6D-21*01
354



A27
IGKV3-20*01
335



A3
IGKV2D-28*01
327



A30
IGKV1-17*01
290



A5
IGKV2D-26*01
325



A7
IGKV2D-24*01
324



B2
IGKV5-2*01
352



B3
IGKV4-1*01
351



L1
IGKV1-16*01
289



L10
IGKV3-7*01
337



L10a
IGKV3-7*02
338



L11
IGKV1-6*01
299



L12
IGKV1-5*01
296



L12a
IGKV1-5*03
298



L14
IGKV1D-17*01
310



L15
IGKV1D-16*01
308



L15a
IGKV1D-16*02
309



L16
IGKV3D-15*01
348



L18
IGKV1D-13*01
307



L19
IGKV1D-12*01
305



L2
IGKV3-15*01
334



L20
IGKV3D-11*01
347



L22
IGKV1D-42*01
314



L23
IGKV1D-43*01
315



L24
IGKV1D-8*01
316



L25
IGKV3D-7*01
350



L4/18a
IGKV1-13*02
288



L5
IGKV1-12*01
286



L6
IGKV3-11*01
332



L8
IGKV1-9*01
301



L9
IGKV1-8*01
300



O1
IGKV2D-40*01
331



O11
IGKV2-40*01
322



O11a
IGKV2-40*02
323



O12
IGKV1-39*01
295



O12a
IGKV1-39*02
868



O14
IGKV1-37*01
294



O18
IGKV1-33*01
293



O2
IGKV1D-39*01
313



O4
IGKV1D-37*01
312



O8
IGKV1D-33*01
311



Z0
IGKV1/OR2-0*01
303

















TABLE 8







Human Light Chain Lambda V Genes











Kawasaki
IMGT
SEQ ID



Nomenclature
Nomenclature
No.







V1-11
IGLV1-36*01
365



V1-13
IGLV1-40*01
366



V1-16
IGLV1-44*01
369



V1-17
IGLV1-47*02
372



V1-18
IGLV1-50*01
373



V1-19
IGLV1-51*01
374



V1-2
IGLV2-8*01
397



V1-20
IGLV10-54*02
377



V1-22
IGLV6-57*01
432



V1-3
IGLV2-11*01
380



V1-4
IGLV2-14*01
383



V1-5
IGLV2-18*01
387



V1-7
IGLV2-23*03
393



V1-9
IGLV2-33*01
394



V2-1
IGLV3-1*01
400



V2-11
IGLV3-16*01
405



V2-13
IGLV3-19*01
406



V2-14
IGLV3-21*02
408



V2-15
IGLV3-22*01
410



V2-17
IGLV3-25*02
412



V2-19
IGLV3-27*01
414



V2-6
IGLV3-9*01
416



V2-7
IGLV3-10*01
401



V2-8
IGLV3-12*02
404



V3-2
IGLV7-43*01
433



V3-3
IGLV7-46*02
435



V3-4
IGLV8-61*01
436



V4-1
IGLV5-37*01
424



V4-2
IGLV5-45*03
429



V4-3
IGLV5-48*01
430



V4-4
IGLV5-52*01
431



V4-6
IGLV11-55*01
379



V5-1
IGLV4-3*01
418



V5-2
IGLV9-49*2
440



V5-4
IGLV4-60*02
420



V5-6
IGLV4-69*01
422










ii. Modified Germline Segments
It is contemplated herein that the practice of the method is not limited to germline segment sequences. Hence, any modified VH, DH, JH, Vκ, Jκ, Vλ and or Jλ segment sequences, or any sequence analogous thereto, can be used in the practice of the method. By virtue of adding to the repertoire of segment sequences by modification thereto, the diversity of the library and the permutations of compiled segments can be further increased. The germline segments can be modified randomly or empirically. The germline segments can be modified to generate further diversity in the library. Alternatively or in addition, the germline segments can be modified to facilitate generation of the recombined full-length nucleic acid molecules by the introduction of linkers, restriction enzyme sites or other sequences of nucleotides required for practice of the method described herein.
Generally, the modified germline segments include those that are derived from germline sequences. The germline segments can be modified by introducing mutations into the germline sequence, randomly or empirically, or can be modified to generate germline consensus sequences. For example, modified JH germline segments are set forth in SEQ ID NOS: 3450-3455.
In another example, additional modifications of the germline segments include the addition of flanking sequences at one or both of the 5′ and 3′ termini of individual germline segments that provide restriction sites. Such modifications can be incorporated into the germline sequences using DNA synthesis, or by PCR, for example using primers that incorporate the restriction enzyme sites. In one example, as discussed below, the addition of such restriction sites facilitate joining of germline segments. In some cases, however, modifications of germline segments include the removal of restrictions sites. Restriction sites include any restriction site known in the art. Exemplary restriction site sequences are set forth in Table 15. Generally, the restriction site chosen is compatible with the subsequent compilation of germline segment sequences and can be chosen to facilitate blunt-ended ligation or sticky-ended ligation. The choice of restriction enzyme is routine and is well within the level of one of skill in the art.
In some examples, sequences of known antibodies, including monoclonal antibodies, particularly therapeutic antibodies, that are derived from germline sequences can be used in the methods herein. Since monoclonal antibodies already have a recognized antigen specificity, it is contemplated herein that incorporation of such derived sequences into the methods will permit the identification of antibodies with improved specificity and functionality against a target antigen. The nucleotide sequences derived from germline sequences, for example corresponding to any one or more of a VH, DH, JH, Vκ, Jκ, Vλ and or Jλ can themselves be combined with germline segment sequences. One of skill in the art can identify the corresponding sequences in a nucleic acid molecule encoding a particular antibody that are derived from germline sequences. Table 9 below identifies V, D and J regions that correlate with the derived germline sequences.







TABLE 9







Exemplary Monoclonal Antibody Sequences (V regions, normal font;


D regions, underlined; J regions, boldface)














Protein






SEQ ID
Nucleotide


TARGET
ANTIBODY

NO:
SEQ ID NO:







HEAVY CHAIN SEQUENCE




CD20
Rituxan
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW
1027
1043



(rituximab)
VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADK






SSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVW






GAGTTVTVSA




 


EGFR
Erbitux
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWV
1028
1044



(cetuximab
RQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSK






SQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG






TLVTVSA




 


Her2/Neu
Herceptin
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR
1029
 452



(trastuzumab)
QAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSK






NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ






GTLVTVSS




 


VEGFA
Avastin
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNW
1030
1045



(bevacizumab)
VRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLD






TSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYF






DVWGQGTLVTVSS




 


VEGFA
Lucentis
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNW
1031
1046



(ranibizumab)
VRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLD






TSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYF






DVWGQGTLVTVSS




 


CD52
Campath
QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMNWV
1032
1047



(alemtuzumab)
RQPPGRGLEWIGFIRDKAKGYTTEYNPSVKGRVTMLVD






TSKNQFSLRLSSVTAADTAVYYCAREGHTAAPFDYWG






QGSLVTVSS




 


TNF
Humira
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWV
1033
1048



(adalimumab)
RQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNA






KNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWG






QGTLVTVSS




 


EGFR
Vectibix
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWT
1034
1049



(panitumumab)
WIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTS






KTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTM






VTSS




 




LIGHT CHAIN SEQUENCE




CD20
Rituxan
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKP
1035
1050



(rituximab)
GSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRV






EAEDAATYYCQQWTSNPPTFGGGTKLEIK




 


EGFR
Erbitux
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQR
1036
1051



(cetuximab
TNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVE






SEDIADYYCQQNNNWPTTFGAGTKLELK




 


Her2/Neu
Herceptin
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ
1037
 818



(trastuzumab)
QKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTIS






SLQPEDFATYYCQQHYTTPPTFGQGTKVEIK




 


VEGFA
Avastin
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQ
1038
1052



(bevacizumab)
KPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISS






LQPEDFATYYCQQYSTVPWTFGQGTKVEIK




 


VEGFA
Lucentis
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQK
1039
1053



(ranibizumab)
PGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSL






QPEDFATYYCQQYSTVPWTFGQGTKVEIK




 


CD52
Campath
DIQMTQSPSSLSASVGDRVTITCKASQNIDKYLNWYQQ
1040
1054



(alemtuzumab)
KPGKAPKLLIYNTNNLQTGVPSRFSGSGSGTDFTFTISS






LQPEDIATYYCLQHISRPRTFGQGTKVEIK




 


TNF
Humira
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQ
1041
1055



(adalimumab)
KPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISS






LQPEDVATYYCQRYNRAPYTFGQGTKVEIK




 


EGFR
Vectibix
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQ
1042
1056



(panitumumab)
KPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISS






LQPEDIATYFCQHFDHLPLAFGGGTKVEIK









In some examples, the modified germline sequence can include a sequence of nucleotides that replaces all or some of the nucleotides of a germline segment. For example, it is further contemplated herein, that the modified germline sequences designated DH can be any sequence of nucleotides. The DH segment of a nucleic acid molecule encodes the central portion of the CDRH3 and is largely responsible for the antigen specificity and variability among antibodies. Since this region is the most variable among antibodies, it can tolerate more modification. Also, it is the region most responsible for antigen specificity. Generally, a segment designated DH includes 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. The sequence of nucleotides is chosen such that once compiled, in-frame, with a VH and JH as discussed below, an antibody molecule or fragment or portion thereof is produced that contains a sufficient antigen-binding site. The nucleotides are chosen randomly or empirically. In some examples, a segment of nucleotides designated DH can include a random sequence of nucleotides. In other examples, the segment of nucleotides can be selected to be targeted against a specific antigen. For example, the segment of nucleotides can encode a peptide mimetic (see e.g. Table 16 below). In additional examples, a segment of nucleotides designated DH can include nucleotides that are the reverse complement (i.e. inverted) compared to a known DH germline segment. This is exemplified in Example 14.
In other examples, germline segment sequences can be modified to provide a consensus sequence between and among germline segments. Generally, due to the variability between and among the CDR regions, consensus sequences are generated in framework regions. Such modifications aid in the practice of the method by facilitating manipulation of a common sequence, for example, where the method of generating a combinatorial antibody library is performed manually. This is exemplified in Example 1 where each of the JH contain a common F4 framework region.
b. Choosing Germline Segments or Modified Segments thereof
As described herein above, each VH and VL chain is encoded by a nucleic acid molecule combinatorially generated from gene segments, generally germline segments or modified forms thereof. The members of the resulting library can be chosen by selecting, randomly or empirically, the gene segments that can be recombined. One of skill in the art can select any desired V(D)J gene segment or subsets thereof for recombination to generate in-frame nucleic acid molecules encoding VH or VL.
In one example, the germline V(D)J segment sequences can be recombined randomly, whereby all known germline sequences (e.g. any described in the Sequence Listing herein or any available in public databases or known to those of skill in the art, and any modified forms thereof) are recombined together in all possible permutations. In such an example, every VH gene segment is recombined with every DH which is recombined with every JH. Similarly, every VL(κ or λ). is recombined with every JL(κ or λ). In such an example, the resulting recombined germline nucleic acid molecules represent the complete repertoire of naïve VH and VL. For example, if germline segments are recombined based on known germline segment sequences set forth in Tables 3-5, greater then or about 100,000 different recombined nucleic acid molecules encoding VH can be generated, greater then or about 600 different recombined nucleic acids encoding VLκ can be generated, and greater then or about 700 different recombined nucleic acid molecules encoding VLλ can be generated. Thus, libraries of nucleic acids encoding variable heavy and light chains provided herein can encode for every possible recombined antibody variable region. In addition, further diversity can be introduced by modification, such as by mutagenesis, by introducing directed peptides, or by using inverted DH sequences, as described herein above.
Alternatively, the V(D)J segments can be recombined using rational or semi-rational approaches such that a specific germline segment sequence or subset of sequences used are restricted in generating the members of the library. For example, as described in Example 14 herein, all members of the library contain a VH germline segment that is an IGHV3-23*01. In other examples, germline segment sequences can be selected that contain modifications, for example, those that contain mutations to a specific region or region generated randomly (e.g. by site-directed mutagenesis to a particular CDR) or empirically (e.g. modified to contain directed peptide mimetics). By permitting selection of germline segment sequences, the libraries provided herein are versatile and can be rationally designed based on the application of the library.
For example, antibody germline segments can be selected wherein the resulting nucleic acid sequences are restricted based on sequence similarities or differences or other shared characteristics. For example, germline segment sequences can be selected based on sequence similarities or differences or based on shared characteristics (e.g., a V region family, length, CDR3 length or composition, species, functionality, specificity, group, subgroup, pattern within the CDR, specific amino acids or other biochemical attribute). Antibody structure databases (e.g. CATH database: available at cathwww.biochem.ucl.ac.uk/; SACS database: available at bioinf.org.uk/abs/sacs/; IMGT 3D structure database: available at imgt3d.igh.cnrs.fr/) or other databases are available to sort germline segments based on a selected criteria. Alternatively, such selection can be done manually, for example, using sequence alignments or other manual sorting.
In one example, germline segments can be selected based on their sequence similarity or differences. One of skill in the art knows or can determine the sequence identity between and among germline segments, and identify germline segments that have a particular sequence identity. In one example, germline segment sequences from one or more of a VH, DH, JH, Vκ, Jκ, Vλ and or Jλ group can be selected based on sequence similarity. Sequence similarity between selected segments can include, but is not limited to, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. For example, subsets of germline segment belonging to the same subgroup or gene family can be selected, which generally share a high degree (e.g. greater then 70%, typically 75% or more) sequence identity. Tables 3-5 above identify germline segments belonging to the same subgroup or gene family. For example, in Table 3, IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6 and IGHV7 each represent a subgroup of a VH segment, and germline segments within a subgroup share at least 75% sequence identity. Thus, all germline segments in the IGHV1 can be selected, or all germline segments in IGHV2 can be selected, or all germline segments in IGHV3 can be selected, etc. In another example, in Table 3 IGHV1-18*01 and IGHV1-18*02 represent a gene family having germline segments that are alleles. Thus, all germline segments that are related by virtue of being in the same family can be selected as a subset of germline sequences.
In another example, germline segments can be selected based on sequence differences so that the resulting subset represents a diverse repertoire of sequences. One of skill in the art knows or can identify germline segments that have a particular sequence identity. Sequence differences between selected segments can include those that exhibit 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, or less sequence similarity. For example, subsets of germline segments, each from a different subgroup can be selected. Thus, in one example, VH segments can be selected from each of the IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6 and IGHV7 subgroups; for example, a subset can include IGHV1-18*01, IGHV1-26*01, IGHV3-11*01, IGHV4-28*01, IGHV6-1*01, IGHV7-4-1*03. In another example, a VH segment can be selected from each gene family; for example, a subset can include IGHV1-18*01, IGHV1-2*01, IGHV1-3*01, IGHV1-45*01, IGHV1-46*01, IGHV1-58*01, IGHV1-69*01, IGHV1-8*01, IGHV1-c*01, IGHV1-f*01, IGHV2-26*01, IGHV2-5*01, IGHV2-70*01, IGHV3-11*01, IGHV3-13*01, IGHV3-15*01, IGHV3-16*01, IGHV3-20*01, IGHV3-21*01, IGHV3-23*01, IGHV3-30*01, IGHV3-33*01, IGHV3-38*01, IGHV3-43*01, IGHV3-48*01, IGHV3-49*01, IGHV3-53*01, IGHV3-64*01, IGHV3-66*01, IGHV3-7*01, IGHV3-72*01, IGHV3-73*01, IGHV3-74*01, IGHV3-9*01; IGHV4-28*01, IGHV4-30-2*01, IGHV4-31*01, IGHV4-34*01, IGHV4-39*01, IGHV4-59*01, IGHV4-61*01, IGHV5-51*01, IGHV6-1*01 and IGHV7-4-1*01. One of skill of the art is able to select any subset of germline sequences as desired based on sequence differences. Subsets for other germline segments sequences also can be selected based on sequence differences. Tables 10-12 sets forth exemplary selected VH, DH, JH, Vκ, Jκ, Vλ and or Jλ germline segments representing selection of at least one germline segment from each gene family.







TABLE 10







Selected Human Heavy Chain Germline Segments









SEQ ID NO.














V Segments




IGHV1-18*01
10



IGHV1-2*02
13



IGHV1-24*01
16



IGHV1-3*02
18



IGHV1-45*02
20



IGHV1-46*01
22



IGHV1-58*02
26



IGHV1-69*06
32



IGHV1-8*01
40



IGHV2-26*01
44



IGHV2-5*01
45



IGHV2-70*13
67



IGHV3-11*01
68



IGHV3-13*01
70



IGHV3-15*01
73



IGHV3-16*02
82



IGHV3-20*01
83



IGHV3-21*01
84



IGHV3-23*01
86



IGHV3-30*03
93



IGHV3-35*01
117



IGHV3-38*02
119



IGHV3-43*01
120



IGHV3-48*02
123



IGHV3-49*03
127



IGHV3-53*01
130



V or D Segments



IGHV3-64*02
134



IGHV3-66*03
140



IGHV3-7*01
142



IGHV3-72*01
144



IGHV3-73*02
147



IGHV3-74*01
148



IGHV3-9*01
151



IGHV4-28*01
153



IGHV4-31*02
169



IGHV4-34*01
178



IGHV4-39*01
191



IGHV4-4*07
204



IGHV4-59*01
205



IGHV5-51*03
227



IGHV6-1*01
233



IGHV7-81*01
238



D Segments



IGHD1-1*01
239



IGHD1-14*01
240



IGHD1-20*01
241



IGHD1-26*01
242



IGHD1-7*01
243



IGHD2-15*01
244



IGHD2-2*01
245



IGHD2-21*01
248



IGHD2-8*01
250



D or J Segments



IGHD3-10*01
252



IGHD3-16*01
254



IGHD3-22*01
256



IGHD3-3*01
257



IGHD3-9*01
259



IGHD4-11*01
260



IGHD4-17*01
261



IGHD4-23*01
262



IGHD4-4*01
263



IGHD5-12*01
264



IGHD5-18*01
265



IGHD5-24*01
266



IGHD5-5*01
267



IGHD6-13*01
268



IGHD6-19*01
269



IGHD6-25*01
270



IGHD6-6*01
271



IGHD7-27*01
272



J Segments



IGHJ1*01
273



IGHJ2*01
274



IGHJ3*01
275



IGHJ4*01
277



IGHJ5*01
280



IGHJ6*01
282

















TABLE 11







Human Light Chain Kappa Germline Segments Selected for


Manual Compilation









SEQ ID NO.














V Segments




IGKV2D-30*01
330



IGKV2-30*01
321



IGKV2D-29*01
328



IGKV1-27*01
292



IGKV2-24*01
317



IGKV6D-21*01
354



IGKV3-20*01
335



IGKV1-17*01
290



IGKV5-2*01
352



IGKV4-1*01
351



IGKV1-6*01
299



IGKV1-5*01
296



IGKV1D-17*01
310



IGKV3-15*01
334



IGKV1D-42*01
314



IGKV1D-43*01
315



IGKV3D-7*01
350



IGKV1-13*02
288



IGKV1-12*01
286



IGKV3-11*01
332



V or J Segments



IGKV1-9*01
301



IGKV1-8*01
300



IGKV2D-40*01
331



IGKV1-39*01
295



IGKV1-33*01
293



J Segments



IGKJ1*01
356



IGKJ2*01
357



IGKJ3*01
361

















TABLE 12







Human Light Chain Lambda Germline Segments Selected for


Manual Compilation









SEQ ID NO.














V Segments




IGLV1-36*01
365



IGLV1-40*01
366



IGLV1-44*01
369



IGLV1-50*01
373



IGLV2-8*01
397



IGLV10-54*02
377



IGLV6-57*01
432



IGLV2-11*01
380



IGLV2-14*01
383



IGLV2-18*01
387



IGLV2-23*03
393



IGLV2-33*01
394



IGLV3-1*01
400



J Segments



IGLV3-16*01
405



IGLV3-19*01
406



IGLV3-21*02
408



IGLV3-22*01
410



IGLV3-25*02
412



IGLV3-27*01
414



IGLV3-9*01
416



IGLV3-10*01
401



IGLV3-12*02
404



IGLV7-43*01
433



IGLV7-46*02
435



IGLV8-61*01
436



IGLV5-37*01
424



IGLV5-45*03
429



IGLV5-48*01
430



IGLV5-52*01
431



IGLV11-55*01
379



IGLV4-60*02
420



IGLV4-69*01
422



IGLJ1*01
442



IGLJ2*01
443



IGLJ4*01
446



IGLJ5*01
447



IGLJ6*01
449



GLJ7*01
450










In all of the examples above, selection of germline segments based on sequence similarity or differences or other characteristics can be restricted for only one group of germline segments (from among any of VH, DH, JH, Vκ, Jκ, Vλ and or Jλ), 2 groups, 3 groups, 4 groups, 5 groups, 6 groups or all 7 groups. Thus, for example, in recombining the gene segments to encode for a plurality of VH chains, only the VH germline segment sequences can be restricted based on sequence similarity or differences or other characteristic, and the DH and JH segment sequences can represent all known DH and JH germline segment sequences. In another example, each of the VH, DH and JH segment sequences can be selected based on sequence similarity or differences or other characteristic, thereby resulting in a restricted subset of germline segment sequences for compilation herein. In yet another example, in recombining the gene segments to encode for a plurality of VH chains, the DH segment is restricted based on its modification to include nucleotides encoding a particular peptide mimetic or mimetics against a target. In such an example, the VH and JH segment sequences can represent all known germline segment sequences to be recombined with the restricted subset of DH (e.g. modified) segment. The choice of germline segments selected for use in the compilation method provided herein depends on a variety of factors including, but not limited to, the diversity of the resulting library, knowledge regarding preference for a particular germline segment sequence for a target from an initial screen of a library (as described herein below under Section G.4 entitled iterative screening), and the size of the library.
c. Sequence Compilation
In the methods provided herein, the variable gene segment sequences are recombined to generate heavy chain variable regions (5′-VHDHJH-3′), kappa light chain variable regions (5′-VκJκ-3′), and lambda light chain variable regions (5′-VλJλ-3′) as described below. The gene segments can be recombined to generate full-length variable regions, or variable regions that are less then full length (i.e. portion thereof of full length), so long as the portion is sufficient to form an antigen binding site when expressed. The nucleic acid sequences are combined so that the resulting nucleic acid molecule is in-frame and encodes a functional VH or VL polypeptide, i.e. a full-length polypeptide or a portion thereof that is sufficient to form an antigen binding site.
The compilation method provided herein can be implemented by any procedures known to one of skill in the art. For example, it can be implemented manually, in silico (e.g. through computer software) or combinations thereof. In some examples, as described elsewhere herein below, the method can be implemented using sequence compilation software. In addition, public databases, such as those providing germline segment sequences or other public bioinformatics tools can be use to aid practice of the method.
Generally, full length heavy chain variable regions (5′-VHDHJH-3′), or portions thereof, are recombined such that a VH segment is combined with a DH segment which is combined with a JH segment. Heavy chain segments are always combined such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment. The exact VHDHJH can be chosen randomly or empirically and can represent germline segments, or modified forms thereof as discussed above. It is understood that when the method is performed manually using molecular biology techniques, restriction enzymes can be added to the ends of germline segments to facilitate joining of segments. Hence, in some examples, the resulting VH chain can contain additional amino acids between germline segments. For example, as described in the examples herein, nucleic acid molecule library members encoding a VH chain can encode sequences that contain amino acids SY at 5′end of the JH region between the joined DH region.
In one example of the methods herein, all permutations of VHDHJH gene segments can be recombined to generate a nucleic acid molecule encoding a variable heavy chain. Thus, every VH segment (e.g., set forth in any of SEQ ID NOS:10-238), is combined with every DH segment (e.g., any set forth in SEQ ID NOS: 239-272), which is combined with every JH segment (e.g. any set forth in SEQ ID NOS: 273-285). In such an example, based on the exemplary heavy chain germline segments set forth in Table 3, greater then or about 100,000 nucleic acid molecules encoding a variable heavy (VH) chain can be generated. In other examples, the VHDHJH gene segments can be recombined empirically (e.g. using rational or semi-rational approaches as discussed below). For example, as discussed below any subset of VH, DH and/or JH gene segment can be chosen to generate a recombined nucleic acid molecule. In some examples, individual gene segments are selected because of a shared characteristic including, but not limited to, diversity, same V region family, CDR3 length, composition or other biochemical attribute.
Full length kappa light chain variable regions (5′-VκJκ-3′), or portions thereof, are recombined such that a Vκ segment is combined with a Jκ segment. Full length lambda light chain variable regions (5′-VλJλ-3′) are recombined such that a Vλ segment is combined with a Jλ segment. Light chain segments are always combined such that the VL segment is 5′ to the JL segment. The exact VκJκ or VλJλ can be chosen randomly or empirically and can represent germline segments, or modified forms thereof as discussed above. It is understood that when the method is performed manually using molecular biology techniques, restriction enzymes can be added to the ends of germline segments to facilitate joining of segments. Hence, in some examples, the resulting VL chain can contain additional amino acids between germline segments.
In one example of the methods herein, all permutations of Vκ Jκ can be recombined to generate a nucleic acid molecule encoding a variable kappa light chain. Thus, every Vκ (e.g. any set forth in SEQ ID NOS:286-355, 868) is combined with every Jκ (e.g. any set forth in SEQ ID NOS: 356-364). In such an example, based onthe exemplary kappa light chain germ line segments set forth in Table 4, greater then or about 600 nucleic acid molecules encoding a variable kappa light chain can be generated. In another example, all permutations of VλJλ can be recombined to generate a nucleic acid molecule encoding a variable lambda light chain. Thus, every Vλ (e.g. any set forth in any of SEQ ID NOS:365-441) is combined with every Jλ (e.g. any set forth in any of SEQ ID NOS: 442-451). In such an example, based on the exemplary lambda light chain germline segment set forth in Table 5, greater then or about 700 nucleic acid molecules encoding a variable lambda light chain can be generated. In another example, the VκJκ or VλJλ gene segments can be recombined empirically as described herein below.
In all of the examples above, recombined segments are joined such that the recombined full length nucleic acid is in frame with the 5′ start codon (ATG), thereby allowing expression of a full length polypeptide. Any combination of a V(D)J can be made, and junctions modified accordingly in order to generate a compiled V(D)J sequence that is in-frame, while preserving reading frames of each segment. The choice of junction modification is a function of the combination of V(D)J that will be joined, and the proper reading frame of each gene segment. For example, any of the variable gene segments can exist in reading frame 1, 2 or 3 when compiled. Generally, however, for the practice of the method herein, the V sequence (VH, Vκ or Vλ) is always reading frame 1. Also, the reading frame of the J sequence is set to be either reading frame 1, 2 or 3 such that the resulting gene segment encodes the correct amino acids. Table 13 below sets forth the reading frames of the exemplary J germline sequences.







TABLE 13







J Germline Segment Sequences in Coding Frames











SEQ




ID



SEQUENCE
NO





Heavy




IGHJ1*01
GCT GAA TAC TTC CAG CAC TGG GGC CAG GGC ACC CTG GTC ACC GTC TCC TCA G
273


 


IGHJ2*01
C TAC TGG TAC TTC GAT CTC TGG GGC CGT GGC ACC CTG GTC ACT GTC TCC TCA G
274


 


IGHJ3*01
T GAT GCT TTT GAT GTC TGG GGC CAA GGG ACA ATG GTC ACC GTC TCT TCA G
275


 


IGHJ3*02
T GAT GCT TTT GAT ATC TGG GGC CAA GGG ACA ATG GTC ACC GTC TCT TCA G
276


 


IGHJ4*01
AC TAC TTT GAC TAC TGG GGC CAA GGA ACC CTG GTC ACC GTC TCC TCA G
277


 


IGHJ4*02
AC TAC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G
278


 


IGHJ4*03
GC TAC TTT GAC TAC TGG GGC CAA GGG ACC CTG GTC ACC GTC TCC TCA G
279


 


IGHJ5*01
AC AAC TGG TTC GAC TCC TGG GGC CAA GGA ACC CTG GTC ACC GTC TCC TCA G
280


 


IGHJ5*02
AC AAC TGG TTC GAC CCC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA G
281


 


IGHJ6*01
AT TAC TAC TAC TAC TAC GGT ATG GAC GTC TGG GGG CAA GGG ACC ACG GTC ACC GTC
282



TCC TCA G



 


IGHJ6*02
AT TAC TAC TAC TAC TAC GGT ATG GAC GTC TGG GGC CAA GGG ACC ACG GTC ACC GTC
283



TCC TCA



 


IGHJ6*03
AT TAC TAC TAC TAC TAC TAC ATG GAC GTC TGG GGC AAA GGG ACC ACG GTC ACC GTC
284



TCC TCA



 


IGHJ6*04
AT TAC TAC TAC TAC TAC GGT ATG GAC GTC TGG GGC AAA GGG ACC ACG GTC ACC GTC
285



TCC TCA G



 


Light Kappa




IGKJ1*01
G TGG ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA C
356


 


IGKJ2*01
TG TAC ACT TTT GGC CAG GGG ACC AAG CTG GAG ATC AAA C
357


 


IGKJ2*02
G TGC ACT TTT GGC CAG GGG ACC AAG CTG GAG ATC AAA C
358


 


IGKJ2*03
TG TAC AGT TTT GGC CAG GGG ACC AAG CTG GAG ATC AAA C
359


 


IGKJ2*04
TG TGC AGT TTT GGC CAG GGG ACC AAG CTG GAG ATC AAA C
360


 


IGKJ3*01
A TTC ACT TTC GGC CCT GGG ACC AAA GTG GAT ATC AAA C
361


 


IGKJ4*01
G CTC ACT TTC GGC GGA GGG ACC AAG GTG GAG ATC AAA C
362


 


IGKJ4*02
G CTC ACG TTC GGC GGA GGG ACC AAG GTG GAG ATC AAA C
363


 


IGKJ5*01
G ATC ACC TTC GGC CAA GGG ACA CGA CTG GAG ATT AAA C
364


 


Light Lambda




IGLJ1*01
T TAT GTC TTC GGA ACT GGG ACC AAG GTC ACC GTC CTA G
442


 


IGLJ2*01
T GTG GTA TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA G
443


 


IGLJ3*01
T GTG GTA TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA G
444


 


IGLJ3*02
T TGG GTG TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA G
445


 


IGLJ4*01
T TTT GTA TTT GGT GGA GGA ACC CAG CTG ATC ATT TTA G
446


 


IGLJ5*01
C TGG GTG TTT GGT GAG GGG ACC GAG CTG ACC GTC CTA G
447


 


IGLJ5*02
C TGG GTG TTT GGT GAG GGG ACG GAG CTG ACC GTC CTA G
448


 


IGLJ6*01
T AAT GTG TTC GGC AGT GGC ACC AAG GTG ACC GTC CTC G
449


 


IGLJ7*01
T GCT GTG TTC GGA GGA GGC ACC CAG CTG ACC GTC CTC G
450


 


IGLJ7*02
T GCT GTG TTC GGA GGA GGC ACC CAG CTG ACC GCC CTC G
451









For the heavy chain, the reading frame of the D variable gene segment sequence chosen is less rigid then for the V or J germline segments. This is because the D gene sequence is responsible for encoding the central portion of the CDRH3, which plays a prominent role in antigen specificity. Hence, variation of amino acids is expected in the D gene segment sequence. Thus, for example, the DH gene segment can be any DH gene segment in any reading frame, an inverted or reverse complement thereof, or a modified form thereof, or any sequence of nucleotides designated as the DH. In some examples, however, the reading frame of the D germline sequence is chosen so that the resulting encoded amino acids are predominately hydrophilic. CDR3 is an antigen-binding site, and thereby is rich in hydrophilic residues that are surface exposed (see e.g., Zanetti and Billetta, Antigenized Antibodies from Concepts to Applications (1996), In The Antibodies, Volume 2 (pp. 75-122), Harwood Academic Publishers; Pommie et al. (2004) J Mol. Recognition, 17:17-32). One of skill in the art is familiar with techniques to assess the hydrophobicity/hydrophilicity of sequences. For example, hydrophilicity can be measured using protein grand average of hydropathy (GRAVY), which gives hydropathy value for a sequence by adding the hydropathy value for each residue and dividing by the length of the sequence (see e.g., Kyte and Doolittle (1982 and bioinformatics.org/sms2/protein_gravy.html). The lower the GRAVY value, the more hydrophilic a sequence is.
In some instances, compilation of variable gene segments in-frame, while preserving reading frames, requires no manipulation, i.e. no modification of joint regions. In other instances, however, simply compiling V(D)J sequences does not conserve reading frames. Thus, where the junctions between gene segments are not in the desired frame, modifications are made to nucleotides within the junctions between the segments so that each gene segment is in its desired reading frame, and the full length sequence is in-frame. Nucleic acid modifications include replacements or substitutions, insertions, or deletions of nucleotides, or any combination thereof. For example, at the V-D junction, one or more nucleotide can be deleted from the 5′ end of the D, one or more nucleotide can be deleted from the 3′ end of the V or one or more nucleotides can be inserted between the V and D (e.g. a nucleotide can be added to the 3′ end of the V). In another example, at the D-J junction, one or more nucleotides can be deleted from the 5′ end of the J, one or more nucleotides can be deleted from the 3′ end of the D, or one or more nucleotides can be inserted between the D and J (e.g., a nucleotide can be added to the 3′ end of the D). In a further example, at the V-J junction, as occurs in generation of a light chain, one or more nucleotides can be deleted from the 5′ end of the J, one or more nucleotides can be deleted from the 3′ end of the V or one or more nucleotides can be inserted between the V and J (e.g. a nucleotide can be added to the 3′ end of the V). In such examples where nucleotides are inserted, any nucleotide insertion from among one or more of a guanine (G), adenine (A), cytosine (C) and thymine (T) is contemplated. In some examples, guanine (G) is chosen as the inserted nucleotide because of the slight preference of terminal deoxynucleotidyl transferase (TdT) for guanine residues (Alt et al. 1982).
In the methods, heavy chain segments are recombined separately from light chain gene segment sequences. Thus, an individual nucleic acid molecule encodes for either a heavy chain (VH) or a light chain (VL) variable region. In the methods, a plurality of VH nucleic acid molecules encoding a VH chain and a plurality of nucleic acid molecules encoding a VL chain are generated. The number of such sequences can be up to all possible permutations depending on the number of V, D or J gene segments available for combination. For example, where all known germline segment sequences are used for practice of the method, a fully naïve antibody library is generated. In other examples, modified gene segments can also be used for practice of the method. Alternatively, the number of permutations is a function of the selected V, D and J, which can be a subset of all germline segments or modified forms thereof.
Once a nucleic acid sequence is compiled, it is further modified to remove stop codons so that the resulting molecule is functional, i.e. encodes a polypeptide that is not truncated early. For example, modifications to remove stop codons include substitutions of nucleotides. Exemplary of such modifications, include, but are not limited to, stop codon TAA replaced by codons TAT; stop codon TAG replaced by codons TAT, and stop codon TGA replaced by codons TCA.
d. Further Sequence Modification of Recombined Nucleic Acid Sequences As discussed above, germline segment sequences can be modified before performing compilation as described herein. In addition or alternatively, modification can be made directly to the recombined nucleic acid sequence. Hence, it is understood that any of the modifications described below also can be made to individual germline segment sequences before compilation so long as the reading frames are maintained and the rules governing compilation as described herein are observed to generate in-frame recombined nucleic acid sequences.
Thus, any of the plurality of recombined nucleic acids encoding a VH chain or a VL chain can be further modified. Modifications of the nucleic acid sequences include replacements or substitutions, insertions, or deletions of nucleotides, or any combination thereof. Any modification contemplated by one of skill in the art can be made to the nucleic acid molecule, so long as the modification(s) do not interfere with or alter the junction joints made to maintain reading frames of the V(D)J segments achieved by virtue of practice of the method (as discussed in the Section entitled “Sequence Compilation” above). Any modification should be checked to confirm that all reading frames are intact to ensure that the resulting full length nucleic acid is in frame with the 5′ start codon (ATG) thereby allowing expression of a full length VH or VL polypeptide, or a portion thereof that is sufficient to form an antigen binding site.
The resulting recombined germline variable heavy and light chain nucleic acid sequences can be further modified through DNA synthesis (i.e. modifications introduced upon synthesis of the nucleic acid molecule) or by using standard molecular biology techniques. Thus, in one example, any desired modification contemplated can be made to a nucleic acid molecule encoding a recombined variable heavy or variable light chain and the resulting nucleic acid molecule including any modifications synthesized as described in sub-section e.iii below. Due to the degeneracy of the genetic code nucleic acid sequences can be designed to avoid unwanted nucleotide sequences, including unwanted restriction sites, splicing donor or acceptor sites, or other nucleotide sequences potentially detrimental to efficient translation. Additionally, organisms sometimes favor particular codon usage and/or a defined ratio of GC to AT nucleotides. Thus, degeneracy of the genetic code permits design of nucleic acid sequences tailored for expression in particular organisms or groups of organisms. Additionally, nucleic acid molecules can be designed for different levels of expression based on optimizing (or non-optimizing) of the sequences. In another example, generated recombined germline VH and VL nucleic acid molecules as described in sub-section e.iii below, can be further modified using using standard molecular biology techniques, such as PCR, site-directed mutagenesis, restriction enzyme digestion, ligation, cloning and any combination thereof. The choice of whether to generate such modifications during DNA synthesis or using molecular biology techniques is dependent on the end user and can be influenced by factors such as the purpose of the modification, the extent of the modification and timing considerations.
Modifications of recombined germline nucleic acid molecules encoding VH or VL can be generated randomly or empirically. For example, random mutation of one or more regions can increase diversity of the library, particularly where modifications are made to any of the CDR-loop regions, which contribute to the specificity and affinity of the antibody. This library with increased diversity permits the generation of antibodies, derivatives thereof or portions or fragments thereof, which potentially can bind to any desired antigen with a high affinity. In another example, modifications can be empirically generated using rational or semi-rational approaches. Among such empirical modifications of nucleic acid molecules encoding VH and VL chains contemplated herein include, but are not limited to, modifications of the CDR regions, for example for the generation of directed libraries, modifications to optimize codon usage, and/or modifications to introduce restriction sites or detectable moieties. Modifications also can include a combinations of random and empirical modifications.
i. Codon Usage
For example, nucleic acid sequences can be modified to adapt the codon usage for expression such as, for example, bacterial expression. Codon usage is degenerate in that multiple codons encode for the same amino acid. Thus a single amino acid is therefore encoded by multiple codons, however within any organism, codon usage varies for any given amino acid. The full length nucleic acids provided herein are modified to replace rare codons with more abundant codons utilized in the particular expression system. Typically, modifications include silent mutations, such that the substitutions do not alter the specificity of the codon. Codon usage tables are known to those of skill in the art, particularly for common expression systems. For example, for expression in bacteria E. coli K12, codon usage Tables are known (see, e.g., Grantham, R. et al., Nuc. Acids Res., 8:1892-1912 (1980); Grantham, R. et al., Nuc. Acids Res., 9:r43-r74 (1981) and also Table 14). The codon usage table lists all of the sixty four possible three nucleotide codons for DNA or RNA with their frequency of usage in the bacteria E. coli K12. The Table shows that while a single amino acid is encoded by multiple codons (redundancy), these codons are not used at the same rate for any given amino acid. For example, the amino acid arginine is coded for by six different codons: CGT, CGC, CGA, CGG, AGA and AGG. The codon AGA has a frequency of 2.0% while the codon CGC has a frequency of 22%.







TABLE 14





Codon Usage in E. coli K12


fields: [triplet] [frequency: per thousand] ([number])


















TTT 22.2 (30361)
TCT 8.4 (11498)
TAT 16.1 (22071)
TGT 5.1 (7020)


TTC 16.6 (22649)
TCC 8.6 (11804)
TAC 12.2 (16734)
TGC 6.4 (8787)


TTA 13.8 (18915)
TCA 7.1 (9706)
TAA 2.0 (2752)
TGA 0.9 (1261)


TTG 13.6 (18601)
TCG 8.9 (12156)
TAG 0.2 (321)
TGG 15.2 (20835)


CTT 11.0 (15043)
CCT 7.0 (9554)
CAT 12.9 (17656)
CGT 21.0 (28700)


CTC 11.1 (15183)
CCC 5.4 (7448)
CAC 9.7 (13329)
CGC 22.0 (30159)


CTA 3.9 (5303)
CCA 8.4 (11518)
CAA 15.3 (20970)
CGA 3.5 (4787)


CTG 52.9 (72403)
CCG 23.3 (31869)
CAG 28.9 (39560)
CGG 5.4 (7320)


ATT 30.4 (41551)
ACT 8.9 (12197)
AAT 17.7 (24192)
AGT 8.7 (11917)


ATC 25.2 (34426)
ACC 23.5 (32101)
AAC 21.7 (29656)
AGC 16.1 (21961)


ATA 4.3 (5827)
ACA 7.0 (9564)
AAA 33.7 (46044)
AGA 2.0 (2783)


ATG 27.8 (38012)
AGC 14.4 (19743)
AAG 10.3 (14043)
AGG 1.1 (1533)


GTT 18.3 (25079)
GCT 15.3 (20863)
GAT 32.2 (44103)
GGT 24.9 (34009)


GTC 15.3 (20913)
GCC 25.6 (35018)
GAC 19.2 (26201)
GGC 29.8 (40725)


GTA 10.9 (14885)
GCA 20.2 (27638)
GAA 39.7 (54267)
GGA 7.9 (10817)


GTG 26.3 (35960)
GCG 33.8 (46222)
GAG 17.8 (24414)
GGG 11.0 (15116)









ii. Adding or Removing Restriction Enzyme Sites
In another example, additional modifications of the nucleic acids include the addition of flanking sequences at one or both of the 5′ and 3′ termini of recombined VH or VL nucleic acid sequences that provide restriction sites. Such modifications can be incorporated into the germline recombined nucleic acid molecules during DNA synthesis, or by PCR, for example using primers that incorporate the restriction enzyme sites. In some examples, addition of such restriction sites facilitate cloning of the nucleic acids into a selected vector. For example, restriction sites include any restriction site known in the art. Exemplary restriction site sequences are set forth in Table 15. Generally, the restriction site chosen is compatible with the expression vector and can be chosen to facilitate blunt-ended ligation or sticky-ended ligation. The choice of restriction enzyme is routine and is well within the level of one of skill in the art.







TABLE 15







Common Restriction Enzyme Cleavage


Recognition Sites











Restriction
Cleavage
SEQ ID



Enzyme
Sequence
NO







NcoI
CCATGG
 977



 



NheI
GCTAGC
 978



 



AvrII
CCTAGG
 979



 



BsiWI
CGTACG
 980



 



SfiI
GGCCNNNNNGGCC
1889



 



NotI
GCGGCCGC
1890



 



HindIII
AAGCTT
1891



 



EcoRI
GAATTC
1892



 



BamHI
GGATCC
1893



 



EcoRV
GATATC
1894



 



PstI
CTGCAG
1895



 



SalI
GTCGAC
1896



 



SmaI
CCCGGG
1897



 



XmaI
CCCGGG
1898



 



BglI
GCCNNNNNGGC
1899



 



MfeI
CAATTG
1900



 



BsaI
GGTCTCN
3719










In some examples, nucleic acids can be modified to remove any restriction sites that occur within the nucleic acid sequence. In particular, removal of restriction sites is desired so that such sites do not interfere with subsequent digestion, ligation and cloning procedures. For example, as discussed above, recombined nucleic acid molecules can be modified to contain terminal flanking restriction sites to facilitate cloning into expression vectors. Generally, such restriction sites are chosen to be unique so that the presence of the site exists only at the terminal flanking end(s). If the site is not unique, modifications can be made to the sequence of the nucleic acid molecule to remove any conflicting restriction sites. One of skill in the art is familiar with restriction sites and can identify such sites within a nucleic acid sequence. Table 15 lists exemplary restriction sites that can be removed.
In some instances, a single nucleotide change is possible to effect change of the restriction site. In other instances, two or three nucleotide changes are necessary to remove a restriction site. Typically, modification of restriction sites existing internally in a recombined nucleic acid molecule are made in view of the codon usage as discussed above. For example, if a Sal I restriction sites (GTCGAC; SEQ ID NO:1896) exists internally in a nucleic acid molecule, the GTC codon that codes for valine (V) can be modified to GTA, GTG, GTT or GTC codons. Simply changing the last C to G correlates to changing the GTC codon (15.3% frequency of usage) to GTG (26.3% frequency of usage), which is an 11% increase in frequency of codon usage. Alternatively, the GAC codon (19.2% frequency of usage) that codes for asparagine (D) can be modified to GAT (32.2% frequency of usage) by changing the last C to T, which is a 13% increase in codon usage. In this example, either of the above modifications can be made. Typically, modifications are made to convey the highest absolute beneficial increase in frequency of codon usage.
iii. Linkers
In additional examples, nucleic acid molecules can be modified with a linker sequence. For example, where a single-chain antibody is desired (e.g. an scFv antibody) the variable heavy and light chains can first be joined by a linker. The linkage can be direct or via a linker. For example, nucleic acids encoding peptide linkers can be added during DNA synthesis or using molecular biology techniques to the 5′ end of a first sequence (e.g. variable heavy chain) and the 3′ terminus of a second nucleic acid sequence (e.g. variable light chain). Typically, the linker is of sufficient length so that the resulting polypeptide is soluble. Nucleic acid sequences for use as linkers can encode peptide linkers from about 2 or 2 to about 60 or 60 amino acid residues, for example from about 5 to 40, or from about 10 to 30, 2 to 6, 7, or 8 amino acid residues. Examples of known linker moieties include, but are not limited to, peptides, such as (GlymSer)n and (SermGly)n, in which n is 1 to 6, including 1 to 4 and 2 to 4, and m is 1 to 6, including 1 to 4, and 2 to 4. Exemplary of such linkers include any that encode peptide linkers such as glycine serine polypeptides, such as -Gly-Gly-, GGGGG (SEQ ID NO:981), GGGGS (SEQ ID NO:982) or (GGGGS)n (SEQ ID NO:985), SSSSG (SEQ ID NO:983) or (SSSSG)n (SEQ ID NO:1996). Linking moieties are described, for example, in Huston et al. (1988) PNAS 85:5879-5883, Whitlow et al. (1993) Protein Engineering 6:989-995, and Newton et al., (1996) Biochemistry 35:545-553. Other suitable linkers include any encoding a peptide linker, such as any of those described in U.S. Pat. Nos. 4,751,180 or 4,935,233, which are hereby incorporated by reference. A polynucleotide encoding a desired peptide linker can be inserted anywhere in variable heavy or light chain sequence or at the 5′- or 3′-terminus, in frame, using any suitable conventional technique. For example, restriction sites can be added to the 5′ terminus of the heavy chain sequence and to the 3′ terminus of the light chain sequence while a nucleic acid encoding a linker segment (e.g. (Gly4Ser)3; SEQ ID NO:984) can be added to the 3′ terminus of the heavy chain sequence connecting it to the 5′ terminus of the light chain sequence. Upon expression, such a nucleic acid molecule encodes an scFv antibody where the heavy chain variable region is operably linked to the light chain variable region.
iv. Tags or Detectable Moieties
Additionally, a small epitope tag, such as a myc tag, His tag, Flag tag or other small epitope tag, and/or any other additional DNA sequence can be added for incorporation into a nucleic acid sequence encoding a variable heavy chain or variable light chain (Arnau et al. (2006) Protein Expression and Purification, 48:1-13). In some instances, for example, a tag that permit attachment, for example, an LPETG tag, can be added that allows for site specific modification using the protein ligase, sortase (Chan et al. (2007) PLoS ONE, 2:e1164). Hence, inclusion of such a tag permits immobilization (e.g. on a BIAcore chip) and/or selective sorting in the presence of a sortase. Generally, the additional DNA sequence is added to the 3′ or 5′ terminus of the nucleic acid molecule encoding the recombined variable sequence directly or indirectly using a linker. Alternatively, the additional DNA sequence can be included in the expression vector of choice, such that, upon expression, the resulting antibody contains the additional sequence. For example, plasmid A set forth in SEQ ID NO:1 contains a His-Flag Tag corresponding to nucleotides 3265-3306 (Flag corresponds to nucleotides 3265-3288; His corresponds to nucleotides 3289-3306). In another example, Plasmid D set forth in SEQ ID NO: 2 contains a Flag tag corresponding to nucleotides 3265-3288, an LPETG tag corresponding to nucleotides 3289-3303. Thus, upon expression of the heavy chains, alone or together with a variable light chain, resulting antibodies can be detected using anti-Flag or anti-His tag reagents. This is described in Example 10. One of skill in the art can add any desired detectable sequence or other identifiable moiety to a nucleic acid molecule encoding a recombined variable heavy or light chain sequence to facilitate identification and/or purification of the resulting antibodies.
v. Mutational Diversity
In other examples, modifications can be made to introduce mutational diversity into the resulting nucleic acid molecules. Any modification can be made, such as by replacement, substitution, deletion or addition of amino acids, either randomly or empirically (i.e. into any region or segment of the recombined nucleic acid molecule). The modifications can be made during DNA synthesis or using routine molecular biology techniques such as site-directed mutagenesis, digestion with restriction enzymes and/or cloning.
For example, modification(s) can be introduced into a nucleic acid molecule encoding the VH chain, a nucleic acid molecule encoding the VL chain, or both. The modification(s) can be introduced in the region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3 or FR4. For example, modifications can be introduced into one, two or all three of the three CDRs of a given variable domain (VH, VL or both). In one example, modifications are introduced into CDR1 and CDR2, e.g. of a heavy chain variable domain. Typically, modification(s) are introduced into the CDR3 of the heavy chain (CDRH3). Any combination is contemplated. One of skill in the art knows and can identify CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4 regions in a nucleic acid molecule encoding a VH or VL (see e.g., Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al. (1997) J Mol. Biol., 273:927-948); WO/2007/137616; bioinf.org.uk/abs/; bioc.unizh.ch/antibody/Numbering/NumFrame.html; Martin et al., Bioinformatics Tools for Antibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH (2007), pp. 96-103). For example, CDRs can be identified in VH and VL chains using Kabat numbering based on sequence alignment or the Chothia numbering scheme based on structural topology. Since the Kabat numbering scheme was developed from sequence alignment, insertions in the sequence relative to the numbering scheme by alignment are indicated by letters (e.g. 27, 27A, 27B, 27C, etc. . . . ) and deletions have the corresponding number skipped. The residues corresponding to the six CDRs of the light and heavy chains based on Kabat numbering are CDR-L1: L24-L34; CDR-L2: L50-L56; CDR-L3: L89-L97; CDR-H1; H31-H35B; CDR-H2: HSO-H65; CDR-H3: H95-H102. One of skill in the art knows that CDR lengths can vary and can identify corresponding residues, for example, by alignment and use of kabat numbering.
vi. Directed Peptides
In some cases, modifications include rationally generated modifications to generate antibodies and portions or fragments thereof that mimic the activity of biologically active peptides against known targets (see e.g., International published PCT Application No. WO 2004/050017). Important biological functions, such as receptor binding, activation and enzymatic activity, are often attributable to discrete regions of larger protein molecules, containing a limited number of amino acid residues termed peptide epitopes and mimitopes. These peptide epitopes and mimitopes can be used as therapeutics, but due to their small size, are typically unstable in vivo due to rapid degradation. The peptide epitopes, however, can be introduced into variable regions of antibodies, which can act to mimic the activity of the biologically active peptide. Such antibodies are more stable, and exhibit increased half-life. Thus, antibodies or portions thereof can be directed toward a known target or function by incorporating sequences into the variable regions of an antibody that correspond to a polynucleotide target of interest. Often, structure and or function information of the known targets is available. These libraries are useful in providing lead antibodies for future antibody libraries.
Hence, included in the modifications herein are nucleic acid sequences encoding germline recombined VH and VL, wherein nucleotides corresponding to one or more CDR is replaced with nucleotides encoding one or more amino acid residues for a peptide of choice. In one example, the modifications in a nucleic acid molecule encoding a germline recombined VH and/or VL can be generated during DNA synthesis. Alternatively, the modification can be introduced into a nucleic acid molecule encoding a germline recombined VH and/or VL by restriction digestion followed by ligation with a peptide of choice. If necessary, restriction sites can be created in a CDR, such as by site-directed mutagenesis or PCR, to facilitate ligation of the peptide. This latter method is described in Example 12 herein.
The nucleotides can encode for peptides of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 16, 20, 25 or more amino acids. Any peptide that exhibits a useful property is suitable for insertion into an antibody scaffold. Generally, the peptide is one that specifically binds a target molecule. The peptides also include those that exhibit a specific activity, for example, an agonist or antagonist activity upon binding to the target. Peptide activities and uses include, but are not limited to, binding a receptor, binding a membrane bound surface molecule, binding a ligand, binding an enzyme or structural protein, activating or inhibiting a receptor, target drug delivery or any enzymatic activity. Exemplary of peptides are those that bind to a cell surface receptor such as a receptor for a cytokine, growth factor or growth inhibitor. Peptide mimetics for incorporation into a recombined germline VH or VL include any set forth in U.S. Pat. Nos. 7,169,905; 7,396,917, 7,272,508, 7,019,017; U.S. published Patent Appl. No. US200701344; published International Appl. No. WO2005060642; Johnson et al. (2000) Nephrol Dial. Transplant, 15:1274-1277. Exemplary of such peptides are set forth in Table 16. Other peptides for incorporation into the VH and VL encoded by the recombined germline nucleic acids provided herein are known in the art (see e.g., any of the references cited above) and/or can be identified depending on the desired target.







TABLE 16







Exemplary Peptide Mimetics











SEQ ID


Peptide
Sequence
NO:





TPO
IEGPTLRQWLAARA
 987


 



GGCADGPTLREWISFCGGK
 988


 



GGCADGPTLREWISFCGG
 989


 



LAI EGPTLRQWLHGNGRDT
 990


 



GNADGPTLRQWLEGRRPKN
 991


 



TIKGPTLRQWLKSREHTS
 992


 


EPO
TYSCHFGPLTWVCKPQ
 891


 



DYHCRMGPLTWVCKPLGG
 993


 



GGTYSCHFGPLTWVCKPQGG
 994


 



DREGCRRGWVGQCKAWFN
 995


 



QRVEILEGRTECVLSNLRGRTRY
 996


 


G-CSF
EEDCK
 997


 


IL-5
TGGGDGYVCVEWARCPTCK
 998


 



EGYVCVEWAACPTCR
 999


 


human brain 
CFGRKMDRISSSSGLGC
1000


natriuretic peptide




(hBNP-32)




 



FGRKMDRISSSSGLG
1001


 


Exendin 4
HGEGRFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
1002


 


GLP-1
HAEGTFTSDVSSYLEGQMKEFIAWLVKGR
1003


 


GLP-2
HADGSFSDEMNTILDNLAARDFINWLIQTKITDR
1004


 


Glucagon
HSQGTFTSDYSKYLDSRRAQDRVQWLMNT
1005


 


PACAP-38
HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK
1006


 


CD209L
RYWNSGEPNNSGNEDCAEFSGSGWNCNRCDVDN
1007


 


TNF
YCFTASENHCY
1008


 



YCFTNSENHCY
1009


 


VEGF
VEPNCDIHVMWEWECFERL
1010


 



GERWCFDGPLTWVCGEES
1011


 


MMP inhibitor
CTTHWGFTLC
1012


 


CTLA-4
CSLHWGFWWC
1013


 



GFVCSGIFAVGVGRC
1014









Nucleic acid molecules encoding for recombined germline VH or VL can be modified by replacement or introduction of nucleotides encoding a peptide into one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3 or FR4. For example, nucleic acid molecules encoding for recombined germline VH or VL can be modified by replacement of an entire CDR with nucleotides encoding a peptide. The CDR replaced by a peptide can be CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3. For example, in the resulting VH or VL chain one or more CDRs is replaced by a peptide. The peptides can be the same or different. In another example, nucleic acid molecules encoding for recombined human germline VH and VL are modified by replacement of a portion of a CDR with nucleotides encoding a peptide. The portion of the CDR replaced by the nucleotides is a portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3. The portion of the CDR replaced by the nucleotides can encode for a peptide that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 16, 20 or 25 or more amino acids. In an additional example, one or more portions of two or more CDRs are replaced by nucleotides encoding a peptide. The resulting peptides can be the same or different. In a further example, nucleic acid molecules encoding for recombined human germline VH or VL can be modified by insertion of nucleotides encoding a peptide between two nucleotides of a CDR of the antibody. The CDR with a peptide inserted is CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3. In some instances, the resulting VH or VL chain includes one or more peptides inserted in one or more CDRs. The peptides inserted into the CDRs can be the same or different.
The addition of flanking sequences at the carboxy or N-terminal ends of a peptide have been shown to increase biological activity, by altering the presentation of the peptide within the antibody scaffold. Hence, nucleic acid molecules can be modified to encode peptides having adding flanking sequences at the carboxy or N-terminal ends of the peptides. Flanking sequences can encode for 1, 2, 3, 4, 5 or more amino acids. Flanking sequences can encode for any amino acid or any combinations of amino acids. Glycine is the smallest and simplest of the amino acids, containing only a single hydrogen atom in its side chain. Due to its small size, glycine can fit into small spaces and can adopt particular conformations that other amino acids can not. Proline is a sterically constrained amino acid that has been shown to increase activity of a peptide when flanking the peptide sequence (REF). Generally, flanking sequences encode for glycine or proline. Typically, flanking sequences encode for proline. For example, a nucleic acid molecule can encode a peptide containing proline and/or glycine added to the N- or C-terminus of the EPO peptide set forth in SEQ ID NO:891. Exemplary of nucleic acid molecules containing flanking sequences encode any of the EPO peptides set forth in SEQ ID NOS: 874-895.
e. Generating Variable Heavy and Light Chain Sequences and Nucleic Acid Molecules
The sequences for recombined nucleic acid molecules encoding VH and VL chain compiled by practice of the method herein are collected and stored. The collected sequences can be analyzed for any particular characteristic, such as for example, sequence similarity between and among other recombined sequences. The sequences then can be ranked based on sequence diversity. All recombined sequences, or a subset thereof, can be generated into recombined nucleic acid molecules using DNA synthesis and/or recombinant DNA technology. For example, a subset of sequences can be selected based on their sequence similarity or difference for generation of an antibody library.
i. Storage and Collection
Sequences recombined by the method herein are collected and stored. Typically, collection and storage is in an addressable format, such that the identity of each sequence is known by its locus. For example, the sequences can be stored in a database or in a list. Further, the individual gene segment components of each nucleic acid sequence are known, and the recombined nucleic acid sequence identified by the component segments. For example, a nucleic acid sequence named VH1-18_IGHD1-26*01_IGHJ2*01 identifies a nucleic acid sequence encoding a variable heavy chain containing the VH germline segment VH1-18 (also called VH1-18*01 by some nomenclature standards), the DH germline segment IGHD1-26*01, and the JH germline segment IGHJ2*01. One of skill in the art can identify a nucleic acid sequence using any desired naming convention, so long as the component segments are easily identified.
Generally sequences encoding VH chains are recombined, collected and stored separately from VL chains. Further, among VL chains, sequences encoding V-kappa light chains are recombined, collected and stored separate from sequences encoding V-lambda chains. The identity of the nucleic acid sequence at each locus is known and can be mapped to an output file that contains the sequences for all the nucleic acid molecules within the addressable format.
For purposes herein, the sequences are addressably stored such that each sequence can be easily identified, including by its component parts (e.g. the individual compiled segments). By practice of the methods above, a plurality of different recombined nucleic acid sequences encoding a VH chain can be generated, which can represent all possible permutations of recombined segments or subsets thereof. For example, 10, 100, 500, 1000 (103), 2×103, 4×103, 6×103, 8×103, 104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 106, 107 or more VH nucleic acid sequences can be generated. By practice of the methods above, a plurality of different recombined nucleic acid sequences encoding a VL chain can be generated, which can represent all possible permutations of recombined segments or subsets thereof. For example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 (103), 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 104, 5×104, 105 or more VL nucleic acid sequences can be generated.
The examples exemplify collection and storage of sequences compiled by practice of the method in a SequenceHistory.txt file. Such a file represents sequences generated in the Examples by the exemplified software and ordered for DNA synthesis. Sequences also can be stored manually, for example, in spreadsheets or lists.
ii. Determining Sequence Diversity of Collected Nucleic Acid Sequences
In some examples, recombined nucleic acid molecules can be collected and stored based on their sequence diversity. It is contemplated herein that knowledge of the sequence diversity of library members can be employed to select a restricted subset of nucleic acid sequences encoding VH chain and VL chain for synthesis and expression as described herein below. Hence, resulting antibody libraries can be made to maximize sequence diversity among members due to sequence differences. Alternatively, resulting antibody libraries can be made to minimize sequence diversity among members due to sequence similarities. Thus, for example, the sequence of a selected recombined nucleic acid can be compared to all other sequences in the libraries, and those sequences that are different (e.g. having sequence similarity that is less then 70%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 65%) can be selected. In another example, if a “Hit” is identified in an initial screen, a further library can be created where all members have a high sequence similarity (e.g. 70%, 75%, 80%, 85%, 90%, 95% or more) to the identified “Hit.” The percentages given are for exemplification only. One of skill in the art can choose any desired limit of sequence similarity by which to select sequences for inclusion in a particular library.
To determine the sequence similarity or difference between and among recombined nucleic acid sequences sequence diversity based on sequence similarity of all collected nucleic acid sequences is assessed. Typically, due to the degeneracy of the genetic code, recombined nucleic acid sequences are first translated to give an amino acid sequence, and then sequence similarity between and among the resulting amino acid sequences is determined. Translation is performed based on the genetic code, whereby 64 codons encode the 20 amino acids plus three stop codons (see Table 20). Translation of each sequence can be performed manually or by other computer-based or automated methods. One of skill in the art is familiar with methods of translating proteins. The sequences can be grouped or stored based on their sequence diversity.
Typically, sequence diversity is assessed based on sequence similarity of two or more sequences, such as for example, as determined by alignment. One of skill in the art is familiar with various techniques to determine the sequence similarity (e.g. identity) between and among sequences. For example, sequence similarity can be determined manually by determining nucleotide differences between and among sequences. Sequence similarity or sequence identity of nucleotide or amino acid sequences also can be determined using conventional software or computer programs. Such algorithms are well known to one of skill in the art. For example, to find the best segment of identity or similarity of sequences, BLAST (Altschul et al (1990) J. Mol. Biol. 215:403-410 and Lipman et al (1990) J. Mol. Biol. 215:403-410), FASTA (Lipman et al (1985) Science 227:1435-1441), or Smith and Waterman (Smith and Waterman (1981) J. Mol. Biol. 147:195-197) homology search programs can be used. To perform global alignments, sequence alignment programs such as the CLUSTAL W (Thompson et al (1994) Nucleic Acids Research 22:4673-4680) can be used.
For example, nucleic acid or amino acid sequences can be assessed for sequence similarity using BLAST. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity. Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and the following parameters: Match: 1; mismatch:-2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: I 1; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences. The BLAST program provides an output indicator, the BLAST bit score, which is a value calculated from the number of gaps and substitutions associated with each aligned sequence. The higher the score, the more significant the alignment. The bit score can be used to select sequences that have either the most sequence diversity or alternatively, the least sequence diversity to every other selected sequence.
In another example, sequence diversity also can be assessed by comparison of two or more amino acid or nucleic acid sequences by alignment methods, e.g., the CLUSTAL method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The CLUSTAL algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Sequence similarity (e.g. sequence identity) between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions. BLASTclust is another program that can be used for cluster analysis. BLASTclust is used in the software compilation program described in the Examples.
Diversity and cluster information as well as BLAST bit score information provide the user with several options when selecting sequences. For example, the user can create an antibody library where the selected sequences are as diverse as possible. To do this, the user can use the diversity score and cluster information, and select sequences from different clusters that have the highest diversity. Alternatively, for example, the user can create a an antibody library where one sequence is initially selected and all subsequent sequences are as similar as possible to the first sequence. This can be accomplished by using the BLAST function. The user can BLAST the selected first sequence, and then select all the other sequences for the library using the BLAST bit score, choosing sequences with the highest score and therefore the highest sequence similarity. For example, Example 5 describes implementation of assessing sequence sequence diversity between and among recombined sequences using Software Compilation software. The Example illustrates that BLAST can be performed on all sequences and Blast bit scores calculated to identify the sequence similarity or differences between sequences.
iii. Generating Nucleic Acid Molecules from Recombined Sequences a) Synthesis
Where desired, the sequences can be individually synthesized into nucleic acid molecules. All collected sequences can be synthesized, or a subset of sequences synthesized. Nucleic acid molecules encoding VH or VL chain can be synthesized by methods known to one of skill in the art using synthetic gene synthesis (see e.g., U.S. Pat. Nos. 4,652,639; 5,132,215; 5,093,251; 6,110,668; 6,472,184; published U.S. application Nos. US20060281113; US20070004041; US20070122817; and International PCT Published Application Nos. WO98/15567; WO99/47536; WO00/75364; WO2004035781; WO2005071077). These include standard solid phase polypeptide synthesis methods involving synthesis of single stranded oligos that are ligated together. Methods also include methods using standard triplets that act as universal building blocks that represent all possible sequence combinations, and can be combined in a series of reaction steps (Slonomics®).
Nucleic acids can be synthesized that are 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more base pairs in length. Gene synthesis can be performed by automated methods. Any of the known synthesis methods can be used to produce the nucleic acid molecules. For example, companies exist for the purpose of synthesizing oligonucleotides and genes, for example, Integrated DNA Technologies (IDT) (Coralville, Iowa), TriLink Biotechnologies (San Diego, Calif.), Blue Heron Gene Synthesis (Bothell, Wash.), and Sloning Biotechnology (Puchheim, Germany).
The nucleotide monomers used in the synthesis can be purine and pyrimidine deoxyribonucleotides (adenosine (A), cytidine (C), guanosine (G) and thymidine (T)) or ribonucleotides (A, G, C and U (uridine)), or they can analogs or derivatives of these nucleotides, such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Other nucleotide analogs are well known in the art and can be used in synthesizing the oligonucleotides provided herein.
The nucleic acid molecules can be synthesized with nucleotide modifications. In one example, each oligonucleotide contains a terminal phosphate group, for example, a 5′ phosphate group. For example, when it is desired to seal nicks between two adjacent oligonucleotides, e.g. following hybridization of the two oligonucleotides to a common opposite strand polynucleotide according to the methods herein, a 5′ phosphate group is added to the end of the oligonucleotide whose 5′ terminus will be joined with the 3′ terminus of another oligonucleotide to seal the nick. In one example, a 5′ phosphate (PO4) group is added during oligonucleotide synthesis. In another example, a kinase, such as T4 polynucleotide kinase (T4 PK) is added to the oligonucleotide for addition of the 5′ phosphate group. Other oligonucleotide modifications are well-known and can be used with the provided methods.
The synthetic oligonucleotides can be chemically synthesized. Methods for chemical synthesis of oligonucleotides are well-known and involve the addition of nucleotide monomers or trimers to a growing oligonucleotide chain. Typically, synthetic oligonucleotides are made by chemically joining single nucleotide monomers or nucleotide trimers containing protective groups. For example, phosphoramidites, single nucleotides containing protective groups, can be added one at a time. Synthesis typically begins with the 3′ end of the oligonucleotide. The 3′ most phosphoramidite is attached to a solid support and synthesis proceeds by adding each phosphoramidite to the 5′ end of the last. After each addition, the protective group is removed from the 5′ phosphate group on the most recently added base, allowing addition of another phosphoramidite. See, for example, Behlke et al. “Chemical Synthesis of Oligonucleotides” Integrated DNA Technologies (2005), 1-12; Allen et al. “UltramersTM—The Longest Oligonucleotides Available with Mass Spectrometry” Integrated DNA Technologies, Technical Report (2007); and McBride and Caruthers Tetrahedron Lett 24:245-248, which describe synthesizing oligonucleotides using standard cyanoethyl chemistry (using phosphoramidite monomers and tetrazole catalysis). Such methods typically result in generation of oligonucleotides of 100-200 bases.
Thus, to synthesize larger genes, methods include annealing of a series of smaller oligonucleotides. In such a method, individually designed oligonucleotides are made, such as by using an automated DNA synthesizer, purified and connected by specific annealing using standard ligation or polymerase reactions. Generally, the oligos are designed with overlapping stretches of common sequence to permit annealing. Several methods of gene synthesis have been described, including, but not limited to the ligation of phosphorylated overlapping oligonucleotides (Gupta, N. K. et al. (1968) Studies on polynucleotides, 88. Enzymatic joining of chemically synthesized segments corresponding to the gene for alanine-tRNA. Proc. Natl Acad. Sci. USA, 60, 1338-1344; Fuhrmann M et al., A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J. 1999 August; 19(3):353-61); de novo gene construction using Ultramers (Allen et al. “UltramersTM—The Longest Oligonucleotides Avialable with Mass Spectrometry” Integrated DNA Technologies, Technical Report (2007); the Fok I method (Mandecki, W. and Bolling, T. J. (1988) FokI method of gene synthesis. Gene, 68, 101-107); a modified form of ligase chain reaction for gene synthesis; PCR assembly whereby the full-length molecule is generated progressively by overlap extension (Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M. and Heyneker, H. L. (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164, 49-53), thermodynamically balanced inside-out (Gao X, Yo P, Keith A, Ragan T J, Harris T K. Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. Nucleic Acids Res. 2003 Nov. 15; 31(22):e143) or combined approaches (Young L, Dong Q. Two-step total gene synthesis method. Nucleic Acids Res. 2004 Apr. 15; 32(7):e59). Since the error frequency increases with longer oligonucleotides, methods typically include using shorter oligonucleotides (200 base pairs or less) assembled together.
The synthesized molecules can be purified by a number of well-known methods, for example, high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), Polyacrylamide Gel Electrophoresis (PAGE) and desalting.
In one embodiment, the synthesized nucleic acids are arrayed in multiwell plates, with each individual well of a plate corresponding to one individual nucleic acid. More specifically, each individual locus of a plate contains an nucleic acid encoding an antibody variable region, either heavy or light. The identity of the nucleic acid contained within each well of the multiwell plate is known and mapped to an output file that contains the nucleic acid sequences for all of the nucleic acids within the plate. Multiwell plates can include but are not limited to 96-well plates, 384-well plates, and 1536-well plates. In an exemplary embodiment, the nucleic acids are spatially arrayed in a 96-well plate.
Upon synthesis, the resulting nucleic acid molecules are individually addressed into a locus (e.g. a well, chip, tag, and other addressable formats). Each individual locus of a plate can contain a different recombined and synthesized nucleic acid molecule encoding for either a heavy chain variable region or a light chain variable region or portion thereof compared to all other addresses. The identity of the nucleic acid molecule at each locus is known and can be mapped to an output file that contains the sequences for all the nucleic acid molecules within the addressable format. For example, nucleic acid molecules can be addressed by spatial array into multiwell plates, with each individual locus of a plate containing one individual nucleic acid molecule. Multiwell plates can include but are not limited to 12-well plates, 24-well plates, 96-well plates, 384-well plates, and 1536-well plates.
b) Recombinant Generation
In some examples, recombined VH and/or VL sequences, or a subset thereof, can be generated into recombined nucleic acid molecules using recombinant DNA technology. One of skill in the art is familiar with general recombinant DNA techniques, including but not limited to, PCR, cloning and restriction enzyme digestion. Such techniques can be used to combine germline segments as discussed herein above to generate recombined nucleic acid molecules that are in-frame. Generally, each vector is generated individually, such that the identity of the sequence of each vector is known through the cloning process. Thus, the recombinant generation of a combinatorial antibody library is addressable.
In the methods of generating combinatorial antibody libraries using recombinant DNA techniques, germline segments can be linked directly or indirectly by a linker so long as the resulting nucleic acid molecule is in-frame, resulting in a functional and productive antibody. The linker can be a peptide, polypeptide or an amino acid. For example, it is understood that by virtue of using recombinant DNA technologies, including the use of restriction enzymes, that amino acids can be inserted between V-D, D-J and V-J junctions in order to facilitate joining of germline segments. Exemplary of a linker as described herein in Example 14 is a sequence of nucleotides encoding an SY between the 3′end of the DH germline segment and the 5′end of the JH germline segment.
In methods of generating a combinatorial antibody library by recombinant DNA techniques, a parent vector or vectors can be generated that contain common nucleic acid sequences between and among members of the library. For example, a vector can be generated that contains nucleic acid sequence for a VH, DH and/or JH germline segment, modified forms thereof, or portions thereof and/or a VL and/or JL that are common between all members of the library. It is understood that introduction of segments is with reference to the reading frames as described herein above, such that the resulting compiled nucleic acid molecule is in-frame. The description below provides a general summary of a method of generating a combinatorial antibody library using recombinant DNA techniques. It is understood that the reference to the examples is for exemplification only. Using the description provided herein one of skill in the art can generate similar vectors containing nucleic acid compiled from germline segments or modified forms thereof to generate recombined VH or VL chains that are in-frame. For example, it is understood that the order of addition of VH/DH/JH or VL/JL segments or portions thereof to the recombinant vectors can occur in any order, so long as the resulting cloned nucleotide sequence encodes a recombined VH or VL chain that is in-frame.
Thus, a parent vector is typically generated containing a sequence common to all members of the library. For example, if all vectors share a common VH germline sequence, a vector can be generated carrying the VH germline sequence in its correct reading frame, which can be manipulated for subsequent inclusion of other germline segments. For example, the VH germline sequence can be modified to include restriction enzyme sites on the 3′end for subsequent joining with a DH germline sequence. In another example, vectors can be generated containing a portion of a VH, DH or JH germline sequence. For example, a vector can be generated containing a common framework consensus sequence as described elsewhere herein. This is exemplified in Example 14 where a modified Plasmid A vector was generated to contain a common framework 4 region of a JH germline segment. An exemplary parent vector for use in generating a combinatorial antibody library using the methods herein is set forth in SEQ ID NO:2051, which contains a common VH germline segment (VH3-23 (IGHV3-23*01) that is modified to remove an internal restriction site and add additional restriction sites at the 3′end) and a common framework 4 region of a JH germline segment.
The parent vector can then be used to further introduce remaining germline segments, modified forms thereof, or portions thereof such that a plurality of final vectors are generated each containing a nucleic acid sequence encoding a recombined VH and/or VL chain. The generation from a parent vector to a final vector can occur in steps, thereby resulting in intermediate vectors, generally at least one intermediate vector. Generally, nucleic acid sequences for subsequent germline segments, modified forms thereof or portions thereof are generated as oligonucleotides for subsequent cloning into the parent vector or an intermediate vector. It is understood that if a stop codon is inserted at any step, the stop codon is either removed as described herein above, or the particular segment containing the stop codon is not cloned. To facilitate joining with adjacent nucleic acid sequences, the oligonucleotides are generated to contain complementary restriction enzyme sites at the 3′and/or 5′ends.
For example, depending on the components contained in the parent vector, an intermediate vector can be generated to contain remaining germline segments, modified forms thereof or portions thereof. For example, intermediate vectors can be generated from the parent vector above (set forth in SEQ ID NO:2051), whereby each intermediate vector contains a different JH segment in its correct reading frame (see e.g. Table 13). The JH segment can be a germline segment or a modified form thereof. Exemplary of modified JH segments are any set forth in SEQ ID NOS: 3450-3455 and encoding JH regions set forth in any of SEQ ID NOS: 3456-3461. The entire JH segment or a portion of a JH segment can be added to an existing parent or intermediate vector. For example, if a parent vector is made to contain a consensus framework 4 region as described above, a portion of a JH segment containing nucleotides corresponding to the last portion of a CDR3 in the JH segment can be introduced. By virtue of the addition of different segments, for example different JH segments, in the intermediate vectors, the diversity of the library can be increased. Thus, generally, a plurality of intermediate vectors are generated. For example, Example 14 describes the generation of six intermediate vectors (having a sequence set forth in any of SEQ ID NOS: 2064-2069).
A plurality of final vectors are generated that contain the all components of a compiled germline sequence. As above, the remaining nucleotide to be inserted into the vector are generated as oligonucleotides, and typically contain complementary restriction enzyme sites at the 3′and/or 5′ends. As noted, the oligonucleotides are generated to provide the correct reading frame for the inserted segment and do not contain stop codons. In addition, the oligonucleotides are generated to preserve existing reading frames for the segments contained in the parent or intermediate vectors. For example, as described elsewhere herein, it is understood that the reading frame of the DH region is not critical. Thus, DH segments, including DH germline segments, can be inserted in any reading frame, or can be a random sequence of nucleotides. Example 14 exemplifies generation of a plurality of final vectors by introduction of DH germline segments (e.g., any set forth in any of SEQ ID NOS: 239-245, 248, 250, 252, 254, 256, 258-272), or inverted segments thereof (e.g., any set forth in any of SEQ ID NOS: 3462-3488), in all three reading frames. In generating the oligonucleotides, however, one or more nucleotides are removed or added from the 3′or 5′ends in order to preserve reading frames of the adjacent JH segments. This is exemplified in Example 14, which sets forth conditions for removing or adding nucleotides in order to preserve reading frames.
The resulting final vectors contain compiled VH/DH/JH or VL/JL germline segments, or modified forms thereof, that encode a recombined VH chain or VL chain. Each final vector is different and contains a different nucleic acid sequence encoding a different recombined VH chain or VL chain. Since the nucleic acid molecules are already cloned into an expression vector, they can be directly transformed into an expression system as discussed in Section f below.
f. Expressing and Producing Antibodies or Portions or Fragments Thereof
In the methods provided herein, recombined nucleic acid molecules, such as synthetic recombined nucleic acid molecules or recombined nucleic acid molecules generated recombinantly, are cloned into an expression vector. The polynucleotides typically are inserted into the vectors using restriction digest and ligation. Any conventional vector known to one of skill in the art can be used for expression in eukaryotic or prokaryotic cells. Exemplary vectors include plasmid A, C and D described herein below. The vector can be used to transform any expression system compatible therewith for amplification of the nucleic acid and/or expression of the encoded variable heavy or variable light chain polypeptide.
Typically, ligation into a vector is in an addressable format such that the identity of the recombined polypeptide expressed therefrom is known. For example, the vectors containing nucleic acids are spatially arrayed in multiwell plates, with each individual locus of a plate containing a vector with one individual nucleic acid inserted. More specifically, each individual locus of a plate contains a vector encoding for either a heavy chain or a light chain. The identity of the nucleic acid contained within each well of the multiwell plate is known, for example, by mapping to stored sequences collected from the compilation or synthesis above. For example, ligation into vectors can be performed directly into multiwall plates already containing synthesized nucleic acid molecules from above. Multiwell plates can include but are not limited to 96-well plates, 384-well plates, and 1536-well plates. In an exemplary embodiment, the nucleic acids are spatially arrayed in a 96-well plate.
Generally in practicing the methods, a nucleic acid molecules encoding a variable heavy chain is ligated into a first vector. A nucleic acid molecule encoding a variable light chain is ligated into a second vector. The first vector and second vector can be co-transformed into the same expression host for co-expression of a variable heavy chain and a variable light chain. The polypeptides, upon expression, will become operably joined by virtue of interactions between the heavy and light chain polypeptides. In some examples, it is possible to operably join the nucleic acid molecules directly prior to expression, such as by including a linker. In such examples, a single nucleic acid molecule encodes a variable heavy chain and a variable light chain, and can be ligated into a single vector for expression thereof.
In all methods herein, the expressed antibodies minimally include a VH chain and a VL chain, or portions thereof sufficient to form an antigen-binding site. In addition, if desired, a constant chain can be included for expression in operative linkage with the variable chains. In all examples of the methods, the recombined nucleic acid molecules, upon expression and ligation, encode for antibodies or fragments thereof, including, but not limited to an IgG, a Fab fragment, a F(ab′)2 fragment or a Fv fragment, such as a disulfide-linked Fv or a single chain Fv. An exemplary antibody is a Fab fragment. Such antibodies or fragments thereof can be purified by any methods known to one of skill in the art.
Section F describes methods of expressing and purifying antibodies or fragments thereof.
2. Automation
Any of the steps of the method described above can be automated and/or made high-throughput and/or otherwise rendered more efficient or fast. One of skill in the art is familiar with methods of automation of systems and processes, including the implementation of in silico databases, application of computer programs, robotics and/or other high-throughput methods that can be used in practice of the method. It is contemplated that the entire process of the method can be automated or only a few steps can be automated. The choice of automation is up to the user. The description below and examples exemplify automation of various processes of the method.
a. User-Created Database
To practice the methods herein, sequences of germline segments or modified forms thereof must be obtained. Such sequences are known to one of skill in the art and can be obtained from commercially available databases, such as described above. Such germline segment sequences are set forth in the sequence listing as set forth in Tables 3-5 above. Exemplary of modified JH germline segments are set forth in SEQ ID NOS: 3450-3455. The sequences can be compiled into a user-created database for ease of access. Generation of a file or database containing all of the sequence information provides immediate access to these sequences. In addition the sequence file can be linked to other systems and processes to facilitate performance of the method. For example, as exemplified in FIG. 9, in Example 4, a database file is linked to the Sequence Compilation Alogrithm as an input file for identification of V(D)J heavy and light chain sequences for sequence compilation.
The database file contains sequences for germline VH, DH, JH, Vκ, Jκ, Vλ and Jλ segments. In particular, the database file can contain sequences of nucleic acids set forth in any of SEQ ID NOS:10-451, 868, or a subset thereof. It is helpful if the sequences are specified using FASTA format and all sequences contain a blank line between them. For purposes of practice of the method, the JH, Jκ and Jλ segment sequences are set forth in the database file in coding frame triplets corresponding to their optimal reading frame, which is set forth in Table 13 above. The sequences in the database file are named for identification. For example, germline segments are identified by section title headings [VH], [DH], [JH], [VK], [JK], [VL], and [JL]. Such a databased file is described in the Examples (e.g. Example 3) as a SequenceDatabase.txt.file. FIG. 11 provides a schematic illustration of a Sequence Database file format.
In addition, the database can contain other sequences used in practicing the method. For example, the database can contain nucleic acid sequences for restriction sites, and can be identified in the database under the section title heading [Restriction Sites]. These sequences can be accessed by particular program processes as described below to identify nucleic acid sequences corresponding to restriction sites within a recombined nucleic acid molecule. Restriction site sequences contained in the database include any of SEQ ID NOS:977-980, 1889-1900. Any restriction site sequence known to one of skill in the art can be contained in the database file. For example, the schematic illustration of a database file in FIG. 11 includes a sequence for the restriction enzyme Mfe I.
It is contemplated that the database file can be periodically updated to contain additional sequences. The database file also can be updated to include any sequence contemplated for practice of the method. For example, nucleic acid sequences that encode for proteins other than antibody germline segments can be entered into the database, using FASTA format, under an appropriate heading. These sequences are then available to be recombined into the germline antibody sequences. For example, one can insert peptide sequences into an antibody at DH by including nucleic acid sequences encoding for the peptide under the section title [DH].
b. Sequence Compilation
The method of compilation of sequence can be performed in silico, for example, using software. Any software programmed to perform an algorithm or process allowing for compiling germline segments in accordance with the method herein or any suitable method, can be used. One of skill in the art familiar with software programming can generate a computer program capable of performing such an algorithm or process. Generally, the software is programmed to perform any one or more of the following processes:

    (a) accessing a user-created in silico database of all available antibody germline segments (VH, DH, JH, Vκ, Jκ, Vλ and Jλ);
    (b) applying an algorithm to generate every possible recombined full length nucleic acid sequence for heavy chains (5′-VH-DH-JH-3′ combinations), every possible recombined full length nucleic acid sequence for kappa light chains (5′-Vκ-Jκ-3′ combinations) and every possible recombined full length nucleic acid sequence for lambda light chains combinations);
    (c) applying an algorithm to modify the nucleic acid sequences of the joints so that the resulting nucleic acids sequences are in frame;
    (d) modifying the resulting nucleic acid sequences of the joints to remove any inadvertently generated stop codons;
    (e) modifying the resulting full length nucleic acid to optimize codon usage for bacterial expression;
    (f) modifying the resulting nucleic acid to remove any undesired restriction sites;
    (g) inserting flanking nucleic acids containing restriction sites for cloning at the 5′ and 3′ termini of the optimized full length nucleic acid sequences;
    (h) ranking recombined nucleic acid sequences based on their sequence diversity;
    (g) selecting recombined nucleic acid sequence(s) (encoding either a heavy chain variable region or a light chain variable region) from the library of recombined nucleic acid sequences;
    (h) assigning the selected nucleic acid sequence to a unique locus of an addressable format;
    (i) generating an output file that contains all recombined nucleic acid sequences in the form of the addressed format that lists the distinct heavy chain or light chain sequences such that each locus is addressed and corresponds to a locus of the addressed format (e.g. 96-well plate).


Provided herein are software, computer-readable media, and computer systems for performing the method as described herein. The Examples describe an exemplary software, computer-readable media, computer system and systems. It is understood that those of skill in the art can modify such software, computer-readable media, computer systems and systems based upon this disclosure and that such modifications are included herein.
For example, each of these processes of the method described herein is performed by an exemplary computer software exemplified in the Examples herein. For example, Example 2 and FIGS. 8 and 9 describe an exemplary process for sequence compilation of germline segments. The flow chart in FIG. 9 describes processes for each of steps (a)-(d) and (f) above. In addition, Example 2 and FIG. 10 describes an exemplary process for ranking sequences based on sequence diversity. The flow chart in FIG. 10 describes the process used to rank sequences after determination of diversity scores and an example of the ranking is set forth in FIG. 17.
c. Automation of Protein Expression and Purification
Methods of automating protein expression and purification are known to one of skill in the art (see, e.g., Lesley et al. (2001) Protein Expression and Purification, 22:159-164; Acton T B et al. (2005) Methods Enzymol., 394:210-43; Nguyen et al. (2004) Journal of Structural and Functional Genomics, 5:23-27). Such processes typically include robotic methods.
Exemplary of a high-throughput automated method of protein expression and purification is PiccoloTM (Wollerton et al. (2006) JALA, 11:291-303). The PiccoloTM system automates protein expression and purification of proteins from both E. coli and baculovirus mediated insect cell expression systems. Piccolo is able to perform multiple different protein expression and purifications in parallel. The Piccolo system utilizes a 24-position culture vessel block (CVB) in an aeration assembly that supports the expression and purification of multiple samples at once. The Piccolo system contains four modules that perform these functions: a liquid handling module, CVB incubators, a centrifuge and storage carousels. A rail mounted 6-axis RX60L Spinal Transfer Robot (ST Robot; Staubli, Horgen, Switzerland) moves the lab ware between the liquid handling module, incubators, centrifuge and storage carousels. The system is controlled by software that permits the user to control expression and purification conditions.
Expression can be initiated by inoculation of CVB plates containing appropriate growth medium with an an input inoculum, such as a bacterial culture. A total of 576 individual cultures can be grown at any one time, corresponding to 24 culture vessel blocks. The plates can be incubated under user-specified periods and conditions. Typical growth and induction temperatures range from 16° C. to 37° C. Selection of optimal temperatures for growth and induction is well within the level of skill of the skilled artisan. Bacterial growth can be monitored. If desired, protein expression can be induced by adding an appropriate amount of inducer into the CVB plate assembly and further grown under appropriate conditions. Protein expression can be induced by addition of any inducer compatible with the expression vector, including isopropyl -D-1-thiogalactopyranoside (IPTG) and arabinose. Expression times range from 2 hours to 48 hours. Selection of optimal expression times is well within the level of skill of the skilled artisan. Following expression, plates can be stored under cooling conditions. For example, as set forth in Example 9, spatially arrayed transformed cells are mapped to a 24-well culture vessel block for cell growth and protein expression. For each 96-well plate of transformed cells, four culture vessel blocks are generated, thereby allowing the growth of Fabs corresponding to every well of the 96-well plate.
Following expression of the desired protein, the PiccoloTM system also can be used to purify the resulting proteins. The Piccolo machine is programmed to perform lysis and purification steps. The cells are harvested and lysed using an appropriate lysis buffer that is compatible with the purification technique. Selection of a lysis buffer is well within the level of skill of the skilled artisan. The resulting supernatant is then purified by column chromatography with an appropriately modified resin, such as an anti-flag resin or Ni-charged resin. One of skill in the art can identify an appropriate resin for protein purification as described elsewhere herein. The resin should be manually equilibrated in an appropriate wash buffer before starting the run. The bound protein can be eluted with an appropriate elution buffer and the eluate collected in an output plate. The output plate can be stored at cool temperatures (e.g. 6° C.) until collected by the operator.
Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques. Additional purification methods can be combined with Piccolo such as are described elsewhere herein. For example, proteins can be further purified using an orthogonal secondary high throughput method of protein purification (see e.g. Example 10). Additional column chromatography can be performed using a compatible resin, an Aktapurifier (GE Healthcare) and an autosampler. Exemplary of purifying antibodies, a protein G resin can be utilized.
E. Libraries
Provided herein are libraries. The libraries include nucleic acid libraries encoding VH or VL chains, vector libraries transformed with recombined nucleic acid molecules, and antibody libraries. In some examples, the members of each of the libraries are addressed in an addressable format, such as any discussed in Section E.2. The members of the libraries and the resulting libraries can be produced by the methods described herein above.
1. VH Nucleic Acid Libraries and Vector Libraries thereof
Provided herein are recombined nucleic acid libraries encoding VH chains. The libraries provided herein include recombined nucleic acid molecules made up entirely of VH, DH and JH germline segments or modified forms thereof. The VH, DH and JH germline segments include any set forth in Table 3 above, modified forms thereof, or a subset thereof. Any permutation is possible. The resulting nucleic acid molecule in the library have a sequence such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment. The segments can be linked directly or indirectly by a peptide linker.
Because the nucleic acid molecules in the library are derived from germline segments, members of such a nucleic acid library are capable of encoding a naïve antibody when co-expressed with a nucleic acid encoding a VL chain. It is understood that the library is considered to be naïve and derived from germline even though, in practicing the method herein, the joint regions of the segments are altered to render the resulting encoding nucleic acid molecules in frame. Such alterations, however, are minor and variously include insertion or deletion generally of only a single nucleotide of a germline segment. In addition, other modification made to the recombined nucleic acid sequence by virtue of practice of the method herein, such as removal of stop codons and restriction enzyme site sequences, also result in naïve antibodies.
It is understood that libraries can be generated that are compiled from sequences that include modified germline segments. In some examples of the libraries, the libraries include recombined nucleic acid molecules made up entirely of a VH and a JH germline segment, and also any sequence of nucleotides between the VH and JH germline segment. This is the region that includes the central portion of the CDRH3, which is largely responsible for the antigen specificity of the resulting antibody. The sequence of nucleotides can be any random sequence of nucleotides. In some instances, the sequence of nucleotides is a sequence that encodes a peptide mimetic against any desired target, for example, a cell surface receptor. Exemplary peptide mimetics are set forth in Table 16. Generally, the sequence of nucleotides is or is about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more nucleotides in length. The resulting nucleic acid molecule in the library have a sequence such that the VH segment is 5′ to the random sequence of nucleotides which is 5′ to the JH segment. In some examples, the random sequence of nucleotides is a DH germline segment.
In other examples, the libraries provided herein include recombined nucleic acid molecules where at least one, two or all three of a VH, DH and JH germline segment contained therein are modified, for example, due to modification by insertion, deletion or addition of amino acids. For example, the libraries include nucleic acid molecules containing sequences encoding a directed peptide. The libraries also include recombined nucleic acid molecules containing nucleotide mutations encoding amino acid replacements, for example, of one or more amino acids of a CDR. In an additional example, the libraries provided herein include recombined nucleic acid molecules where at least a portion of the nucleic acid molecule, such as the entire nucleic acid molecule encoding the VH chain, or at least one or more of a VH, DH and JH are derived from an existing monoclonal antibody, including, but not limited to, any monoclonal antibody set forth in Table 9. For example, exemplary libraries provided herein can include a nucleic acid molecule encoding the VH chain of an anti-CD20 antibody such as is set forth in SEQ ID NO:1043 or SEQ ID NO:1058 (SEQ ID NO:453 including terminal restriction site sequences) or Herceptin such as is set forth in SEQ ID NO:1057 (SEQ ID NO:452 including terminal restriction site sequences)
Libraries of recombined nucleic acid molecules provided herein can include members that represent one, some or all of the above examples. Any of the libraries provided herein also can include members whose sequences include heterologous sequence, for example, restriction site sequences, linker sequences, sequences encoding tags or other detectable moieties or other sequences.
In the VH nucleic acid libraries provided herein, each recombined nucleic acid molecule member of the library is productive and, when co-expressed with a nucleic acid molecule encoding a VH chain, generates a functional antibody or portion thereof that is sufficient to form an antigen-binding site. In addition, in the VH nucleic acid libraries provided herein, each nucleic acid member of the library is different. The VH nucleic acid libraries provided herein can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 (104), 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 106 or more different members. The nucleic acid members are provided in addressed formats, such that when addressed the identity of each nucleic acid is known by its location in the array.
For example, an exemplary VH nucleic acid library includes members set forth in SEQ ID NOS: 454-805, each representing a different recombined nucleic acid molecule of VH, DH and JH germline segments. Such a library includes members containing heterologous sequence for restriction sites at the 3′ and 5′ ends. It is understood that members of the library also can include those having sequences not including the heterologous sequence, such as is set forth in any of SEQ ID NOS: 1059-1410.
In an additional example, an exemplary VH nucleic acid library includes members set forth in SEQ ID NOS: 2070-2759, each representing a different recombined nucleic acid molecule of VH, DH and JH germline segments or modified forms thereof. Such a library includes members containing heterologous sequence for restriction sites at the 3′end corresponding to CTAGC (set forth in SEQ ID NO:1903) and at the 5′end corresponding to CCATGGCA (set forth in SEQ ID NO:1901). It is understood that members of the library also can include those having sequences set forth in any of SEQ ID NOS: 2070-2759 that do no include the heterologous sequences at one or both of the 3′and 5′ends.
A VH nucleic acid library can include members from any of the libraries provided herein, or a subset thereof. For example, a VH nucleic acid library includes members set forth in any of SEQ ID NOS:454-805 and 2070-2759, or a subset thereof. The library members can include those containing heterologous sequences at the 3′ or 5′ends, and/or members that do not include heterologous sequences.
In some examples, any of the nucleic acid sequences in the libraries provided herein can be included in a vector to generate vector libraries. Exemplary of vector libraries are libraries of recombined nucleic acid molecules encoding VH chain included in backbone Plasmid A or Plasmid D.
2. VL Nucleic Acid Libraries and Vector Libraries Thereof
Provided herein are recombined nucleic acid libraries encoding VL chains. The libraries include those encoding for lambda or gamma light chains, or combinations thereof. Thus, the libraries provided herein include recombined nucleic acid molecules made up entirely of Vκ and Jκ germline segments and/or Vλ and Jλ germline segments. The Vκ and Jκ germline segments and/or Vλ and Jλ germline segments include any set forth in Tables 4-5 above, or a subset thereof. Any permutation is possible. The resulting nucleic acid molecules in the library have a sequence such that the VL segment (Vκ or Vλ) is 5′ to the JL segment (Jκ or Jλ).
Because the nucleic acid molecules in the library are derived from germline segments, members of such a nucleic acid library are capable of encoding a naïve antibody when co-expressed with a nucleic acid encoding a VH chain. It is understood that the library is considered to be naïve and derived from germline even though, in practicing the method herein, the joint regions of the segments are altered to render the resulting encoding nucleic acid molecules in frame. Such alterations, however, are minor and variously include insertion or deletion generally of only a single nucleotide of a germline segment. In addition, other modification made to the recombined nucleic acid sequence by virtue of practice of the method herein, such as removal of stop codons and restriction enzyme site sequences, also result in naïve antibodies.
In some examples, the libraries provided herein include recombined nucleic acid molecules where at least one or both a Vκ, and Jκ germline segment or a Vλ and Jλ germline segment contained therein are modified, for example, due to modification by insertion, deletion or addition of amino acids. For example, the libraries include nucleic acid molecules containing sequences encoding a directed peptide. The libraries also include recombined nucleic acid molecules containing nucleotide mutations encoding amino acid replacements, for example, of one or more amino acids of a CDR. In an additional example, the libraries provided herein include recombined nucleic acid molecules where at least a portion of the nucleic acid molecule, such as the entire nucleic acid molecule encoding the VH chain, or at least one or more of a Vκ, and Jκ or Vλ and Jλ are derived from an existing monoclonal antibody including, but not limited to, any monoclonal antibody set forth in Table 9. For example, exemplary libraries provided herein can include a nucleic acid molecule encoding the VL chain of Herceptin such as is set forth in SEQ ID NO: 1423 (SEQ ID NO:818 including terminal restriction site sequences) or the VL chain of an anti-CD20 antibody such as is set forth in SEQ ID NO: 1050 or SEQ ID NO:1440 (SEQ ID NO:835 including terminal restriction site sequences).
Libraries of recombined nucleic acid molecules provided herein can include members that represent one, some or all of the above examples. Any of the libraries provided herein also can include members whose sequences include heterologous sequence, for example, restriction site sequences, linker sequences, sequences encoding tags or other detectable moieties or other sequences.
In the VL nucleic acid libraries provided herein, each recombined nucleic acid molecule member of the library is productive and, when co-expressed with a nucleic acid molecule encoding a VH chain, generates a functional antibody or portion thereof that contains a sufficient antigen-binding site. In addition, in the VL nucleic acid libraries provided herein, each nucleic acid member of the library is different. The VL nucleic acid libraries provided herein can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 (104), 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 106 or more different members. The nucleic acid members are provided in addressed formats, such that when addressed the identity of each nucleic acid is known by its location in the array.
For example, an exemplary VL nucleic acid library includes members set forth in SEQ ID NOS: 806-815, 817, 819-834, 836-867 each representing a different recombined nucleic acid molecule of Vκ, and Jκ germline segments. Such a library includes members containing heterologous sequence for restriction sites at the 3′ and 5′ ends. It is understood that members of the library also can include those having sequences not including the heterologous sequence, such as is set forth in any of SEQ ID NOS: 1411-1422, 1424-1439, 1441-1472.
In some examples, any of the nucleic acid sequences in the libraries provided herein can be included in a vector to generate vector libraries. Exemplary of vector libraries are libraries of recombined nucleic acid molecules encoding VL chain included in backbone Plasmid C or Plasmid E.
3. Paired Nucleic Acid Libraries or Vector Libraries Thereof
Also provided herein are libraries containing both recombined nucleic acid molecules encoding a VH chain and nucleic acid molecules encoding a VL chain, i.e. paired nucleic acid libraries. The paired libraries provided herein can contain a first nucleic acid molecule that is any of the nucleic acid members of the VH nucleic acid library in Section E.1 above and a second nucleic acid molecule that is any of the nucleic acid members of the VL nucleic acid library in Section E.2 above. The nucleic acid members in the paired libraries include those having heterologous sequence and those not having heterologous sequence. In some examples, one of the nucleic acid molecules in the pair can contain a heterologous sequence (e.g. a tag or other detectable moiety), while the other paired molecule at the locus in the library does not contain any heterologous sequence.
The paired nucleic acid libraries can be provided as addressed libraries. In such libraries, each locus of an addressed format contains one nucleic acid molecule encoding a VH chain and one nucleic acid molecule encoding a VL chain. Each nucleic acid pair (i.e. the combination of the nucleic acid molecule encoding the VH chain and the nucleic acid molecule encoding the VL chain) is different compared to all other pairs at all other addressed loci.
In some examples, the nucleic acid molecules can be contained in vectors to generate paired vector libraries. In such libraries, each locus of the addressed library includes a vector containing a nucleic acid molecule encoding a VL chain and a vector containing a nucleic acid molecule encoding a VH chain.
In some examples, the paired nucleic acid libraries can contain a common nucleic acid molecule encoding a VL chain such that each locus in the library contains the same nucleic acid molecule. In other examples, the paired nucleic acid libraries can contain a common nucleic acid molecule encoding a VH chain such that each locus in the library contains the same nucleic acid molecule. Generally, a library contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5,000, 10,000 or more nucleic acid molecules encoding a VL chain and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5,000, 10,000 or more nucleic acid molecules encoding a VH chain. The resulting paired library contains 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 (104), 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 106, 107, 108, 109, 1010 or more different paired members.
As described herein below, upon co-expression of a first and second nucleic acid molecule (e.g a nucleic acid molecule encoding a VH chain and a nucleic acid encoding a light chain), a library of antibodies can be generated. If the nucleic acid molecule further contains a sequence encoding a CH, a Fab library can be generated, whereby each member in the library contains a VH chain and a VL chain linked by a CH.
For example, an exemplary nucleic acid paired library includes those where a first nucleic acid is any one or more nucleic acids molecules set forth in any of SEQ ID NOS: 454-805 or 2070-2759 (each encoding a VH chain), and a second nucleic acid molecule set forth in any of SEQ ID NOS: 806-815, 817, 819-834, and 836-867 (each encoding a VL chain). The sequences set forth above contain heterologous sequence for restriction sites at the 3′ and 5′ ends. It is understood that nucleic acid libraries can be generated without the heterologus sequences. Thus, in some examples, members of the library also can include those having sequences not including a heterologous sequence. For example, an exemplary nucleic acid paired library includes nucleic acid sequences not containing heterologous sequence for restriction sites at the 3′ and 5′ ends from a first nucleic acid set forth in any of SEQ ID NOS: 1059-1410 or 2070-2759 (not including the 3′and 5′restriction sites) (each encoding a VH chain) and a second nucleic acid molecule set forth in SEQ ID NOS: 1411-1422, 1424-1439 and 1441-1471. Such a library can include all permutations of any of the above paired nucleic acid sequences. Thus, the paired library can contain at or about 1.5 ×105, 2.1×105, 2.5×105, 3.5×105, 4×105, 4.2×105, 4.4×105, 4.6×105, 4.8×105, 5×105, 5.2×105, 5.4×105, 5.6×105, 5.8×105, 6×10′, or more members, or a subset thereof such as 500, 600, 700, 800, 900, 103, 5×103, 104, 5×104, 105 or more members.
Exemplary of a paired library is set forth in Table 17, where each row sets forth a different loci of the library. In the Table, SEQ ID NOS for the nucleic acid molecules are set forth as “RS” (containing a heterologous restriction site sequence) and “NO RS” (not containing a heterologous restriction site sequence).







TABLE 17







Exemplary Nucleic Acid Paired Library















RS
NO RS

RS
NO RS



Heavy Chain Name
SEQ ID NO
SEQ ID NO
Light Chain Name
SEQ ID NO
SEQ ID NO

















1
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|O12_IGKJ1*01
833
1438


2
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|O12_IGKJ1*01
833
1438


3
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|O12_IGKJ1*01
833
1438


4
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|O12_IGKJ1*01
833
1438


5
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|O12_IGKJ1*01
833
1438


6
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|O12_IGKJ1*01
833
1438


7
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|O12_IGKJ1*01
833
1438


8
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|O12_IGKJ1*01
833
1438


9
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|O12_IGKJ1*01
833
1438


10
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|O12_IGKJ1*01
833
1438


11
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|O12_IGKJ1*01
833
1438


12
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|O12_IGKJ1*01
833
1438


13
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|O12_IGKJ1*01
833
1438


14
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|O12_IGKJ1*01
833
1438


15
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|O12_IGKJ1*01
833
1438


16
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|O12_IGKJ1*01
833
1438


17
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|O12_IGKJ1*01
833
1438


18
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|O12_IGKJ1*01
833
1438


19
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|O12_IGKJ1*01
833
1438


20
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|O12_IGKJ1*01
833
1438


21
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|O12_IGKJ1*01
833
1438


22
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|O12_IGKJ1*01
833
1438


23
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|O12_IGKJ1*01
833
1438


24
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|O12_IGKJ1*01
833
1438


25
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|O12_IGKJ1*01
833
1438


26
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|O12_IGKJ1*01
833
1438


27
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|O12_IGKJ1*01
833
1438


28
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|O12_IGKJ1*01
833
1438


29
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|O12_IGKJ1*01
833
1438


30
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|O12_IGKJ1*01
833
1438


31
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|O12_IGKJ1*01
833
1438


32
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|O12_IGKJ1*01
833
1438


33
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|O12_IGKJ1*01
833
1438


34
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|O12_IGKJ1*01
833
1438


35
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|O12_IGKJ1*01
833
1438


36
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|O12_IGKJ1*01
833
1438


37
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|O12_IGKJ1*01
833
1438


38
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|O12_IGKJ1*01
833
1438


39
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|O12_IGKJ1*01
833
1438


40
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|O12_IGKJ1*01
833
1438


41
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|O12_IGKJ1*01
833
1438


42
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|O12_IGKJ1*01
833
1438


43
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|O12_IGKJ1*01
833
1438


44
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|O12_IGKJ1*01
833
1438


45
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|O12_IGKJ1*01
833
1438


46
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|O12_IGKJ1*01
833
1438


47
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|O12_IGKJ1*01
833
1438


48
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|O12_IGKJ1*01
833
1438


49
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|O12_IGKJ1*01
833
1438


50
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|O12_IGKJ1*01
833
1438


51
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|O12_IGKJ1*01
833
1438


52
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|O12_IGKJ1*01
833
1438


53
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|O12_IGKJ1*01
833
1438


54
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|O12_IGKJ1*01
833
1438


55
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|O12_IGKJ1*01
833
1438


56
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|O12_IGKJ1*01
833
1438


57
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|O12_IGKJ1*01
833
1438


58
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|O12_IGKJ1*01
833
1438


59
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|O12_IGKJ1*01
833
1438


60
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|O12_IGKJ1*01
833
1438


61
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|O12_IGKJ1*01
833
1438


62
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|O12_IGKJ1*01
833
1438


63
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|O12_IGKJ1*01
833
1438


64
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|O12_IGKJ1*01
833
1438


65
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|O12_IGKJ1*01
833
1438


66
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|O12_IGKJ1*01
833
1438


67
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|O12_IGKJ1*01
833
1438


68
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|O12_IGKJ1*01
833
1438


69
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|O12_IGKJ1*01
833
1438


70
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|O12_IGKJ1*01
833
1438


71
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|O12_IGKJ1*01
833
1438


72
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|O12_IGKJ1*01
833
1438


73
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|O12_IGKJ1*01
833
1438


74
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|O12_IGKJ1*01
833
1438


75
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|O12_IGKJ1*01
833
1438


76
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|O12_IGKJ1*01
833
1438


77
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|O12_IGKJ1*01
833
1438


78
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|O12_IGKJ1*01
833
1438


79
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|O12_IGKJ1*01
833
1438


80
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|O12_IGKJ1*01
833
1438


81
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|O12_IGKJ1*01
833
1438


82
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|O12_IGKJ1*01
833
1438


83
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|O12_IGKJ1*01
833
1438


84
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|O12_IGKJ1*01
833
1438


85
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|O12_IGKJ1*01
833
1438


86
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|O12_IGKJ1*01
833
1438


87
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|O12_IGKJ1*01
833
1438


88
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|O12_IGKJ1*01
833
1438


89
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|O12_IGKJ1*01
833
1438


90
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|O12_IGKJ1*01
833
1438


91
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|O12_IGKJ1*01
833
1438


92
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|O12_IGKJ1*01
833
1438


93
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|O12_IGKJ1*01
833
1438


94
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|O12_IGKJ1*01
833
1438


95
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|O12_IGKJ1*01
833
1438


96
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|O12_IGKJ1*01
833
1438


97
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|O18_IGKJ1*01
834
1439


98
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|O18_IGKJ1*01
834
1439


99
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|O18_IGKJ1*01
834
1439


100
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|O18_IGKJ1*01
834
1439


101
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|O18_IGKJ1*01
834
1439


102
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|O18_IGKJ1*01
834
1439


103
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|O18_IGKJ1*01
834
1439


104
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|O18_IGKJ1*01
834
1439


105
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|O18_IGKJ1*01
834
1439


106
gnl|Fabrus|VH1-69_GHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|O18_IGKJ1*01
834
1439


107
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|O18_IGKJ1*01
834
1439


108
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|O18_IGKJ1*01
834
1439


109
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|O18_IGKJ1*01
834
1439


110
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|O18_IGKJ1*01
834
1439


111
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|O18_IGKJ1*01
834
1439


112
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|O18_IGKJ1*01
834
1439


113
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|O18_IGKJ1*01
834
1439


114
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|O18_IGKJ1*01
834
1439


115
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|O18_IGKJ1*01
834
1439


116
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|O18_IGKJ1*01
834
1439


117
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|O18_IGKJ1*01
834
1439


118
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|O18_IGKJ1*01
834
1439


119
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|O18_IGKJ1*01
834
1439


120
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|O18_IGKJ1*01
834
1439


121
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|O18_IGKJ1*01
834
1439


122
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|O18_IGKJ1*01
834
1439


123
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|O18_IGKJ1*01
834
1439


124
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|O18_IGKJ1*01
834
1439


125
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|O18_IGKJ1*01
834
1439


126
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|O18_IGKJ1*01
834
1439


127
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|O18_IGKJ1*01
834
1439


128
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|O18_IGKJ1*01
834
1439


129
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|O18_IGKJ1*01
834
1439


130
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|O18_IGKJ1*01
834
1439


131
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|O18_IGKJ1*01
834
1439


132
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|O18_IGKJ1*01
834
1439


133
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|O18_IGKJ1*01
834
1439


134
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|O18_IGKJ1*01
834
1439


135
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|O18_IGKJ1*01
834
1439


136
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|O18_IGKJ1*01
834
1439


137
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|O18_IGKJ1*01
834
1439


138
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|O18_IGKJ1*01
834
1439


139
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|O18_IGKJ1*01
834
1439


140
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|O18_IGKJ1*01
834
1439


141
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|O18_IGKJ1*01
834
1439


142
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|O18_IGKJ1*01
834
1439


143
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|O18_IGKJ1*01
834
1439


144
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|O18_IGKJ1*01
834
1439


145
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|O18_IGKJ1*01
834
1439


146
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|O18_IGKJ1*01
834
1439


147
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|O18_IGKJ1*01
834
1439


148
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|O18_IGKJ1*01
834
1439


149
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|O18_IGKJ1*01
834
1439


150
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|O18_IGKJ1*01
834
1439


151
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|O18_IGKJ1*01
834
1439


152
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|O18_IGKJ1*01
834
1439


153
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|O18_IGKJ1*01
834
1439


154
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|O18_IGKJ1*01
834
1439


155
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|O18_IGKJ1*01
834
1439


156
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|O18_IGKJ1*01
834
1439


157
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|O18_IGKJ1*01
834
1439


158
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|O18_IGKJ1*01
834
1439


159
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|O18_IGKJ1*01
834
1439


160
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|O18_IGKJ1*01
834
1439


161
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|O18_IGKJ1*01
834
1439


162
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|O18_IGKJ1*01
834
1439


163
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|O18_IGKJ1*01
834
1439


164
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|O18_IGKJ1*01
834
1439


165
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|O18_IGKJ1*01
834
1439


166
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|O18_IGKJ1*01
834
1439


167
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|O18_IGKJ1*01
834
1439


168
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|O18_IGKJ1*01
834
1439


169
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|O18_IGKJ1*01
834
1439


170
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|O18_IGKJ1*01
834
1439


171
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|O18_IGKJ1*01
834
1439


172
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|O18_IGKJ1*01
834
1439


173
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|O18_IGKJ1*01
834
1439


174
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|O18_IGKJ1*01
834
1439


175
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|O18_IGKJ1*01
834
1439


176
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|O18_IGKJ1*01
834
1439


177
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|O18_IGKJ1*01
834
1439


178
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|O18_IGKJ1*01
834
1439


179
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|O18_IGKJ1*01
834
1439


180
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|O18_IGKJ1*01
834
1439


181
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|O18_IGKJ1*01
834
1439


182
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|O18_IGKJ1*01
834
1439


183
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|O18_IGKJ1*01
834
1439


184
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|O18_IGKJ1*01
834
1439


185
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|O18_IGKJ1*01
834
1439


186
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|O18_IGKJ1*01
834
1439


187
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|O18_IGKJ1*01
834
1439


188
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|O18_IGKJ1*01
834
1439


189
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|O18_IGKJ1*01
834
1439


190
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|O18_IGKJ1*01
834
1439


191
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|O18_IGKJ1*01
834
1439


192
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|O18_IGKJ1*01
834
1439


193
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|A20_IGKJ1*01
809
1414


194
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|A20_IGKJ1*01
809
1414


195
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|A20_IGKJ1*01
809
1414


196
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|A20_IGKJ1*01
809
1414


197
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|A20_IGKJ1*01
809
1414


198
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|A20_IGKJ1*01
809
1414


199
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|A20_IGKJ1*01
809
1414


200
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|A20_IGKJ1*01
809
1414


201
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|A20_IGKJ1*01
809
1414


202
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|A20_IGKJ1*01
809
1414


203
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|A20_IGKJ1*01
809
1414


204
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|A20_IGKJ1*01
809
1414


205
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|A20_IGKJ1*01
809
1414


206
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|A20_IGKJ1*01
809
1414


207
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|A20_IGKJ1*01
809
1414


208
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|A20_IGKJ1*01
809
1414


209
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|A20_IGKJ1*01
809
1414


210
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|A20_IGKJ1*01
809
1414


211
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|A20_IGKJ1*01
809
1414


212
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|A20_IGKJ1*01
809
1414


213
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|A20_IGKJ1*01
809
1414


214
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|A20_IGKJ1*01
809
1414


215
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|A20_IGKJ1*01
809
1414


216
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|A20_IGKJ1*01
809
1414


217
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|A20_IGKJ1*01
809
1414


218
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|A20_IGKJ1*01
809
1414


219
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|A20_IGKJ1*01
809
1414


220
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|A20_IGKJ1*01
809
1414


221
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|A20_IGKJ1*01
809
1414


222
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|A20_IGKJ1*01
809
1414


223
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|A20_IGKJ1*01
809
1414


224
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|A20_IGKJ1*01
809
1414


225
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|A20_IGKJ1*01
809
1414


226
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|A20_IGKJ1*01
809
1414


227
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|A20_IGKJ1*01
809
1414


228
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|A20_IGKJ1*01
809
1414


229
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|A20_IGKJ1*01
809
1414


230
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|A20_IGKJ1*01
809
1414


231
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|A20_IGKJ1*01
809
1414


232
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|A20_IGKJ1*01
809
1414


233
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|A20_IGKJ1*01
809
1414


234
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|A20_IGKJ1*01
809
1414


235
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|A20_IGKJ1*01
809
1414


236
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|A20_IGKJ1*01
809
1414


237
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|A20_IGKJ1*01
809
1414


238
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|A20_IGKJ1*01
809
1414


239
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|A20_IGKJ1*01
809
1414


240
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|A20_IGKJ1*01
809
1414


241
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|A20_IGKJ1*01
809
1414


242
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|A20_IGKJ1*01
809
1414


243
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|A20_IGKJ1*01
809
1414


244
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|A20_IGKJ1*01
809
1414


245
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|A20_IGKJ1*01
809
1414


246
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|A20_IGKJ1*01
809
1414


247
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|A20_IGKJ1*01
809
1414


248
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|A20_IGKJ1*01
809
1414


249
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|A20_IGKJ1*01
809
1414


250
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|A20_IGKJ1*01
809
1414


251
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|A20_IGKJ1*01
809
1414


252
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|A20_IGKJ1*01
809
1414


253
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|A20_IGKJ1*01
809
1414


254
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|A20_IGKJ1*01
809
1414


255
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|A20_IGKJ1*01
809
1414


256
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|A20_IGKJ1*01
809
1414


257
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|A20_IGKJ1*01
809
1414


258
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|A20_IGKJ1*01
809
1414


259
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|A20_IGKJ1*01
809
1414


260
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|A20_IGKJ1*01
809
1414


261
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|A20_IGKJ1*01
809
1414


262
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|A20_IGKJ1*01
809
1414


263
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|A20_IGKJ1*01
809
1414


264
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|A20_IGKJ1*01
809
1414


265
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|A20_IGKJ1*01
809
1414


266
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|A20_IGKJ1*01
809
1414


267
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|A20_IGKJ1*01
809
1414


268
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|A20_IGKJ1*01
809
1414


269
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|A20_IGKJ1*01
809
1414


270
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|A20_IGKJ1*01
809
1414


271
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|A20_IGKJ1*01
809
1414


272
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|A20_IGKJ1*01
809
1414


273
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|A20_IGKJ1*01
809
1414


274
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|A20_IGKJ1*01
809
1414


275
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|A20_IGKJ1*01
809
1414


276
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|A20_IGKJ1*01
809
1414


277
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|A20_IGKJ1*01
809
1414


278
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|A20_IGKJ1*01
809
1414


279
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|A20_IGKJ1*01
809
1414


280
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|A20_IGKJ1*01
809
1414


281
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|A20_IGKJ1*01
809
1414


282
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|A20_IGKJ1*01
809
1414


283
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|A20_IGKJ1*01
809
1414


284
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|A20_IGKJ1*01
809
1414


285
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|A20_IGKJ1*01
809
1414


286
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|A20_IGKJ1*01
809
1414


287
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|A20_IGKJ1*01
809
1414


288
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|A20_IGKJ1*01
809
1414


289
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|A30_IGKJ1*01
814
1419


290
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|A30_IGKJ1*01
814
1419


291
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|A30_IGKJ1*01
814
1419


292
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|A30_IGKJ1*01
814
1419


293
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|A30_IGKJ1*01
814
1419


294
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|A30_IGKJ1*01
814
1419


295
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|A30_IGKJ1*01
814
1419


296
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|A30_IGKJ1*01
814
1419


297
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|A30_IGKJ1*01
814
1419


298
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|A30_IGKJ1*01
814
1419


299
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|A30_IGKJ1*01
814
1419


300
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|A30_IGKJ1*01
814
1419


301
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|A30_IGKJ1*01
814
1419


302
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|A30_IGKJ1*01
814
1419


303
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|A30_IGKJ1*01
814
1419


304
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|A30_IGKJ1*01
814
1419


305
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|A30_IGKJ1*01
814
1419


306
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|A30_IGKJ1*01
814
1419


307
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|A30_IGKJ1*01
814
1419


308
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|A30_IGKJ1*01
814
1419


309
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|A30_IGKJ1*01
814
1419


310
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|A30_IGKJ1*01
814
1419


311
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|A30_IGKJ1*01
814
1419


312
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|A30_IGKJ1*01
814
1419


313
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|A30_IGKJ1*01
814
1419


314
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|A30_IGKJ1*01
814
1419


315
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|A30_IGKJ1*01
814
1419


316
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|A30_IGKJ1*01
814
1419


317
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|A30_IGKJ1*01
814
1419


318
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|A30_IGKJ1*01
814
1419


319
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|A30_IGKJ1*01
814
1419


320
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|A30_IGKJ1*01
814
1419


321
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|A30_IGKJ1*01
814
1419


322
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|A30_IGKJ1*01
814
1419


323
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|A30_IGKJ1*01
814
1419


324
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|A30_IGKJ1*01
814
1419


325
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|A30_IGKJ1*01
814
1419


326
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|A30_IGKJ1*01
814
1419


327
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|A30_IGKJ1*01
814
1419


328
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|A30_IGKJ1*01
814
1419


329
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|A30_IGKJ1*01
814
1419


330
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|A30_IGKJ1*01
814
1419


331
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|A30_IGKJ1*01
814
1419


332
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|A30_IGKJ1*01
814
1419


333
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|A30_IGKJ1*01
814
1419


334
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|A30_IGKJ1*01
814
1419


335
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|A30_IGKJ1*01
814
1419


336
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|A30_IGKJ1*01
814
1419


337
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|A30_IGKJ1*01
814
1419


338
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|A30_IGKJ1*01
814
1419


339
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|A30_IGKJ1*01
814
1419


340
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|A30_IGKJ1*01
814
1419


341
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|A30_IGKJ1*01
814
1419


342
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|A30_IGKJ1*01
814
1419


343
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|A30_IGKJ1*01
814
1419


344
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|A30_IGKJ1*01
814
1419


345
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|A30_IGKJ1*01
814
1419


346
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|A30_IGKJ1*01
814
1419


347
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|A30_IGKJ1*01
814
1419


348
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|A30_IGKJ1*01
814
1419


349
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|A30_IGKJ1*01
814
1419


350
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|A30_IGKJ1*01
814
1419


351
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|A30_IGKJ1*01
814
1419


352
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|A30_IGKJ1*01
814
1419


353
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|A30_IGKJ1*01
814
1419


354
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|A30_IGKJ1*01
814
1419


355
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|A30_IGKJ1*01
814
1419


356
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|A30_IGKJ1*01
814
1419


357
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|A30_IGKJ1*01
814
1419


358
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|A30_IGKJ1*01
814
1419


359
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|A30_IGKJ1*01
814
1419


360
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|A30_IGKJ1*01
814
1419


361
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|A30_IGKJ1*01
814
1419


362
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|A30_IGKJ1*01
814
1419


363
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|A30_IGKJ1*01
814
1419


364
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|A30_IGKJ1*01
814
1419


365
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|A30_IGKJ1*01
814
1419


366
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|A30_IGKJ1*01
814
1419


367
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|A30_IGKJ1*01
814
1419


368
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|A30_IGKJ1*01
814
1419


369
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|A30_IGKJ1*01
814
1419


370
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|A30_IGKJ1*01
814
1419


371
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|A30_IGKJ1*01
814
1419


372
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|A30_IGKJ1*01
814
1419


373
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|A30_IGKJ1*01
814
1419


374
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|A30_IGKJ1*01
814
1419


375
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|A30_IGKJ1*01
814
1419


376
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|A30_IGKJ1*01
814
1419


377
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|A30_IGKJ1*01
814
1419


378
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|A30_IGKJ1*01
814
1419


379
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|A30_IGKJ1*01
814
1419


380
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|A30_IGKJ1*01
814
1419


381
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|A30_IGKJ1*01
814
1419


382
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|A30_IGKJ1*01
814
1419


383
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|A30_IGKJ1*01
814
1419


384
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|A30_IGKJ1*01
814
1419


385
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


386
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


387
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


388
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


389
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


390
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


391
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


392
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


393
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


394
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


395
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


396
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


397
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


398
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


399
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


400
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


401
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


402
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


403
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


404
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


405
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


406
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


407
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


408
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


409
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


410
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


411
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


412
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


413
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


414
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


415
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


416
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


417
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


418
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


419
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


420
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


421
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


422
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


423
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


424
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


425
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


426
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


427
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


428
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


429
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


430
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


431
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


432
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


433
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


434
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


435
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


436
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


437
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


438
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


439
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


440
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


441
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


442
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


443
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


444
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


445
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


446
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


447
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


448
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


449
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


450
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


451
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


452
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


453
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


454
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


455
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


456
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


457
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


458
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


459
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


460
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


461
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


462
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


463
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


464
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


465
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


466
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


467
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


468
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


469
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


470
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


471
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


472
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


473
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


474
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


475
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


476
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


477
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


478
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


479
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


480
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


481
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|L5_IGKJ1*01
828
1433


482
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|L5_IGKJ1*01
828
1433


483
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|L5_IGKJ1*01
828
1433


484
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|L5_IGKJ1*01
828
1433


485
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|L5_IGKJ1*01
828
1433


486
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|L5_IGKJ1*01
828
1433


487
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|L5_IGKJ1*01
828
1433


488
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|L5_IGKJ1*01
828
1433


489
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|L5_IGKJ1*01
828
1433


490
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|L5_IGKJ1*01
828
1433


491
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|L5_IGKJ1*01
828
1433


492
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|L5_IGKJ1*01
828
1433


493
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|L5_IGKJ1*01
828
1433


494
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|L5_IGKJ1*01
828
1433


495
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|L5_IGKJ1*01
828
1433


496
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|L5_IGKJ1*01
828
1433


497
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|L5_IGKJ1*01
828
1433


498
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|L5_IGKJ1*01
828
1433


499
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|L5_IGKJ1*01
828
1433


500
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|L5_IGKJ1*01
828
1433


501
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|L5_IGKJ1*01
828
1433


502
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|L5_IGKJ1*01
828
1433


503
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|L5_IGKJ1*01
828
1433


504
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|L5_IGKJ1*01
828
1433


505
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|L5_IGKJ1*01
828
1433


506
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|L5_IGKJ1*01
828
1433


507
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|L5_IGKJ1*01
828
1433


508
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|L5_IGKJ1*01
828
1433


509
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|L5_IGKJ1*01
828
1433


510
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|L5_IGKJ1*01
828
1433


511
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|L5_IGKJ1*01
828
1433


512
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|L5_IGKJ1*01
828
1433


513
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|L5_IGKJ1*01
828
1433


514
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|L5_IGKJ1*01
828
1433


515
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|L5_IGKJ1*01
828
1433


516
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|L5_IGKJ1*01
828
1433


517
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|L5_IGKJ1*01
828
1433


518
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|L5_IGKJ1*01
828
1433


519
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|L5_IGKJ1*01
828
1433


520
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|L5_IGKJ1*01
828
1433


521
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|L5_IGKJ1*01
828
1433


522
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|L5_IGKJ1*01
828
1433


523
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|L5_IGKJ1*01
828
1433


524
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|L5_IGKJ1*01
828
1433


525
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|L5_IGKJ1*01
828
1433


526
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|L5_IGKJ1*01
828
1433


527
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|L5_IGKJ1*01
828
1433


528
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|L5_IGKJ1*01
828
1433


529
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|L5_IGKJ1*01
828
1433


530
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|L5_IGKJ1*01
828
1433


531
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|L5_IGKJ1*01
828
1433


532
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|L5_IGKJ1*01
828
1433


533
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|L5_IGKJ1*01
828
1433


534
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|L5_IGKJ1*01
828
1433


535
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|L5_IGKJ1*01
828
1433


536
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|L5_IGKJ1*01
828
1433


537
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|L5_IGKJ1*01
828
1433


538
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|L5_IGKJ1*01
828
1433


539
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|L5_IGKJ1*01
828
1433


540
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|L5_IGKJ1*01
828
1433


541
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|L5_IGKJ1*01
828
1433


542
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|L5_IGKJ1*01
828
1433


543
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|L5_IGKJ1*01
828
1433


544
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|L5_IGKJ1*01
828
1433


545
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|L5_IGKJ1*01
828
1433


546
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|L5_IGKJ1*01
828
1433


547
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|L5_IGKJ1*01
828
1433


548
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|L5_IGKJ1*01
828
1433


549
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|L5_IGKJ1*01
828
1433


550
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|L5_IGKJ1*01
828
1433


551
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|L5_IGKJ1*01
828
1433


552
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|L5_IGKJ1*01
828
1433


553
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|L5_IGKJ1*01
828
1433


554
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|L5_IGKJ1*01
828
1433


555
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|L5_IGKJ1*01
828
1433


556
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|L5_IGKJ1*01
828
1433


557
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|L5_IGKJ1*01
828
1433


558
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|L5_IGKJ1*01
828
1433


559
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|L5_IGKJ1*01
828
1433


560
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|L5_IGKJ1*01
828
1433


561
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|L5_IGKJ1*01
828
1433


562
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|L5_IGKJ1*01
828
1433


563
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|L5_IGKJ1*01
828
1433


564
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|L5_IGKJ1*01
828
1433


565
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|L5_IGKJ1*01
828
1433


566
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|L5_IGKJ1*01
828
1433


567
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|L5_IGKJ1*01
828
1433


568
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|L5_IGKJ1*01
828
1433


569
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|L5_IGKJ1*01
828
1433


570
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|L5_IGKJ1*01
828
1433


571
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|L5_IGKJ1*01
828
1433


572
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|L5_IGKJ1*01
828
1433


573
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|L5_IGKJ1*01
828
1433


574
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|L5_IGKJ1*01
828
1433


575
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|L5_IGKJ1*01
828
1433


576
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|L5_IGKJ1*01
828
1433


577
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|L8_IGKJ1*01
830
1435


578
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|L8_IGKJ1*01
830
1435


579
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|L8_IGKJ1*01
830
1435


580
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|L8_IGKJ1*01
830
1435


581
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|L8_IGKJ1*01
830
1435


582
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|L8_IGKJ1*01
830
1435


583
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|L8_IGKJ1*01
830
1435


584
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|L8_IGKJ1*01
830
1435


585
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|L8_IGKJ1*01
830
1435


586
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|L8_IGKJ1*01
830
1435


587
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|L8_IGKJ1*01
830
1435


588
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|L8_IGKJ1*01
830
1435


589
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|L8_IGKJ1*01
830
1435


590
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|L8_IGKJ1*01
830
1435


591
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|L8_IGKJ1*01
830
1435


592
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|L8_IGKJ1*01
830
1435


593
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|L8_IGKJ1*01
830
1435


594
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|L8_IGKJ1*01
830
1435


595
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|L8_IGKJ1*01
830
1435


596
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|L8_IGKJ1*01
830
1435


597
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|L8_IGKJ1*01
830
1435


598
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|L8_IGKJ1*01
830
1435


599
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|L8_IGKJ1*01
830
1435


600
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|L8_IGKJ1*01
830
1435


601
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|L8_IGKJ1*01
830
1435


602
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|L8_IGKJ1*01
830
1435


603
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|L8_IGKJ1*01
830
1435


604
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|L8_IGKJ1*01
830
1435


605
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|L8_IGKJ1*01
830
1435


606
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|L8_IGKJ1*01
830
1435


607
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|L8_IGKJ1*01
830
1435


608
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|L8_IGKJ1*01
830
1435


609
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|L8_IGKJ1*01
830
1435


610
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|L8_IGKJ1*01
830
1435


611
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|L8_IGKJ1*01
830
1435


612
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|L8_IGKJ1*01
830
1435


613
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|L8_IGKJ1*01
830
1435


614
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|L8_IGKJ1*01
830
1435


615
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|L8_IGKJ1*01
830
1435


616
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|L8_IGKJ1*01
830
1435


617
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|L8_IGKJ1*01
830
1435


618
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|L8_IGKJ1*01
830
1435


619
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|L8_IGKJ1*01
830
1435


620
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|L8_IGKJ1*01
830
1435


621
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|L8_IGKJ1*01
830
1435


622
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|L8_IGKJ1*01
830
1435


623
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|L8_IGKJ1*01
830
1435


624
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|L8_IGKJ1*01
830
1435


625
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|L8_IGKJ1*01
830
1435


626
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|L8_IGKJ1*01
830
1435


627
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|L8_IGKJ1*01
830
1435


628
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|L8_IGKJ1*01
830
1435


629
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|L8_IGKJ1*01
830
1435


630
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|L8_IGKJ1*01
830
1435


631
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|L8_IGKJ1*01
830
1435


632
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|L8_IGKJ1*01
830
1435


633
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|L8_IGKJ1*01
830
1435


634
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|L8_IGKJ1*01
830
1435


635
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|L8_IGKJ1*01
830
1435


636
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|L8_IGKJ1*01
830
1435


637
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|L8_IGKJ1*01
830
1435


638
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|L8_IGKJ1*01
830
1435


639
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|L8_IGKJ1*01
830
1435


640
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|L8_IGKJ1*01
830
1435


641
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|L8_IGKJ1*01
830
1435


642
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|L8_IGKJ1*01
830
1435


643
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|L8_IGKJ1*01
830
1435


644
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|L8_IGKJ1*01
830
1435


645
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|L8_IGKJ1*01
830
1435


646
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|L8_IGKJ1*01
830
1435


647
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|L8_IGKJ1*01
830
1435


648
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|L8_IGKJ1*01
830
1435


649
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|L8_IGKJ1*01
830
1435


650
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|L8_IGKJ1*01
830
1435


651
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|L8_IGKJ1*01
830
1435


652
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|L8_IGKJ1*01
830
1435


653
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|L8_IGKJ1*01
830
1435


654
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|L8_IGKJ1*01
830
1435


655
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|L8_IGKJ1*01
830
1435


656
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|L8_IGKJ1*01
830
1435


657
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|L8_IGKJ1*01
830
1435


658
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|L8_IGKJ1*01
830
1435


659
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|L8_IGKJ1*01
830
1435


660
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|L8_IGKJ1*01
830
1435


661
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|L8_IGKJ1*01
830
1435


662
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|L8_IGKJ1*01
830
1435


663
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|L8_IGKJ1*01
830
1435


664
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|L8_IGKJ1*01
830
1435


665
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|L8_IGKJ1*01
830
1435


666
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|L8_IGKJ1*01
830
1435


667
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|L8_IGKJ1*01
830
1435


668
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|L8_IGKJ1*01
830
1435


669
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|L8_IGKJ1*01
830
1435


670
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|L8_IGKJ1*01
830
1435


671
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|L8_IGKJ1*01
830
1435


672
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|L8_IGKJ1*01
830
1435


673
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|L11_IGKJ1*01
819
1424


674
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|L11_IGKJ1*01
819
1424


675
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|L11_IGKJ1*01
819
1424


676
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|L11_IGKJ1*01
819
1424


677
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|L11_IGKJ1*01
819
1424


678
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|L11_IGKJ1*01
819
1424


679
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|L11_IGKJ1*01
819
1424


680
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|L11_IGKJ1*01
819
1424


681
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|L11_IGKJ1*01
819
1424


682
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|L11_IGKJ1*01
819
1424


683
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|L11_IGKJ1*01
819
1424


684
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|L11_IGKJ1*01
819
1424


685
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|L11_IGKJ1*01
819
1424


686
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|L11_IGKJ1*01
819
1424


687
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|L11_IGKJ1*01
819
1424


688
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|L11_IGKJ1*01
819
1424


689
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|L11_IGKJ1*01
819
1424


690
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|L11_IGKJ1*01
819
1424


691
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|L11_IGKJ1*01
819
1424


692
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|L11_IGKJ1*01
819
1424


693
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|L11_IGKJ1*01
819
1424


694
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|L11_IGKJ1*01
819
1424


695
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|L11_IGKJ1*01
819
1424


696
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|L11_IGKJ1*01
819
1424


697
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|L11_IGKJ1*01
819
1424


698
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|L11_IGKJ1*01
819
1424


699
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|L11_IGKJ1*01
819
1424


700
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|L11_IGKJ1*01
819
1424


701
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|L11_IGKJ1*01
819
1424


702
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|L11_IGKJ1*01
819
1424


703
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|L11_IGKJ1*01
819
1424


704
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|L11_IGKJ1*01
819
1424


705
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|L11_IGKJ1*01
819
1424


706
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|L11_IGKJ1*01
819
1424


707
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|L11_IGKJ1*01
819
1424


708
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|L11_IGKJ1*01
819
1424


709
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|L11_IGKJ1*01
819
1424


710
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|L11_IGKJ1*01
819
1424


711
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|L11_IGKJ1*01
819
1424


712
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|L11_IGKJ1*01
819
1424


713
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|L11_IGKJ1*01
819
1424


714
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|L11_IGKJ1*01
819
1424


715
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|L11_IGKJ1*01
819
1424


716
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|L11_IGKJ1*01
819
1424


717
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|L11_IGKJ1*01
819
1424


718
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|L11_IGKJ1*01
819
1424


719
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|L11_IGKJ1*01
819
1424


720
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|L11_IGKJ1*01
819
1424


721
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|L11_IGKJ1*01
819
1424


722
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|L11_IGKJ1*01
819
1424


723
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|L11_IGKJ1*01
819
1424


724
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|L11_IGKJ1*01
819
1424


725
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|L11_IGKJ1*01
819
1424


726
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|L11_IGKJ1*01
819
1424


727
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|L11_IGKJ1*01
819
1424


728
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|L11_IGKJ1*01
819
1424


729
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|L11_IGKJ1*01
819
1424


730
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|L11_IGKJ1*01
819
1424


731
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|L11_IGKJ1*01
819
1424


732
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|L11_IGKJ1*01
819
1424


733
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|L11_IGKJ1*01
819
1424


734
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|L11_IGKJ1*01
819
1424


735
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|L11_IGKJ1*01
819
1424


736
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|L11_IGKJ1*01
819
1424


737
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|L11_IGKJ1*01
819
1424


738
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|L11_IGKJ1*01
819
1424


739
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|L11_IGKJ1*01
819
1424


740
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|L11_IGKJ1*01
819
1424


741
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|L11_IGKJ1*01
819
1424


742
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|L11_IGKJ1*01
819
1424


743
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|L11_IGKJ1*01
819
1424


744
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|L11_IGKJ1*01
819
1424


745
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|L11_IGKJ1*01
819
1424


746
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|L11_IGKJ1*01
819
1424


747
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|L11_IGKJ1*01
819
1424


748
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|L11_IGKJ1*01
819
1424


749
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|L11_IGKJ1*01
819
1424


750
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|L11_IGKJ1*01
819
1424


751
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|L11_IGKJ1*01
819
1424


752
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|L11_IGKJ1*01
819
1424


753
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|L11_IGKJ1*01
819
1424


754
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|L11_IGKJ1*01
819
1424


755
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|L11_IGKJ1*01
819
1424


756
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|L11_IGKJ1*01
819
1424


757
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|L11_IGKJ1*01
819
1424


758
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|L11_IGKJ1*01
819
1424


759
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|L11_IGKJ1*01
819
1424


760
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|L11_IGKJ1*01
819
1424


761
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|L11_IGKJ1*01
819
1424


762
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|L11_IGKJ1*01
819
1424


763
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|L11_IGKJ1*01
819
1424


764
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|L11_IGKJ1*01
819
1424


765
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|L11_IGKJ1*01
819
1424


766
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|L11_IGKJ1*01
819
1424


767
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|L11_IGKJ1*01
819
1424


768
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|L11_IGKJ1*01
819
1424


769
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|L12_IGKJ1*01
820
1425


770
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|L12_IGKJ1*01
820
1425


771
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|L12_IGKJ1*01
820
1425


772
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|L12_IGKJ1*01
820
1425


773
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|L12_IGKJ1*01
820
1425


774
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|L12_IGKJ1*01
820
1425


775
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|L12_IGKJ1*01
820
1425


776
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|L12_IGKJ1*01
820
1425


777
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|L12_IGKJ1*01
820
1425


778
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|L12_IGKJ1*01
820
1425


779
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|L12_IGKJ1*01
820
1425


780
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|L12_IGKJ1*01
820
1425


781
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|L12_IGKJ1*01
820
1425


782
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|L12_IGKJ1*01
820
1425


783
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|L12_IGKJ1*01
820
1425


784
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|L12_IGKJ1*01
820
1425


785
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|L12_IGKJ1*01
820
1425


786
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|L12_IGKJ1*01
820
1425


787
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|L12_IGKJ1*01
820
1425


788
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|L12_IGKJ1*01
820
1425


789
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|L12_IGKJ1*01
820
1425


790
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|L12_IGKJ1*01
820
1425


791
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|L12_IGKJ1*01
820
1425


792
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|L12_IGKJ1*01
820
1425


793
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|L12_IGKJ1*01
820
1425


794
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|L12_IGKJ1*01
820
1425


795
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|L12_IGKJ1*01
820
1425


796
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|L12_IGKJ1*01
820
1425


797
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|L12_IGKJ1*01
820
1425


798
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|L12_IGKJ1*01
820
1425


799
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|L12_IGKJ1*01
820
1425


800
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|L12_IGKJ1*01
820
1425


801
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|L12_IGKJ1*01
820
1425


802
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|L12_IGKJ1*01
820
1425


803
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|L12_IGKJ1*01
820
1425


804
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|L12_IGKJ1*01
820
1425


805
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|L12_IGKJ1*01
820
1425


806
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|L12_IGKJ1*01
820
1425


807
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|L12_IGKJ1*01
820
1425


808
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|L12_IGKJ1*01
820
1425


809
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|L12_IGKJ1*01
820
1425


810
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|L12_IGKJ1*01
820
1425


811
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|L12_IGKJ1*01
820
1425


812
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|L12_IGKJ1*01
820
1425


813
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|L12_IGKJ1*01
820
1425


814
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|L12_IGKJ1*01
820
1425


815
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|L12_IGKJ1*01
820
1425


816
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|L12_IGKJ1*01
820
1425


817
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|L12_IGKJ1*01
820
1425


818
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|L12_IGKJ1*01
820
1425


819
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|L12_IGKJ1*01
820
1425


820
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|L12_IGKJ1*01
820
1425


821
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|L12_IGKJ1*01
820
1425


822
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|L12_IGKJ1*01
820
1425


823
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|L12_IGKJ1*01
820
1425


824
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|L12_IGKJ1*01
820
1425


825
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|L12_IGKJ1*01
820
1425


826
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|L12_IGKJ1*01
820
1425


827
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|L12_IGKJ1*01
820
1425


828
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|L12_IGKJ1*01
820
1425


829
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|L12_IGKJ1*01
820
1425


830
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|L12_IGKJ1*01
820
1425


831
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|L12_IGKJ1*01
820
1425


832
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|L12_IGKJ1*01
820
1425


833
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|L12_IGKJ1*01
820
1425


834
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|L12_IGKJ1*01
820
1425


835
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|L12_IGKJ1*01
820
1425


836
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|L12_IGKJ1*01
820
1425


837
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|L12_IGKJ1*01
820
1425


838
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|L12_IGKJ1*01
820
1425


839
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|L12_IGKJ1*01
820
1425


840
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|L12_IGKJ1*01
820
1425


841
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|L12_IGKJ1*01
820
1425


842
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|L12_IGKJ1*01
820
1425


843
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|L12_IGKJ1*01
820
1425


844
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|L12_IGKJ1*01
820
1425


845
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|L12_IGKJ1*01
820
1425


846
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|L12_IGKJ1*01
820
1425


847
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|L12_IGKJ1*01
820
1425


848
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|L12_IGKJ1*01
820
1425


849
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|L12_IGKJ1*01
820
1425


850
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|L12_IGKJ1*01
820
1425


851
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|L12_IGKJ1*01
820
1425


852
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|L12_IGKJ1*01
820
1425


853
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|L12_IGKJ1*01
820
1425


854
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|L12_IGKJ1*01
820
1425


855
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|L12_IGKJ1*01
820
1425


856
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|L12_IGKJ1*01
820
1425


857
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|L12_IGKJ1*01
820
1425


858
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|L12_IGKJ1*01
820
1425


859
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|L12_IGKJ1*01
820
1425


860
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|L12_IGKJ1*01
820
1425


861
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|L12_IGKJ1*01
820
1425


862
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|L12_IGKJ1*01
820
1425


863
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|L12_IGKJ1*01
820
1425


864
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|L12_IGKJ1*01
820
1425


865
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|O1_IGKJ1*01
832
1437


866
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|O1_IGKJ1*01
832
1437


867
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|O1_IGKJ1*01
832
1437


868
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|O1_IGKJ1*01
832
1437


869
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|O1_IGKJ1*01
832
1437


870
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|O1_IGKJ1*01
832
1437


871
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|O1_IGKJ1*01
832
1437


872
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|O1_IGKJ1*01
832
1437


873
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|O1_IGKJ1*01
832
1437


874
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|O1_IGKJ1*01
832
1437


875
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|O1_IGKJ1*01
832
1437


876
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|O1_IGKJ1*01
832
1437


877
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|O1_IGKJ1*01
832
1437


878
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|O1_IGKJ1*01
832
1437


879
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|O1_IGKJ1*01
832
1437


880
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|O1_IGKJ1*01
832
1437


881
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|O1_IGKJ1*01
832
1437


882
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|O1_IGKJ1*01
832
1437


883
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|O1_IGKJ1*01
832
1437


884
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|O1_IGKJ1*01
832
1437


885
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|O1_IGKJ1*01
832
1437


886
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|O1_IGKJ1*01
832
1437


887
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|O1_IGKJ1*01
832
1437


888
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|O1_IGKJ1*01
832
1437


889
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|O1_IGKJ1*01
832
1437


890
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|O1_IGKJ1*01
832
1437


891
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|O1_IGKJ1*01
832
1437


892
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|O1_IGKJ1*01
832
1437


893
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|O1_IGKJ1*01
832
1437


894
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|O1_IGKJ1*01
832
1437


895
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|O1_IGKJ1*01
832
1437


896
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|O1_IGKJ1*01
832
1437


897
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|O1_IGKJ1*01
832
1437


898
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|O1_IGKJ1*01
832
1437


899
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|O1_IGKJ1*01
832
1437


900
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|O1_IGKJ1*01
832
1437


901
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|O1_IGKJ1*01
832
1437


902
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|O1_IGKJ1*01
832
1437


903
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|O1_IGKJ1*01
832
1437


904
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|O1_IGKJ1*01
832
1437


905
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|O1_IGKJ1*01
832
1437


906
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|O1_IGKJ1*01
832
1437


907
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|O1_IGKJ1*01
832
1437


908
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|O1_IGKJ1*01
832
1437


909
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|O1_IGKJ1*01
832
1437


910
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|O1_IGKJ1*01
832
1437


911
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|O1_IGKJ1*01
832
1437


912
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|O1_IGKJ1*01
832
1437


913
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|O1_IGKJ1*01
832
1437


914
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|O1_IGKJ1*01
832
1437


915
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|O1_IGKJ1*01
832
1437


916
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|O1_IGKJ1*01
832
1437


917
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|O1_IGKJ1*01
832
1437


918
gnl|Fabrus|VH3-86_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|O1_IGKJ1*01
832
1437


919
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|O1_IGKJ1*01
832
1437


920
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|O1_IGKJ1*01
832
1437


921
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|O1_IGKJ1*01
832
1437


922
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|O1_IGKJ1*01
832
1437


923
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|O1_IGKJ1*01
832
1437


924
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|O1_IGKJ1*01
832
1437


925
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|O1_IGKJ1*01
832
1437


926
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|O1_IGKJ1*01
832
1437


927
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|O1_IGKJ1*01
832
1437


928
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|O1_IGKJ1*01
832
1437


929
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|O1_IGKJ1*01
832
1437


930
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|O1_IGKJ1*01
832
1437


931
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|O1_IGKJ1*01
832
1437


932
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|O1_IGKJ1*01
832
1437


933
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|O1_IGKJ1*01
832
1437


934
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|O1_IGKJ1*01
832
1437


935
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|O1_IGKJ1*01
832
1437


936
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|O1_IGKJ1*01
832
1437


937
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|O1_IGKJ1*01
832
1437


938
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|O1_IGKJ1*01
832
1437


939
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|O1_IGKJ1*01
832
1437


940
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|O1_IGKJ1*01
832
1437


941
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|O1_IGKJ1*01
832
1437


942
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|O1_IGKJ1*01
832
1437


943
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|O1_IGKJ1*01
832
1437


944
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|O1_IGKJ1*01
832
1437


945
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|O1_IGKJ1*01
832
1437


946
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|O1_IGKJ1*01
832
1437


947
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|O1_IGKJ1*01
832
1437


948
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|O1_IGKJ1*01
832
1437


949
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|O1_IGKJ1*01
832
1437


950
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|O1_IGKJ1*01
832
1437


951
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|O1_IGKJ1*01
832
1437


952
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|O1_IGKJ1*01
832
1437


953
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|O1_IGKJ1*01
832
1437


954
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|O1_IGKJ1*01
832
1437


955
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|O1_IGKJ1*01
832
1437


956
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|O1_IGKJ1*01
832
1437


957
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|O1_IGKJ1*01
832
1437


958
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|O1_IGKJ1*01
832
1437


959
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|O1_IGKJ1*01
832
1437


960
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|O1_IGKJ1*01
832
1437


961
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|L25_IGKJ3*01
826
1431


962
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|L25_IGKJ3*01
826
1431


963
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|L25_IGKJ3*01
826
1431


964
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|L25_IGKJ3*01
826
1431


965
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|L25_IGKJ3*01
826
1431


966
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|L25_IGKJ3*01
826
1431


967
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|L25_IGKJ3*01
826
1431


968
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|L25_IGKJ3*01
826
1431


969
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|L25_IGKJ3*01
826
1431


970
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|L25_IGKJ3*01
826
1431


971
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|L25_IGKJ3*01
826
1431


972
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|L25_IGKJ3*01
826
1431


973
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|L25_IGKJ3*01
826
1431


974
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|L25_IGKJ3*01
826
1431


975
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|L25_IGKJ3*01
826
1431


976
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|L25_IGKJ3*01
826
1431


977
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|L25_IGKJ3*01
826
1431


978
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|L25_IGKJ3*01
826
1431


979
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|L25_IGKJ3*01
826
1431


980
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|L25_IGKJ3*01
826
1431


981
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|L25_IGKJ3*01
826
1431


982
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|L25_IGKJ3*01
826
1431


983
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|L25_IGKJ3*01
826
1431


984
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|L25_IGKJ3*01
826
1431


985
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|L25_IGKJ3*01
826
1431


986
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|L25_IGKJ3*01
826
1431


987
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|L25_IGKJ3*01
826
1431


988
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|L25_IGKJ3*01
826
1431


989
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|L25_IGKJ3*01
826
1431


990
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|L25_IGKJ3*01
826
1431


991
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|L25_IGKJ3*01
826
1431


992
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|L25_IGKJ3*01
826
1431


993
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|L25_IGKJ3*01
826
1431


994
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|L25_IGKJ3*01
826
1431


995
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|L25_IGKJ3*01
826
1431


996
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|L25_IGKJ3*01
826
1431


997
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|L25_IGKJ3*01
826
1431


998
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|L25_IGKJ3*01
826
1431


999
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|L25_IGKJ3*01
826
1431


1000
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|L25_IGKJ3*01
826
1431


1001
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|L25_IGKJ3*01
826
1431


1002
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|L25_IGKJ3*01
826
1431


1003
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|L25_IGKJ3*01
826
1431


1004
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|L25_IGKJ3*01
826
1431


1005
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|L25_IGKJ3*01
826
1431


1006
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|L25_IGKJ3*01
826
1431


1007
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|L25_IGKJ3*01
826
1431


1008
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|L25_IGKJ3*01
826
1431


1009
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|L25_IGKJ3*01
826
1431


1010
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|L25_IGKJ3*01
826
1431


1011
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|L25_IGKJ3*01
826
1431


1012
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|L25_IGKJ3*01
826
1431


1013
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|L25_IGKJ3*01
826
1431


1014
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|L25_IGKJ3*01
826
1431


1015
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|L25_IGKJ3*01
826
1431


1016
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|L25_IGKJ3*01
826
1431


1017
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|L25_IGKJ3*01
826
1431


1018
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|L25_IGKJ3*01
826
1431


1019
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|L25_IGKJ3*01
826
1431


1020
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|L25_IGKJ3*01
826
1431


1021
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|L25_IGKJ3*01
826
1431


1022
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|L25_IGKJ3*01
826
1431


1023
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|L25_IGKJ3*01
826
1431


1024
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|L25_IGKJ3*01
826
1431


1025
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|L25_IGKJ3*01
826
1431


1026
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|L25_IGKJ3*01
826
1431


1027
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|L25_IGKJ3*01
826
1431


1028
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|L25_IGKJ3*01
826
1431


1029
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|L25_IGKJ3*01
826
1431


1030
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|L25_IGKJ3*01
826
1431


1031
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|L25_IGKJ3*01
826
1431


1032
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|L25_IGKJ3*01
826
1431


1033
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|L25_IGKJ3*01
826
1431


1034
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|L25_IGKJ3*01
826
1431


1035
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|L25_IGKJ3*01
826
1431


1036
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|L25_IGKJ3*01
826
1431


1037
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|L25_IGKJ3*01
826
1431


1038
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|L25_IGKJ3*01
826
1431


1039
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|L25_IGKJ3*01
826
1431


1040
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|L25_IGKJ3*01
826
1431


1041
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|L25_IGKJ3*01
826
1431


1042
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|L25_IGKJ3*01
826
1431


1043
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|L25_IGKJ3*01
826
1431


1044
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|L25_IGKJ3*01
826
1431


1045
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|L25_IGKJ3*01
826
1431


1046
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|L25_IGKJ3*01
826
1431


1047
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|L25_IGKJ3*01
826
1431


1048
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|L25_IGKJ3*01
826
1431


1049
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|L25_IGKJ3*01
826
1431


1050
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|L25_IGKJ3*01
826
1431


1051
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|L25_IGKJ3*01
826
1431


1052
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|L25_IGKJ3*01
826
1431


1053
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|L25_IGKJ3*01
826
1431


1054
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|L25_IGKJ3*01
826
1431


1055
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|L25_IGKJ3*01
826
1431


1056
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|L25_IGKJ3*01
826
1431


1057
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|A27_IGKJ1*01
812
1417


1058
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|A27_IGKJ1*01
812
1417


1059
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|A27_IGKJ1*01
812
1417


1060
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|A27_IGKJ1*01
812
1417


1061
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|A27_IGKJ1*01
812
1417


1062
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|A27_IGKJ1*01
812
1417


1063
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|A27_IGKJ1*01
812
1417


1064
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|A27_IGKJ1*01
812
1417


1065
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|A27_IGKJ1*01
812
1417


1066
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|A27_IGKJ1*01
812
1417


1067
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|A27_IGKJ1*01
812
1417


1068
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|A27_IGKJ1*01
812
1417


1069
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|A27_IGKJ1*01
812
1417


1070
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|A27_IGKJ1*01
812
1417


1071
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|A27_IGKJ1*01
812
1417


1072
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|A27_IGKJ1*01
812
1417


1073
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|A27_IGKJ1*01
812
1417


1074
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|A27_IGKJ1*01
812
1417


1075
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|A27_IGKJ1*01
812
1417


1076
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|A27_IGKJ1*01
812
1417


1077
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|A27_IGKJ1*01
812
1417


1078
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|A27_IGKJ1*01
812
1417


1079
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|A27_IGKJ1*01
812
1417


1080
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|A27_IGKJ1*01
812
1417


1081
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|A27_IGKJ1*01
812
1417


1082
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|A27_IGKJ1*01
812
1417


1083
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|A27_IGKJ1*01
812
1417


1084
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|A27_IGKJ1*01
812
1417


1085
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|A27_IGKJ1*01
812
1417


1086
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|A27_IGKJ1*01
812
1417


1087
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|A27_IGKJ1*01
812
1417


1088
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|A27_IGKJ1*01
812
1417


1089
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|A27_IGKJ1*01
812
1417


1090
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|A27_IGKJ1*01
812
1417


1091
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|A27_IGKJ1*01
812
1417


1092
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|A27_IGKJ1*01
812
1417


1093
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|A27_IGKJ1*01
812
1417


1094
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|A27_IGKJ1*01
812
1417


1095
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|A27_IGKJ1*01
812
1417


1096
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|A27_IGKJ1*01
812
1417


1097
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|A27_IGKJ1*01
812
1417


1098
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|A27_IGKJ1*01
812
1417


1099
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|A27_IGKJ1*01
812
1417


1100
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|A27_IGKJ1*01
812
1417


1101
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|A27_IGKJ1*01
812
1417


1102
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|A27_IGKJ1*01
812
1417


1103
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|A27_IGKJ1*01
812
1417


1104
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|A27_IGKJ1*01
812
1417


1105
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|A27_IGKJ1*01
812
1417


1106
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|A27_IGKJ1*01
812
1417


1107
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|A27_IGKJ1*01
812
1417


1108
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|A27_IGKJ1*01
812
1417


1109
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|A27_IGKJ1*01
812
1417


1110
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|A27_IGKJ1*01
812
1417


1111
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|A27_IGKJ1*01
812
1417


1112
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|A27_IGKJ1*01
812
1417


1113
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|A27_IGKJ1*01
812
1417


1114
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|A27_IGKJ1*01
812
1417


1115
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|A27_IGKJ1*01
812
1417


1116
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|A27_IGKJ1*01
812
1417


1117
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|A27_IGKJ1*01
812
1417


1118
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|A27_IGKJ1*01
812
1417


1119
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|A27_IGKJ1*01
812
1417


1120
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|A27_IGKJ1*01
812
1417


1121
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|A27_IGKJ1*01
812
1417


1122
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|A27_IGKJ1*01
812
1417


1123
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|A27_IGKJ1*01
812
1417


1124
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|A27_IGKJ1*01
812
1417


1125
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|A27_IGKJ1*01
812
1417


1126
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|A27_IGKJ1*01
812
1417


1127
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|A27_IGKJ1*01
812
1417


1128
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|A27_IGKJ1*01
812
1417


1129
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|A27_IGKJ1*01
812
1417


1130
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|A27_IGKJ1*01
812
1417


1131
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|A27_IGKJ1*01
812
1417


1132
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|A27_IGKJ1*01
812
1417


1133
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|A27_IGKJ1*01
812
1417


1134
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|A27_IGKJ1*01
812
1417


1135
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|A27_IGKJ1*01
812
1417


1136
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|A27_IGKJ1*01
812
1417


1137
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|A27_IGKJ1*01
812
1417


1138
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|A27_IGKJ1*01
812
1417


1139
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|A27_IGKJ1*01
812
1417


1140
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|A27_IGKJ1*01
812
1417


1141
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|A27_IGKJ1*01
812
1417


1142
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|A27_IGKJ1*01
812
1417


1143
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|A27_IGKJ1*01
812
1417


1144
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|A27_IGKJ1*01
812
1417


1145
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|A27_IGKJ1*01
812
1417


1146
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|A27_IGKJ1*01
812
1417


1147
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|A27_IGKJ1*01
812
1417


1148
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|A27_IGKJ1*01
812
1417


1149
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|A27_IGKJ1*01
812
1417


1150
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|A27_IGKJ1*01
812
1417


1151
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|A27_IGKJ1*01
812
1417


1152
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|A27_IGKJ1*01
812
1417


1153
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|A2_IGKJ1*01
808
1413


1154
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|A2_IGKJ1*01
808
1413


1155
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|A2_IGKJ1*01
808
1413


1156
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|A2_IGKJ1*01
808
1413


1157
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|A2_IGKJ1*01
808
1413


1158
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|A2_IGKJ1*01
808
1413


1159
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|A2_IGKJ1*01
808
1413


1160
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|A2_IGKJ1*01
808
1413


1161
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|A2_IGKJ1*01
808
1413


1162
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|A2_IGKJ1*01
808
1413


1163
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|A2_IGKJ1*01
808
1413


1164
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|A2_IGKJ1*01
808
1413


1165
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|A2_IGKJ1*01
808
1413


1166
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|A2_IGKJ1*01
808
1413


1167
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|A2_IGKJ1*01
808
1413


1168
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|A2_IGKJ1*01
808
1413


1169
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|A2_IGKJ1*01
808
1413


1170
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|A2_IGKJ1*01
808
1413


1171
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|A2_IGKJ1*01
808
1413


1172
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|A2_IGKJ1*01
808
1413


1173
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|A2_IGKJ1*01
808
1413


1174
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|A2_IGKJ1*01
808
1413


1175
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|A2_IGKJ1*01
808
1413


1176
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|A2_IGKJ1*01
808
1413


1177
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|A2_IGKJ1*01
808
1413


1178
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|A2_IGKJ1*01
808
1413


1179
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|A2_IGKJ1*01
808
1413


1180
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|A2_IGKJ1*01
808
1413


1181
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|A2_IGKJ1*01
808
1413


1182
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|A2_IGKJ1*01
808
1413


1183
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|A2_IGKJ1*01
808
1413


1184
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|A2_IGKJ1*01
808
1413


1185
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|A2_IGKJ1*01
808
1413


1186
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|A2_IGKJ1*01
808
1413


1187
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|A2_IGKJ1*01
808
1413


1188
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|A2_IGKJ1*01
808
1413


1189
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|A2_IGKJ1*01
808
1413


1190
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|A2_IGKJ1*01
808
1413


1191
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|A2_IGKJ1*01
808
1413


1192
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|A2_IGKJ1*01
808
1413


1193
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|A2_IGKJ1*01
808
1413


1194
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|A2_IGKJ1*01
808
1413


1195
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|A2_IGKJ1*01
808
1413


1196
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|A2_IGKJ1*01
808
1413


1197
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|A2_IGKJ1*01
808
1413


1198
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|A2_IGKJ1*01
808
1413


1199
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|A2_IGKJ1*01
808
1413


1200
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|A2_IGKJ1*01
808
1413


1201
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|A2_IGKJ1*01
808
1413


1202
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|A2_IGKJ1*01
808
1413


1203
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|A2_IGKJ1*01
808
1413


1204
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|A2_IGKJ1*01
808
1413


1205
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|A2_IGKJ1*01
808
1413


1206
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|A2_IGKJ1*01
808
1413


1207
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|A2_IGKJ1*01
808
1413


1208
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|A2_IGKJ1*01
808
1413


1209
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|A2_IGKJ1*01
808
1413


1210
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|A2_IGKJ1*01
808
1413


1211
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|A2_IGKJ1*01
808
1413


1212
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|A2_IGKJ1*01
808
1413


1213
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|A2_IGKJ1*01
808
1413


1214
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|A2_IGKJ1*01
808
1413


1215
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|A2_IGKJ1*01
808
1413


1216
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|A2_IGKJ1*01
808
1413


1217
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|A2_IGKJ1*01
808
1413


1218
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|A2_IGKJ1*01
808
1413


1219
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|A2_IGKJ1*01
808
1413


1220
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|A2_IGKJ1*01
808
1413


1221
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|A2_IGKJ1*01
808
1413


1222
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|A2_IGKJ1*01
808
1413


1223
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|A2_IGKJ1*01
808
1413


1224
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|A2_IGKJ1*01
808
1413


1225
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|A2_IGKJ1*01
808
1413


1226
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|A2_IGKJ1*01
808
1413


1227
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|A2_IGKJ1*01
808
1413


1228
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|A2_IGKJ1*01
808
1413


1229
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|A2_IGKJ1*01
808
1413


1230
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|A2_IGKJ1*01
808
1413


1231
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|A2_IGKJ1*01
808
1413


1232
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|A2_IGKJ1*01
808
1413


1233
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|A2_IGKJ1*01
808
1413


1234
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|A2_IGKJ1*01
808
1413


1235
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|A2_IGKJ1*01
808
1413


1236
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|A2_IGKJ1*01
808
1413


1237
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|A2_IGKJ1*01
808
1413


1238
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|A2_IGKJ1*01
808
1413


1239
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|A2_IGKJ1*01
808
1413


1240
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|A2_IGKJ1*01
808
1413


1241
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|A2_IGKJ1*01
808
1413


1242
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|A2_IGKJ1*01
808
1413


1243
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|A2_IGKJ1*01
808
1413


1244
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|A2_IGKJ1*01
808
1413


1245
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|A2_IGKJ1*01
808
1413


1246
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|A2_IGKJ1*01
808
1413


1247
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|A2_IGKJ1*01
808
1413


1248
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|A2_IGKJ1*01
808
1413


1249
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595
1200
gnl|Fabrus|HerceptinLC
818
1423


1250
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598
1203
gnl|Fabrus|HerceptinLC
818
1423


1251
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602
1207
gnl|Fabrus|HerceptinLC
818
1423


1252
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604
1209
gnl|Fabrus|HerceptinLC
818
1423


1253
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606
1211
gnl|Fabrus|HerceptinLC
818
1423


1254
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608
1213
gnl|Fabrus|HerceptinLC
818
1423


1255
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609
1214
gnl|Fabrus|HerceptinLC
818
1423


1256
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612
1217
gnl|Fabrus|HerceptinLC
818
1423


1257
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613
1218
gnl|Fabrus|HerceptinLC
818
1423


1258
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502
1107
gnl|Fabrus|HerceptinLC
818
1423


1259
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503
1108
gnl|Fabrus|HerceptinLC
818
1423


1260
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504
1109
gnl|Fabrus|HerceptinLC
818
1423


1261
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505
1110
gnl|Fabrus|HerceptinLC
818
1423


1262
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506
1111
gnl|Fabrus|HerceptinLC
818
1423


1263
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508
1113
gnl|Fabrus|HerceptinLC
818
1423


1264
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509
1114
gnl|Fabrus|HerceptinLC
818
1423


1265
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511
1116
gnl|Fabrus|HerceptinLC
818
1423


1266
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513
1118
gnl|Fabrus|HerceptinLC
818
1423


1267
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749
1354
gnl|Fabrus|HerceptinLC
818
1423


1268
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750
1355
gnl|Fabrus|HerceptinLC
818
1423


1269
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751
1356
gnl|Fabrus|HerceptinLC
818
1423


1270
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753
1358
gnl|Fabrus|HerceptinLC
818
1423


1271
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754
1359
gnl|Fabrus|HerceptinLC
818
1423


1272
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755
1360
gnl|Fabrus|HerceptinLC
818
1423


1273
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756
1361
gnl|Fabrus|HerceptinLC
818
1423


1274
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758
1363
gnl|Fabrus|HerceptinLC
818
1423


1275
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521
1126
gnl|Fabrus|HerceptinLC
818
1423


1276
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523
1128
gnl|Fabrus|HerceptinLC
818
1423


1277
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524
1129
gnl|Fabrus|HerceptinLC
818
1423


1278
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526
1131
gnl|Fabrus|HerceptinLC
818
1423


1279
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528
1133
gnl|Fabrus|HerceptinLC
818
1423


1280
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529
1134
gnl|Fabrus|HerceptinLC
818
1423


1281
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530
1135
gnl|Fabrus|HerceptinLC
818
1423


1282
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776
1381
gnl|Fabrus|HerceptinLC
818
1423


1283
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778
1383
gnl|Fabrus|HerceptinLC
818
1423


1284
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780
1385
gnl|Fabrus|HerceptinLC
818
1423


1285
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781
1386
gnl|Fabrus|HerceptinLC
818
1423


1286
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782
1387
gnl|Fabrus|HerceptinLC
818
1423


1287
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783
1388
gnl|Fabrus|HerceptinLC
818
1423


1288
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784
1389
gnl|Fabrus|HerceptinLC
818
1423


1289
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785
1390
gnl|Fabrus|HerceptinLC
818
1423


1290
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786
1391
gnl|Fabrus|HerceptinLC
818
1423


1291
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788
1393
gnl|Fabrus|HerceptinLC
818
1423


1292
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791
1396
gnl|Fabrus|HerceptinLC
818
1423


1293
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793
1398
gnl|Fabrus|HerceptinLC
818
1423


1294
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792
1397
gnl|Fabrus|HerceptinLC
818
1423


1295
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794
1399
gnl|Fabrus|HerceptinLC
818
1423


1296
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795
1400
gnl|Fabrus|HerceptinLC
818
1423


1297
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796
1401
gnl|Fabrus|HerceptinLC
818
1423


1298
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797
1402
gnl|Fabrus|HerceptinLC
818
1423


1299
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775
1380
gnl|Fabrus|HerceptinLC
818
1423


1300
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655
1260
gnl|Fabrus|HerceptinLC
818
1423


1301
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625
1230
gnl|Fabrus|HerceptinLC
818
1423


1302
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681
1286
gnl|Fabrus|HerceptinLC
818
1423


1303
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670
1275
gnl|Fabrus|HerceptinLC
818
1423


1304
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536
1141
gnl|Fabrus|HerceptinLC
818
1423


1305
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543
1148
gnl|Fabrus|HerceptinLC
818
1423


1306
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567
1172
gnl|Fabrus|HerceptinLC
818
1423


1307
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565
1170
gnl|Fabrus|HerceptinLC
818
1423


1308
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662
1267
gnl|Fabrus|HerceptinLC
818
1423


1309
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663
1268
gnl|Fabrus|HerceptinLC
818
1423


1310
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699
1304
gnl|Fabrus|HerceptinLC
818
1423


1311
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701
1306
gnl|Fabrus|HerceptinLC
818
1423


1312
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709
1314
gnl|Fabrus|HerceptinLC
818
1423


1313
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708
1313
gnl|Fabrus|HerceptinLC
818
1423


1314
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650
1255
gnl|Fabrus|HerceptinLC
818
1423


1315
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653
1258
gnl|Fabrus|HerceptinLC
818
1423


1316
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724
1329
gnl|Fabrus|HerceptinLC
818
1423


1317
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721
1326
gnl|Fabrus|HerceptinLC
818
1423


1318
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727
1332
gnl|Fabrus|HerceptinLC
818
1423


1319
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762
1367
gnl|Fabrus|HerceptinLC
818
1423


1320
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766
1371
gnl|Fabrus|HerceptinLC
818
1423


1321
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460
1065
gnl|Fabrus|HerceptinLC
818
1423


1322
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467
1072
gnl|Fabrus|HerceptinLC
818
1423


1323
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461
1066
gnl|Fabrus|HerceptinLC
818
1423


1324
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475
1080
gnl|Fabrus|HerceptinLC
818
1423


1325
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480
1085
gnl|Fabrus|HerceptinLC
818
1423


1326
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486
1091
gnl|Fabrus|HerceptinLC
818
1423


1327
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800
1405
gnl|Fabrus|HerceptinLC
818
1423


1328
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542
1147
gnl|Fabrus|HerceptinLC
818
1423


1329
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496
1101
gnl|Fabrus|HerceptinLC
818
1423


1330
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799
1404
gnl|Fabrus|HerceptinLC
818
1423


1331
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734
1339
gnl|Fabrus|HerceptinLC
818
1423


1332
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740
1345
gnl|Fabrus|HerceptinLC
818
1423


1333
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535
1140
gnl|Fabrus|HerceptinLC
818
1423


1334
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515
1120
gnl|Fabrus|HerceptinLC
818
1423


1335
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540
1145
gnl|Fabrus|HerceptinLC
818
1423


1336
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639
1244
gnl|Fabrus|HerceptinLC
818
1423


1337
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570
1175
gnl|Fabrus|HerceptinLC
818
1423


1338
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706
1311
gnl|Fabrus|HerceptinLC
818
1423


1339
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548
1153
gnl|Fabrus|HerceptinLC
818
1423


1340
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552
1157
gnl|Fabrus|HerceptinLC
818
1423


1341
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584
1189
gnl|Fabrus|HerceptinLC
818
1423


1342
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571
1176
gnl|Fabrus|HerceptinLC
818
1423


1343
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692
1297
gnl|Fabrus|HerceptinLC
818
1423


1344
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576
1181
gnl|Fabrus|HerceptinLC
818
1423


1345
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|O12_IGKJ1*01
833
1438


1346
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|O18_IGKJ1*01
834
1439


1347
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A20_IGKJ1*01
809
1414


1348
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A30_IGKJ1*01
814
1419


1349
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L14_IGKJ1*01
821
1426


1350
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


1351
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L5_IGKJ1*01
828
1433


1352
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L8_IGKJ1*01
830
1435


1353
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L23_IGKJ1*01
824
1429


1354
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L11_IGKJ1*01
819
1424


1355
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L12_IGKJ1*01
820
1425


1356
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|O1_IGKJ1*01
832
1437


1357
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A17_IGKJ1*01
807
1412


1358
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A2_IGKJ1*01
808
1413


1359
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A23_IGKJ1*01
810
1415


1360
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A27_IGKJ3*01
813
1418


1361
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L2_IGKJ1*01
822
1427


1362
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L6_IGKJ1*01
829
1434


1363
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L25_IGKJ1*01
825
1430


1364
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|B3_IGKJ1*01
817
1422


1365
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|B2_IGKJ1*01
815
1420


1366
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A26_IGKJ1*01
811
1416


1367
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A14_IGKJ1*01
806
1411


1368
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L9_IGKJ2*01
831
1436


1369
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|A27_IGKJ1*01
812
1417


1370
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|B2_IGKJ3*01
816
1421


1371
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L25_IGKJ3*01
826
1431


1372
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|RituxanLC
835
1440


1373
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|L22_IGKJ3*01
823
1428


1374
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600
1205
gnl|Fabrus|HerceptinLC
818
1423


1375
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|O12_IGKJ1*01
833
1438


1376
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|O18_IGKJ1*01
834
1439


1377
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A20_IGKJ1*01
809
1414


1378
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A30_IGKJ1*01
814
1419


1379
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L14_IGKJ1*01
821
1426


1380
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


1381
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L5_IGKJ1*01
828
1433


1382
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L8_IGKJ1*01
830
1435


1383
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L23_IGKJ1*01
824
1429


1384
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L11_IGKJ1*01
819
1424


1385
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L12_IGKJ1*01
820
1425


1386
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|O1_IGKJ1*01
832
1437


1387
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A17_IGKJ1*01
807
1412


1388
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A2_IGKJ1*01
808
1413


1389
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A23_IGKJ1*01
810
1415


1390
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A27_IGKJ3*01
813
1418


1391
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L2_IGKJ1*01
822
1427


1392
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L6_IGKJ1*01
829
1434


1393
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L25_IGKJ1*01
825
1430


1394
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|B3_IGKJ1*01
817
1422


1395
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|B2_IGKJ1*01
815
1420


1396
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A26_IGKJ1*01
811
1416


1397
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A14_IGKJ1*01
806
1411


1398
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L9_IGKJ2*01
831
1436


1399
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|A27_IGKJ1*01
812
1417


1400
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|B2_IGKJ3*01
816
1421


1401
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L25_IGKJ3*01
826
1431


1402
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|RituxanLC
835
1440


1403
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|L22_IGKJ3*01
823
1428


1404
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747
1352
gnl|Fabrus|HerceptinLC
818
1423


1405
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|O12_IGKJ1*01
833
1438


1406
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|O18_IGKJ1*01
834
1439


1407
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A20_IGKJ1*01
809
1414


1408
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A30_IGKJ1*01
814
1419


1409
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L14_IGKJ1*01
821
1426


1410
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


1411
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L5_IGKJ1*01
828
1433


1412
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L8_IGKJ1*01
830
1435


1413
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L23_IGKJ1*01
824
1429


1414
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L11_IGKJ1*01
819
1424


1415
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L12_IGKJ1*01
820
1425


1416
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|O1_IGKJ1*01
832
1437


1417
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A17_IGKJ1*01
807
1412


1418
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A2_IGKJ1*01
808
1413


1419
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A23_IGKJ1*01
810
1415


1420
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A27_IGKJ3*01
813
1418


1421
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L2_IGKJ1*01
822
1427


1422
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L6_IGKJ1*01
829
1434


1423
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L25_IGKJ1*01
825
1430


1424
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|B3_IGKJ1*01
817
1422


1425
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|B2_IGKJ1*01
815
1420


1426
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A26_IGKJ1*01
811
1416


1427
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A14_IGKJ1*01
806
1411


1428
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L9_IGKJ2*01
831
1436


1429
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|A27_IGKJ1*01
812
1417


1430
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|B2_IGKJ3*01
816
1421


1431
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L25_IGKJ3*01
826
1431


1432
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|RituxanLC
835
1440


1433
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|L22_IGKJ3*01
823
1428


1434
gnl|Fabrus|RituxanHC
453
1058
gnl|Fabrus|HerceptinLC
818
1423


1435
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|O12_IGKJ1*01
833
1438


1436
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|O18_IGKJ1*01
834
1439


1437
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A20_IGKJ1*01
809
1414


1438
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A30_IGKJ1*01
814
1419


1439
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L14_IGKJ1*01
821
1426


1440
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L4/18a_IGKJ1*01
827
1432


1441
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L5_IGKJ1*01
828
1433


1442
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L8_IGKJ1*01
830
1435


1443
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L23_IGKJ1*01
824
1429


1444
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L11_IGKJ1*01
819
1424


1445
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L12_IGKJ1*01
820
1425


1446
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|O1_IGKJ1*01
832
1437


1447
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A17_IGKJ1*01
807
1412


1448
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A2_IGKJ1*01
808
1413


1449
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A23_IGKJ1*01
810
1415


1450
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A27_IGKJ3*01
813
1418


1451
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L2_IGKJ1*01
822
1427


1452
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L6_IGKJ1*01
829
1434


1453
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L25_IGKJ1*01
825
1430


1454
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|B3_IGKJ1*01
817
1422


1455
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|B2_IGKJ1*01
815
1420


1456
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A26_IGKJ1*01
811
1416


1457
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A14_IGKJ1*01
806
1411


1458
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L9_IGKJ2*01
831
1436


1459
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|A27_IGKJ1*01
812
1417


1460
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|B2_IGKJ3*01
816
1421


1461
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L25_IGKJ3*01
826
1431


1462
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|RituxanLC
835
1440


1463
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|L22_IGKJ3*01
823
1428


1464
gnl|Fabrus|HerceptinHC
452
1057
gnl|Fabrus|HerceptinLC
818
1423









An additional exemplary paired library is set forth in Table 17A, where each row sets forth a different loci of the library.







TABLE 17A







Exemplary Nucleic Acid Paired Library














RS

RS
NO




SEQ

SEQ
RS



HEAVY CHAIN
ID
LIGHT CHAIN
ID
SEQ



Name
NO
Name
NO
ID NO
















1
VH3-23_IGHD1-1*01>1_IGHJ1*01
2070
O12_IGKJ1*01
833
1438


2
VH3-23_IGHD1-1*01>2_IGHJ1*01
2071
O12_IGKJ1*01
833
1438


3
VH3-23_IGHD1-1*01>3_IGHJ1*01
2072
O12_IGKJ1*01
833
1438


4
VH3-23_IGHD1-7*01>1_IGHJ1*01
2073
O12_IGKJ1*01
833
1438


5
VH3-23_IGHD1-7*01>3_IGHJ1*01
2074
O12_IGKJ1*01
833
1438


6
VH3-23_IGHD1-14*01>1_IGHJ1*01
2075
O12_IGKJ1*01
833
1438


7
VH3-23_IGHD1-14*01>3_IGHJ1*01
2076
O12_IGKJ1*01
833
1438


8
VH3-23_IGHD1-20*01>1_IGHJ1*01
2077
O12_IGKJ1*01
833
1438


9
VH3-23_IGHD1-20*01>3_IGHJ1*01
2078
O12_IGKJ1*01
833
1438


10
VH3-23_IGHD1-26*01>1_IGHJ1*01
2079
O12_IGKJ1*01
833
1438


11
VH3-23_IGHD1-26*01>3_IGHJ1*01
2080
O12_IGKJ1*01
833
1438


12
VH3-23_IGHD2-2*01>2_IGHJ1*01
2081
O12_IGKJ1*01
833
1438


13
VH3-23_IGHD2-2*01>3_IGHJ1*01
2082
O12_IGKJ1*01
833
1438


14
VH3-23_IGHD2-8*01>2_IGHJ1*01
2083
O12_IGKJ1*01
833
1438


15
VH3-23_IGHD2-8*01>3_IGHJ1*01
2084
O12_IGKJ1*01
833
1438


16
VH3-23_IGHD2-15*01>2_IGHJ1*01
2085
O12_IGKJ1*01
833
1438


17
VH3-23_IGHD2-15*01>3_IGHJ1*01
2086
O12_IGKJ1*01
833
1438


18
VH3-23_IGHD2-21*01>2_IGHJ1*01
2087
O12_IGKJ1*01
833
1438


19
VH3-23_IGHD2-21*01>3_IGHJ1*01
2088
O12_IGKJ1*01
833
1438


20
VH3-23_IGHD3-3*01>1_IGHJ1*01
2089
O12_IGKJ1*01
833
1438


21
VH3-23_IGHD3-3*01>2_IGHJ1*01
2090
O12_IGKJ1*01
833
1438


22
VH3-23_IGHD3-3*01>3_IGHJ1*01
2091
O12_IGKJ1*01
833
1438


23
VH3-23_IGHD3-9*01>2_IGHJ1*01
2092
O12_IGKJ1*01
833
1438


24
VH3-23_IGHD3-10*01>2_IGHJ1*01
2093
O12_IGKJ1*01
833
1438


25
VH3-23_IGHD3-10*01>3_IGHJ1*01
2094
O12_IGKJ1*01
833
1438


26
VH3-23_IGHD3-16*01>2_IGHJ1*01
2095
O12_IGKJ1*01
833
1438


27
VH3-23_IGHD3-16*01>3_IGHJ1*01
2096
O12_IGKJ1*01
833
1438


28
VH3-23_IGHD3-22*01>2_IGHJ1*01
2097
O12_IGKJ1*01
833
1438


29
VH3-23_IGHD3-22*01>3_IGHJ1*01
2098
O12_IGKJ1*01
833
1438


30
VH3-23_IGHD4-4*01 (1) >2_IGHJ1*01
2099
O12_IGKJ1*01
833
1438


31
VH3-23_IGHD4-4*01 (1) >3_IGHJ1*01
2100
O12_IGKJ1*01
833
1438


32
VH3-23_IGHD4-11*01 (1) >2_IGHJ1*01
2101
O12_IGKJ1*01
833
1438


33
VH3-23_IGHD4-11*01 (1) >3_IGHJ1*01
2102
O12_IGKJ1*01
833
1438


34
VH3-23_IGHD4-17*01>2_IGHJ1*01
2103
O12_IGKJ1*01
833
1438


35
VH3-23_IGHD4-17*01>3_IGHJ1*01
2104
O12_IGKJ1*01
833
1438


36
VH3-23_IGHD4-23*01>2_IGHJ1*01
2105
O12_IGKJ1*01
833
1438


37
VH3-23_IGHD4-23*01>3_IGHJ1*01
2106
O12_IGKJ1*01
833
1438


38
VH3-23_IGHD5-5*01 (2) >1_IGHJ1*01
2107
O12_IGKJ1*01
833
1438


39
VH3-23_IGHD5-5*01 (2) >2_IGHJ1*01
2108
O12_IGKJ1*01
833
1438


40
VH3-23_IGHD5-5*01 (2) >3_IGHJ1*01
2109
O12_IGKJ1*01
833
1438


41
VH3-23_IGHD5-12*01>1_IGHJ1*01
2110
O12_IGKJ1*01
833
1438


42
VH3-23_IGHD5-12*01>3_IGHJ1*01
2111
O12_IGKJ1*01
833
1438


43
VH3-23_IGHD5-18*01 (2) >1_IGHJ1*01
2112
O12_IGKJ1*01
833
1438


44
VH3-23_IGHD5-18*01 (2) >2_IGHJ1*01
2113
O12_IGKJ1*01
833
1438


45
VH3-23_IGHD5-18*01 (2) >3_IGHJ1*01
2114
O12_IGKJ1*01
833
1438


46
VH3-23_IGHD5-24*01>1_IGHJ1*01
2115
O12_IGKJ1*01
833
1438


47
VH3-23_IGHD5-24*01>3_IGHJ1*01
2116
O12_IGKJ1*01
833
1438


48
VH3-23_IGHD6-6*01>1_IGHJ1*01
2117
O12_IGKJ1*01
833
1438


49
VH3-23_IGHD1-1*01>1′_IGHJ1*01
2127
O12_IGKJ1*01
833
1438


50
VH3-23_IGHD1-1*01>2′_IGHJ1*01
2128
O12_IGKJ1*01
833
1438


51
VH3-23_IGHD1-1*01>3′_IGHJ1*01
2129
O12_IGKJ1*01
833
1438


52
VH3-23_IGHD1-7*01>1′_IGHJ1*01
2130
O12_IGKJ1*01
833
1438


53
VH3-23_IGHD1-7*01>3′_IGHJ1*01
2131
O12_IGKJ1*01
833
1438


54
VH3-23_IGHD1-14*01>1′_IGHJ1*01
2132
O12_IGKJ1*01
833
1438


55
VH3-23_IGHD1-14*01>2′_IGHJ1*01
2133
O12_IGKJ1*01
833
1438


56
VH3-23_IGHD1-14*01>3′_IGHJ1*01
2134
O12_IGKJ1*01
833
1438


57
VH3-23_IGHD1-20*01>1′_IGHJ1*01
2135
O12_IGKJ1*01
833
1438


58
VH3-23_IGHD1-20*01>2′_IGHJ1*01
2136
O12_IGKJ1*01
833
1438


59
VH3-23_IGHD1-20*01>3′_IGHJ1*01
2137
O12_IGKJ1*01
833
1438


60
VH3-23_IGHD1-26*01>1′_IGHJ1*01
2138
O12_IGKJ1*01
833
1438


61
VH3-23_IGHD1-26*01>3′_IGHJ1*01
2139
O12_IGKJ1*01
833
1438


62
VH3-23_IGHD2-2*01>1′_IGHJ1*01
2140
O12_IGKJ1*01
833
1438


63
VH3-23_IGHD2-2*01>3′_IGHJ1*01
2141
O12_IGKJ1*01
833
1438


64
VH3-23_IGHD2-8*01>1′_IGHJ1*01
2142
O12_IGKJ1*01
833
1438


65
VH3-23_IGHD2-15*01>1′_IGHJ1*01
2143
O12_IGKJ1*01
833
1438


66
VH3-23_IGHD2-15*01>3′_IGHJ1*01
2144
O12_IGKJ1*01
833
1438


67
VH3-23_IGHD2-21*01>1′_IGHJ1*01
2145
O12_IGKJ1*01
833
1438


68
VH3-23_IGHD2-21*01>3′_IGHJ1*01
2146
O12_IGKJ1*01
833
1438


69
VH3-23_IGHD3-3*01>1′_IGHJ1*01
2147
O12_IGKJ1*01
833
1438


70
VH3-23_IGHD3-3*01>3′_IGHJ1*01
2148
O12_IGKJ1*01
833
1438


71
VH3-23_IGHD3-9*01>1′_IGHJ1*01
2149
O12_IGKJ1*01
833
1438


72
VH3-23_IGHD3-9*01>3′_IGHJ1*01
2150
O12_IGKJ1*01
833
1438


73
VH3-23_IGHD3-10*01>1′_IGHJ1*01
2151
O12_IGKJ1*01
833
1438


74
VH3-23_IGHD3-10*01>3′_IGHJ1*01
2152
O12_IGKJ1*01
833
1438


75
VH3-23_IGHD3-16*01>1′_IGHJ1*01
2153
O12_IGKJ1*01
833
1438


76
VH3-23_IGHD3-16*01>3′_IGHJ1*01
2154
O12_IGKJ1*01
833
1438


77
VH3-23_IGHD3-22*01>1′_IGHJ1*01
2155
O12_IGKJ1*01
833
1438


78
VH3-23_IGHD4-4*01 (1) >1′_IGHJ1*01
2156
O12_IGKJ1*01
833
1438


79
VH3-23_IGHD4-4*01 (1) >3′_IGHJ1*01
2157
O12_IGKJ1*01
833
1438


80
VH3-23_IGHD4-11*01 (1) >1′_IGHJ1*01
2158
O12_IGKJ1*01
833
1438


81
VH3-23_IGHD4-11*01 (1) >3′_IGHJ1*01
2159
O12_IGKJ1*01
833
1438


82
VH3-23_IGHD4-17*01>1′_IGHJ1*01
2160
O12_IGKJ1*01
833
1438


83
VH3-23_IGHD4-17*01>3′_IGHJ1*01
2161
O12_IGKJ1*01
833
1438


84
VH3-23_IGHD4-23*01>1′_IGHJ1*01
2162
O12_IGKJ1*01
833
1438


85
VH3-23_IGHD4-23*01>3′_IGHJ1*01
2163
O12_IGKJ1*01
833
1438


86
VH3-23_IGHD5-5*01 (2) >1′_IGHJ1*01
2164
O12_IGKJ1*01
833
1438


87
VH3-23_IGHD5-5*01 (2) >3′_IGHJ1*01
2165
O12_IGKJ1*01
833
1438


88
VH3-23_IGHD5-12*01>1′_IGHJ1*01
2166
O12_IGKJ1*01
833
1438


89
VH3-23_IGHD5-12*01>3′_IGHJ1*01
2167
O12_IGKJ1*01
833
1438


90
VH3-23_IGHD5-18*01 (2) >1′_IGHJ1*01
2168
O12_IGKJ1*01
833
1438


91
VH3-23_IGHD5-18*01 (2) >3′_IGHJ1*01
2169
O12_IGKJ1*01
833
1438


92
VH3-23_IGHD5-24*01>1′_IGHJ1*01
2170
O12_IGKJ1*01
833
1438


93
VH3-23_IGHD5-24*01>3′_IGHJ1*01
2171
O12_IGKJ1*01
833
1438


94
VH3-23_IGHD6-6*01>1′_IGHJ1*01
2172
O12_IGKJ1*01
833
1438


95
VH3-23_IGHD6-6*01>2′_IGHJ1*01
2173
O12_IGKJ1*01
833
1438


96
VH3-23_IGHD6-6*01>3′_IGHJ1*01
2174
O12_IGKJ1*01
833
1438


97
VH3-23_IGHD6-6*01>2_IGHJ1*01
2118
O12_IGKJ1*01
833
1438


98
VH3-23_IGHD6-13*01>1_IGHJ1*01
2119
O12_IGKJ1*01
833
1438


99
VH3-23_IGHD6-13*01>2_IGHJ1*01
2120
O12_IGKJ1*01
833
1438


100
VH3-23_IGHD6-19*01>1_IGHJ1*01
2121
O12_IGKJ1*01
833
1438


101
VH3-23_IGHD6-19*01>2_IGHJ1*01
2122
O12_IGKJ1*01
833
1438


102
VH3-23_IGHD6-25*01>1_IGHJ1*01
2123
O12_IGKJ1*01
833
1438


103
VH3-23_IGHD6-25*01>2_IGHJ1*01
2124
O12_IGKJ1*01
833
1438


104
VH3-23_IGHD7-27*01>1_IGHJ1*01
2125
O12_IGKJ1*01
833
1438


105
VH3-23_IGHD7-27*01>3_IGHJ1*01
2126
O12_IGKJ1*01
833
1438


106
VH3-23_IGHD6-13*01>1′_IGHJ1*01
2175
O12_IGKJ1*01
833
1438


107
VH3-23_IGHD6-13*01>2′_IGHJ1*01
2176
O12_IGKJ1*01
833
1438


108
VH3-23_IGHD6-13*01>2_IGHJ1*01_B
2177
O12_IGKJ1*01
833
1438


109
VH3-23_IGHD6-19*01>1′_IGHJ1*01
2178
O12_IGKJ1*01
833
1438


110
VH3-23_IGHD6-19*01>2′_IGHJ1*01
2179
O12_IGKJ1*01
833
1438


111
VH3-23_IGHD6-19*01>2_IGHJ1*01_B
2180
O12_IGKJ1*01
833
1438


112
VH3-23_IGHD6-25*01>1′_IGHJ1*01
2181
O12_IGKJ1*01
833
1438


113
VH3-23_IGHD6-25*01>3′_IGHJ1*01
2182
O12_IGKJ1*01
833
1438


114
VH3-23_IGHD7-27*01>1′_IGHJ1*01_B
2183
O12_IGKJ1*01
833
1438


115
VH3-23_IGHD7-27*01>2′_IGHJ1*01
2184
O12_IGKJ1*01
833
1438


116
VH3-23_IGHD1-1*01>1_IGHJ2*01
2185
O12_IGKJ1*01
833
1438


117
VH3-23_IGHD1-1*01>2_IGHJ2*01
2186
O12_IGKJ1*01
833
1438


118
VH3-23_IGHD1-1*01>3_IGHJ2*01
2187
O12_IGKJ1*01
833
1438


119
VH3-23_IGHD1-7*01>1_IGHJ2*01
2188
O12_IGKJ1*01
833
1438


120
VH3-23_IGHD1-7*01>3_IGHJ2*01
2189
O12_IGKJ1*01
833
1438


121
VH3-23_IGHD1-14*01>1_IGHJ2*01
2190
O12_IGKJ1*01
833
1438


122
VH3-23_IGHD1-14*01>3_IGHJ2*01
2191
O12_IGKJ1*01
833
1438


123
VH3-23_IGHD1-20*01>1_IGHJ2*01
2192
O12_IGKJ1*01
833
1438


124
VH3-23_IGHD1-20*01>3_IGHJ2*01
2193
O12_IGKJ1*01
833
1438


125
VH3-23_IGHD1-26*01>1_IGHJ2*01
2194
O12_IGKJ1*01
833
1438


126
VH3-23_IGHD1-26*01>3_IGHJ2*01
2195
O12_IGKJ1*01
833
1438


127
VH3-23_IGHD2-2*01>2_IGHJ2*01
2196
O12_IGKJ1*01
833
1438


128
VH3-23_IGHD2-2*01>3_IGHJ2*01
2197
O12_IGKJ1*01
833
1438


129
VH3-23_IGHD2-8*01>2_IGHJ2*01
2198
O12_IGKJ1*01
833
1438


130
VH3-23_IGHD2-8*01>3_IGHJ2*01
2199
O12_IGKJ1*01
833
1438


131
VH3-23_IGHD2-15*01>2_IGHJ2*01
2200
O12_IGKJ1*01
833
1438


132
VH3-23_IGHD2-15*01>3_IGHJ2*01
2201
O12_IGKJ1*01
833
1438


133
VH3-23_IGHD2-21*01>2_IGHJ2*01
2202
O12_IGKJ1*01
833
1438


134
VH3-23_IGHD2-21*01>3_IGHJ2*01
2203
O12_IGKJ1*01
833
1438


135
VH3-23_IGHD3-3*01>1_IGHJ2*01
2204
O12_IGKJ1*01
833
1438


136
VH3-23_IGHD3-3*01>2_IGHJ2*01
2205
O12_IGKJ1*01
833
1438


137
VH3-23_IGHD3-3*01>3_IGHJ2*01
2206
O12_IGKJ1*01
833
1438


138
VH3-23_IGHD3-9*01>2_IGHJ2*01
2207
O12_IGKJ1*01
833
1438


139
VH3-23_IGHD3-10*01>2_IGHJ2*01
2208
O12_IGKJ1*01
833
1438


140
VH3-23_IGHD3-10*01>3_IGHJ2*01
2209
O12_IGKJ1*01
833
1438


141
VH3-23_IGHD3-16*01>2_IGHJ2*01
2210
O12_IGKJ1*01
833
1438


142
VH3-23_IGHD3-16*01>3_IGHJ2*01
2211
O12_IGKJ1*01
833
1438


143
VH3-23_IGHD3-22*01>2_IGHJ2*01
2212
O12_IGKJ1*01
833
1438


144
VH3-23_IGHD3-22*01>3_IGHJ2*01
2213
O12_IGKJ1*01
833
1438


145
VH3-23_IGHD4-4*01 (1) >2_IGHJ2*01
2214
O12_IGKJ1*01
833
1438


146
VH3-23_IGHD4-4*01 (1) >3_IGHJ2*01
2215
O12_IGKJ1*01
833
1438


147
VH3-23_IGHD4-11*01 (1) >2_IGHJ2*01
2216
O12_IGKJ1*01
833
1438


148
VH3-23_IGHD4-11*01 (1) >3_IGHJ2*01
2217
O12_IGKJ1*01
833
1438


149
VH3-23_IGHD4-17*01>2_IGHJ2*01
2218
O12_IGKJ1*01
833
1438


150
VH3-23_IGHD4-17*01>3_IGHJ2*01
2219
O12_IGKJ1*01
833
1438


151
VH3-23_IGHD4-23*01>2_IGHJ2*01
2220
O12_IGKJ1*01
833
1438


152
VH3-23_IGHD4-23*01>3_IGHJ2*01
2221
O12_IGKJ1*01
833
1438


153
VH3-23_IGHD5-5*01 (2) >1_IGHJ2*01
2222
O12_IGKJ1*01
833
1438


154
VH3-23_IGHD5-5*01 (2) >2_IGHJ2*01
2223
O12_IGKJ1*01
833
1438


155
VH3-23_IGHD5-5*01 (2) >3_IGHJ2*01
2224
O12_IGKJ1*01
833
1438


156
VH3-23_IGHD5-12*01>1_IGHJ2*01
2225
O12_IGKJ1*01
833
1438


157
VH3-23_IGHD5-12*01>3_IGHJ2*01
2226
O12_IGKJ1*01
833
1438


158
VH3-23_IGHD5-18*01 (2) >1_IGHJ2*01
2227
O12_IGKJ1*01
833
1438


159
VH3-23_IGHD5-18*01 (2) >2_IGHJ2*01
2228
O12_IGKJ1*01
833
1438


160
VH3-23_IGHD5-18*01 (2) >3_IGHJ2*01
2229
O12_IGKJ1*01
833
1438


161
VH3-23_IGHD5-24*01>1_IGHJ2*01
2230
O12_IGKJ1*01
833
1438


162
VH3-23_IGHD5-24*01>3_IGHJ2*01
2231
O12_IGKJ1*01
833
1438


163
VH3-23_IGHD6-6*01>1_IGHJ2*01
2232
O12_IGKJ1*01
833
1438


164
VH3-23_IGHD1-1*01>1′_IGHJ2*01
2242
O12_IGKJ1*01
833
1438


165
VH3-23_IGHD1-1*01>2′_IGHJ2*01
2243
O12_IGKJ1*01
833
1438


166
VH3-23_IGHD1-1*01>3′_IGHJ2*01
2244
O12_IGKJ1*01
833
1438


167
VH3-23_IGHD1-7*01>1′_IGHJ2*01
2245
O12_IGKJ1*01
833
1438


168
VH3-23_IGHD1-7*01>3′_IGHJ2*01
2246
O12_IGKJ1*01
833
1438


169
VH3-23_IGHD1-14*01>1′_IGHJ2*01
2247
O12_IGKJ1*01
833
1438


170
VH3-23_IGHD1-14*01>2′_IGHJ2*01
2248
O12_IGKJ1*01
833
1438


171
VH3-23_IGHD1-14*01>3′_IGHJ2*01
2249
O12_IGKJ1*01
833
1438


172
VH3-23_IGHD1-20*01>1′_IGHJ2*01
2250
O12_IGKJ1*01
833
1438


173
VH3-23_IGHD1-20*01>2′_IGHJ2*01
2251
O12_IGKJ1*01
833
1438


174
VH3-23_IGHD1-20*01>3′_IGHJ2*01
2252
O12_IGKJ1*01
833
1438


175
VH3-23_IGHD1-26*01>1′_IGHJ2*01
2253
O12_IGKJ1*01
833
1438


176
VH3-23_IGHD1-26*01>1_IGHJ2*01_B
2254
O12_IGKJ1*01
833
1438


177
VH3-23_IGHD2-2*01>1′_IGHJ2*01
2255
O12_IGKJ1*01
833
1438


178
VH3-23_IGHD2-2*01>3′_IGHJ2*01
2256
O12_IGKJ1*01
833
1438


179
VH3-23_IGHD2-8*01>1′_IGHJ2*01
2257
O12_IGKJ1*01
833
1438


180
VH3-23_IGHD2-15*01>1′_IGHJ2*01
2258
O12_IGKJ1*01
833
1438


181
VH3-23_IGHD2-15*01>3′_IGHJ2*01
2259
O12_IGKJ1*01
833
1438


182
VH3-23_IGHD2-21*01>1′_IGHJ2*01
2260
O12_IGKJ1*01
833
1438


183
VH3-23_IGHD2-21*01>3′_IGHJ2*01
2261
O12_IGKJ1*01
833
1438


184
VH3-23_IGHD3-3*01>1′_IGHJ2*01
2262
O12_IGKJ1*01
833
1438


185
VH3-23_IGHD3-3*01>3′_IGHJ2*01
2263
O12_IGKJ1*01
833
1438


186
VH3-23_IGHD3-9*01>1′_IGHJ2*01
2264
O12_IGKJ1*01
833
1438


187
VH3-23_IGHD3-9*01>3′_IGHJ2*01
2265
O12_IGKJ1*01
833
1438


188
VH3-23_IGHD3-10*01>1′_IGHJ2*01
2266
O12_IGKJ1*01
833
1438


189
VH3-23_IGHD3-10*01>3′_IGHJ2*01
2267
O12_IGKJ1*01
833
1438


190
VH3-23_IGHD3-16*01>1′_IGHJ2*01
2268
O12_IGKJ1*01
833
1438


191
VH3-23_IGHD3-16*01>3′_IGHJ2*01
2269
O12_IGKJ1*01
833
1438


192
VH3-23_IGHD3-22*01>1′_IGHJ2*01
2270
O12_IGKJ1*01
833
1438


193
VH3-23_IGHD4-4*01 (1) >1′_IGHJ2*01
2271
O12_IGKJ1*01
833
1438


194
VH3-23_IGHD4-4*01 (1) >3′_IGHJ2*01
2272
O12_IGKJ1*01
833
1438


195
VH3-23_IGHD4-11*01 (1) >1′_IGHJ2*01
2273
O12_IGKJ1*01
833
1438


196
VH3-23_IGHD4-11*01 (1) >3′_IGHJ2*01
2274
O12_IGKJ1*01
833
1438


197
VH3-23_IGHD4-17*01>1′_IGHJ2*01
2275
O12_IGKJ1*01
833
1438


198
VH3-23_IGHD4-17*01>3′_IGHJ2*01
2276
O12_IGKJ1*01
833
1438


199
VH3-23_IGHD4-23*01>1′_IGHJ2*01
2277
O12_IGKJ1*01
833
1438


200
VH3-23_IGHD4-23*01>3′_IGHJ2*01
2278
O12_IGKJ1*01
833
1438


201
VH3-23_IGHD5-5*01 (2) >1′_IGHJ2*01
2279
O12_IGKJ1*01
833
1438


202
VH3-23_IGHD5-5*01 (2) >3′_IGHJ2*01
2280
O12_IGKJ1*01
833
1438


203
VH3-23_IGHD5-12*01>1′_IGHJ2*01
2281
O12_IGKJ1*01
833
1438


204
VH3-23_IGHD5-12*01>3′_IGHJ2*01
2282
O12_IGKJ1*01
833
1438


205
VH3-23_IGHD5-18*01 (2) >1′_IGHJ2*01
2283
O12_IGKJ1*01
833
1438


206
VH3-23_IGHD5-18*01 (2) >3′_IGHJ2*01
2284
O12_IGKJ1*01
833
1438


207
VH3-23_IGHD5-24*01>1′_IGHJ2*01
2285
O12_IGKJ1*01
833
1438


208
VH3-23_IGHD5-24*01>3′_IGHJ2*01
2286
O12_IGKJ1*01
833
1438


209
VH3-23_IGHD6-6*01>1′_IGHJ2*01
2287
O12_IGKJ1*01
833
1438


210
VH3-23_IGHD6-6*01>2′_IGHJ2*01
2288
O12_IGKJ1*01
833
1438


211
VH3-23_IGHD6-6*01>3′_IGHJ2*01
2289
O12_IGKJ1*01
833
1438


212
VH3-23_IGHD6-6*01>2_IGHJ2*01
2233
O12_IGKJ1*01
833
1438


213
VH3-23_IGHD6-13*01>1_IGHJ2*01
2234
O12_IGKJ1*01
833
1438


214
VH3-23_IGHD6-13*01>2_IGHJ2*01
2235
O12_IGKJ1*01
833
1438


215
VH3-23_IGHD6-19*01>1_IGHJ2*01
2236
O12_IGKJ1*01
833
1438


216
VH3-23_IGHD6-19*01>2_IGHJ2*01
2237
O12_IGKJ1*01
833
1438


217
VH3-23_IGHD6-25*01>1_IGHJ2*01
2238
O12_IGKJ1*01
833
1438


218
VH3-23_IGHD6-25*01>2_IGHJ2*01
2239
O12_IGKJ1*01
833
1438


219
VH3-23_IGHD7-27*01>1_IGHJ2*01
2240
O12_IGKJ1*01
833
1438


220
VH3-23_IGHD7-27*01>3_IGHJ2*01
2241
O12_IGKJ1*01
833
1438


221
VH3-23_IGHD6-13*01>1′_IGHJ2*01
2290
O12_IGKJ1*01
833
1438


222
VH3-23_IGHD6-13*01>2′_IGHJ2*01
2291
O12_IGKJ1*01
833
1438


223
VH3-23_IGHD6-13*01>2_IGHJ2*01_B
2292
O12_IGKJ1*01
833
1438


224
VH3-23_IGHD6-19*01>1′_IGHJ2*01
2293
O12_IGKJ1*01
833
1438


225
VH3-23_IGHD6-19*01>2′_IGHJ2*01
2294
O12_IGKJ1*01
833
1438


226
VH3-23_IGHD6-19*01>2_IGHJ2*01_B
2295
O12_IGKJ1*01
833
1438


227
VH3-23_IGHD6-25*01>1′_IGHJ2*01
2296
O12_IGKJ1*01
833
1438


228
VH3-23_IGHD6-25*01>3′_IGHJ2*01
2297
O12_IGKJ1*01
833
1438


229
VH3-23_IGHD7-27*01>1′_IGHJ2*01
2298
O12_IGKJ1*01
833
1438


230
VH3-23_IGHD7-27*01>2′_IGHJ2*01
2299
O12_IGKJ1*01
833
1438


231
VH3-23_IGHD1-1*01>1_IGHJ3*01
2300
O12_IGKJ1*01
833
1438


232
VH3-23_IGHD1-1*01>2_IGHJ3*01
2301
O12_IGKJ1*01
833
1438


233
VH3-23_IGHD1-1*01>3_IGHJ3*01
2302
O12_IGKJ1*01
833
1438


234
VH3-23_IGHD1-7*01>1_IGHJ3*01
2303
O12_IGKJ1*01
833
1438


235
VH3-23_IGHD1-7*01>3_IGHJ3*01
2304
O12_IGKJ1*01
833
1438


236
VH3-23_IGHD1-14*01>1_IGHJ3*01
2305
O12_IGKJ1*01
833
1438


237
VH3-23_IGHD1-14*01>3_IGHJ3*01
2306
O12_IGKJ1*01
833
1438


238
VH3-23_IGHD1-20*01>1_IGHJ3*01
2307
O12_IGKJ1*01
833
1438


239
VH3-23_IGHD1-20*01>3_IGHJ3*01
2308
O12_IGKJ1*01
833
1438


240
VH3-23_IGHD1-26*01>1_IGHJ3*01
2309
O12_IGKJ1*01
833
1438


241
VH3-23_IGHD1-26*01>3_IGHJ3*01
2310
O12_IGKJ1*01
833
1438


242
VH3-23_IGHD2-2*01>2_IGHJ3*01
2311
O12_IGKJ1*01
833
1438


243
VH3-23_IGHD2-2*01>3_IGHJ3*01
2312
O12_IGKJ1*01
833
1438


244
VH3-23_IGHD2-8*01>2_IGHJ3*01
2313
O12_IGKJ1*01
833
1438


245
VH3-23_IGHD2-8*01>3_IGHJ3*01
2314
O12_IGKJ1*01
833
1438


246
VH3-23_IGHD2-15*01>2_IGHJ3*01
2315
O12_IGKJ1*01
833
1438


247
VH3-23_IGHD2-15*01>3_IGHJ3*01
2316
O12_IGKJ1*01
833
1438


248
VH3-23_IGHD2-21*01>2_IGHJ3*01
2317
O12_IGKJ1*01
833
1438


249
VH3-23_IGHD2-21*01>3_IGHJ3*01
2318
O12_IGKJ1*01
833
1438


250
VH3-23_IGHD3-3*01>1_IGHJ3*01
2319
O12_IGKJ1*01
833
1438


251
VH3-23_IGHD3-3*01>2_IGHJ3*01
2320
O12_IGKJ1*01
833
1438


252
VH3-23_IGHD3-3*01>3_IGHJ3*01
2321
O12_IGKJ1*01
833
1438


253
VH3-23_IGHD3-9*01>2_IGHJ3*01
2322
O12_IGKJ1*01
833
1438


254
VH3-23_IGHD3-10*01>2_IGHJ3*01
2323
O12_IGKJ1*01
833
1438


255
VH3-23_IGHD3-10*01>3_IGHJ3*01
2324
O12_IGKJ1*01
833
1438


256
VH3-23_IGHD3-16*01>2_IGHJ3*01
2325
O12_IGKJ1*01
833
1438


257
VH3-23_IGHD3-16*01>3_IGHJ3*01
2326
O12_IGKJ1*01
833
1438


258
VH3-23_IGHD3-22*01>2_IGHJ3*01
2327
O12_IGKJ1*01
833
1438


259
VH3-23_IGHD3-22*01>3_IGHJ3*01
2328
O12_IGKJ1*01
833
1438


260
VH3-23_IGHD4-4*01 (1) >2_IGHJ3*01
2329
O12_IGKJ1*01
833
1438


261
VH3-23_IGHD4-4*01 (1) >3_IGHJ3*01
2330
O12_IGKJ1*01
833
1438


262
VH3-23_IGHD4-11*01 (1) >2_IGHJ3*01
2331
O12_IGKJ1*01
833
1438


263
VH3-23_IGHD4-11*01 (1) >3_IGHJ3*01
2332
O12_IGKJ1*01
833
1438


264
VH3-23_IGHD4-17*01>2_IGHJ3*01
2333
O12_IGKJ1*01
833
1438


265
VH3-23_IGHD4-17*01>3_IGHJ3*01
2334
O12_IGKJ1*01
833
1438


266
VH3-23_IGHD4-23*01>2_IGHJ3*01
2335
O12_IGKJ1*01
833
1438


267
VH3-23_IGHD4-23*01>3_IGHJ3*01
2336
O12_IGKJ1*01
833
1438


268
VH3-23_IGHD5-5*01 (2) >1_IGHJ3*01
2337
O12_IGKJ1*01
833
1438


269
VH3-23_IGHD5-5*01 (2) >2_IGHJ3*01
2338
O12_IGKJ1*01
833
1438


270
VH3-23_IGHD5-5*01 (2) >3_IGHJ3*01
2339
O12_IGKJ1*01
833
1438


271
VH3-23_IGHD5-12*01>1_IGHJ3*01
2340
O12_IGKJ1*01
833
1438


272
VH3-23_IGHD5-12*01>3_IGHJ3*01
2341
O12_IGKJ1*01
833
1438


273
VH3-23_IGHD5-18*01 (2) >1_IGHJ3*01
2342
O12_IGKJ1*01
833
1438


274
VH3-23_IGHD5-18*01 (2) >2_IGHJ3*01
2343
O12_IGKJ1*01
833
1438


275
VH3-23_IGHD5-18*01 (2) >3_IGHJ3*01
2344
O12_IGKJ1*01
833
1438


276
VH3-23_IGHD5-24*01>1_IGHJ3*01
2345
O12_IGKJ1*01
833
1438


277
VH3-23_IGHD5-24*01>3_IGHJ3*01
2346
O12_IGKJ1*01
833
1438


278
VH3-23_IGHD6-6*01>1_IGHJ3*01
2347
O12_IGKJ1*01
833
1438


279
VH3-23_IGHD1-1*01>1′_IGHJ3*01
2357
O12_IGKJ1*01
833
1438


280
VH3-23_IGHD1-1*01>2′_IGHJ3*01
2358
O12_IGKJ1*01
833
1438


281
VH3-23_IGHD1-1*01>3′_IGHJ3*01
2359
O12_IGKJ1*01
833
1438


282
VH3-23_IGHD1-7*01>1′_IGHJ3*01
2360
O12_IGKJ1*01
833
1438


283
VH3-23_IGHD1-7*01>3′_IGHJ3*01
2361
O12_IGKJ1*01
833
1438


284
VH3-23_IGHD1-14*01>1′_IGHJ3*01
2362
O12_IGKJ1*01
833
1438


285
VH3-23_IGHD1-14*01>2′_IGHJ3*01
2363
O12_IGKJ1*01
833
1438


286
VH3-23_IGHD1-14*01>3′_IGHJ3*01
2364
O12_IGKJ1*01
833
1438


287
VH3-23_IGHD1-20*01>1′_IGHJ3*01
2365
O12_IGKJ1*01
833
1438


288
VH3-23_IGHD1-20*01>2′_IGHJ3*01
2366
O12_IGKJ1*01
833
1438


289
VH3-23_IGHD1-20*01>3′_IGHJ3*01
2367
O12_IGKJ1*01
833
1438


290
VH3-23_IGHD1-26*01>1′_IGHJ3*01
2368
O12_IGKJ1*01
833
1438


291
VH3-23_IGHD1-26*01>3′_IGHJ3*01
2369
O12_IGKJ1*01
833
1438


292
VH3-23_IGHD2-2*01>1′_IGHJ3*01
2370
O12_IGKJ1*01
833
1438


293
VH3-23_IGHD2-2*01>3′_IGHJ3*01
2371
O12_IGKJ1*01
833
1438


294
VH3-23_IGHD2-8*01>1′_IGHJ3*01
2372
O12_IGKJ1*01
833
1438


295
VH3-23_IGHD2-15*01>1′_IGHJ3*01
2373
O12_IGKJ1*01
833
1438


296
VH3-23_IGHD2-15*01>3′_IGHJ3*01
2374
O12_IGKJ1*01
833
1438


297
VH3-23_IGHD2-21*01>1′_IGHJ3*01
2375
O12_IGKJ1*01
833
1438


298
VH3-23_IGHD2-21*01>3′_IGHJ3*01
2376
O12_IGKJ1*01
833
1438


299
VH3-23_IGHD3-3*01>1′_IGHJ3*01
2377
O12_IGKJ1*01
833
1438


300
VH3-23_IGHD3-3*01>3′_IGHJ3*01
2378
O12_IGKJ1*01
833
1438


301
VH3-23_IGHD3-9*01>1′_IGHJ3*01
2379
O12_IGKJ1*01
833
1438


302
VH3-23_IGHD3-9*01>3′_IGHJ3*01
2380
O12_IGKJ1*01
833
1438


303
VH3-23_IGHD3-10*01>1′_IGHJ3*01
2381
O12_IGKJ1*01
833
1438


304
VH3-23_IGHD3-10*01>3′_IGHJ3*01
2382
O12_IGKJ1*01
833
1438


305
VH3-23_IGHD3-16*01>1′_IGHJ3*01
2383
O12_IGKJ1*01
833
1438


306
VH3-23_IGHD3-16*01>3′_IGHJ3*01
2384
O12_IGKJ1*01
833
1438


307
VH3-23_IGHD3-22*01>1′_IGHJ3*01
2385
O12_IGKJ1*01
833
1438


308
VH3-23_IGHD4-4*01 (1) >1′_IGHJ3*01
2386
O12_IGKJ1*01
833
1438


309
VH3-23_IGHD4-4*01 (1) >3′_IGHJ3*01
2387
O12_IGKJ1*01
833
1438


310
VH3-23_IGHD4-11*01 (1) >1′_IGHJ3*01
2388
O12_IGKJ1*01
833
1438


311
VH3-23_IGHD4-11*01 (1) >3′_IGHJ3*01
2389
O12_IGKJ1*01
833
1438


312
VH3-23_IGHD4-17*01>1′_IGHJ3*01
2390
O12_IGKJ1*01
833
1438


313
VH3-23_IGHD4-17*01>3′_IGHJ3*01
2391
O12_IGKJ1*01
833
1438


314
VH3-23_IGHD4-23*01>1′_IGHJ3*01
2392
O12_IGKJ1*01
833
1438


315
VH3-23_IGHD4-23*01>3′_IGHJ3*01
2393
O12_IGKJ1*01
833
1438


316
VH3-23_IGHD5-5*01 (2) >1′_IGHJ3*01
2394
O12_IGKJ1*01
833
1438


317
VH3-23_IGHD5-5*01 (2) >3′_IGHJ3*01
2395
O12_IGKJ1*01
833
1438


318
VH3-23_IGHD5-12*01>1′_IGHJ3*01
2396
O12_IGKJ1*01
833
1438


319
VH3-23_IGHD5-12*01>3′_IGHJ3*01
2397
O12_IGKJ1*01
833
1438


320
VH3-23_IGHD5-18*01 (2) >1′_IGHJ3*01
2398
O12_IGKJ1*01
833
1438


321
VH3-23_IGHD5-18*01 (2) >3′_IGHJ3*01
2399
O12_IGKJ1*01
833
1438


322
VH3-23_IGHD5-24*01>1′_IGHJ3*01
2400
O12_IGKJ1*01
833
1438


323
VH3-23_IGHD5-24*01>3′_IGHJ3*01
2401
O12_IGKJ1*01
833
1438


324
VH3-23_IGHD6-6*01>1′_IGHJ3*01
2402
O12_IGKJ1*01
833
1438


325
VH3-23_IGHD6-6*01>2′_IGHJ3*01
2403
O12_IGKJ1*01
833
1438


326
VH3-23_IGHD6-6*01>3′_IGHJ3*01
2404
O12_IGKJ1*01
833
1438


327
VH3-23_IGHD6-6*01>2_IGHJ3*01
2348
O12_IGKJ1*01
833
1438


328
VH3-23_IGHD6-13*01>1_IGHJ3*01
2349
O12_IGKJ1*01
833
1438


329
VH3-23_IGHD6-13*01>2_IGHJ3*01
2350
O12_IGKJ1*01
833
1438


330
VH3-23_IGHD6-19*01>1_IGHJ3*01
2351
O12_IGKJ1*01
833
1438


331
VH3-23_IGHD6-19*01>2_IGHJ3*01
2352
O12_IGKJ1*01
833
1438


332
VH3-23_IGHD6-25*01>1_IGHJ3*01
2353
O12_IGKJ1*01
833
1438


333
VH3-23_IGHD6-25*01>2_IGHJ3*01
2354
O12_IGKJ1*01
833
1438


334
VH3-23_IGHD7-27*01>1_IGHJ3*01
2355
O12_IGKJ1*01
833
1438


335
VH3-23_IGHD7-27*01>3_IGHJ3*01
2356
O12_IGKJ1*01
833
1438


336
VH3-23_IGHD6-13*01>1′_IGHJ3*01
2405
O12_IGKJ1*01
833
1438


337
VH3-23_IGHD6-13*01>2′_IGHJ3*01
2406
O12_IGKJ1*01
833
1438


338
VH3-23_IGHD6-13*01>1_IGHJ6*01
2407
O12_IGKJ1*01
833
1438


339
VH3-23_IGHD6-19*01>1′_IGHJ3*01
2408
O12_IGKJ1*01
833
1438


340
VH3-23_IGHD6-19*01>2′_IGHJ3*01
2409
O12_IGKJ1*01
833
1438


341
VH3-23_IGHD6-19*01>3′_IGHJ3*01
2410
O12_IGKJ1*01
833
1438


342
VH3-23_IGHD6-25*01>1′_IGHJ3*01
2411
O12_IGKJ1*01
833
1438


343
VH3-23_IGHD6-25*01>3′_IGHJ3*01
2412
O12_IGKJ1*01
833
1438


344
VH3-23_IGHD7-27*01>1′_IGHJ3*01
2413
O12_IGKJ1*01
833
1438


345
VH3-23_IGHD7-27*01>2′_IGHJ3*01
2414
O12_IGKJ1*01
833
1438


346
VH3-23_IGHD1-1*01>1_IGHJ4*01
2415
O12_IGKJ1*01
833
1438


347
VH3-23_IGHD1-1*01>2_IGHJ4*01
2416
O12_IGKJ1*01
833
1438


348
VH3-23_IGHD1-1*01>3_IGHJ4*01
2417
O12_IGKJ1*01
833
1438


349
VH3-23_IGHD1-7*01>1_IGHJ4*01
2418
O12_IGKJ1*01
833
1438


350
VH3-23_IGHD1-7*01>3_IGHJ4*01
2419
O12_IGKJ1*01
833
1438


351
VH3-23_IGHD1-14*01>1_IGHJ4*01
2420
O12_IGKJ1*01
833
1438


352
VH3-23_IGHD1-14*01>3_IGHJ4*01
2421
O12_IGKJ1*01
833
1438


353
VH3-23_IGHD1-20*01>1_IGHJ4*01
2422
O12_IGKJ1*01
833
1438


354
VH3-23_IGHD1-20*01>3_IGHJ4*01
2423
O12_IGKJ1*01
833
1438


355
VH3-23_IGHD1-26*01>1_IGHJ4*01
2424
O12_IGKJ1*01
833
1438


356
VH3-23_IGHD1-26*01>3_IGHJ4*01
2425
O12_IGKJ1*01
833
1438


357
VH3-23_IGHD2-2*01>2_IGHJ4*01
2426
O12_IGKJ1*01
833
1438


358
VH3-23_IGHD2-2*01>3_IGHJ4*01
2427
O12_IGKJ1*01
833
1438


359
VH3-23_IGHD2-8*01>2_IGHJ4*01
2428
O12_IGKJ1*01
833
1438


360
VH3-23_IGHD2-8*01>3_IGHJ4*01
2429
O12_IGKJ1*01
833
1438


361
VH3-23_IGHD2-15*01>2_IGHJ4*01
2430
O12_IGKJ1*01
833
1438


362
VH3-23_IGHD2-15*01>3_IGHJ4*01
2431
O12_IGKJ1*01
833
1438


363
VH3-23_IGHD2-21*01>2_IGHJ4*01
2432
O12_IGKJ1*01
833
1438


364
VH3-23_IGHD2-21*01>3_IGHJ4*01
2433
O12_IGKJ1*01
833
1438


365
VH3-23_IGHD3-3*01>1_IGHJ4*01
2434
O12_IGKJ1*01
833
1438


366
VH3-23_IGHD3-3*01>2_IGHJ4*01
2435
O12_IGKJ1*01
833
1438


367
VH3-23_IGHD3-3*01>3_IGHJ4*01
2436
O12_IGKJ1*01
833
1438


368
VH3-23_IGHD3-9*01>2_IGHJ4*01
2437
O12_IGKJ1*01
833
1438


369
VH3-23_IGHD3-10*01>2_IGHJ4*01
2438
O12_IGKJ1*01
833
1438


370
VH3-23_IGHD3-10*01>3_IGHJ4*01
2439
O12_IGKJ1*01
833
1438


371
VH3-23_IGHD3-16*01>2_IGHJ4*01
2440
O12_IGKJ1*01
833
1438


372
VH3-23_IGHD3-16*01>3_IGHJ4*01
2441
O12_IGKJ1*01
833
1438


373
VH3-23_IGHD3-22*01>2_IGHJ4*01
2442
O12_IGKJ1*01
833
1438


374
VH3-23_IGHD3-22*01>3_IGHJ4*01
2443
O12_IGKJ1*01
833
1438


375
VH3-23_IGHD4-4*01 (1) >2_IGHJ4*01
2444
O12_IGKJ1*01
833
1438


376
VH3-23_IGHD4-4*01 (1) >3_IGHJ4*01
2445
O12_IGKJ1*01
833
1438


377
VH3-23_IGHD4-11*01 (1) >2_IGHJ4*01
2446
O12_IGKJ1*01
833
1438


378
VH3-23_IGHD4-11*01 (1) >3_IGHJ4*01
2447
O12_IGKJ1*01
833
1438


379
VH3-23_IGHD4-17*01>2_IGHJ4*01
2448
O12_IGKJ1*01
833
1438


380
VH3-23_IGHD4-17*01>3_IGHJ4*01
2449
O12_IGKJ1*01
833
1438


381
VH3-23_IGHD4-23*01>2_IGHJ4*01
2450
O12_IGKJ1*01
833
1438


382
VH3-23_IGHD4-23*01>3_IGHJ4*01
2451
O12_IGKJ1*01
833
1438


383
VH3-23_IGHD5-5*01 (2) >1_IGHJ4*01
2452
O12_IGKJ1*01
833
1438


384
VH3-23_IGHD5-5*01 (2) >2_IGHJ4*01
2453
O12_IGKJ1*01
833
1438


385
VH3-23_IGHD5-5*01 (2) >3_IGHJ4*01
2454
O12_IGKJ1*01
833
1438


386
VH3-23_IGHD5-12*01>1_IGHJ4*01
2455
O12_IGKJ1*01
833
1438


387
VH3-23_IGHD5-12*01>3_IGHJ4*01
2456
O12_IGKJ1*01
833
1438


388
VH3-23_IGHD5-18*01 (2) >1_IGHJ4*01
2457
O12_IGKJ1*01
833
1438


389
VH3-23_IGHD5-18*01 (2) >2_IGHJ4*01
2458
O12_IGKJ1*01
833
1438


390
VH3-23_IGHD5-18*01 (2) >3_IGHJ4*01
2459
O12_IGKJ1*01
833
1438


391
VH3-23_IGHD5-24*01>1_IGHJ4*01
2460
O12_IGKJ1*01
833
1438


392
VH3-23_IGHD5-24*01>3_IGHJ4*01
2461
O12_IGKJ1*01
833
1438


393
VH3-23_IGHD6-6*01>1_IGHJ4*01
2462
O12_IGKJ1*01
833
1438


394
VH3-23_IGHD1-1*01>1′_IGHJ4*01
2472
O12_IGKJ1*01
833
1438


395
VH3-23_IGHD1-1*01>2′_IGHJ4*01
2473
O12_IGKJ1*01
833
1438


396
VH3-23_IGHD1-1*01>3′_IGHJ4*01
2474
O12_IGKJ1*01
833
1438


397
VH3-23_IGHD1-7*01>1′_IGHJ4*01
2475
O12_IGKJ1*01
833
1438


398
VH3-23_IGHD1-7*01>3′_IGHJ4*01
2476
O12_IGKJ1*01
833
1438


399
VH3-23_IGHD1-14*01>1′_IGHJ4*01
2477
O12_IGKJ1*01
833
1438


400
VH3-23_IGHD1-14*01>2′_IGHJ4*01
2478
O12_IGKJ1*01
833
1438


401
VH3-23_IGHD1-14*01>3′_IGHJ4*01
2479
O12_IGKJ1*01
833
1438


402
VH3-23_IGHD1-20*01>1′_IGHJ4*01
2480
O12_IGKJ1*01
833
1438


403
VH3-23_IGHD1-20*01>2′_IGHJ4*01
2481
O12_IGKJ1*01
833
1438


404
VH3-23_IGHD1-20*01>3′_IGHJ4*01
2482
O12_IGKJ1*01
833
1438


405
VH3-23_IGHD1-26*01>1′_IGHJ4*01
2483
O12_IGKJ1*01
833
1438


406
VH3-23_IGHD1-26*01>1_IGHJ4*01_B
2484
O12_IGKJ1*01
833
1438


407
VH3-23_IGHD2-2*01>1′_IGHJ4*01
2485
O12_IGKJ1*01
833
1438


408
VH3-23_IGHD2-2*01>3′_IGHJ4*01
2486
O12_IGKJ1*01
833
1438


409
VH3-23_IGHD2-8*01>1′_IGHJ4*01
2487
O12_IGKJ1*01
833
1438


410
VH3-23_IGHD2-15*01>1′_IGHJ4*01
2488
O12_IGKJ1*01
833
1438


411
VH3-23_IGHD2-15*01>3′_IGHJ4*01
2489
O12_IGKJ1*01
833
1438


412
VH3-23_IGHD2-21*01>1′_IGHJ4*01
2490
O12_IGKJ1*01
833
1438


413
VH3-23_IGHD2-21*01>3′_IGHJ4*01
2491
O12_IGKJ1*01
833
1438


414
VH3-23_IGHD3-3*01>1′_IGHJ4*01
2492
O12_IGKJ1*01
833
1438


415
VH3-23_IGHD3-3*01>3′_IGHJ4*01
2493
O12_IGKJ1*01
833
1438


416
VH3-23_IGHD3-9*01>1′_IGHJ4*01
2494
O12_IGKJ1*01
833
1438


417
VH3-23_IGHD3-9*01>3′_IGHJ4*01
2495
O12_IGKJ1*01
833
1438


418
VH3-23_IGHD3-10*01>1′_IGHJ4*01
2496
O12_IGKJ1*01
833
1438


419
VH3-23_IGHD3-10*01>3′_IGHJ4*01
2497
O12_IGKJ1*01
833
1438


420
VH3-23_IGHD3-16*01>1′_IGHJ4*01
2498
O12_IGKJ1*01
833
1438


421
VH3-23_IGHD3-16*01>3′_IGHJ4*01
2499
O12_IGKJ1*01
833
1438


422
VH3-23_IGHD3-22*01>1′_IGHJ4*01
2500
O12_IGKJ1*01
833
1438


423
VH3-23_IGHD4-4*01 (1) >1′_IGHJ4*01
2501
O12_IGKJ1*01
833
1438


424
VH3-23_IGHD4-4*01 (1) >3′_IGHJ4*01
2502
O12_IGKJ1*01
833
1438


425
VH3-23_IGHD4-11*01 (1) >1′_IGHJ4*01
2503
O12_IGKJ1*01
833
1438


426
VH3-23_IGHD4-11*01 (1) >3′_IGHJ4*01
2504
O12_IGKJ1*01
833
1438


427
VH3-23_IGHD4-17*01>1′_IGHJ4*01
2505
O12_IGKJ1*01
833
1438


428
VH3-23_IGHD4-17*01>3′_IGHJ4*01
2506
O12_IGKJ1*01
833
1438


429
VH3-23_IGHD4-23*01>1′_IGHJ4*01
2507
O12_IGKJ1*01
833
1438


430
VH3-23_IGHD4-23*01>3′_IGHJ4*01
2508
O12_IGKJ1*01
833
1438


431
VH3-23_IGHD5-5*01 (2) >1′_IGHJ4*01
2509
O12_IGKJ1*01
833
1438


432
VH3-23_IGHD5-5*01 (2) >3′_IGHJ4*01
2510
O12_IGKJ1*01
833
1438


433
VH3-23_IGHD5-12*01>1′_IGHJ4*01
2511
O12_IGKJ1*01
833
1438


434
VH3-23_IGHD5-12*01>3′_IGHJ4*01
2512
O12_IGKJ1*01
833
1438


435
VH3-23_IGHD5-18*01 (2) >1′_IGHJ4*01
2513
O12_IGKJ1*01
833
1438


436
VH3-23_IGHD5-18*01 (2) >3′_IGHJ4*01
2514
O12_IGKJ1*01
833
1438


437
VH3-23_IGHD5-24*01>1′_IGHJ4*01
2515
O12_IGKJ1*01
833
1438


438
VH3-23_IGHD5-24*01>3′_IGHJ4*01
2516
O12_IGKJ1*01
833
1438


439
VH3-23_IGHD6-6*01>1′_IGHJ4*01
2517
O12_IGKJ1*01
833
1438


440
VH3-23_IGHD6-6*01>2′_IGHJ4*01
2518
O12_IGKJ1*01
833
1438


441
VH3-23_IGHD6-6*01>3′_IGHJ4*01
2519
O12_IGKJ1*01
833
1438


442
VH3-23_IGHD6-6*01>2_IGHJ4*01
2463
O12_IGKJ1*01
833
1438


443
VH3-23_IGHD6-13*01>1_IGHJ4*01
2464
O12_IGKJ1*01
833
1438


444
VH3-23_IGHD6-13*01>2_IGHJ4*01
2465
O12_IGKJ1*01
833
1438


445
VH3-23_IGHD6-19*01>1_IGHJ4*01
2466
O12_IGKJ1*01
833
1438


446
VH3-23_IGHD6-19*01>2_IGHJ4*01
2467
O12_IGKJ1*01
833
1438


447
VH3-23_IGHD6-25*01>1_IGHJ4*01
2468
O12_IGKJ1*01
833
1438


448
VH3-23_IGHD6-25*01>2_IGHJ4*01
2469
O12_IGKJ1*01
833
1438


449
VH3-23_IGHD7-27*01>1_IGHJ4*01
2470
O12_IGKJ1*01
833
1438


450
VH3-23_IGHD7-27*01>3_IGHJ4*01
2471
O12_IGKJ1*01
833
1438


451
VH3-23_IGHD6-13*01>1′_IGHJ4*01
2520
O12_IGKJ1*01
833
1438


452
VH3-23_IGHD6-13*01>2′_IGHJ4*01
2521
O12_IGKJ1*01
833
1438


453
VH3-23_IGHD6-13*01>2_IGHJ4*01_B
2522
O12_IGKJ1*01
833
1438


454
VH3-23_IGHD6-19*01>1′_IGHJ4*01
2523
O12_IGKJ1*01
833
1438


455
VH3-23_IGHD6-19*01>2′_IGHJ4*01
2524
O12_IGKJ1*01
833
1438


456
VH3-23_IGHD6-19*01>2_IGHJ4*01_B
2525
O12_IGKJ1*01
833
1438


457
VH3-23_IGHD6-25*01>1′_IGHJ4*01
2526
O12_IGKJ1*01
833
1438


458
VH3-23_IGHD6-25*01>3′_IGHJ4*01
2527
O12_IGKJ1*01
833
1438


459
VH3-23_IGHD7-27*01>1′_IGHJ4*01
2528
O12_IGKJ1*01
833
1438


460
VH3-23_IGHD7-27*01>2′_IGHJ4*01
2529
O12_IGKJ1*01
833
1438


461
VH3-23_IGHD1-1*01>1_IGHJ5*01
2530
O12_IGKJ1*01
833
1438


462
VH3-23_IGHD1-1*01>2_IGHJ5*01
2531
O12_IGKJ1*01
833
1438


463
VH3-23_IGHD1-1*01>3_IGHJ5*01
2532
O12_IGKJ1*01
833
1438


464
VH3-23_IGHD1-7*01>1_IGHJ5*01
2533
O12_IGKJ1*01
833
1438


465
VH3-23_IGHD1-7*01>3_IGHJ5*01
2534
O12_IGKJ1*01
833
1438


466
VH3-23_IGHD1-14*01>1_IGHJ5*01
2535
O12_IGKJ1*01
833
1438


467
VH3-23_IGHD1-14*01>3_IGHJ5*01
2536
O12_IGKJ1*01
833
1438


468
VH3-23_IGHD1-20*01>1_IGHJ5*01
2537
O12_IGKJ1*01
833
1438


469
VH3-23_IGHD1-20*01>3_IGHJ5*01
2538
O12_IGKJ1*01
833
1438


470
VH3-23_IGHD1-26*01>1_IGHJ5*01
2539
O12_IGKJ1*01
833
1438


471
VH3-23_IGHD1-26*01>3_IGHJ5*01
2540
O12_IGKJ1*01
833
1438


472
VH3-23_IGHD2-2*01>2_IGHJ5*01
2541
O12_IGKJ1*01
833
1438


473
VH3-23_IGHD2-2*01>3_IGHJ5*01
2542
O12_IGKJ1*01
833
1438


474
VH3-23_IGHD2-8*01>2_IGHJ5*01
2543
O12_IGKJ1*01
833
1438


475
VH3-23_IGHD2-8*01>3_IGHJ5*01
2544
O12_IGKJ1*01
833
1438


476
VH3-23_IGHD2-15*01>2_IGHJ5*01
2545
O12_IGKJ1*01
833
1438


477
VH3-23_IGHD2-15*01>3_IGHJ5*01
2546
O12_IGKJ1*01
833
1438


478
VH3-23_IGHD2-21*01>2_IGHJ5*01
2547
O12_IGKJ1*01
833
1438


479
VH3-23_IGHD2-21*01>3_IGHJ5*01
2548
O12_IGKJ1*01
833
1438


480
VH3-23_IGHD3-3*01>1_IGHJ5*01
2549
O12_IGKJ1*01
833
1438


481
VH3-23_IGHD3-3*01>2_IGHJ5*01
2550
O12_IGKJ1*01
833
1438


482
VH3-23_IGHD3-3*01>3_IGHJ5*01
2551
O12_IGKJ1*01
833
1438


483
VH3-23_IGHD3-9*01>2_IGHJ5*01
2552
O12_IGKJ1*01
833
1438


484
VH3-23_IGHD3-10*01>2_IGHJ5*01
2553
O12_IGKJ1*01
833
1438


485
VH3-23_IGHD3-10*01>3_IGHJ5*01
2554
O12_IGKJ1*01
833
1438


486
VH3-23_IGHD3-16*01>2_IGHJ5*01
2555
O12_IGKJ1*01
833
1438


487
VH3-23_IGHD3-16*01>3_IGHJ5*01
2556
O12_IGKJ1*01
833
1438


488
VH3-23_IGHD3-22*01>2_IGHJ5*01
2557
O12_IGKJ1*01
833
1438


489
VH3-23_IGHD3-22*01>3_IGHJ5*01
2558
O12_IGKJ1*01
833
1438


490
VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01
2559
O12_IGKJ1*01
833
1438


491
VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01
2560
O12_IGKJ1*01
833
1438


492
VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01
2561
O12_IGKJ1*01
833
1438


493
VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01
2562
O12_IGKJ1*01
833
1438


494
VH3-23_IGHD4-17*01>2_IGHJ5*01
2563
O12_IGKJ1*01
833
1438


495
VH3-23_IGHD4-17*01>3_IGHJ5*01
2564
O12_IGKJ1*01
833
1438


496
VH3-23_IGHD4-23*01>2_IGHJ5*01
2565
O12_IGKJ1*01
833
1438


497
VH3-23_IGHD4-23*01>3_IGHJ5*01
2566
O12_IGKJ1*01
833
1438


498
VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01
2567
O12_IGKJ1*01
833
1438


499
VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01
2568
O12_IGKJ1*01
833
1438


500
VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01
2569
O12_IGKJ1*01
833
1438


501
VH3-23_IGHD5-12*01>1_IGHJ5*01
2570
O12_IGKJ1*01
833
1438


502
VH3-23_IGHD5-12*01>3_IGHJ5*01
2571
O12_IGKJ1*01
833
1438


503
VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01
2572
O12_IGKJ1*01
833
1438


504
VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01
2573
O12_IGKJ1*01
833
1438


505
VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01
2574
O12_IGKJ1*01
833
1438


506
VH3-23_IGHD5-24*01>1_IGHJ5*01
2575
O12_IGKJ1*01
833
1438


507
VH3-23_IGHD5-24*01>3_IGHJ5*01
2576
O12_IGKJ1*01
833
1438


508
VH3-23_IGHD6-6*01>1_IGHJ5*01
2577
O12_IGKJ1*01
833
1438


509
VH3-23_IGHD1-1*01>1′_IGHJ5*01
2587
O12_IGKJ1*01
833
1438


510
VH3-23_IGHD1-1*01>2′_IGHJ5*01
2588
O12_IGKJ1*01
833
1438


511
VH3-23_IGHD1-1*01>3′_IGHJ5*01
2589
O12_IGKJ1*01
833
1438


512
VH3-23_IGHD1-7*01>1′_IGHJ5*01
2590
O12_IGKJ1*01
833
1438


513
VH3-23_IGHD1-7*01>3′_IGHJ5*01
2591
O12_IGKJ1*01
833
1438


514
VH3-23_IGHD1-14*01>1′_IGHJ5*01
2592
O12_IGKJ1*01
833
1438


515
VH3-23_IGHD1-14*01>2′_IGHJ5*01
2593
O12_IGKJ1*01
833
1438


516
VH3-23_IGHD1-14*01>3′_IGHJ5*01
2594
O12_IGKJ1*01
833
1438


517
VH3-23_IGHD1-20*01>1′_IGHJ5*01
2595
O12_IGKJ1*01
833
1438


518
VH3-23_IGHD1-20*01>2′_IGHJ5*01
2596
O12_IGKJ1*01
833
1438


519
VH3-23_IGHD1-20*01>3′_IGHJ5*01
2597
O12_IGKJ1*01
833
1438


520
VH3-23_IGHD1-26*01>1′_IGHJ5*01
2598
O12_IGKJ1*01
833
1438


521
VH3-23_IGHD1-26*01>3′_IGHJ5*01
2599
O12_IGKJ1*01
833
1438


522
VH3-23_IGHD2-2*01>1′_IGHJ5*01
2600
O12_IGKJ1*01
833
1438


523
VH3-23_IGHD2-2*01>3′_IGHJ5*01
2601
O12_IGKJ1*01
833
1438


524
VH3-23_IGHD2-8*01>1′_IGHJ5*01
2602
O12_IGKJ1*01
833
1438


525
VH3-23_IGHD2-15*01>1′_IGHJ5*01
2603
O12_IGKJ1*01
833
1438


526
VH3-23_IGHD2-15*01>3′_IGHJ5*01
2604
O12_IGKJ1*01
833
1438


527
VH3-23_IGHD2-21*01>1′_IGHJ5*01
2605
O12_IGKJ1*01
833
1438


528
VH3-23_IGHD2-21*01>3′_IGHJ5*01
2606
O12_IGKJ1*01
833
1438


529
VH3-23_IGHD3-3*01>1′_IGHJ5*01
2607
O12_IGKJ1*01
833
1438


530
VH3-23_IGHD3-3*01>3′_IGHJ5*01
2608
O12_IGKJ1*01
833
1438


531
VH3-23_IGHD3-9*01>1′_IGHJ5*01
2609
O12_IGKJ1*01
833
1438


532
VH3-23_IGHD3-9*01>3′_IGHJ5*01
2610
O12_IGKJ1*01
833
1438


533
VH3-23_IGHD3-10*01>1′_IGHJ5*01
2611
O12_IGKJ1*01
833
1438


534
VH3-23_IGHD3-10*01>3′_IGHJ5*01
2612
O12_IGKJ1*01
833
1438


535
VH3-23_IGHD3-16*01>1′_IGHJ5*01
2613
O12_IGKJ1*01
833
1438


536
VH3-23_IGHD3-16*01>3′_IGHJ5*01
2614
O12_IGKJ1*01
833
1438


537
VH3-23_IGHD3-22*01>1′_IGHJ5*01
2615
O12_IGKJ1*01
833
1438


538
VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01
2616
O12_IGKJ1*01
833
1438


539
VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01
2617
O12_IGKJ1*01
833
1438


540
VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01
2618
O12_IGKJ1*01
833
1438


541
VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01
2619
O12_IGKJ1*01
833
1438


542
VH3-23_IGHD4-17*01>1′_IGHJ5*01
2620
O12_IGKJ1*01
833
1438


543
VH3-23_IGHD4-17*01>3′_IGHJ5*01
2621
O12_IGKJ1*01
833
1438


544
VH3-23_IGHD4-23*01>1′_IGHJ5*01
2622
O12_IGKJ1*01
833
1438


545
VH3-23_IGHD4-23*01>3′_IGHJ5*01
2623
O12_IGKJ1*01
833
1438


546
VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01
2624
O12_IGKJ1*01
833
1438


547
VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01
2625
O12_IGKJ1*01
833
1438


548
VH3-23_IGHD5-12*01>1′_IGHJ5*01
2626
O12_IGKJ1*01
833
1438


549
VH3-23_IGHD5-12*01>3′_IGHJ5*01
2627
O12_IGKJ1*01
833
1438


550
VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01
2628
O12_IGKJ1*01
833
1438


551
VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01
2629
O12_IGKJ1*01
833
1438


552
VH3-23_IGHD5-24*01>1′_IGHJ5*01
2630
O12_IGKJ1*01
833
1438


553
VH3-23_IGHD5-24*01>3′_IGHJ5*01
2631
O12_IGKJ1*01
833
1438


554
VH3-23_IGHD6-6*01>1′_IGHJ5*01
2632
O12_IGKJ1*01
833
1438


555
VH3-23_IGHD6-6*01>2′_IGHJ5*01
2633
O12_IGKJ1*01
833
1438


556
VH3-23_IGHD6-6*01>3′_IGHJ5*01
2634
O12_IGKJ1*01
833
1438


557
VH3-23_IGHD6-6*01>2_IGHJ5*01
2578
O12_IGKJ1*01
833
1438


558
VH3-23_IGHD6-13*01>1_IGHJ5*01
2579
O12_IGKJ1*01
833
1438


559
VH3-23_IGHD6-13*01>2_IGHJ5*01
2580
O12_IGKJ1*01
833
1438


560
VH3-23_IGHD6-19*01>1_IGHJ5*01
2581
O12_IGKJ1*01
833
1438


561
VH3-23_IGHD6-19*01>2_IGHJ5*01
2582
O12_IGKJ1*01
833
1438


562
VH3-23_IGHD6-25*01>1_IGHJ5*01
2583
O12_IGKJ1*01
833
1438


563
VH3-23_IGHD6-25*01>2_IGHJ5*01
2584
O12_IGKJ1*01
833
1438


564
VH3-23_IGHD7-27*01>1_IGHJ5*01
2585
O12_IGKJ1*01
833
1438


565
VH3-23_IGHD7-27*01>3_IGHJ5*01
2586
O12_IGKJ1*01
833
1438


566
VH3-23_IGHD6-13*01>1′_IGHJ5*01
2635
O12_IGKJ1*01
833
1438


567
VH3-23_IGHD6-13*01>2′_IGHJ5*01
2636
O12_IGKJ1*01
833
1438


568
VH3-23_IGHD6-13*01>3′_IGHJ5*01
2637
O12_IGKJ1*01
833
1438


569
VH3-23_IGHD6-19*01>1′_IGHJ5*01
2638
O12_IGKJ1*01
833
1438


570
VH3-23_IGHD6-19*01>2′_IGHJ5*01
2639
O12_IGKJ1*01
833
1438


571
VH3-23_IGHD6-19*01>2_IGHJ5*01_B
2640
O12_IGKJ1*01
833
1438


572
VH3-23_IGHD6-25*01>1′_IGHJ5*01
2641
O12_IGKJ1*01
833
1438


573
VH3-23_IGHD6-25*01>3′_IGHJ5*01
2642
O12_IGKJ1*01
833
1438


574
VH3-23_IGHD7-27*01>1′_IGHJ5*01
2643
O12_IGKJ1*01
833
1438


575
VH3-23_IGHD7-27*01>2′_IGHJ5*01
2644
O12_IGKJ1*01
833
1438


576
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
O12_IGKJ1*01
833
1438


577
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
O12_IGKJ1*01
833
1438


578
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
O12_IGKJ1*01
833
1438


579
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
O12_IGKJ1*01
833
1438


580
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
O12_IGKJ1*01
833
1438


581
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
O12_IGKJ1*01
833
1438


582
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
O12_IGKJ1*01
833
1438


583
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
O12_IGKJ1*01
833
1438


584
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
O12_IGKJ1*01
833
1438


585
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
O12_IGKJ1*01
833
1438


586
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
O12_IGKJ1*01
833
1438


587
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
O12_IGKJ1*01
833
1438


588
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
O12_IGKJ1*01
833
1438


589
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
O12_IGKJ1*01
833
1438


590
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
O12_IGKJ1*01
833
1438


591
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
O12_IGKJ1*01
833
1438


592
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
O12_IGKJ1*01
833
1438


593
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
O12_IGKJ1*01
833
1438


594
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
O12_IGKJ1*01
833
1438


595
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
O12_IGKJ1*01
833
1438


596
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
O12_IGKJ1*01
833
1438


597
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
O12_IGKJ1*01
833
1438


598
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
O12_IGKJ1*01
833
1438


599
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
O12_IGKJ1*01
833
1438


600
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
O12_IGKJ1*01
833
1438


601
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
O12_IGKJ1*01
833
1438


602
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
O12_IGKJ1*01
833
1438


603
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
O12_IGKJ1*01
833
1438


604
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
O12_IGKJ1*01
833
1438


605
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
O12_IGKJ1*01
833
1438


606
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
O12_IGKJ1*01
833
1438


607
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
O12_IGKJ1*01
833
1438


608
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
O12_IGKJ1*01
833
1438


609
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
O12_IGKJ1*01
833
1438


610
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
O12_IGKJ1*01
833
1438


611
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
O12_IGKJ1*01
833
1438


612
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
O12_IGKJ1*01
833
1438


613
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
O12_IGKJ1*01
833
1438


614
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
O12_IGKJ1*01
833
1438


615
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
O12_IGKJ1*01
833
1438


616
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
O12_IGKJ1*01
833
1438


617
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
O12_IGKJ1*01
833
1438


618
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
O12_IGKJ1*01
833
1438


619
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
O12_IGKJ1*01
833
1438


620
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
O12_IGKJ1*01
833
1438


621
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
O12_IGKJ1*01
833
1438


622
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
O12_IGKJ1*01
833
1438


623
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
O12_IGKJ1*01
833
1438


624
VH3-23_IGHD6-6*01>2_IGHJ6*01
2693
O12_IGKJ1*01
833
1438


625
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
O12_IGKJ1*01
833
1438


626
VH3-23_IGHD5-18*01(2)>1′_IGHJ6*01
2743
O12_IGKJ1*01
833
1438


627
VH3-23_IGHD5-18*01(2)>3′_IGHJ6*01
2744
O12_IGKJ1*01
833
1438


628
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
O12_IGKJ1*01
833
1438


629
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
O12_IGKJ1*01
833
1438


630
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
O12_IGKJ1*01
833
1438


631
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
O12_IGKJ1*01
833
1438


632
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
O12_IGKJ1*01
833
1438


633
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
O12_IGKJ1*01
833
1438


634
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
O12_IGKJ1*01
833
1438


635
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
O12_IGKJ1*01
833
1438


636
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
O12_IGKJ1*01
833
1438


637
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
O12_IGKJ1*01
833
1438


638
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
O12_IGKJ1*01
833
1438


639
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
O12_IGKJ1*01
833
1438


640
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
O12_IGKJ1*01
833
1438


641
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
O12_IGKJ1*01
833
1438


642
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
O12_IGKJ1*01
833
1438


643
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
O12_IGKJ1*01
833
1438


644
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
O12_IGKJ1*01
833
1438


645
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
O12_IGKJ1*01
833
1438


646
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
O12_IGKJ1*01
833
1438


647
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
O12_IGKJ1*01
833
1438


648
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
O12_IGKJ1*01
833
1438


649
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
O12_IGKJ1*01
833
1438


650
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
O12_IGKJ1*01
833
1438


651
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
O12_IGKJ1*01
833
1438


652
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
O12_IGKJ1*01
833
1438


653
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
O12_IGKJ1*01
833
1438


654
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
O12_IGKJ1*01
833
1438


655
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
O12_IGKJ1*01
833
1438


656
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
O12_IGKJ1*01
833
1438


657
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
O12_IGKJ1*01
833
1438


658
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
O12_IGKJ1*01
833
1438


659
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
O12_IGKJ1*01
833
1438


660
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
O12_IGKJ1*01
833
1438


661
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
O12_IGKJ1*01
833
1438


662
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
O12_IGKJ1*01
833
1438


663
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
O12_IGKJ1*01
833
1438


664
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
O12_IGKJ1*01
833
1438


665
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
O12_IGKJ1*01
833
1438


666
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
O12_IGKJ1*01
833
1438


667
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
O12_IGKJ1*01
833
1438


668
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
O12_IGKJ1*01
833
1438


669
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
O12_IGKJ1*01
833
1438


670
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
O12_IGKJ1*01
833
1438


671
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
O12_IGKJ1*01
833
1438


672
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
O12_IGKJ1*01
833
1438


673
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
O12_IGKJ1*01
833
1438


674
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
O12_IGKJ1*01
833
1438


675
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
O12_IGKJ1*01
833
1438


676
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
O12_IGKJ1*01
833
1438


677
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
O12_IGKJ1*01
833
1438


678
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
O12_IGKJ1*01
833
1438


679
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
O12_IGKJ1*01
833
1438


680
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
O12_IGKJ1*01
833
1438


681
VH3-23_IGHD6-13*01>1′_IGHJ6*01
2750
O12_IGKJ1*01
833
1438


682
VH3-23_IGHD6-13*01>2′_IGHJ6*01
2751
O12_IGKJ1*01
833
1438


683
VH3-23_IGHD6-13*01>3′_IGHJ6*01
2752
O12_IGKJ1*01
833
1438


684
VH3-23_IGHD6-19*01>1′_IGHJ6*01
2753
O12_IGKJ1*01
833
1438


685
VH3-23_IGHD6-19*01>2′_IGHJ6*01
2754
O12_IGKJ1*01
833
1438


686
VH3-23_IGHD6-19*01>3′_IGHJ6*01
2755
O12_IGKJ1*01
833
1438


687
VH3-23_IGHD6-25*01>1′_IGHJ6*01
2756
O12_IGKJ1*01
833
1438


688
VH3-23_IGHD6-25*01>3′_IGHJ6*01
2757
O12_IGKJ1*01
833
1438


689
VH3-23_IGHD7-27*01>1′_IGHJ6*01
2758
O12_IGKJ1*01
833
1438


690
VH3-23_IGHD7-27*01>2′_IGHJ6*01
2759
O12_IGKJ1*01
833
1438


691
VH3-23_IGHD1-1*01>1_IGHJ1*01
2070
O18_IGKJ1*01
834
1439


692
VH3-23_IGHD1-1*01>2_IGHJ1*01
2071
O18_IGKJ1*01
834
1439


693
VH3-23_IGHD1-1*01>3_IGHJ1*01
2072
O18_IGKJ1*01
834
1439


694
VH3-23_IGHD1-7*01>1_IGHJ1*01
2073
O18_IGKJ1*01
834
1439


695
VH3-23_IGHD1-7*01>3_IGHJ1*01
2074
O18_IGKJ1*01
834
1439


696
VH3-23_IGHD1-14*01>1_IGHJ1*01
2075
O18_IGKJ1*01
834
1439


697
VH3-23_IGHD1-14*01>3_IGHJ1*01
2076
O18_IGKJ1*01
834
1439


698
VH3-23_IGHD1-20*01>1_IGHJ1*01
2077
O18_IGKJ1*01
834
1439


699
VH3-23_IGHD1-20*01>3_IGHJ1*01
2078
O18_IGKJ1*01
834
1439


700
VH3-23_IGHD1-26*01>1_IGHJ1*01
2079
O18_IGKJ1*01
834
1439


701
VH3-23_IGHD1-26*01>3_IGHJ1*01
2080
O18_IGKJ1*01
834
1439


702
VH3-23_IGHD2-2*01>2_IGHJ1*01
2081
O18_IGKJ1*01
834
1439


703
VH3-23_IGHD2-2*01>3_IGHJ1*01
2082
O18_IGKJ1*01
834
1439


704
VH3-23_IGHD2-8*01>2_IGHJ1*01
2083
O18_IGKJ1*01
834
1439


705
VH3-23_IGHD2-8*01>3_IGHJ1*01
2084
O18_IGKJ1*01
834
1439


706
VH3-23_IGHD2-15*01>2_IGHJ1*01
2085
O18_IGKJ1*01
834
1439


707
VH3-23_IGHD2-15*01>3_IGHJ1*01
2086
O18_IGKJ1*01
834
1439


708
VH3-23_IGHD2-21*01>2_IGHJ1*01
2087
O18_IGKJ1*01
834
1439


709
VH3-23_IGHD2-21*01>3_IGHJ1*01
2088
O18_IGKJ1*01
834
1439


710
VH3-23_IGHD3-3*01>1_IGHJ1*01
2089
O18_IGKJ1*01
834
1439


711
VH3-23_IGHD3-3*01>2_IGHJ1*01
2090
O18_IGKJ1*01
834
1439


712
VH3-23_IGHD3-3*01>3_IGHJ1*01
2091
O18_IGKJ1*01
834
1439


713
VH3-23_IGHD3-9*01>2_IGHJ1*01
2092
O18_IGKJ1*01
834
1439


714
VH3-23_IGHD3-10*01>2_IGHJ1*01
2093
O18_IGKJ1*01
834
1439


715
VH3-23_IGHD3-10*01>3_IGHJ1*01
2094
O18_IGKJ1*01
834
1439


716
VH3-23_IGHD3-16*01>2_IGHJ1*01
2095
O18_IGKJ1*01
834
1439


717
VH3-23_IGHD3-16*01>3_IGHJ1*01
2096
O18_IGKJ1*01
834
1439


718
VH3-23_IGHD3-22*01>2_IGHJ1*01
2097
O18_IGKJ1*01
834
1439


719
VH3-23_IGHD3-22*01>3_IGHJ1*01
2098
O18_IGKJ1*01
834
1439


720
VH3-23_IGHD4-4*01 (1) >2_IGHJ1*01
2099
O18_IGKJ1*01
834
1439


721
VH3-23_IGHD4-4*01 (1) >3_IGHJ1*01
2100
O18_IGKJ1*01
834
1439


722
VH3-23_IGHD4-11*01 (1) >2_IGHJ1*01
2101
O18_IGKJ1*01
834
1439


723
VH3-23_IGHD4-11*01 (1) >3_IGHJ1*01
2102
O18_IGKJ1*01
834
1439


724
VH3-23_IGHD4-17*01>2_IGHJ1*01
2103
O18_IGKJ1*01
834
1439


725
VH3-23_IGHD4-17*01>3_IGHJ1*01
2104
O18_IGKJ1*01
834
1439


726
VH3-23_IGHD4-23*01>2_IGHJ1*01
2105
O18_IGKJ1*01
834
1439


727
VH3-23_IGHD4-23*01>3_IGHJ1*01
2106
O18_IGKJ1*01
834
1439


728
VH3-23_IGHD5-5*01 (2) >1_IGHJ1*01
2107
O18_IGKJ1*01
834
1439


729
VH3-23_IGHD5-5*01 (2) >2_IGHJ1*01
2108
O18_IGKJ1*01
834
1439


730
VH3-23_IGHD5-5*01 (2) >3_IGHJ1*01
2109
O18_IGKJ1*01
834
1439


731
VH3-23_IGHD5-12*01>1_IGHJ1*01
2110
O18_IGKJ1*01
834
1439


732
VH3-23_IGHD5-12*01>3_IGHJ1*01
2111
O18_IGKJ1*01
834
1439


733
VH3-23_IGHD5-18*01 (2) >1_IGHJ1*01
2112
O18_IGKJ1*01
834
1439


734
VH3-23_IGHD5-18*01 (2) >2_IGHJ1*01
2113
O18_IGKJ1*01
834
1439


735
VH3-23_IGHD5-18*01 (2) >3_IGHJ1*01
2114
O18_IGKJ1*01
834
1439


736
VH3-23_IGHD5-24*01>1_IGHJ1*01
2115
O18_IGKJ1*01
834
1439


737
VH3-23_IGHD5-24*01>3_IGHJ1*01
2116
O18_IGKJ1*01
834
1439


738
VH3-23_IGHD6-6*01>1_IGHJ1*01
2117
O18_IGKJ1*01
834
1439


739
VH3-23_IGHD1-1*01>1′_IGHJ1*01
2127
O18_IGKJ1*01
834
1439


740
VH3-23_IGHD1-1*01>2′_IGHJ1*01
2128
O18_IGKJ1*01
834
1439


741
VH3-23_IGHD1-1*01>3′_IGHJ1*01
2129
O18_IGKJ1*01
834
1439


742
VH3-23_IGHD1-7*01>1′_IGHJ1*01
2130
O18_IGKJ1*01
834
1439


743
VH3-23_IGHD1-7*01>3′_IGHJ1*01
2131
O18_IGKJ1*01
834
1439


744
VH3-23_IGHD1-14*01>1′_IGHJ1*01
2132
O18_IGKJ1*01
834
1439


745
VH3-23_IGHD1-14*01>2′_IGHJ1*01
2133
O18_IGKJ1*01
834
1439


746
VH3-23_IGHD1-14*01>3′_IGHJ1*01
2134
O18_IGKJ1*01
834
1439


747
VH3-23_IGHD1-20*01>1′_IGHJ1*01
2135
O18_IGKJ1*01
834
1439


748
VH3-23_IGHD1-20*01>2′_IGHJ1*01
2136
O18_IGKJ1*01
834
1439


749
VH3-23_IGHD1-20*01>3′_IGHJ1*01
2137
O18_IGKJ1*01
834
1439


750
VH3-23_IGHD1-26*01>1′_IGHJ1*01
2138
O18_IGKJ1*01
834
1439


751
VH3-23_IGHD1-26*01>3′_IGHJ1*01
2139
O18_IGKJ1*01
834
1439


752
VH3-23_IGHD2-2*01>1′_IGHJ1*01
2140
O18_IGKJ1*01
834
1439


753
VH3-23_IGHD2-2*01>3′_IGHJ1*01
2141
O18_IGKJ1*01
834
1439


754
VH3-23_IGHD2-8*01>1′_IGHJ1*01
2142
O18_IGKJ1*01
834
1439


755
VH3-23_IGHD2-15*01>1′_IGHJ1*01
2143
O18_IGKJ1*01
834
1439


756
VH3-23_IGHD2-15*01>3′_IGHJ1*01
2144
O18_IGKJ1*01
834
1439


757
VH3-23_IGHD2-21*01>1′_IGHJ1*01
2145
O18_IGKJ1*01
834
1439


758
VH3-23_IGHD2-21*01>3′_IGHJ1*01
2146
O18_IGKJ1*01
834
1439


759
VH3-23_IGHD3-3*01>1′_IGHJ1*01
2147
O18_IGKJ1*01
834
1439


760
VH3-23_IGHD3-3*01>3′_IGHJ1*01
2148
O18_IGKJ1*01
834
1439


761
VH3-23_IGHD3-9*01>1′_IGHJ1*01
2149
O18_IGKJ1*01
834
1439


762
VH3-23_IGHD3-9*01>3′_IGHJ1*01
2150
O18_IGKJ1*01
834
1439


763
VH3-23_IGHD3-10*01>1′_IGHJ1*01
2151
O18_IGKJ1*01
834
1439


764
VH3-23_IGHD3-10*01>3′_IGHJ1*01
2152
O18_IGKJ1*01
834
1439


765
VH3-23_IGHD3-16*01>1′_IGHJ1*01
2153
O18_IGKJ1*01
834
1439


766
VH3-23_IGHD3-16*01>3′_IGHJ1*01
2154
O18_IGKJ1*01
834
1439


767
VH3-23_IGHD3-22*01>1′_IGHJ1*01
2155
O18_IGKJ1*01
834
1439


768
VH3-23_IGHD4-4*01 (1) >1′_IGHJ1*01
2156
O18_IGKJ1*01
834
1439


769
VH3-23_IGHD4-4*01 (1) >3′_IGHJ1*01
2157
O18_IGKJ1*01
834
1439


770
VH3-23_IGHD4-11*01 (1) >1′_IGHJ1*01
2158
O18_IGKJ1*01
834
1439


771
VH3-23_IGHD4-11*01 (1) >3′_IGHJ1*01
2159
O18_IGKJ1*01
834
1439


772
VH3-23_IGHD4-17*01>1′_IGHJ1*01
2160
O18_IGKJ1*01
834
1439


773
VH3-23_IGHD4-17*01>3′_IGHJ1*01
2161
O18_IGKJ1*01
834
1439


774
VH3-23_IGHD4-23*01>1′_IGHJ1*01
2162
O18_IGKJ1*01
834
1439


775
VH3-23_IGHD4-23*01>3′_IGHJ1*01
2163
O18_IGKJ1*01
834
1439


776
VH3-23_IGHD5-5*01 (2) >1′_IGHJ1*01
2164
O18_IGKJ1*01
834
1439


777
VH3-23_IGHD5-5*01 (2) >3′_IGHJ1*01
2165
O18_IGKJ1*01
834
1439


778
VH3-23_IGHD5-12*01>1′_IGHJ1*01
2166
O18_IGKJ1*01
834
1439


779
VH3-23_IGHD5-12*01>3′_IGHJ1*01
2167
O18_IGKJ1*01
834
1439


780
VH3-23_IGHD5-18*01 (2) >1′_IGHJ1*01
2168
O18_IGKJ1*01
834
1439


781
VH3-23_IGHD5-18*01 (2) >3′_IGHJ1*01
2169
O18_IGKJ1*01
834
1439


782
VH3-23_IGHD5-24*01>1′_IGHJ1*01
2170
O18_IGKJ1*01
834
1439


783
VH3-23_IGHD5-24*01>3′_IGHJ1*01
2171
O18_IGKJ1*01
834
1439


784
VH3-23_IGHD6-6*01>1′_IGHJ1*01
2172
O18_IGKJ1*01
834
1439


785
VH3-23_IGHD6-6*01>2′_IGHJ1*01
2173
O18_IGKJ1*01
834
1439


786
VH3-23_IGHD6-6*01>3′_IGHJ1*01
2174
O18_IGKJ1*01
834
1439


787
VH3-23_IGHD7-27*01>1′_IGHJ6*01
2758
O18_IGKJ1*01
834
1439


788
VH3-23_IGHD6-13*01>2_IGHJ6*01
2695
O18_IGKJ1*01
834
1439


789
VH3-23_IGHD6-19*01>1_IGHJ6*01
2696
O18_IGKJ1*01
834
1439


790
VH3-23_IGHD6-19*01>2_IGHJ6*01
2697
O18_IGKJ1*01
834
1439


791
VH3-23_IGHD6-25*01>1_IGHJ6*01
2698
O18_IGKJ1*01
834
1439


792
VH3-23_IGHD6-25*01>2_IGHJ6*01
2699
O18_IGKJ1*01
834
1439


793
VH3-23_IGHD7-27*01>1_IGHJ6*01
2700
O18_IGKJ1*01
834
1439


794
VH3-23_IGHD7-27*01>3_IGHJ6*01
2701
O18_IGKJ1*01
834
1439


795
VH3-23_IGHD6-13*01>1′_IGHJ6*01
2750
O18_IGKJ1*01
834
1439


796
VH3-23_IGHD6-13*01>2′_IGHJ6*01
2751
O18_IGKJ1*01
834
1439


797
VH3-23_IGHD6-13*01>2_IGHJ6*01_B
2695
O18_IGKJ1*01
834
1439


798
VH3-23_IGHD6-19*01>1′_IGHJ6*01
2753
O18_IGKJ1*01
834
1439


799
VH3-23_IGHD6-19*01>2′_IGHJ6*01
2754
O18_IGKJ1*01
834
1439


800
VH3-23_IGHD6-25*01>1_IGHJ6*01_B
2698
O18_IGKJ1*01
834
1439


801
VH3-23_IGHD6-25*01>1′_IGHJ6*01
2756
O18_IGKJ1*01
834
1439


802
VH3-23_IGHD6-25*01>3′_IGHJ6*01
2757
O18_IGKJ1*01
834
1439


803
VH3-23_IGHD7-27*01>1′_IGHJ6*01
2758
O18_IGKJ1*01
834
1439


804
VH3-23_IGHD7-27*01>2′_IGHJ6*01
2759
O18_IGKJ1*01
834
1439


805
VH3-23_IGHD7-27*01>1′_IGHJ6*01
2758
A20_IGKJ1*01
809
1414


806
VH3-23_IGHD6-13*01>2_IGHJ6*01
2695
A20_IGKJ1*01
809
1414


807
VH3-23_IGHD6-19*01>1_IGHJ6*01
2696
A20_IGKJ1*01
809
1414


808
VH3-23_IGHD6-19*01>2_IGHJ6*01
2697
A20_IGKJ1*01
809
1414


809
VH3-23_IGHD6-25*01>1_IGHJ6*01
2698
A20_IGKJ1*01
809
1414


810
VH3-23_IGHD6-25*01>2_IGHJ6*01
2699
A20_IGKJ1*01
809
1414


811
VH3-23_IGHD7-27*01>1_IGHJ6*01
2700
A20_IGKJ1*01
809
1414


812
VH3-23_IGHD7-27*01>3_IGHJ6*01
2701
A20_IGKJ1*01
809
1414


813
VH3-23_IGHD6-13*01>1′_IGHJ6*01
2750
A20_IGKJ1*01
809
1414


814
VH3-23_IGHD6-13*01>2′_IGHJ6*01
2751
A20_IGKJ1*01
809
1414


815
VH3-23_IGHD6-13*01>2_IGHJ6*01_B
2695
A20_IGKJ1*01
809
1414


816
VH3-23_IGHD6-19*01>1′_IGHJ6*01
2753
A20_IGKJ1*01
809
1414


817
VH3-23_IGHD6-19*01>2′_IGHJ6*01
2754
A20_IGKJ1*01
809
1414


818
VH3-23_IGHD6-25*01>1_IGHJ6*01_B
2698
A20_IGKJ1*01
809
1414


819
VH3-23_IGHD6-25*01>1′_IGHJ6*01
2756
A20_IGKJ1*01
809
1414


820
VH3-23_IGHD6-25*01>3′_IGHJ6*01
2757
A20_IGKJ1*01
809
1414


821
VH3-23_IGHD7-27*01>1′_IGHJ6*01
2758
A20_IGKJ1*01
809
1414


822
VH3-23_IGHD7-27*01>2′_IGHJ6*01
2759
A20_IGKJ1*01
809
1414


823
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
L11_IGKJ1*01
819
1424


824
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
L11_IGKJ1*01
819
1424


825
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
L11_IGKJ1*01
819
1424


826
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
L11_IGKJ1*01
819
1424


827
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
L11_IGKJ1*01
819
1424


828
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
L11_IGKJ1*01
819
1424


829
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
L11_IGKJ1*01
819
1424


830
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
L11_IGKJ1*01
819
1424


831
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
L11_IGKJ1*01
819
1424


832
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
L11_IGKJ1*01
819
1424


833
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
L11_IGKJ1*01
819
1424


834
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
L11_IGKJ1*01
819
1424


835
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
L11_IGKJ1*01
819
1424


836
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
L11_IGKJ1*01
819
1424


837
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
L11_IGKJ1*01
819
1424


838
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
L11_IGKJ1*01
819
1424


839
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
L11_IGKJ1*01
819
1424


840
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
L11_IGKJ1*01
819
1424


841
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
L11_IGKJ1*01
819
1424


842
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
L11_IGKJ1*01
819
1424


843
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
L11_IGKJ1*01
819
1424


844
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
L11_IGKJ1*01
819
1424


845
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
L11_IGKJ1*01
819
1424


846
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
L11_IGKJ1*01
819
1424


847
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
L11_IGKJ1*01
819
1424


848
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
L11_IGKJ1*01
819
1424


849
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
L11_IGKJ1*01
819
1424


850
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
L11_IGKJ1*01
819
1424


851
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
L11_IGKJ1*01
819
1424


852
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
L11_IGKJ1*01
819
1424


853
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
L11_IGKJ1*01
819
1424


854
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
L11_IGKJ1*01
819
1424


855
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
L11_IGKJ1*01
819
1424


856
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
L11_IGKJ1*01
819
1424


857
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
L11_IGKJ1*01
819
1424


858
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
L11_IGKJ1*01
819
1424


859
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
L11_IGKJ1*01
819
1424


860
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
L11_IGKJ1*01
819
1424


861
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
L11_IGKJ1*01
819
1424


862
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
L11_IGKJ1*01
819
1424


863
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
L11_IGKJ1*01
819
1424


864
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
L11_IGKJ1*01
819
1424


865
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
L11_IGKJ1*01
819
1424


866
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
L11_IGKJ1*01
819
1424


867
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
L11_IGKJ1*01
819
1424


868
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
L11_IGKJ1*01
819
1424


869
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
L11_IGKJ1*01
819
1424


870
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
L11_IGKJ1*01
819
1424


871
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
L11_IGKJ1*01
819
1424


872
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
L11_IGKJ1*01
819
1424


873
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
L11_IGKJ1*01
819
1424


874
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
L11_IGKJ1*01
819
1424


875
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
L11_IGKJ1*01
819
1424


876
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
L11_IGKJ1*01
819
1424


877
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
L11_IGKJ1*01
819
1424


878
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
L11_IGKJ1*01
819
1424


879
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
L11_IGKJ1*01
819
1424


880
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
L11_IGKJ1*01
819
1424


881
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
L11_IGKJ1*01
819
1424


882
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
L11_IGKJ1*01
819
1424


883
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
L11_IGKJ1*01
819
1424


884
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
L11_IGKJ1*01
819
1424


885
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
L11_IGKJ1*01
819
1424


886
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
L11_IGKJ1*01
819
1424


887
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
L11_IGKJ1*01
819
1424


888
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
L11_IGKJ1*01
819
1424


889
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
L11_IGKJ1*01
819
1424


890
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
L11_IGKJ1*01
819
1424


891
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
L11_IGKJ1*01
819
1424


892
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
L11_IGKJ1*01
819
1424


893
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
L11_IGKJ1*01
819
1424


894
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
L11_IGKJ1*01
819
1424


895
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
L11_IGKJ1*01
819
1424


896
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
L11_IGKJ1*01
819
1424


897
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
L11_IGKJ1*01
819
1424


898
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
L11_IGKJ1*01
819
1424


899
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
L11_IGKJ1*01
819
1424


900
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
L11_IGKJ1*01
819
1424


901
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
L11_IGKJ1*01
819
1424


902
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
L11_IGKJ1*01
819
1424


903
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
L11_IGKJ1*01
819
1424


904
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
L11_IGKJ1*01
819
1424


905
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
L11_IGKJ1*01
819
1424


906
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
L11_IGKJ1*01
819
1424


907
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
L11_IGKJ1*01
819
1424


908
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
L11_IGKJ1*01
819
1424


909
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
L11_IGKJ1*01
819
1424


910
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
L11_IGKJ1*01
819
1424


911
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
L11_IGKJ1*01
819
1424


912
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
L11_IGKJ1*01
819
1424


913
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
L11_IGKJ1*01
819
1424


914
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
L11_IGKJ1*01
819
1424


915
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
L11_IGKJ1*01
819
1424


916
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
L11_IGKJ1*01
819
1424


917
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
L11_IGKJ1*01
819
1424


918
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
L11_IGKJ1*01
819
1424


919
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
L12_IGKJ1*01
820
1425


920
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
L12_IGKJ1*01
820
1425


921
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
L12_IGKJ1*01
820
1425


922
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
L12_IGKJ1*01
820
1425


923
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
L12_IGKJ1*01
820
1425


924
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
L12_IGKJ1*01
820
1425


925
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
L12_IGKJ1*01
820
1425


926
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
L12_IGKJ1*01
820
1425


927
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
L12_IGKJ1*01
820
1425


928
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
L12_IGKJ1*01
820
1425


929
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
L12_IGKJ1*01
820
1425


930
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
L12_IGKJ1*01
820
1425


931
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
L12_IGKJ1*01
820
1425


932
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
L12_IGKJ1*01
820
1425


933
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
L12_IGKJ1*01
820
1425


934
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
L12_IGKJ1*01
820
1425


935
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
L12_IGKJ1*01
820
1425


936
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
L12_IGKJ1*01
820
1425


937
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
L12_IGKJ1*01
820
1425


938
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
L12_IGKJ1*01
820
1425


939
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
L12_IGKJ1*01
820
1425


940
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
L12_IGKJ1*01
820
1425


941
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
L12_IGKJ1*01
820
1425


942
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
L12_IGKJ1*01
820
1425


943
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
L12_IGKJ1*01
820
1425


944
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
L12_IGKJ1*01
820
1425


945
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
L12_IGKJ1*01
820
1425


946
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
L12_IGKJ1*01
820
1425


947
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
L12_IGKJ1*01
820
1425


948
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
L12_IGKJ1*01
820
1425


949
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
L12_IGKJ1*01
820
1425


950
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
L12_IGKJ1*01
820
1425


951
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
L12_IGKJ1*01
820
1425


952
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
L12_IGKJ1*01
820
1425


953
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
L12_IGKJ1*01
820
1425


954
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
L12_IGKJ1*01
820
1425


955
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
L12_IGKJ1*01
820
1425


956
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
L12_IGKJ1*01
820
1425


957
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
L12_IGKJ1*01
820
1425


958
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
L12_IGKJ1*01
820
1425


959
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
L12_IGKJ1*01
820
1425


960
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
L12_IGKJ1*01
820
1425


961
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
L12_IGKJ1*01
820
1425


962
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
L12_IGKJ1*01
820
1425


963
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
L12_IGKJ1*01
820
1425


964
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
L12_IGKJ1*01
820
1425


965
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
L12_IGKJ1*01
820
1425


966
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
L12_IGKJ1*01
820
1425


967
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
L12_IGKJ1*01
820
1425


968
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
L12_IGKJ1*01
820
1425


969
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
L12_IGKJ1*01
820
1425


970
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
L12_IGKJ1*01
820
1425


971
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
L12_IGKJ1*01
820
1425


972
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
L12_IGKJ1*01
820
1425


973
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
L12_IGKJ1*01
820
1425


974
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
L12_IGKJ1*01
820
1425


975
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
L12_IGKJ1*01
820
1425


976
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
L12_IGKJ1*01
820
1425


977
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
L12_IGKJ1*01
820
1425


978
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
L12_IGKJ1*01
820
1425


979
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
L12_IGKJ1*01
820
1425


980
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
L12_IGKJ1*01
820
1425


981
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
L12_IGKJ1*01
820
1425


982
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
L12_IGKJ1*01
820
1425


983
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
L12_IGKJ1*01
820
1425


984
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
L12_IGKJ1*01
820
1425


985
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
L12_IGKJ1*01
820
1425


986
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
L12_IGKJ1*01
820
1425


987
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
L12_IGKJ1*01
820
1425


988
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
L12_IGKJ1*01
820
1425


989
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
L12_IGKJ1*01
820
1425


990
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
L12_IGKJ1*01
820
1425


991
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
L12_IGKJ1*01
820
1425


992
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
L12_IGKJ1*01
820
1425


993
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
L12_IGKJ1*01
820
1425


994
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
L12_IGKJ1*01
820
1425


995
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
L12_IGKJ1*01
820
1425


996
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
L12_IGKJ1*01
820
1425


997
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
L12_IGKJ1*01
820
1425


998
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
L12_IGKJ1*01
820
1425


999
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
L12_IGKJ1*01
820
1425


1000
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
L12_IGKJ1*01
820
1425


1001
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
L12_IGKJ1*01
820
1425


1002
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
L12_IGKJ1*01
820
1425


1003
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
L12_IGKJ1*01
820
1425


1004
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
L12_IGKJ1*01
820
1425


1005
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
L12_IGKJ1*01
820
1425


1006
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
L12_IGKJ1*01
820
1425


1007
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
L12_IGKJ1*01
820
1425


1008
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
L12_IGKJ1*01
820
1425


1009
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
L12_IGKJ1*01
820
1425


1010
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
L12_IGKJ1*01
820
1425


1011
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
L12_IGKJ1*01
820
1425


1012
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
L12_IGKJ1*01
820
1425


1013
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
L12_IGKJ1*01
820
1425


1014
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
L12_IGKJ1*01
820
1425


1015
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
O1_IGKJ1*01
832
1437


1016
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
O1_IGKJ1*01
832
1437


1017
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
O1_IGKJ1*01
832
1437


1018
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
O1_IGKJ1*01
832
1437


1019
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
O1_IGKJ1*01
832
1437


1020
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
O1_IGKJ1*01
832
1437


1021
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
O1_IGKJ1*01
832
1437


1022
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
O1_IGKJ1*01
832
1437


1023
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
O1_IGKJ1*01
832
1437


1024
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
O1_IGKJ1*01
832
1437


1025
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
O1_IGKJ1*01
832
1437


1026
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
O1_IGKJ1*01
832
1437


1027
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
O1_IGKJ1*01
832
1437


1028
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
O1_IGKJ1*01
832
1437


1029
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
O1_IGKJ1*01
832
1437


1030
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
O1_IGKJ1*01
832
1437


1031
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
O1_IGKJ1*01
832
1437


1032
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
O1_IGKJ1*01
832
1437


1033
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
O1_IGKJ1*01
832
1437


1034
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
O1_IGKJ1*01
832
1437


1035
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
O1_IGKJ1*01
832
1437


1036
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
O1_IGKJ1*01
832
1437


1037
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
O1_IGKJ1*01
832
1437


1038
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
O1_IGKJ1*01
832
1437


1039
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
O1_IGKJ1*01
832
1437


1040
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
O1_IGKJ1*01
832
1437


1041
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
O1_IGKJ1*01
832
1437


1042
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
O1_IGKJ1*01
832
1437


1043
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
O1_IGKJ1*01
832
1437


1044
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
O1_IGKJ1*01
832
1437


1045
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
O1_IGKJ1*01
832
1437


1046
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
O1_IGKJ1*01
832
1437


1047
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
O1_IGKJ1*01
832
1437


1048
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
O1_IGKJ1*01
832
1437


1049
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
O1_IGKJ1*01
832
1437


1050
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
O1_IGKJ1*01
832
1437


1051
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
O1_IGKJ1*01
832
1437


1052
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
O1_IGKJ1*01
832
1437


1053
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
O1_IGKJ1*01
832
1437


1054
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
O1_IGKJ1*01
832
1437


1055
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
O1_IGKJ1*01
832
1437


1056
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
O1_IGKJ1*01
832
1437


1057
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
O1_IGKJ1*01
832
1437


1058
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
O1_IGKJ1*01
832
1437


1059
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
O1_IGKJ1*01
832
1437


1060
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
O1_IGKJ1*01
832
1437


1061
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
O1_IGKJ1*01
832
1437


1062
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
O1_IGKJ1*01
832
1437


1063
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
O1_IGKJ1*01
832
1437


1064
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
O1_IGKJ1*01
832
1437


1065
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
O1_IGKJ1*01
832
1437


1066
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
O1_IGKJ1*01
832
1437


1067
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
O1_IGKJ1*01
832
1437


1068
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
O1_IGKJ1*01
832
1437


1069
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
O1_IGKJ1*01
832
1437


1070
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
O1_IGKJ1*01
832
1437


1071
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
O1_IGKJ1*01
832
1437


1072
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
O1_IGKJ1*01
832
1437


1073
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
O1_IGKJ1*01
832
1437


1074
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
O1_IGKJ1*01
832
1437


1075
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
O1_IGKJ1*01
832
1437


1076
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
O1_IGKJ1*01
832
1437


1077
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
O1_IGKJ1*01
832
1437


1078
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
O1_IGKJ1*01
832
1437


1079
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
O1_IGKJ1*01
832
1437


1080
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
O1_IGKJ1*01
832
1437


1081
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
O1_IGKJ1*01
832
1437


1082
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
O1_IGKJ1*01
832
1437


1083
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
O1_IGKJ1*01
832
1437


1084
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
O1_IGKJ1*01
832
1437


1085
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
O1_IGKJ1*01
832
1437


1086
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
O1_IGKJ1*01
832
1437


1087
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
O1_IGKJ1*01
832
1437


1088
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
O1_IGKJ1*01
832
1437


1089
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
O1_IGKJ1*01
832
1437


1090
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
O1_IGKJ1*01
832
1437


1091
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
O1_IGKJ1*01
832
1437


1092
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
O1_IGKJ1*01
832
1437


1093
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
O1_IGKJ1*01
832
1437


1094
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
O1_IGKJ1*01
832
1437


1095
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
O1_IGKJ1*01
832
1437


1096
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
O1_IGKJ1*01
832
1437


1097
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
O1_IGKJ1*01
832
1437


1098
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
O1_IGKJ1*01
832
1437


1099
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
O1_IGKJ1*01
832
1437


1100
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
O1_IGKJ1*01
832
1437


1101
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
O1_IGKJ1*01
832
1437


1102
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
O1_IGKJ1*01
832
1437


1103
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
O1_IGKJ1*01
832
1437


1104
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
O1_IGKJ1*01
832
1437


1105
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
O1_IGKJ1*01
832
1437


1106
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
O1_IGKJ1*01
832
1437


1107
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
O1_IGKJ1*01
832
1437


1108
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
O1_IGKJ1*01
832
1437


1109
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
O1_IGKJ1*01
832
1437


1110
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
O1_IGKJ1*01
832
1437


1111
VH3-23_IGHD1-1*01>1_IGHJ5*01
2530
A2_IGKJ1*01
808
1413


1112
VH3-23_IGHD1-1*01>2_IGHJ5*01
2531
A2_IGKJ1*01
808
1413


1113
VH3-23_IGHD1-1*01>3_IGHJ5*01
2532
A2_IGKJ1*01
808
1413


1114
VH3-23_IGHD1-7*01>1_IGHJ5*01
2533
A2_IGKJ1*01
808
1413


1115
VH3-23_IGHD1-7*01>3_IGHJ5*01
2534
A2_IGKJ1*01
808
1413


1116
VH3-23_IGHD1-14*01>1_IGHJ5*01
2535
A2_IGKJ1*01
808
1413


1117
VH3-23_IGHD1-14*01>3_IGHJ5*01
2536
A2_IGKJ1*01
808
1413


1118
VH3-23_IGHD1-20*01>1_IGHJ5*01
2537
A2_IGKJ1*01
808
1413


1119
VH3-23_IGHD1-20*01>3_IGHJ5*01
2538
A2_IGKJ1*01
808
1413


1120
VH3-23_IGHD1-26*01>1_IGHJ5*01
2539
A2_IGKJ1*01
808
1413


1121
VH3-23_IGHD1-26*01>3_IGHJ5*01
2540
A2_IGKJ1*01
808
1413


1122
VH3-23_IGHD2-2*01>2_IGHJ5*01
2541
A2_IGKJ1*01
808
1413


1123
VH3-23_IGHD2-2*01>3_IGHJ5*01
2542
A2_IGKJ1*01
808
1413


1124
VH3-23_IGHD2-8*01>2_IGHJ5*01
2543
A2_IGKJ1*01
808
1413


1125
VH3-23_IGHD2-8*01>3_IGHJ5*01
2544
A2_IGKJ1*01
808
1413


1126
VH3-23_IGHD2-15*01>2_IGHJ5*01
2545
A2_IGKJ1*01
808
1413


1127
VH3-23_IGHD2-15*01>3_IGHJ5*01
2546
A2_IGKJ1*01
808
1413


1128
VH3-23_IGHD2-21*01>2_IGHJ5*01
2547
A2_IGKJ1*01
808
1413


1129
VH3-23_IGHD2-21*01>3_IGHJ5*01
2548
A2_IGKJ1*01
808
1413


1130
VH3-23_IGHD3-3*01>1_IGHJ5*01
2549
A2_IGKJ1*01
808
1413


1131
VH3-23_IGHD3-3*01>2_IGHJ5*01
2550
A2_IGKJ1*01
808
1413


1132
VH3-23_IGHD3-3*01>3_IGHJ5*01
2551
A2_IGKJ1*01
808
1413


1133
VH3-23_IGHD3-9*01>2_IGHJ5*01
2552
A2_IGKJ1*01
808
1413


1134
VH3-23_IGHD3-10*01>2_IGHJ5*01
2553
A2_IGKJ1*01
808
1413


1135
VH3-23_IGHD3-10*01>3_IGHJ5*01
2554
A2_IGKJ1*01
808
1413


1136
VH3-23_IGHD3-16*01>2_IGHJ5*01
2555
A2_IGKJ1*01
808
1413


1137
VH3-23_IGHD3-16*01>3_IGHJ5*01
2556
A2_IGKJ1*01
808
1413


1138
VH3-23_IGHD3-22*01>2_IGHJ5*01
2557
A2_IGKJ1*01
808
1413


1139
VH3-23_IGHD3-22*01>3_IGHJ5*01
2558
A2_IGKJ1*01
808
1413


1140
VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01
2559
A2_IGKJ1*01
808
1413


1141
VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01
2560
A2_IGKJ1*01
808
1413


1142
VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01
2561
A2_IGKJ1*01
808
1413


1143
VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01
2562
A2_IGKJ1*01
808
1413


1144
VH3-23_IGHD4-17*01>2_IGHJ5*01
2563
A2_IGKJ1*01
808
1413


1145
VH3-23_IGHD4-17*01>3_IGHJ5*01
2564
A2_IGKJ1*01
808
1413


1146
VH3-23_IGHD4-23*01>2_IGHJ5*01
2565
A2_IGKJ1*01
808
1413


1147
VH3-23_IGHD4-23*01>3_IGHJ5*01
2566
A2_IGKJ1*01
808
1413


1148
VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01
2567
A2_IGKJ1*01
808
1413


1149
VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01
2568
A2_IGKJ1*01
808
1413


1150
VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01
2569
A2_IGKJ1*01
808
1413


1151
VH3-23_IGHD5-12*01>1_IGHJ5*01
2570
A2_IGKJ1*01
808
1413


1152
VH3-23_IGHD5-12*01>3_IGHJ5*01
2571
A2_IGKJ1*01
808
1413


1153
VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01
2572
A2_IGKJ1*01
808
1413


1154
VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01
2573
A2_IGKJ1*01
808
1413


1155
VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01
2574
A2_IGKJ1*01
808
1413


1156
VH3-23_IGHD5-24*01>1_IGHJ5*01
2575
A2_IGKJ1*01
808
1413


1157
VH3-23_IGHD5-24*01>3_IGHJ5*01
2576
A2_IGKJ1*01
808
1413


1158
VH3-23_IGHD6-6*01>1_IGHJ5*01
2577
A2_IGKJ1*01
808
1413


1159
VH3-23_IGHD1-1*01>1′_IGHJ5*01
2587
A2_IGKJ1*01
808
1413


1160
VH3-23_IGHD1-1*01>2′_IGHJ5*01
2588
A2_IGKJ1*01
808
1413


1161
VH3-23_IGHD1-1*01>3′_IGHJ5*01
2589
A2_IGKJ1*01
808
1413


1162
VH3-23_IGHD1-7*01>1′_IGHJ5*01
2590
A2_IGKJ1*01
808
1413


1163
VH3-23_IGHD1-7*01>3′_IGHJ5*01
2591
A2_IGKJ1*01
808
1413


1164
VH3-23_IGHD1-14*01>1′_IGHJ5*01
2592
A2_IGKJ1*01
808
1413


1165
VH3-23_IGHD1-14*01>2′_IGHJ5*01
2593
A2_IGKJ1*01
808
1413


1166
VH3-23_IGHD1-14*01>3′_IGHJ5*01
2594
A2_IGKJ1*01
808
1413


1167
VH3-23_IGHD1-20*01>1′_IGHJ5*01
2595
A2_IGKJ1*01
808
1413


1168
VH3-23_IGHD1-20*01>2′_IGHJ5*01
2596
A2_IGKJ1*01
808
1413


1169
VH3-23_IGHD1-20*01>3′_IGHJ5*01
2597
A2_IGKJ1*01
808
1413


1170
VH3-23_IGHD1-26*01>1′_IGHJ5*01
2598
A2_IGKJ1*01
808
1413


1171
VH3-23_IGHD1-26*01>3′_IGHJ5*01
2599
A2_IGKJ1*01
808
1413


1172
VH3-23_IGHD2-2*01>1′_IGHJ5*01
2600
A2_IGKJ1*01
808
1413


1173
VH3-23_IGHD2-2*01>3′_IGHJ5*01
2601
A2_IGKJ1*01
808
1413


1174
VH3-23_IGHD2-8*01>1′_IGHJ5*01
2602
A2_IGKJ1*01
808
1413


1175
VH3-23_IGHD2-15*01>1′_IGHJ5*01
2603
A2_IGKJ1*01
808
1413


1176
VH3-23_IGHD2-15*01>3′_IGHJ5*01
2604
A2_IGKJ1*01
808
1413


1177
VH3-23_IGHD2-21*01>1′_IGHJ5*01
2605
A2_IGKJ1*01
808
1413


1178
VH3-23_IGHD2-21*01>3′_IGHJ5*01
2606
A2_IGKJ1*01
808
1413


1179
VH3-23_IGHD3-3*01>1′_IGHJ5*01
2607
A2_IGKJ1*01
808
1413


1180
VH3-23_IGHD3-3*01>3′_IGHJ5*01
2608
A2_IGKJ1*01
808
1413


1181
VH3-23_IGHD3-9*01>1′_IGHJ5*01
2609
A2_IGKJ1*01
808
1413


1182
VH3-23_IGHD3-9*01>3′_IGHJ5*01
2610
A2_IGKJ1*01
808
1413


1183
VH3-23_IGHD3-10*01>1′_IGHJ5*01
2611
A2_IGKJ1*01
808
1413


1184
VH3-23_IGHD3-10*01>3′_IGHJ5*01
2612
A2_IGKJ1*01
808
1413


1185
VH3-23_IGHD3-16*01>1′_IGHJ5*01
2613
A2_IGKJ1*01
808
1413


1186
VH3-23_IGHD3-16*01>3′_IGHJ5*01
2614
A2_IGKJ1*01
808
1413


1187
VH3-23_IGHD3-22*01>1′_IGHJ5*01
2615
A2_IGKJ1*01
808
1413


1188
VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01
2616
A2_IGKJ1*01
808
1413


1189
VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01
2617
A2_IGKJ1*01
808
1413


1190
VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01
2618
A2_IGKJ1*01
808
1413


1191
VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01
2619
A2_IGKJ1*01
808
1413


1192
VH3-23_IGHD4-17*01>1′_IGHJ5*01
2620
A2_IGKJ1*01
808
1413


1193
VH3-23_IGHD4-17*01>3′_IGHJ5*01
2621
A2_IGKJ1*01
808
1413


1194
VH3-23_IGHD4-23*01>1′_IGHJ5*01
2622
A2_IGKJ1*01
808
1413


1195
VH3-23_IGHD4-23*01>3′_IGHJ5*01
2623
A2_IGKJ1*01
808
1413


1196
VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01
2624
A2_IGKJ1*01
808
1413


1197
VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01
2625
A2_IGKJ1*01
808
1413


1198
VH3-23_IGHD5-12*01>1′_IGHJ5*01
2626
A2_IGKJ1*01
808
1413


1199
VH3-23_IGHD5-12*01>3′_IGHJ5*01
2627
A2_IGKJ1*01
808
1413


1200
VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01
2628
A2_IGKJ1*01
808
1413


1201
VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01
2629
A2_IGKJ1*01
808
1413


1202
VH3-23_IGHD5-24*01>1′_IGHJ5*01
2630
A2_IGKJ1*01
808
1413


1203
VH3-23_IGHD5-24*01>3′_IGHJ5*01
2631
A2_IGKJ1*01
808
1413


1204
VH3-23_IGHD6-6*01>1′_IGHJ5*01
2632
A2_IGKJ1*01
808
1413


1205
VH3-23_IGHD6-6*01>2′_IGHJ5*01
2633
A2_IGKJ1*01
808
1413


1206
VH3-23_IGHD6-6*01>3′_IGHJ5*01
2634
A2_IGKJ1*01
808
1413


1207
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
L2_IGKJ1*01
822
1427


1208
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
L2_IGKJ1*01
822
1427


1209
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
L2_IGKJ1*01
822
1427


1210
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
L2_IGKJ1*01
822
1427


1211
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
L2_IGKJ1*01
822
1427


1212
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
L2_IGKJ1*01
822
1427


1213
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
L2_IGKJ1*01
822
1427


1214
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
L2_IGKJ1*01
822
1427


1215
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
L2_IGKJ1*01
822
1427


1216
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
L2_IGKJ1*01
822
1427


1217
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
L2_IGKJ1*01
822
1427


1218
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
L2_IGKJ1*01
822
1427


1219
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
L2_IGKJ1*01
822
1427


1220
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
L2_IGKJ1*01
822
1427


1221
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
L2_IGKJ1*01
822
1427


1222
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
L2_IGKJ1*01
822
1427


1223
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
L2_IGKJ1*01
822
1427


1224
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
L2_IGKJ1*01
822
1427


1225
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
L2_IGKJ1*01
822
1427


1226
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
L2_IGKJ1*01
822
1427


1227
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
L2_IGKJ1*01
822
1427


1228
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
L2_IGKJ1*01
822
1427


1229
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
L2_IGKJ1*01
822
1427


1230
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
L2_IGKJ1*01
822
1427


1231
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
L2_IGKJ1*01
822
1427


1232
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
L2_IGKJ1*01
822
1427


1233
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
L2_IGKJ1*01
822
1427


1234
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
L2_IGKJ1*01
822
1427


1235
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
L2_IGKJ1*01
822
1427


1236
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
L2_IGKJ1*01
822
1427


1237
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
L2_IGKJ1*01
822
1427


1238
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
L2_IGKJ1*01
822
1427


1239
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
L2_IGKJ1*01
822
1427


1240
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
L2_IGKJ1*01
822
1427


1241
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
L2_IGKJ1*01
822
1427


1242
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
L2_IGKJ1*01
822
1427


1243
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
L2_IGKJ1*01
822
1427


1244
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
L2_IGKJ1*01
822
1427


1245
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
L2_IGKJ1*01
822
1427


1246
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
L2_IGKJ1*01
822
1427


1247
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
L2_IGKJ1*01
822
1427


1248
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
L2_IGKJ1*01
822
1427


1249
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
L2_IGKJ1*01
822
1427


1250
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
L2_IGKJ1*01
822
1427


1251
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
L2_IGKJ1*01
822
1427


1252
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
L2_IGKJ1*01
822
1427


1253
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
L2_IGKJ1*01
822
1427


1254
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
L2_IGKJ1*01
822
1427


1255
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
L2_IGKJ1*01
822
1427


1256
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
L2_IGKJ1*01
822
1427


1257
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
L2_IGKJ1*01
822
1427


1258
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
L2_IGKJ1*01
822
1427


1259
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
L2_IGKJ1*01
822
1427


1260
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
L2_IGKJ1*01
822
1427


1261
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
L2_IGKJ1*01
822
1427


1262
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
L2_IGKJ1*01
822
1427


1263
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
L2_IGKJ1*01
822
1427


1264
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
L2_IGKJ1*01
822
1427


1265
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
L2_IGKJ1*01
822
1427


1266
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
L2_IGKJ1*01
822
1427


1267
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
L2_IGKJ1*01
822
1427


1268
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
L2_IGKJ1*01
822
1427


1269
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
L2_IGKJ1*01
822
1427


1270
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
L2_IGKJ1*01
822
1427


1271
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
L2_IGKJ1*01
822
1427


1272
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
L2_IGKJ1*01
822
1427


1273
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
L2_IGKJ1*01
822
1427


1274
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
L2_IGKJ1*01
822
1427


1275
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
L2_IGKJ1*01
822
1427


1276
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
L2_IGKJ1*01
822
1427


1277
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
L2_IGKJ1*01
822
1427


1278
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
L2_IGKJ1*01
822
1427


1279
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
L2_IGKJ1*01
822
1427


1280
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
L2_IGKJ1*01
822
1427


1281
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
L2_IGKJ1*01
822
1427


1282
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
L2_IGKJ1*01
822
1427


1283
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
L2_IGKJ1*01
822
1427


1284
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
L2_IGKJ1*01
822
1427


1285
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
L2_IGKJ1*01
822
1427


1286
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
L2_IGKJ1*01
822
1427


1287
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
L2_IGKJ1*01
822
1427


1288
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
L2_IGKJ1*01
822
1427


1289
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
L2_IGKJ1*01
822
1427


1290
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
L2_IGKJ1*01
822
1427


1291
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
L2_IGKJ1*01
822
1427


1292
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
L2_IGKJ1*01
822
1427


1293
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
L2_IGKJ1*01
822
1427


1294
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
L2_IGKJ1*01
822
1427


1295
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
L2_IGKJ1*01
822
1427


1296
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
L2_IGKJ1*01
822
1427


1297
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
L2_IGKJ1*01
822
1427


1298
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
L2_IGKJ1*01
822
1427


1299
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
L2_IGKJ1*01
822
1427


1300
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
L2_IGKJ1*01
822
1427


1301
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
L2_IGKJ1*01
822
1427


1302
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
L2_IGKJ1*01
822
1427


1303
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
L6_IGKJ1*01
829
1434


1304
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
L6_IGKJ1*01
829
1434


1305
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
L6_IGKJ1*01
829
1434


1306
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
L6_IGKJ1*01
829
1434


1307
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
L6_IGKJ1*01
829
1434


1308
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
L6_IGKJ1*01
829
1434


1309
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
L6_IGKJ1*01
829
1434


1310
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
L6_IGKJ1*01
829
1434


1311
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
L6_IGKJ1*01
829
1434


1312
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
L6_IGKJ1*01
829
1434


1313
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
L6_IGKJ1*01
829
1434


1314
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
L6_IGKJ1*01
829
1434


1315
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
L6_IGKJ1*01
829
1434


1316
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
L6_IGKJ1*01
829
1434


1317
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
L6_IGKJ1*01
829
1434


1318
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
L6_IGKJ1*01
829
1434


1319
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
L6_IGKJ1*01
829
1434


1320
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
L6_IGKJ1*01
829
1434


1321
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
L6_IGKJ1*01
829
1434


1322
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
L6_IGKJ1*01
829
1434


1323
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
L6_IGKJ1*01
829
1434


1324
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
L6_IGKJ1*01
829
1434


1325
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
L6_IGKJ1*01
829
1434


1326
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
L6_IGKJ1*01
829
1434


1327
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
L6_IGKJ1*01
829
1434


1328
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
L6_IGKJ1*01
829
1434


1329
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
L6_IGKJ1*01
829
1434


1330
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
L6_IGKJ1*01
829
1434


1331
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
L6_IGKJ1*01
829
1434


1332
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
L6_IGKJ1*01
829
1434


1333
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
L6_IGKJ1*01
829
1434


1334
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
L6_IGKJ1*01
829
1434


1335
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
L6_IGKJ1*01
829
1434


1336
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
L6_IGKJ1*01
829
1434


1337
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
L6_IGKJ1*01
829
1434


1338
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
L6_IGKJ1*01
829
1434


1339
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
L6_IGKJ1*01
829
1434


1340
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
L6_IGKJ1*01
829
1434


1341
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
L6_IGKJ1*01
829
1434


1342
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
L6_IGKJ1*01
829
1434


1343
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
L6_IGKJ1*01
829
1434


1344
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
L6_IGKJ1*01
829
1434


1345
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
L6_IGKJ1*01
829
1434


1346
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
L6_IGKJ1*01
829
1434


1347
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
L6_IGKJ1*01
829
1434


1348
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
L6_IGKJ1*01
829
1434


1349
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
L6_IGKJ1*01
829
1434


1350
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
L6_IGKJ1*01
829
1434


1351
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
L6_IGKJ1*01
829
1434


1352
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
L6_IGKJ1*01
829
1434


1353
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
L6_IGKJ1*01
829
1434


1354
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
L6_IGKJ1*01
829
1434


1355
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
L6_IGKJ1*01
829
1434


1356
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
L6_IGKJ1*01
829
1434


1357
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
L6_IGKJ1*01
829
1434


1358
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
L6_IGKJ1*01
829
1434


1359
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
L6_IGKJ1*01
829
1434


1360
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
L6_IGKJ1*01
829
1434


1361
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
L6_IGKJ1*01
829
1434


1362
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
L6_IGKJ1*01
829
1434


1363
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
L6_IGKJ1*01
829
1434


1364
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
L6_IGKJ1*01
829
1434


1365
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
L6_IGKJ1*01
829
1434


1366
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
L6_IGKJ1*01
829
1434


1367
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
L6_IGKJ1*01
829
1434


1368
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
L6_IGKJ1*01
829
1434


1369
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
L6_IGKJ1*01
829
1434


1370
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
L6_IGKJ1*01
829
1434


1371
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
L6_IGKJ1*01
829
1434


1372
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
L6_IGKJ1*01
829
1434


1373
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
L6_IGKJ1*01
829
1434


1374
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
L6_IGKJ1*01
829
1434


1375
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
L6_IGKJ1*01
829
1434


1376
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
L6_IGKJ1*01
829
1434


1377
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
L6_IGKJ1*01
829
1434


1378
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
L6_IGKJ1*01
829
1434


1379
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
L6_IGKJ1*01
829
1434


1380
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
L6_IGKJ1*01
829
1434


1381
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
L6_IGKJ1*01
829
1434


1382
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
L6_IGKJ1*01
829
1434


1383
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
L6_IGKJ1*01
829
1434


1384
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
L6_IGKJ1*01
829
1434


1385
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
L6_IGKJ1*01
829
1434


1386
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
L6_IGKJ1*01
829
1434


1387
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
L6_IGKJ1*01
829
1434


1388
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
L6_IGKJ1*01
829
1434


1389
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
L6_IGKJ1*01
829
1434


1390
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
L6_IGKJ1*01
829
1434


1391
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
L6_IGKJ1*01
829
1434


1392
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
L6_IGKJ1*01
829
1434


1393
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
L6_IGKJ1*01
829
1434


1394
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
L6_IGKJ1*01
829
1434


1395
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
L6_IGKJ1*01
829
1434


1396
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
L6_IGKJ1*01
829
1434


1397
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
L6_IGKJ1*01
829
1434


1398
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
L6_IGKJ1*01
829
1434


1399
VH3-23_IGHD1-1*01>1_IGHJ5*01
2530
L25_IGKJ1*01
826
1431


1400
VH3-23_IGHD1-1*01>2_IGHJ5*01
2531
L25_IGKJ1*01
826
1431


1401
VH3-23_IGHD1-1*01>3_IGHJ5*01
2532
L25_IGKJ1*01
826
1431


1402
VH3-23_IGHD1-7*01>1_IGHJ5*01
2533
L25_IGKJ1*01
826
1431


1403
VH3-23_IGHD1-7*01>3_IGHJ5*01
2534
L25_IGKJ1*01
826
1431


1404
VH3-23_IGHD1-14*01>1_IGHJ5*01
2535
L25_IGKJ1*01
826
1431


1405
VH3-23_IGHD1-14*01>3_IGHJ5*01
2536
L25_IGKJ1*01
826
1431


1406
VH3-23_IGHD1-20*01>1_IGHJ5*01
2537
L25_IGKJ1*01
826
1431


1407
VH3-23_IGHD1-20*01>3_IGHJ5*01
2538
L25_IGKJ1*01
826
1431


1408
VH3-23_IGHD1-26*01>1_IGHJ5*01
2539
L25_IGKJ1*01
826
1431


1409
VH3-23_IGHD1-26*01>3_IGHJ5*01
2540
L25_IGKJ1*01
826
1431


1410
VH3-23_IGHD2-2*01>2_IGHJ5*01
2541
L25_IGKJ1*01
826
1431


1411
VH3-23_IGHD2-2*01>3_IGHJ5*01
2542
L25_IGKJ1*01
826
1431


1412
VH3-23_IGHD2-8*01>2_IGHJ5*01
2543
L25_IGKJ1*01
826
1431


1413
VH3-23_IGHD2-8*01>3_IGHJ5*01
2544
L25_IGKJ1*01
826
1431


1414
VH3-23_IGHD2-15*01>2_IGHJ5*01
2545
L25_IGKJ1*01
826
1431


1415
VH3-23_IGHD2-15*01>3_IGHJ5*01
2546
L25_IGKJ1*01
826
1431


1416
VH3-23_IGHD2-21*01>2_IGHJ5*01
2547
L25_IGKJ1*01
826
1431


1417
VH3-23_IGHD2-21*01>3_IGHJ5*01
2548
L25_IGKJ1*01
826
1431


1418
VH3-23_IGHD3-3*01>1_IGHJ5*01
2549
L25_IGKJ1*01
826
1431


1419
VH3-23_IGHD3-3*01>2_IGHJ5*01
2550
L25_IGKJ1*01
826
1431


1420
VH3-23_IGHD3-3*01>3_IGHJ5*01
2551
L25_IGKJ1*01
826
1431


1421
VH3-23_IGHD3-9*01>2_IGHJ5*01
2552
L25_IGKJ1*01
826
1431


1422
VH3-23_IGHD3-10*01>2_IGHJ5*01
2553
L25_IGKJ1*01
826
1431


1423
VH3-23_IGHD3-10*01>3_IGHJ5*01
2554
L25_IGKJ1*01
826
1431


1424
VH3-23_IGHD3-16*01>2_IGHJ5*01
2555
L25_IGKJ1*01
826
1431


1425
VH3-23_IGHD3-16*01>3_IGHJ5*01
2556
L25_IGKJ1*01
826
1431


1426
VH3-23_IGHD3-22*01>2_IGHJ5*01
2557
L25_IGKJ1*01
826
1431


1427
VH3-23_IGHD3-22*01>3_IGHJ5*01
2558
L25_IGKJ1*01
826
1431


1428
VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01
2559
L25_IGKJ1*01
826
1431


1429
VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01
2560
L25_IGKJ1*01
826
1431


1430
VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01
2561
L25_IGKJ1*01
826
1431


1431
VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01
2562
L25_IGKJ1*01
826
1431


1432
VH3-23_IGHD4-17*01>2_IGHJ5*01
2563
L25_IGKJ1*01
826
1431


1433
VH3-23_IGHD4-17*01>3_IGHJ5*01
2564
L25_IGKJ1*01
826
1431


1434
VH3-23_IGHD4-23*01>2_IGHJ5*01
2565
L25_IGKJ1*01
826
1431


1435
VH3-23_IGHD4-23*01>3_IGHJ5*01
2566
L25_IGKJ1*01
826
1431


1436
VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01
2567
L25_IGKJ1*01
826
1431


1437
VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01
2568
L25_IGKJ1*01
826
1431


1438
VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01
2569
L25_IGKJ1*01
826
1431


1439
VH3-23_IGHD5-12*01>1_IGHJ5*01
2570
L25_IGKJ1*01
826
1431


1440
VH3-23_IGHD5-12*01>3_IGHJ5*01
2571
L25_IGKJ1*01
826
1431


1441
VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01
2572
L25_IGKJ1*01
826
1431


1442
VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01
2573
L25_IGKJ1*01
826
1431


1443
VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01
2574
L25_IGKJ1*01
826
1431


1444
VH3-23_IGHD5-24*01>1_IGHJ5*01
2575
L25_IGKJ1*01
826
1431


1445
VH3-23_IGHD5-24*01>3_IGHJ5*01
2576
L25_IGKJ1*01
826
1431


1446
VH3-23_IGHD6-6*01>1_IGHJ5*01
2577
L25_IGKJ1*01
826
1431


1447
VH3-23_IGHD1-1*01>1′_IGHJ5*01
2587
L25_IGKJ1*01
826
1431


1448
VH3-23_IGHD1-1*01>2′_IGHJ5*01
2588
L25_IGKJ1*01
826
1431


1449
VH3-23_IGHD1-1*01>3′_IGHJ5*01
2589
L25_IGKJ1*01
826
1431


1450
VH3-23_IGHD1-7*01>1′_IGHJ5*01
2590
L25_IGKJ1*01
826
1431


1451
VH3-23_IGHD1-7*01>3′_IGHJ5*01
2591
L25_IGKJ1*01
826
1431


1452
VH3-23_IGHD1-14*01>1′_IGHJ5*01
2592
L25_IGKJ1*01
826
1431


1453
VH3-23_IGHD1-14*01>2′_IGHJ5*01
2593
L25_IGKJ1*01
826
1431


1454
VH3-23_IGHD1-14*01>3′_IGHJ5*01
2594
L25_IGKJ1*01
826
1431


1455
VH3-23_IGHD1-20*01>1′_IGHJ5*01
2595
L25_IGKJ1*01
826
1431


1456
VH3-23_IGHD1-20*01>2′_IGHJ5*01
2596
L25_IGKJ1*01
826
1431


1457
VH3-23_IGHD1-20*01>3′_IGHJ5*01
2597
L25_IGKJ1*01
826
1431


1458
VH3-23_IGHD1-26*01>1′_IGHJ5*01
2598
L25_IGKJ1*01
826
1431


1459
VH3-23_IGHD1-26*01>3′_IGHJ5*01
2599
L25_IGKJ1*01
826
1431


1460
VH3-23_IGHD2-2*01>1′_IGHJ5*01
2600
L25_IGKJ1*01
826
1431


1461
VH3-23_IGHD2-2*01>3′_IGHJ5*01
2601
L25_IGKJ1*01
826
1431


1462
VH3-23_IGHD2-8*01>1′_IGHJ5*01
2602
L25_IGKJ1*01
826
1431


1463
VH3-23_IGHD2-15*01>1′_IGHJ5*01
2603
L25_IGKJ1*01
826
1431


1464
VH3-23_IGHD2-15*01>3′_IGHJ5*01
2604
L25_IGKJ1*01
826
1431


1465
VH3-23_IGHD2-21*01>1′_IGHJ5*01
2605
L25_IGKJ1*01
826
1431


1466
VH3-23_IGHD2-21*01>3′_IGHJ5*01
2606
L25_IGKJ1*01
826
1431


1467
VH3-23_IGHD3-3*01>1′_IGHJ5*01
2607
L25_IGKJ1*01
826
1431


1468
VH3-23_IGHD3-3*01>3′_IGHJ5*01
2608
L25_IGKJ1*01
826
1431


1469
VH3-23_IGHD3-9*01>1′_IGHJ5*01
2609
L25_IGKJ1*01
826
1431


1470
VH3-23_IGHD3-9*01>3′_IGHJ5*01
2610
L25_IGKJ1*01
826
1431


1471
VH3-23_IGHD3-10*01>1′_IGHJ5*01
2611
L25_IGKJ1*01
826
1431


1472
VH3-23_IGHD3-10*01>3′_IGHJ5*01
2612
L25_IGKJ1*01
826
1431


1473
VH3-23_IGHD3-16*01>1′_IGHJ5*01
2613
L25_IGKJ1*01
826
1431


1474
VH3-23_IGHD3-16*01>3′_IGHJ5*01
2614
L25_IGKJ1*01
826
1431


1475
VH3-23_IGHD3-22*01>1′_IGHJ5*01
2615
L25_IGKJ1*01
826
1431


1476
VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01
2616
L25_IGKJ1*01
826
1431


1477
VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01
2617
L25_IGKJ1*01
826
1431


1478
VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01
2618
L25_IGKJ1*01
826
1431


1479
VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01
2619
L25_IGKJ1*01
826
1431


1480
VH3-23_IGHD4-17*01>1′_IGHJ5*01
2620
L25_IGKJ1*01
826
1431


1481
VH3-23_IGHD4-17*01>3′_IGHJ5*01
2621
L25_IGKJ1*01
826
1431


1482
VH3-23_IGHD4-23*01>1′_IGHJ5*01
2622
L25_IGKJ1*01
826
1431


1483
VH3-23_IGHD4-23*01>3′_IGHJ5*01
2623
L25_IGKJ1*01
826
1431


1484
VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01
2624
L25_IGKJ1*01
826
1431


1485
VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01
2625
L25_IGKJ1*01
826
1431


1486
VH3-23_IGHD5-12*01>1′_IGHJ5*01
2626
L25_IGKJ1*01
826
1431


1487
VH3-23_IGHD5-12*01>3′_IGHJ5*01
2627
L25_IGKJ1*01
826
1431


1488
VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01
2628
L25_IGKJ1*01
826
1431


1489
VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01
2629
L25_IGKJ1*01
826
1431


1490
VH3-23_IGHD5-24*01>1′_IGHJ5*01
2630
L25_IGKJ1*01
826
1431


1491
VH3-23_IGHD5-24*01>3′_IGHJ5*01
2631
L25_IGKJ1*01
826
1431


1492
VH3-23_IGHD6-6*01>1′_IGHJ5*01
2632
L25_IGKJ1*01
826
1431


1493
VH3-23_IGHD6-6*01>2′_IGHJ5*01
2633
L25_IGKJ1*01
826
1431


1494
VH3-23_IGHD6-6*01>3′_IGHJ5*01
2634
L25_IGKJ1*01
826
1431


1495
VH3-23_IGHD1-1*01>1_IGHJ5*01
2530
B3_IGKJ1*01
817
1422


1496
VH3-23_IGHD1-1*01>2_IGHJ5*01
2531
B3_IGKJ1*01
817
1422


1497
VH3-23_IGHD1-1*01>3_IGHJ5*01
2532
B3_IGKJ1*01
817
1422


1498
VH3-23_IGHD1-7*01>1_IGHJ5*01
2533
B3_IGKJ1*01
817
1422


1499
VH3-23_IGHD1-7*01>3_IGHJ5*01
2534
B3_IGKJ1*01
817
1422


1500
VH3-23_IGHD1-14*01>1_IGHJ5*01
2535
B3_IGKJ1*01
817
1422


1501
VH3-23_IGHD1-14*01>3_IGHJ5*01
2536
B3_IGKJ1*01
817
1422


1502
VH3-23_IGHD1-20*01>1_IGHJ5*01
2537
B3_IGKJ1*01
817
1422


1503
VH3-23_IGHD1-20*01>3_IGHJ5*01
2538
B3_IGKJ1*01
817
1422


1504
VH3-23_IGHD1-26*01>1_IGHJ5*01
2539
B3_IGKJ1*01
817
1422


1505
VH3-23_IGHD1-26*01>3_IGHJ5*01
2540
B3_IGKJ1*01
817
1422


1506
VH3-23_IGHD2-2*01>2_IGHJ5*01
2541
B3_IGKJ1*01
817
1422


1507
VH3-23_IGHD2-2*01>3_IGHJ5*01
2542
B3_IGKJ1*01
817
1422


1508
VH3-23_IGHD2-8*01>2_IGHJ5*01
2543
B3_IGKJ1*01
817
1422


1509
VH3-23_IGHD2-8*01>3_IGHJ5*01
2544
B3_IGKJ1*01
817
1422


1510
VH3-23_IGHD2-15*01>2_IGHJ5*01
2545
B3_IGKJ1*01
817
1422


1511
VH3-23_IGHD2-15*01>3_IGHJ5*01
2546
B3_IGKJ1*01
817
1422


1512
VH3-23_IGHD2-21*01>2_IGHJ5*01
2547
B3_IGKJ1*01
817
1422


1513
VH3-23_IGHD2-21*01>3_IGHJ5*01
2548
B3_IGKJ1*01
817
1422


1514
VH3-23_IGHD3-3*01>1_IGHJ5*01
2549
B3_IGKJ1*01
817
1422


1515
VH3-23_IGHD3-3*01>2_IGHJ5*01
2550
B3_IGKJ1*01
817
1422


1516
VH3-23_IGHD3-3*01>3_IGHJ5*01
2551
B3_IGKJ1*01
817
1422


1517
VH3-23_IGHD3-9*01>2_IGHJ5*01
2552
B3_IGKJ1*01
817
1422


1518
VH3-23_IGHD3-10*01>2_IGHJ5*01
2553
B3_IGKJ1*01
817
1422


1519
VH3-23_IGHD3-10*01>3_IGHJ5*01
2554
B3_IGKJ1*01
817
1422


1520
VH3-23_IGHD3-16*01>2_IGHJ5*01
2555
B3_IGKJ1*01
817
1422


1521
VH3-23_IGHD3-16*01>3_IGHJ5*01
2556
B3_IGKJ1*01
817
1422


1522
VH3-23_IGHD3-22*01>2_IGHJ5*01
2557
B3_IGKJ1*01
817
1422


1523
VH3-23_IGHD3-22*01>3_IGHJ5*01
2558
B3_IGKJ1*01
817
1422


1524
VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01
2559
B3_IGKJ1*01
817
1422


1525
VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01
2560
B3_IGKJ1*01
817
1422


1526
VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01
2561
B3_IGKJ1*01
817
1422


1527
VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01
2562
B3_IGKJ1*01
817
1422


1528
VH3-23_IGHD4-17*01>2_IGHJ5*01
2563
B3_IGKJ1*01
817
1422


1529
VH3-23_IGHD4-17*01>3_IGHJ5*01
2564
B3_IGKJ1*01
817
1422


1530
VH3-23_IGHD4-23*01>2_IGHJ5*01
2565
B3_IGKJ1*01
817
1422


1531
VH3-23_IGHD4-23*01>3_IGHJ5*01
2566
B3_IGKJ1*01
817
1422


1532
VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01
2567
B3_IGKJ1*01
817
1422


1533
VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01
2568
B3_IGKJ1*01
817
1422


1534
VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01
2569
B3_IGKJ1*01
817
1422


1535
VH3-23_IGHD5-12*01>1_IGHJ5*01
2570
B3_IGKJ1*01
817
1422


1536
VH3-23_IGHD5-12*01>3_IGHJ5*01
2571
B3_IGKJ1*01
817
1422


1537
VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01
2572
B3_IGKJ1*01
817
1422


1538
VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01
2573
B3_IGKJ1*01
817
1422


1539
VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01
2574
B3_IGKJ1*01
817
1422


1540
VH3-23_IGHD5-24*01>1_IGHJ5*01
2575
B3_IGKJ1*01
817
1422


1541
VH3-23_IGHD5-24*01>3_IGHJ5*01
2576
B3_IGKJ1*01
817
1422


1542
VH3-23_IGHD6-6*01>1_IGHJ5*01
2577
B3_IGKJ1*01
817
1422


1543
VH3-23_IGHD1-1*01>1′_IGHJ5*01
2587
B3_IGKJ1*01
817
1422


1544
VH3-23_IGHD1-1*01>2′_IGHJ5*01
2588
B3_IGKJ1*01
817
1422


1545
VH3-23_IGHD1-1*01>3′_IGHJ5*01
2589
B3_IGKJ1*01
817
1422


1546
VH3-23_IGHD1-7*01>1′_IGHJ5*01
2590
B3_IGKJ1*01
817
1422


1547
VH3-23_IGHD1-7*01>3′_IGHJ5*01
2591
B3_IGKJ1*01
817
1422


1548
VH3-23_IGHD1-14*01>1′_IGHJ5*01
2592
B3_IGKJ1*01
817
1422


1549
VH3-23_IGHD1-14*01>2′_IGHJ5*01
2593
B3_IGKJ1*01
817
1422


1550
VH3-23_IGHD1-14*01>3′_IGHJ5*01
2594
B3_IGKJ1*01
817
1422


1551
VH3-23_IGHD1-20*01>1′_IGHJ5*01
2595
B3_IGKJ1*01
817
1422


1552
VH3-23_IGHD1-20*01>2′_IGHJ5*01
2596
B3_IGKJ1*01
817
1422


1553
VH3-23_IGHD1-20*01>3′_IGHJ5*01
2597
B3_IGKJ1*01
817
1422


1554
VH3-23_IGHD1-26*01>1′_IGHJ5*01
2598
B3_IGKJ1*01
817
1422


1555
VH3-23_IGHD1-26*01>3′_IGHJ5*01
2599
B3_IGKJ1*01
817
1422


1556
VH3-23_IGHD2-2*01>1′_IGHJ5*01
2600
B3_IGKJ1*01
817
1422


1557
VH3-23_IGHD2-2*01>3′_IGHJ5*01
2601
B3_IGKJ1*01
817
1422


1558
VH3-23_IGHD2-8*01>1′_IGHJ5*01
2602
B3_IGKJ1*01
817
1422


1559
VH3-23_IGHD2-15*01>1′_IGHJ5*01
2603
B3_IGKJ1*01
817
1422


1560
VH3-23_IGHD2-15*01>3′_IGHJ5*01
2604
B3_IGKJ1*01
817
1422


1561
VH3-23_IGHD2-21*01>1′_IGHJ5*01
2605
B3_IGKJ1*01
817
1422


1562
VH3-23_IGHD2-21*01>3′_IGHJ5*01
2606
B3_IGKJ1*01
817
1422


1563
VH3-23_IGHD3-3*01>1′_IGHJ5*01
2607
B3_IGKJ1*01
817
1422


1564
VH3-23_IGHD3-3*01>3′_IGHJ5*01
2608
B3_IGKJ1*01
817
1422


1565
VH3-23_IGHD3-9*01>1′_IGHJ5*01
2609
B3_IGKJ1*01
817
1422


1566
VH3-23_IGHD3-9*01>3′_IGHJ5*01
2610
B3_IGKJ1*01
817
1422


1567
VH3-23_IGHD3-10*01>1′_IGHJ5*01
2611
B3_IGKJ1*01
817
1422


1568
VH3-23_IGHD3-10*01>3′_IGHJ5*01
2612
B3_IGKJ1*01
817
1422


1569
VH3-23_IGHD3-16*01>1′_IGHJ5*01
2613
B3_IGKJ1*01
817
1422


1570
VH3-23_IGHD3-16*01>3′_IGHJ5*01
2614
B3_IGKJ1*01
817
1422


1571
VH3-23_IGHD3-22*01>1′_IGHJ5*01
2615
B3_IGKJ1*01
817
1422


1572
VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01
2616
B3_IGKJ1*01
817
1422


1573
VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01
2617
B3_IGKJ1*01
817
1422


1574
VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01
2618
B3_IGKJ1*01
817
1422


1575
VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01
2619
B3_IGKJ1*01
817
1422


1576
VH3-23_IGHD4-17*01>1′_IGHJ5*01
2620
B3_IGKJ1*01
817
1422


1577
VH3-23_IGHD4-17*01>3′_IGHJ5*01
2621
B3_IGKJ1*01
817
1422


1578
VH3-23_IGHD4-23*01>1′_IGHJ5*01
2622
B3_IGKJ1*01
817
1422


1579
VH3-23_IGHD4-23*01>3′_IGHJ5*01
2623
B3_IGKJ1*01
817
1422


1580
VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01
2624
B3_IGKJ1*01
817
1422


1581
VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01
2625
B3_IGKJ1*01
817
1422


1582
VH3-23_IGHD5-12*01>1′_IGHJ5*01
2626
B3_IGKJ1*01
817
1422


1583
VH3-23_IGHD5-12*01>3′_IGHJ5*01
2627
B3_IGKJ1*01
817
1422


1584
VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01
2628
B3_IGKJ1*01
817
1422


1585
VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01
2629
B3_IGKJ1*01
817
1422


1586
VH3-23_IGHD5-24*01>1′_IGHJ5*01
2630
B3_IGKJ1*01
817
1422


1587
VH3-23_IGHD5-24*01>3′_IGHJ5*01
2631
B3_IGKJ1*01
817
1422


1588
VH3-23_IGHD6-6*01>1′_IGHJ5*01
2632
B3_IGKJ1*01
817
1422


1589
VH3-23_IGHD6-6*01>2′_IGHJ5*01
2633
B3_IGKJ1*01
817
1422


1590
VH3-23_IGHD6-6*01>3′_IGHJ5*01
2634
B3_IGKJ1*01
817
1422


1591
VH3-23_IGHD1-1*01>1_IGHJ5*01
2530
A26_IGKJ1*01
811
1416


1592
VH3-23_IGHD1-1*01>2_IGHJ5*01
2531
A26_IGKJ1*01
811
1416


1593
VH3-23_IGHD1-1*01>3_IGHJ5*01
2532
A26_IGKJ1*01
811
1416


1594
VH3-23_IGHD1-7*01>1_IGHJ5*01
2533
A26_IGKJ1*01
811
1416


1595
VH3-23_IGHD1-7*01>3_IGHJ5*01
2534
A26_IGKJ1*01
811
1416


1596
VH3-23_IGHD1-14*01>1_IGHJ5*01
2535
A26_IGKJ1*01
811
1416


1597
VH3-23_IGHD1-14*01>3_IGHJ5*01
2536
A26_IGKJ1*01
811
1416


1598
VH3-23_IGHD1-20*01>1_IGHJ5*01
2537
A26_IGKJ1*01
811
1416


1599
VH3-23_IGHD1-20*01>3_IGHJ5*01
2538
A26_IGKJ1*01
811
1416


1600
VH3-23_IGHD1-26*01>1_IGHJ5*01
2539
A26_IGKJ1*01
811
1416


1601
VH3-23_IGHD1-26*01>3_IGHJ5*01
2540
A26_IGKJ1*01
811
1416


1602
VH3-23_IGHD2-2*01>2_IGHJ5*01
2541
A26_IGKJ1*01
811
1416


1603
VH3-23_IGHD2-2*01>3_IGHJ5*01
2542
A26_IGKJ1*01
811
1416


1604
VH3-23_IGHD2-8*01>2_IGHJ5*01
2543
A26_IGKJ1*01
811
1416


1605
VH3-23_IGHD2-8*01>3_IGHJ5*01
2544
A26_IGKJ1*01
811
1416


1606
VH3-23_IGHD2-15*01>2_IGHJ5*01
2545
A26_IGKJ1*01
811
1416


1607
VH3-23_IGHD2-15*01>3_IGHJ5*01
2546
A26_IGKJ1*01
811
1416


1608
VH3-23_IGHD2-21*01>2_IGHJ5*01
2547
A26_IGKJ1*01
811
1416


1609
VH3-23_IGHD2-21*01>3_IGHJ5*01
2548
A26_IGKJ1*01
811
1416


1610
VH3-23_IGHD3-3*01>1_IGHJ5*01
2549
A26_IGKJ1*01
811
1416


1611
VH3-23_IGHD3-3*01>2_IGHJ5*01
2550
A26_IGKJ1*01
811
1416


1612
VH3-23_IGHD3-3*01>3_IGHJ5*01
2551
A26_IGKJ1*01
811
1416


1613
VH3-23_IGHD3-9*01>2_IGHJ5*01
2552
A26_IGKJ1*01
811
1416


1614
VH3-23_IGHD3-10*01>2_IGHJ5*01
2553
A26_IGKJ1*01
811
1416


1615
VH3-23_IGHD3-10*01>3_IGHJ5*01
2554
A26_IGKJ1*01
811
1416


1616
VH3-23_IGHD3-16*01>2_IGHJ5*01
2555
A26_IGKJ1*01
811
1416


1617
VH3-23_IGHD3-16*01>3_IGHJ5*01
2556
A26_IGKJ1*01
811
1416


1618
VH3-23_IGHD3-22*01>2_IGHJ5*01
2557
A26_IGKJ1*01
811
1416


1619
VH3-23_IGHD3-22*01>3_IGHJ5*01
2558
A26_IGKJ1*01
811
1416


1620
VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01
2559
A26_IGKJ1*01
811
1416


1621
VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01
2560
A26_IGKJ1*01
811
1416


1622
VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01
2561
A26_IGKJ1*01
811
1416


1623
VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01
2562
A26_IGKJ1*01
811
1416


1624
VH3-23_IGHD4-17*01>2_IGHJ5*01
2563
A26_IGKJ1*01
811
1416


1625
VH3-23_IGHD4-17*01>3_IGHJ5*01
2564
A26_IGKJ1*01
811
1416


1626
VH3-23_IGHD4-23*01>2_IGHJ5*01
2565
A26_IGKJ1*01
811
1416


1627
VH3-23_IGHD4-23*01>3_IGHJ5*01
2566
A26_IGKJ1*01
811
1416


1628
VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01
2567
A26_IGKJ1*01
811
1416


1629
VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01
2568
A26_IGKJ1*01
811
1416


1630
VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01
2569
A26_IGKJ1*01
811
1416


1631
VH3-23_IGHD5-12*01>1_IGHJ5*01
2570
A26_IGKJ1*01
811
1416


1632
VH3-23_IGHD5-12*01>3_IGHJ5*01
2571
A26_IGKJ1*01
811
1416


1633
VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01
2572
A26_IGKJ1*01
811
1416


1634
VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01
2573
A26_IGKJ1*01
811
1416


1635
VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01
2574
A26_IGKJ1*01
811
1416


1636
VH3-23_IGHD5-24*01>1_IGHJ5*01
2575
A26_IGKJ1*01
811
1416


1637
VH3-23_IGHD5-24*01>3_IGHJ5*01
2576
A26_IGKJ1*01
811
1416


1638
VH3-23_IGHD6-6*01>1_IGHJ5*01
2577
A26_IGKJ1*01
811
1416


1639
VH3-23_IGHD1-1*01>1′_IGHJ5*01
2587
A26_IGKJ1*01
811
1416


1640
VH3-23_IGHD1-1*01>2′_IGHJ5*01
2588
A26_IGKJ1*01
811
1416


1641
VH3-23_IGHD1-1*01>3′_IGHJ5*01
2589
A26_IGKJ1*01
811
1416


1642
VH3-23_IGHD1-7*01>1′_IGHJ5*01
2590
A26_IGKJ1*01
811
1416


1643
VH3-23_IGHD1-7*01>3′_IGHJ5*01
2591
A26_IGKJ1*01
811
1416


1644
VH3-23_IGHD1-14*01>1′_IGHJ5*01
2592
A26_IGKJ1*01
811
1416


1645
VH3-23_IGHD1-14*01>2′_IGHJ5*01
2593
A26_IGKJ1*01
811
1416


1646
VH3-23_IGHD1-14*01>3′_IGHJ5*01
2594
A26_IGKJ1*01
811
1416


1647
VH3-23_IGHD1-20*01>1′_IGHJ5*01
2595
A26_IGKJ1*01
811
1416


1648
VH3-23_IGHD1-20*01>2′_IGHJ5*01
2596
A26_IGKJ1*01
811
1416


1649
VH3-23_IGHD1-20*01>3′_IGHJ5*01
2597
A26_IGKJ1*01
811
1416


1650
VH3-23_IGHD1-26*01>1′_IGHJ5*01
2598
A26_IGKJ1*01
811
1416


1651
VH3-23_IGHD1-26*01>3′_IGHJ5*01
2599
A26_IGKJ1*01
811
1416


1652
VH3-23_IGHD2-2*01>1′_IGHJ5*01
2600
A26_IGKJ1*01
811
1416


1653
VH3-23_IGHD2-2*01>3′_IGHJ5*01
2601
A26_IGKJ1*01
811
1416


1654
VH3-23_IGHD2-8*01>1′_IGHJ5*01
2602
A26_IGKJ1*01
811
1416


1655
VH3-23_IGHD2-15*01>1′_IGHJ5*01
2603
A26_IGKJ1*01
811
1416


1656
VH3-23_IGHD2-15*01>3′_IGHJ5*01
2604
A26_IGKJ1*01
811
1416


1657
VH3-23_IGHD2-21*01>1′_IGHJ5*01
2605
A26_IGKJ1*01
811
1416


1658
VH3-23_IGHD2-21*01>3′_IGHJ5*01
2606
A26_IGKJ1*01
811
1416


1659
VH3-23_IGHD3-3*01>1′_IGHJ5*01
2607
A26_IGKJ1*01
811
1416


1660
VH3-23_IGHD3-3*01>3′_IGHJ5*01
2608
A26_IGKJ1*01
811
1416


1661
VH3-23_IGHD3-9*01>1′_IGHJ5*01
2609
A26_IGKJ1*01
811
1416


1662
VH3-23_IGHD3-9*01>3′_IGHJ5*01
2610
A26_IGKJ1*01
811
1416


1663
VH3-23_IGHD3-10*01>1′_IGHJ5*01
2611
A26_IGKJ1*01
811
1416


1664
VH3-23_IGHD3-10*01>3′_IGHJ5*01
2612
A26_IGKJ1*01
811
1416


1665
VH3-23_IGHD3-16*01>1′_IGHJ5*01
2613
A26_IGKJ1*01
811
1416


1666
VH3-23_IGHD3-16*01>3′_IGHJ5*01
2614
A26_IGKJ1*01
811
1416


1667
VH3-23_IGHD3-22*01>1′_IGHJ5*01
2615
A26_IGKJ1*01
811
1416


1668
VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01
2616
A26_IGKJ1*01
811
1416


1669
VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01
2617
A26_IGKJ1*01
811
1416


1670
VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01
2618
A26_IGKJ1*01
811
1416


1671
VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01
2619
A26_IGKJ1*01
811
1416


1672
VH3-23_IGHD4-17*01>1′_IGHJ5*01
2620
A26_IGKJ1*01
811
1416


1673
VH3-23_IGHD4-17*01>3′_IGHJ5*01
2621
A26_IGKJ1*01
811
1416


1674
VH3-23_IGHD4-23*01>1′_IGHJ5*01
2622
A26_IGKJ1*01
811
1416


1675
VH3-23_IGHD4-23*01>3′_IGHJ5*01
2623
A26_IGKJ1*01
811
1416


1676
VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01
2624
A26_IGKJ1*01
811
1416


1677
VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01
2625
A26_IGKJ1*01
811
1416


1678
VH3-23_IGHD5-12*01>1′_IGHJ5*01
2626
A26_IGKJ1*01
811
1416


1679
VH3-23_IGHD5-12*01>3′_IGHJ5*01
2627
A26_IGKJ1*01
811
1416


1680
VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01
2628
A26_IGKJ1*01
811
1416


1681
VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01
2629
A26_IGKJ1*01
811
1416


1682
VH3-23_IGHD5-24*01>1′_IGHJ5*01
2630
A26_IGKJ1*01
811
1416


1683
VH3-23_IGHD5-24*01>3′_IGHJ5*01
2631
A26_IGKJ1*01
811
1416


1684
VH3-23_IGHD6-6*01>1′_IGHJ5*01
2632
A26_IGKJ1*01
811
1416


1685
VH3-23_IGHD6-6*01>2′_IGHJ5*01
2633
A26_IGKJ1*01
811
1416


1686
VH3-23_IGHD6-6*01>3′_IGHJ5*01
2634
A26_IGKJ1*01
811
1416


1687
VH3-23_IGHD1-1*01>1_IGHJ5*01
2530
A14_IGKJ1*01
806
1411


1688
VH3-23_IGHD1-1*01>2_IGHJ5*01
2531
A14_IGKJ1*01
806
1411


1689
VH3-23_IGHD1-1*01>3_IGHJ5*01
2532
A14_IGKJ1*01
806
1411


1690
VH3-23_IGHD1-7*01>1_IGHJ5*01
2533
A14_IGKJ1*01
806
1411


1691
VH3-23_IGHD1-7*01>3_IGHJ5*01
2534
A14_IGKJ1*01
806
1411


1692
VH3-23_IGHD1-14*01>1_IGHJ5*01
2535
A14_IGKJ1*01
806
1411


1693
VH3-23_IGHD1-14*01>3_IGHJ5*01
2536
A14_IGKJ1*01
806
1411


1694
VH3-23_IGHD1-20*01>1_IGHJ5*01
2537
A14_IGKJ1*01
806
1411


1695
VH3-23_IGHD1-20*01>3_IGHJ5*01
2538
A14_IGKJ1*01
806
1411


1696
VH3-23_IGHD1-26*01>1_IGHJ5*01
2539
A14_IGKJ1*01
806
1411


1697
VH3-23_IGHD1-26*01>3_IGHJ5*01
2540
A14_IGKJ1*01
806
1411


1698
VH3-23_IGHD2-2*01>2_IGHJ5*01
2541
A14_IGKJ1*01
806
1411


1699
VH3-23_IGHD2-2*01>3_IGHJ5*01
2542
A14_IGKJ1*01
806
1411


1700
VH3-23_IGHD2-8*01>2_IGHJ5*01
2543
A14_IGKJ1*01
806
1411


1701
VH3-23_IGHD2-8*01>3_IGHJ5*01
2544
A14_IGKJ1*01
806
1411


1702
VH3-23_IGHD2-15*01>2_IGHJ5*01
2545
A14_IGKJ1*01
806
1411


1703
VH3-23_IGHD2-15*01>3_IGHJ5*01
2546
A14_IGKJ1*01
806
1411


1704
VH3-23_IGHD2-21*01>2_IGHJ5*01
2547
A14_IGKJ1*01
806
1411


1705
VH3-23_IGHD2-21*01>3_IGHJ5*01
2548
A14_IGKJ1*01
806
1411


1706
VH3-23_IGHD3-3*01>1_IGHJ5*01
2549
A14_IGKJ1*01
806
1411


1707
VH3-23_IGHD3-3*01>2_IGHJ5*01
2550
A14_IGKJ1*01
806
1411


1708
VH3-23_IGHD3-3*01>3_IGHJ5*01
2551
A14_IGKJ1*01
806
1411


1709
VH3-23_IGHD3-9*01>2_IGHJ5*01
2552
A14_IGKJ1*01
806
1411


1710
VH3-23_IGHD3-10*01>2_IGHJ5*01
2553
A14_IGKJ1*01
806
1411


1711
VH3-23_IGHD3-10*01>3_IGHJ5*01
2554
A14_IGKJ1*01
806
1411


1712
VH3-23_IGHD3-16*01>2_IGHJ5*01
2555
A14_IGKJ1*01
806
1411


1713
VH3-23_IGHD3-16*01>3_IGHJ5*01
2556
A14_IGKJ1*01
806
1411


1714
VH3-23_IGHD3-22*01>2_IGHJ5*01
2557
A14_IGKJ1*01
806
1411


1715
VH3-23_IGHD3-22*01>3_IGHJ5*01
2558
A14_IGKJ1*01
806
1411


1716
VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01
2559
A14_IGKJ1*01
806
1411


1717
VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01
2560
A14_IGKJ1*01
806
1411


1718
VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01
2561
A14_IGKJ1*01
806
1411


1719
VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01
2562
A14_IGKJ1*01
806
1411


1720
VH3-23_IGHD4-17*01>2_IGHJ5*01
2563
A14_IGKJ1*01
806
1411


1721
VH3-23_IGHD4-17*01>3_IGHJ5*01
2564
A14_IGKJ1*01
806
1411


1722
VH3-23_IGHD4-23*01>2_IGHJ5*01
2565
A14_IGKJ1*01
806
1411


1723
VH3-23_IGHD4-23*01>3_IGHJ5*01
2566
A14_IGKJ1*01
806
1411


1724
VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01
2567
A14_IGKJ1*01
806
1411


1725
VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01
2568
A14_IGKJ1*01
806
1411


1726
VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01
2569
A14_IGKJ1*01
806
1411


1727
VH3-23_IGHD5-12*01>1_IGHJ5*01
2570
A14_IGKJ1*01
806
1411


1728
VH3-23_IGHD5-12*01>3_IGHJ5*01
2571
A14_IGKJ1*01
806
1411


1729
VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01
2572
A14_IGKJ1*01
806
1411


1730
VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01
2573
A14_IGKJ1*01
806
1411


1731
VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01
2574
A14_IGKJ1*01
806
1411


1732
VH3-23_IGHD5-24*01>1_IGHJ5*01
2575
A14_IGKJ1*01
806
1411


1733
VH3-23_IGHD5-24*01>3_IGHJ5*01
2576
A14_IGKJ1*01
806
1411


1734
VH3-23_IGHD6-6*01>1_IGHJ5*01
2577
A14_IGKJ1*01
806
1411


1735
VH3-23_IGHD1-1*01>1′_IGHJ5*01
2587
A14_IGKJ1*01
806
1411


1736
VH3-23_IGHD1-1*01>2′_IGHJ5*01
2588
A14_IGKJ1*01
806
1411


1737
VH3-23_IGHD1-1*01>3′_IGHJ5*01
2589
A14_IGKJ1*01
806
1411


1738
VH3-23_IGHD1-7*01>1′_IGHJ5*01
2590
A14_IGKJ1*01
806
1411


1739
VH3-23_IGHD1-7*01>3′_IGHJ5*01
2591
A14_IGKJ1*01
806
1411


1740
VH3-23_IGHD1-14*01>1′_IGHJ5*01
2592
A14_IGKJ1*01
806
1411


1741
VH3-23_IGHD1-14*01>2′_IGHJ5*01
2593
A14_IGKJ1*01
806
1411


1742
VH3-23_IGHD1-14*01>3′_IGHJ5*01
2594
A14_IGKJ1*01
806
1411


1743
VH3-23_IGHD1-20*01>1′_IGHJ5*01
2595
A14_IGKJ1*01
806
1411


1744
VH3-23_IGHD1-20*01>2′_IGHJ5*01
2596
A14_IGKJ1*01
806
1411


1745
VH3-23_IGHD1-20*01>3′_IGHJ5*01
2597
A14_IGKJ1*01
806
1411


1746
VH3-23_IGHD1-26*01>1′_IGHJ5*01
2598
A14_IGKJ1*01
806
1411


1747
VH3-23_IGHD1-26*01>3′_IGHJ5*01
2599
A14_IGKJ1*01
806
1411


1748
VH3-23_IGHD2-2*01>1′_IGHJ5*01
2600
A14_IGKJ1*01
806
1411


1749
VH3-23_IGHD2-2*01>3′_IGHJ5*01
2601
A14_IGKJ1*01
806
1411


1750
VH3-23_IGHD2-8*01>1′_IGHJ5*01
2602
A14_IGKJ1*01
806
1411


1751
VH3-23_IGHD2-15*01>1′_IGHJ5*01
2603
A14_IGKJ1*01
806
1411


1752
VH3-23_IGHD2-15*01>3′_IGHJ5*01
2604
A14_IGKJ1*01
806
1411


1753
VH3-23_IGHD2-21*01>1′_IGHJ5*01
2605
A14_IGKJ1*01
806
1411


1754
VH3-23_IGHD2-21*01>3′_IGHJ5*01
2606
A14_IGKJ1*01
806
1411


1755
VH3-23_IGHD3-3*01>1′_IGHJ5*01
2607
A14_IGKJ1*01
806
1411


1756
VH3-23_IGHD3-3*01>3′_IGHJ5*01
2608
A14_IGKJ1*01
806
1411


1757
VH3-23_IGHD3-9*01>1′_IGHJ5*01
2609
A14_IGKJ1*01
806
1411


1758
VH3-23_IGHD3-9*01>3′_IGHJ5*01
2610
A14_IGKJ1*01
806
1411


1759
VH3-23_IGHD3-10*01>1′_IGHJ5*01
2611
A14_IGKJ1*01
806
1411


1760
VH3-23_IGHD3-10*01>3′_IGHJ5*01
2612
A14_IGKJ1*01
806
1411


1761
VH3-23_IGHD3-16*01>1′_IGHJ5*01
2613
A14_IGKJ1*01
806
1411


1762
VH3-23_IGHD3-16*01>3′_IGHJ5*01
2614
A14_IGKJ1*01
806
1411


1763
VH3-23_IGHD3-22*01>1′_IGHJ5*01
2615
A14_IGKJ1*01
806
1411


1764
VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01
2616
A14_IGKJ1*01
806
1411


1765
VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01
2617
A14_IGKJ1*01
806
1411


1766
VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01
2618
A14_IGKJ1*01
806
1411


1767
VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01
2619
A14_IGKJ1*01
806
1411


1768
VH3-23_IGHD4-17*01>1′_IGHJ5*01
2620
A14_IGKJ1*01
806
1411


1769
VH3-23_IGHD4-17*01>3′_IGHJ5*01
2621
A14_IGKJ1*01
806
1411


1770
VH3-23_IGHD4-23*01>1′_IGHJ5*01
2622
A14_IGKJ1*01
806
1411


1771
VH3-23_IGHD4-23*01>3′_IGHJ5*01
2623
A14_IGKJ1*01
806
1411


1772
VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01
2624
A14_IGKJ1*01
806
1411


1773
VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01
2625
A14_IGKJ1*01
806
1411


1774
VH3-23_IGHD5-12*01>1′_IGHJ5*01
2626
A14_IGKJ1*01
806
1411


1775
VH3-23_IGHD5-12*01>3′_IGHJ5*01
2627
A14_IGKJ1*01
806
1411


1776
VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01
2628
A14_IGKJ1*01
806
1411


1777
VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01
2629
A14_IGKJ1*01
806
1411


1778
VH3-23_IGHD5-24*01>1′_IGHJ5*01
2630
A14_IGKJ1*01
806
1411


1779
VH3-23_IGHD5-24*01>3′_IGHJ5*01
2631
A14_IGKJ1*01
806
1411


1780
VH3-23_IGHD6-6*01>1′_IGHJ5*01
2632
A14_IGKJ1*01
806
1411


1781
VH3-23_IGHD6-6*01>2′_IGHJ5*01
2633
A14_IGKJ1*01
806
1411


1782
VH3-23_IGHD6-6*01>3′_IGHJ5*01
2634
A14_IGKJ1*01
806
1411


1783
VH3-23_IGHD1-1*01>1_IGHJ5*01
2530
A27_IGKJ1*01
812
1417


1784
VH3-23_IGHD1-1*01>2_IGHJ5*01
2531
A27_IGKJ1*01
812
1417


1785
VH3-23_IGHD1-1*01>3_IGHJ5*01
2532
A27_IGKJ1*01
812
1417


1786
VH3-23_IGHD1-7*01>1_IGHJ5*01
2533
A27_IGKJ1*01
812
1417


1787
VH3-23_IGHD1-7*01>3_IGHJ5*01
2534
A27_IGKJ1*01
812
1417


1788
VH3-23_IGHD1-14*01>1_IGHJ5*01
2535
A27_IGKJ1*01
812
1417


1789
VH3-23_IGHD1-14*01>3_IGHJ5*01
2536
A27_IGKJ1*01
812
1417


1790
VH3-23_IGHD1-20*01>1_IGHJ5*01
2537
A27_IGKJ1*01
812
1417


1791
VH3-23_IGHD1-20*01>3_IGHJ5*01
2538
A27_IGKJ1*01
812
1417


1792
VH3-23_IGHD1-26*01>1_IGHJ5*01
2539
A27_IGKJ1*01
812
1417


1793
VH3-23_IGHD1-26*01>3_IGHJ5*01
2540
A27_IGKJ1*01
812
1417


1794
VH3-23_IGHD2-2*01>2_IGHJ5*01
2541
A27_IGKJ1*01
812
1417


1795
VH3-23_IGHD2-2*01>3_IGHJ5*01
2542
A27_IGKJ1*01
812
1417


1796
VH3-23_IGHD2-8*01>2_IGHJ5*01
2543
A27_IGKJ1*01
812
1417


1797
VH3-23_IGHD2-8*01>3_IGHJ5*01
2544
A27_IGKJ1*01
812
1417


1798
VH3-23_IGHD2-15*01>2_IGHJ5*01
2545
A27_IGKJ1*01
812
1417


1799
VH3-23_IGHD2-15*01>3_IGHJ5*01
2546
A27_IGKJ1*01
812
1417


1800
VH3-23_IGHD2-21*01>2_IGHJ5*01
2547
A27_IGKJ1*01
812
1417


1801
VH3-23_IGHD2-21*01>3_IGHJ5*01
2548
A27_IGKJ1*01
812
1417


1802
VH3-23_IGHD3-3*01>1_IGHJ5*01
2549
A27_IGKJ1*01
812
1417


1803
VH3-23_IGHD3-3*01>2_IGHJ5*01
2550
A27_IGKJ1*01
812
1417


1804
VH3-23_IGHD3-3*01>3_IGHJ5*01
2551
A27_IGKJ1*01
812
1417


1805
VH3-23_IGHD3-9*01>2_IGHJ5*01
2552
A27_IGKJ1*01
812
1417


1806
VH3-23_IGHD3-10*01>2_IGHJ5*01
2553
A27_IGKJ1*01
812
1417


1807
VH3-23_IGHD3-10*01>3_IGHJ5*01
2554
A27_IGKJ1*01
812
1417


1808
VH3-23_IGHD3-16*01>2_IGHJ5*01
2555
A27_IGKJ1*01
812
1417


1809
VH3-23_IGHD3-16*01>3_IGHJ5*01
2556
A27_IGKJ1*01
812
1417


1810
VH3-23_IGHD3-22*01>2_IGHJ5*01
2557
A27_IGKJ1*01
812
1417


1811
VH3-23_IGHD3-22*01>3_IGHJ5*01
2558
A27_IGKJ1*01
812
1417


1812
VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01
2559
A27_IGKJ1*01
812
1417


1813
VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01
2560
A27_IGKJ1*01
812
1417


1814
VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01
2561
A27_IGKJ1*01
812
1417


1815
VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01
2562
A27_IGKJ1*01
812
1417


1816
VH3-23_IGHD4-17*01>2_IGHJ5*01
2563
A27_IGKJ1*01
812
1417


1817
VH3-23_IGHD4-17*01>3_IGHJ5*01
2564
A27_IGKJ1*01
812
1417


1818
VH3-23_IGHD4-23*01>2_IGHJ5*01
2565
A27_IGKJ1*01
812
1417


1819
VH3-23_IGHD4-23*01>3_IGHJ5*01
2566
A27_IGKJ1*01
812
1417


1820
VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01
2567
A27_IGKJ1*01
812
1417


1821
VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01
2568
A27_IGKJ1*01
812
1417


1822
VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01
2569
A27_IGKJ1*01
812
1417


1823
VH3-23_IGHD5-12*01>1_IGHJ5*01
2570
A27_IGKJ1*01
812
1417


1824
VH3-23_IGHD5-12*01>3_IGHJ5*01
2571
A27_IGKJ1*01
812
1417


1825
VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01
2572
A27_IGKJ1*01
812
1417


1826
VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01
2573
A27_IGKJ1*01
812
1417


1827
VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01
2574
A27_IGKJ1*01
812
1417


1828
VH3-23_IGHD5-24*01>1_IGHJ5*01
2575
A27_IGKJ1*01
812
1417


1829
VH3-23_IGHD5-24*01>3_IGHJ5*01
2576
A27_IGKJ1*01
812
1417


1830
VH3-23_IGHD6-6*01>1_IGHJ5*01
2577
A27_IGKJ1*01
812
1417


1831
VH3-23_IGHD1-1*01>1′_IGHJ5*01
2587
A27_IGKJ1*01
812
1417


1832
VH3-23_IGHD1-1*01>2′_IGHJ5*01
2588
A27_IGKJ1*01
812
1417


1833
VH3-23_IGHD1-1*01>3′_IGHJ5*01
2589
A27_IGKJ1*01
812
1417


1834
VH3-23_IGHD1-7*01>1′_IGHJ5*01
2590
A27_IGKJ1*01
812
1417


1835
VH3-23_IGHD1-7*01>3′_IGHJ5*01
2591
A27_IGKJ1*01
812
1417


1836
VH3-23_IGHD1-14*01>1′_IGHJ5*01
2592
A27_IGKJ1*01
812
1417


1837
VH3-23_IGHD1-14*01>2′_IGHJ5*01
2593
A27_IGKJ1*01
812
1417


1838
VH3-23_IGHD1-14*01>3′_IGHJ5*01
2594
A27_IGKJ1*01
812
1417


1839
VH3-23_IGHD1-20*01>1′_IGHJ5*01
2595
A27_IGKJ1*01
812
1417


1840
VH3-23_IGHD1-20*01>2′_IGHJ5*01
2596
A27_IGKJ1*01
812
1417


1841
VH3-23_IGHD1-20*01>3′_IGHJ5*01
2597
A27_IGKJ1*01
812
1417


1842
VH3-23_IGHD1-26*01>1′_IGHJ5*01
2598
A27_IGKJ1*01
812
1417


1843
VH3-23_IGHD1-26*01>3′_IGHJ5*01
2599
A27_IGKJ1*01
812
1417


1844
VH3-23_IGHD2-2*01>1′_IGHJ5*01
2600
A27_IGKJ1*01
812
1417


1845
VH3-23_IGHD2-2*01>3′_IGHJ5*01
2601
A27_IGKJ1*01
812
1417


1846
VH3-23_IGHD2-8*01>1′_IGHJ5*01
2602
A27_IGKJ1*01
812
1417


1847
VH3-23_IGHD2-15*01>1′_IGHJ5*01
2603
A27_IGKJ1*01
812
1417


1848
VH3-23_IGHD2-15*01>3′_IGHJ5*01
2604
A27_IGKJ1*01
812
1417


1849
VH3-23_IGHD2-21*01>1′_IGHJ5*01
2605
A27_IGKJ1*01
812
1417


1850
VH3-23_IGHD2-21*01>3′_IGHJ5*01
2606
A27_IGKJ1*01
812
1417


1851
VH3-23_IGHD3-3*01>1′_IGHJ5*01
2607
A27_IGKJ1*01
812
1417


1852
VH3-23_IGHD3-3*01>3′_IGHJ5*01
2608
A27_IGKJ1*01
812
1417


1853
VH3-23_IGHD3-9*01>1′_IGHJ5*01
2609
A27_IGKJ1*01
812
1417


1854
VH3-23_IGHD3-9*01>3′_IGHJ5*01
2610
A27_IGKJ1*01
812
1417


1855
VH3-23_IGHD3-10*01>1′_IGHJ5*01
2611
A27_IGKJ1*01
812
1417


1856
VH3-23_IGHD3-10*01>3′_IGHJ5*01
2612
A27_IGKJ1*01
812
1417


1857
VH3-23_IGHD3-16*01>1′_IGHJ5*01
2613
A27_IGKJ1*01
812
1417


1858
VH3-23_IGHD3-16*01>3′_IGHJ5*01
2614
A27_IGKJ1*01
812
1417


1859
VH3-23_IGHD3-22*01>1′_IGHJ5*01
2615
A27_IGKJ1*01
812
1417


1860
VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01
2616
A27_IGKJ1*01
812
1417


1861
VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01
2617
A27_IGKJ1*01
812
1417


1862
VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01
2618
A27_IGKJ1*01
812
1417


1863
VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01
2619
A27_IGKJ1*01
812
1417


1864
VH3-23_IGHD4-17*01>1′_IGHJ5*01
2620
A27_IGKJ1*01
812
1417


1865
VH3-23_IGHD4-17*01>3′_IGHJ5*01
2621
A27_IGKJ1*01
812
1417


1866
VH3-23_IGHD4-23*01>1′_IGHJ5*01
2622
A27_IGKJ1*01
812
1417


1867
VH3-23_IGHD4-23*01>3′_IGHJ5*01
2623
A27_IGKJ1*01
812
1417


1868
VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01
2624
A27_IGKJ1*01
812
1417


1869
VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01
2625
A27_IGKJ1*01
812
1417


1870
VH3-23_IGHD5-12*01>1′_IGHJ5*01
2626
A27_IGKJ1*01
812
1417


1871
VH3-23_IGHD5-12*01>3′_IGHJ5*01
2627
A27_IGKJ1*01
812
1417


1872
VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01
2628
A27_IGKJ1*01
812
1417


1873
VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01
2629
A27_IGKJ1*01
812
1417


1874
VH3-23_IGHD5-24*01>1′_IGHJ5*01
2630
A27_IGKJ1*01
812
1417


1875
VH3-23_IGHD5-24*01>3′_IGHJ5*01
2631
A27_IGKJ1*01
812
1417


1876
VH3-23_IGHD6-6*01>1′_IGHJ5*01
2632
A27_IGKJ1*01
812
1417


1877
VH3-23_IGHD6-6*01>2′_IGHJ5*01
2633
A27_IGKJ1*01
812
1417


1878
VH3-23_IGHD6-6*01>3′_IGHJ5*01
2634
A27_IGKJ1*01
812
1417


1879
VH3-23_IGHD6-6*01>2_IGHJ1*01
2118
V1-11_IGLJ2*01
836
1441


1880
VH3-23_IGHD6-13*01>1_IGHJ1*01
2119
V1-11_IGLJ2*01
836
1441


1881
VH3-23_IGHD6-13*01>2_IGHJ1*01
2120
V1-11_IGLJ2*01
836
1441


1882
VH3-23_IGHD6-19*01>1_IGHJ1*01
2121
V1-11_IGLJ2*01
836
1441


1883
VH3-23_IGHD6-19*01>2_IGHJ1*01
2122
V1-11_IGLJ2*01
836
1441


1884
VH3-23_IGHD6-25*01>1_IGHJ1*01
2123
V1-11_IGLJ2*01
836
1441


1885
VH3-23_IGHD6-25*01>2_IGHJ1*01
2124
V1-11_IGLJ2*01
836
1441


1886
VH3-23_IGHD7-27*01>1_IGHJ1*01
2125
V1-11_IGLJ2*01
836
1441


1887
VH3-23_IGHD7-27*01>3_IGHJ1*01
2126
V1-11_IGLJ2*01
836
1441


1888
VH3-23_IGHD6-13*01>1′_IGHJ1*01
2175
V1-11_IGLJ2*01
836
1441


1889
VH3-23_IGHD6-13*01>2′_IGHJ1*01
2176
V1-11_IGLJ2*01
836
1441


1890
VH3-23_IGHD6-13*01>2_IGHJ1*01_B
2177
V1-11_IGLJ2*01
836
1441


1891
VH3-23_IGHD6-19*01>1′_IGHJ1*01
2178
V1-11_IGLJ2*01
836
1441


1892
VH3-23_IGHD6-19*01>2′_IGHJ1*01
2179
V1-11_IGLJ2*01
836
1441


1893
VH3-23_IGHD6-19*01>2_IGHJ1*01_B
2180
V1-11_IGLJ2*01
836
1441


1894
VH3-23_IGHD6-25*01>1′_IGHJ1*01
2181
V1-11_IGLJ2*01
836
1441


1895
VH3-23_IGHD6-25*01>3′_IGHJ1*01
2182
V1-11_IGLJ2*01
836
1441


1896
VH3-23_IGHD7-27*01>1′_IGHJ1*01_B
2183
V1-11_IGLJ2*01
836
1441


1897
VH3-23_IGHD7-27*01>2′_IGHJ1*01
2184
V1-11_IGLJ2*01
836
1441


1898
VH3-23_IGHD6-6*01>2_IGHJ2*01
2233
V1-11_IGLJ2*01
836
1441


1899
VH3-23_IGHD6-13*01>1_IGHJ2*01
2234
V1-11_IGLJ2*01
836
1441


1900
VH3-23_IGHD6-13*01>2_IGHJ2*01
2235
V1-11_IGLJ2*01
836
1441


1901
VH3-23_IGHD6-19*01>1_IGHJ2*01
2236
V1-11_IGLJ2*01
836
1441


1902
VH3-23_IGHD6-19*01>2_IGHJ2*01
2237
V1-11_IGLJ2*01
836
1441


1903
VH3-23_IGHD6-25*01>1_IGHJ2*01
2238
V1-11_IGLJ2*01
836
1441


1904
VH3-23_IGHD6-25*01>2_IGHJ2*01
2239
V1-11_IGLJ2*01
836
1441


1905
VH3-23_IGHD7-27*01>1_IGHJ2*01
2240
V1-11_IGLJ2*01
836
1441


1906
VH3-23_IGHD7-27*01>3_IGHJ2*01
2241
V1-11_IGLJ2*01
836
1441


1907
VH3-23_IGHD6-13*01>1′_IGHJ2*01
2290
V1-11_IGLJ2*01
836
1441


1908
VH3-23_IGHD6-13*01>2′_IGHJ2*01
2291
V1-11_IGLJ2*01
836
1441


1909
VH3-23_IGHD6-13*01>2_IGHJ2*01_B
2292
V1-11_IGLJ2*01
836
1441


1910
VH3-23_IGHD6-19*01>1′_IGHJ2*01
2293
V1-11_IGLJ2*01
836
1441


1911
VH3-23_IGHD6-19*01>2′_IGHJ2*01
2294
V1-11_IGLJ2*01
836
1441


1912
VH3-23_IGHD6-19*01>2_IGHJ2*01_B
2295
V1-11_IGLJ2*01
836
1441


1913
VH3-23_IGHD6-25*01>1′_IGHJ2*01
2296
V1-11_IGLJ2*01
836
1441


1914
VH3-23_IGHD6-25*01>3′_IGHJ2*01
2297
V1-11_IGLJ2*01
836
1441


1915
VH3-23_IGHD7-27*01>1′_IGHJ2*01
2298
V1-11_IGLJ2*01
836
1441


1916
VH3-23_IGHD7-27*01>2′_IGHJ2*01
2299
V1-11_IGLJ2*01
836
1441


1917
VH3-23_IGHD6-6*01>2_IGHJ3*01
2348
V1-11_IGLJ2*01
836
1441


1918
VH3-23_IGHD6-13*01>1_IGHJ3*01
2349
V1-11_IGLJ2*01
836
1441


1919
VH3-23_IGHD6-13*01>2_IGHJ3*01
2350
V1-11_IGLJ2*01
836
1441


1920
VH3-23_IGHD6-19*01>1_IGHJ3*01
2351
V1-11_IGLJ2*01
836
1441


1921
VH3-23_IGHD6-19*01>2_IGHJ3*01
2352
V1-11_IGLJ2*01
836
1441


1922
VH3-23_IGHD6-25*01>1_IGHJ3*01
2353
V1-11_IGLJ2*01
836
1441


1923
VH3-23_IGHD6-25*01>2_IGHJ3*01
2354
V1-11_IGLJ2*01
836
1441


1924
VH3-23_IGHD7-27*01>1_IGHJ3*01
2355
V1-11_IGLJ2*01
836
1441


1925
VH3-23_IGHD7-27*01>3_IGHJ3*01
2356
V1-11_IGLJ2*01
836
1441


1926
VH3-23_IGHD6-13*01>1′_IGHJ3*01
2405
V1-11_IGLJ2*01
836
1441


1927
VH3-23_IGHD6-13*01>2′_IGHJ3*01
2406
V1-11_IGLJ2*01
836
1441


1928
VH3-23_IGHD6-13*01>1_IGHJ6*01
2407
V1-11_IGLJ2*01
836
1441


1929
VH3-23_IGHD6-19*01>1′_IGHJ3*01
2408
V1-11_IGLJ2*01
836
1441


1930
VH3-23_IGHD6-19*01>2′_IGHJ3*01
2409
V1-11_IGLJ2*01
836
1441


1931
VH3-23_IGHD6-19*01>3′_IGHJ3*01
2410
V1-11_IGLJ2*01
836
1441


1932
VH3-23_IGHD6-25*01>1′_IGHJ3*01
2411
V1-11_IGLJ2*01
836
1441


1933
VH3-23_IGHD6-25*01>3′_IGHJ3*01
2412
V1-11_IGLJ2*01
836
1441


1934
VH3-23_IGHD7-27*01>1′_IGHJ3*01
2413
V1-11_IGLJ2*01
836
1441


1935
VH3-23_IGHD7-27*01>2′_IGHJ3*01
2414
V1-11_IGLJ2*01
836
1441


1936
VH3-23_IGHD6-6*01>2_IGHJ4*01
2463
V1-11_IGLJ2*01
836
1441


1937
VH3-23_IGHD6-13*01>1_IGHJ4*01
2464
V1-11_IGLJ2*01
836
1441


1938
VH3-23_IGHD6-13*01>2_IGHJ4*01
2465
V1-11_IGLJ2*01
836
1441


1939
VH3-23_IGHD6-19*01>1_IGHJ4*01
2466
V1-11_IGLJ2*01
836
1441


1940
VH3-23_IGHD6-19*01>2_IGHJ4*01
2467
V1-11_IGLJ2*01
836
1441


1941
VH3-23_IGHD6-25*01>1_IGHJ4*01
2468
V1-11_IGLJ2*01
836
1441


1942
VH3-23_IGHD6-25*01>2_IGHJ4*01
2469
V1-11_IGLJ2*01
836
1441


1943
VH3-23_IGHD7-27*01>1_IGHJ4*01
2470
V1-11_IGLJ2*01
836
1441


1944
VH3-23_IGHD7-27*01>3_IGHJ4*01
2471
V1-11_IGLJ2*01
836
1441


1945
VH3-23_IGHD6-13*01>1′_IGHJ4*01
2520
V1-11_IGLJ2*01
836
1441


1946
VH3-23_IGHD6-13*01>2′_IGHJ4*01
2521
V1-11_IGLJ2*01
836
1441


1947
VH3-23_IGHD6-13*01>2_IGHJ4*01_B
2522
V1-11_IGLJ2*01
836
1441


1948
VH3-23_IGHD6-19*01>1′_IGHJ4*01
2523
V1-11_IGLJ2*01
836
1441


1949
VH3-23_IGHD6-19*01>2′_IGHJ4*01
2524
V1-11_IGLJ2*01
836
1441


1950
VH3-23_IGHD6-19*01>2_IGHJ4*01_B
2525
V1-11_IGLJ2*01
836
1441


1951
VH3-23_IGHD6-25*01>1′_IGHJ4*01
2526
V1-11_IGLJ2*01
836
1441


1952
VH3-23_IGHD6-25*01>3′_IGHJ4*01
2527
V1-11_IGLJ2*01
836
1441


1953
VH3-23_IGHD7-27*01>1′_IGHJ4*01
2528
V1-11_IGLJ2*01
836
1441


1954
VH3-23_IGHD7-27*01>2′_IGHJ4*01
2529
V1-11_IGLJ2*01
836
1441


1955
VH3-23_IGHD6-6*01>2_IGHJ5*01
2578
V1-11_IGLJ2*01
836
1441


1956
VH3-23_IGHD6-13*01>1_IGHJ5*01
2579
V1-11_IGLJ2*01
836
1441


1957
VH3-23_IGHD6-13*01>2_IGHJ5*01
2580
V1-11_IGLJ2*01
836
1441


1958
VH3-23_IGHD6-19*01>1_IGHJ5*01
2581
V1-11_IGLJ2*01
836
1441


1959
VH3-23_IGHD6-19*01>2_IGHJ5*01
2582
V1-11_IGLJ2*01
836
1441


1960
VH3-23_IGHD6-25*01>1_IGHJ5*01
2583
V1-11_IGLJ2*01
836
1441


1961
VH3-23_IGHD6-25*01>2_IGHJ5*01
2584
V1-11_IGLJ2*01
836
1441


1962
VH3-23_IGHD7-27*01>1_IGHJ5*01
2585
V1-11_IGLJ2*01
836
1441


1963
VH3-23_IGHD7-27*01>3_IGHJ5*01
2586
V1-11_IGLJ2*01
836
1441


1964
VH3-23_IGHD6-13*01>1′_IGHJ5*01
2635
V1-11_IGLJ2*01
836
1441


1965
VH3-23_IGHD6-13*01>2′_IGHJ5*01
2636
V1-11_IGLJ2*01
836
1441


1966
VH3-23_IGHD6-13*01>3′_IGHJ5*01
2637
V1-11_IGLJ2*01
836
1441


1967
VH3-23_IGHD6-19*01>1′_IGHJ5*01
2638
V1-11_IGLJ2*01
836
1441


1968
VH3-23_IGHD6-19*01>2′_IGHJ5*01
2639
V1-11_IGLJ2*01
836
1441


1969
VH3-23_IGHD6-19*01>2_IGHJ5*01_B
2640
V1-11_IGLJ2*01
836
1441


1970
VH3-23_IGHD6-25*01>1′_IGHJ5*01
2641
V1-11_IGLJ2*01
836
1441


1971
VH3-23_IGHD6-25*01>3′_IGHJ5*01
2642
V1-11_IGLJ2*01
836
1441


1972
VH3-23_IGHD7-27*01>1′_IGHJ5*01
2643
V1-11_IGLJ2*01
836
1441


1973
VH3-23_IGHD7-27*01>2′_IGHJ5*01
2644
V1-11_IGLJ2*01
836
1441


1974
VH3-23_IGHD6-6*01>2_IGHJ6*01
2693
V1-11_IGLJ2*01
836
1441


1975
VH3-23_IGHD6-6*01>2_IGHJ1*01
2118
V1-13_IGLJ5*01
837
1442


1976
VH3-23_IGHD6-13*01>1_IGHJ1*01
2119
V1-13_IGLJ5*01
837
1442


1977
VH3-23_IGHD6-13*01>2_IGHJ1*01
2120
V1-13_IGLJ5*01
837
1442


1978
VH3-23_IGHD6-19*01>1_IGHJ1*01
2121
V1-13_IGLJ5*01
837
1442


1979
VH3-23_IGHD6-19*01>2_IGHJ1*01
2122
V1-13_IGLJ5*01
837
1442


1980
VH3-23_IGHD6-25*01>1_IGHJ1*01
2123
V1-13_IGLJ5*01
837
1442


1981
VH3-23_IGHD6-25*01>2_IGHJ1*01
2124
V1-13_IGLJ5*01
837
1442


1982
VH3-23_IGHD7-27*01>1_IGHJ1*01
2125
V1-13_IGLJ5*01
837
1442


1983
VH3-23_IGHD7-27*01>3_IGHJ1*01
2126
V1-13_IGLJ5*01
837
1442


1984
VH3-23_IGHD6-13*01>1′_IGHJ1*01
2175
V1-13_IGLJ5*01
837
1442


1985
VH3-23_IGHD6-13*01>2′_IGHJ1*01
2176
V1-13_IGLJ5*01
837
1442


1986
VH3-23_IGHD6-13*01>2_IGHJ1*01_B
2177
V1-13_IGLJ5*01
837
1442


1987
VH3-23_IGHD6-19*01>1′_IGHJ1*01
2178
V1-13_IGLJ5*01
837
1442


1988
VH3-23_IGHD6-19*01>2′_IGHJ1*01
2179
V1-13_IGLJ5*01
837
1442


1989
VH3-23_IGHD6-19*01>2_IGHJ1*01_B
2180
V1-13_IGLJ5*01
837
1442


1990
VH3-23_IGHD6-25*01>1′_IGHJ1*01
2181
V1-13_IGLJ5*01
837
1442


1991
VH3-23_IGHD6-25*01>3′_IGHJ1*01
2182
V1-13_IGLJ5*01
837
1442


1992
VH3-23_IGHD7-27*01>1′_IGHJ1*01_B
2183
V1-13_IGLJ5*01
837
1442


1993
VH3-23_IGHD7-27*01>2′_IGHJ1*01
2184
V1-13_IGLJ5*01
837
1442


1994
VH3-23_IGHD6-6*01>2_IGHJ2*01
2233
V1-13_IGLJ5*01
837
1442


1995
VH3-23_IGHD6-13*01>1_IGHJ2*01
2234
V1-13_IGLJ5*01
837
1442


1996
VH3-23_IGHD6-13*01>2_IGHJ2*01
2235
V1-13_IGLJ5*01
837
1442


1997
VH3-23_IGHD6-19*01>1_IGHJ2*01
2236
V1-13_IGLJ5*01
837
1442


1998
VH3-23_IGHD6-19*01>2_IGHJ2*01
2237
V1-13_IGLJ5*01
837
1442


1999
VH3-23_IGHD6-25*01>1_IGHJ2*01
2238
V1-13_IGLJ5*01
837
1442


2000
VH3-23_IGHD6-25*01>2_IGHJ2*01
2239
V1-13_IGLJ5*01
837
1442


2001
VH3-23_IGHD7-27*01>1_IGHJ2*01
2240
V1-13_IGLJ5*01
837
1442


2002
VH3-23_IGHD7-27*01>3_IGHJ2*01
2241
V1-13_IGLJ5*01
837
1442


2003
VH3-23_IGHD6-13*01>1′_IGHJ2*01
2290
V1-13_IGLJ5*01
837
1442


2004
VH3-23_IGHD6-13*01>2′_IGHJ2*01
2291
V1-13_IGLJ5*01
837
1442


2005
VH3-23_IGHD6-13*01>2_IGHJ2*01_B
2292
V1-13_IGLJ5*01
837
1442


2006
VH3-23_IGHD6-19*01>1′_IGHJ2*01
2293
V1-13_IGLJ5*01
837
1442


2007
VH3-23_IGHD6-19*01>2′_IGHJ2*01
2294
V1-13_IGLJ5*01
837
1442


2008
VH3-23_IGHD6-19*01>2_IGHJ2*01_B
2295
V1-13_IGLJ5*01
837
1442


2009
VH3-23_IGHD6-25*01>1′_IGHJ2*01
2296
V1-13_IGLJ5*01
837
1442


2010
VH3-23_IGHD6-25*01>3′_IGHJ2*01
2297
V1-13_IGLJ5*01
837
1442


2011
VH3-23_IGHD7-27*01>1′_IGHJ2*01
2298
V1-13_IGLJ5*01
837
1442


2012
VH3-23_IGHD7-27*01>2′_IGHJ2*01
2299
V1-13_IGLJ5*01
837
1442


2013
VH3-23_IGHD6-6*01>2_IGHJ3*01
2348
V1-13_IGLJ5*01
837
1442


2014
VH3-23_IGHD6-13*01>1_IGHJ3*01
2349
V1-13_IGLJ5*01
837
1442


2015
VH3-23_IGHD6-13*01>2_IGHJ3*01
2350
V1-13_IGLJ5*01
837
1442


2016
VH3-23_IGHD6-19*01>1_IGHJ3*01
2351
V1-13_IGLJ5*01
837
1442


2017
VH3-23_IGHD6-19*01>2_IGHJ3*01
2352
V1-13_IGLJ5*01
837
1442


2018
VH3-23_IGHD6-25*01>1_IGHJ3*01
2353
V1-13_IGLJ5*01
837
1442


2019
VH3-23_IGHD6-25*01>2_IGHJ3*01
2354
V1-13_IGLJ5*01
837
1442


2020
VH3-23_IGHD7-27*01>1_IGHJ3*01
2355
V1-13_IGLJ5*01
837
1442


2021
VH3-23_IGHD7-27*01>3_IGHJ3*01
2356
V1-13_IGLJ5*01
837
1442


2022
VH3-23_IGHD6-13*01>1′_IGHJ3*01
2405
V1-13_IGLJ5*01
837
1442


2023
VH3-23_IGHD6-13*01>2′_IGHJ3*01
2406
V1-13_IGLJ5*01
837
1442


2024
VH3-23_IGHD6-13*01>1_IGHJ6*01
2407
V1-13_IGLJ5*01
837
1442


2025
VH3-23_IGHD6-19*01>1′_IGHJ3*01
2408
V1-13_IGLJ5*01
837
1442


2026
VH3-23_IGHD6-19*01>2′_IGHJ3*01
2409
V1-13_IGLJ5*01
837
1442


2027
VH3-23_IGHD6-19*01>3′_IGHJ3*01
2410
V1-13_IGLJ5*01
837
1442


2028
VH3-23_IGHD6-25*01>1′_IGHJ3*01
2411
V1-13_IGLJ5*01
837
1442


2029
VH3-23_IGHD6-25*01>3′_IGHJ3*01
2412
V1-13_IGLJ5*01
837
1442


2030
VH3-23_IGHD7-27*01>1′_IGHJ3*01
2413
V1-13_IGLJ5*01
837
1442


2031
VH3-23_IGHD7-27*01>2′_IGHJ3*01
2414
V1-13_IGLJ5*01
837
1442


2032
VH3-23_IGHD6-6*01>2_IGHJ4*01
2463
V1-13_IGLJ5*01
837
1442


2033
VH3-23_IGHD6-13*01>1_IGHJ4*01
2464
V1-13_IGLJ5*01
837
1442


2034
VH3-23_IGHD6-13*01>2_IGHJ4*01
2465
V1-13_IGLJ5*01
837
1442


2035
VH3-23_IGHD6-19*01>1_IGHJ4*01
2466
V1-13_IGLJ5*01
837
1442


2036
VH3-23_IGHD6-19*01>2_IGHJ4*01
2467
V1-13_IGLJ5*01
837
1442


2037
VH3-23_IGHD6-25*01>1_IGHJ4*01
2468
V1-13_IGLJ5*01
837
1442


2038
VH3-23_IGHD6-25*01>2_IGHJ4*01
2469
V1-13_IGLJ5*01
837
1442


2039
VH3-23_IGHD7-27*01>1_IGHJ4*01
2470
V1-13_IGLJ5*01
837
1442


2040
VH3-23_IGHD7-27*01>3_IGHJ4*01
2471
V1-13_IGLJ5*01
837
1442


2041
VH3-23_IGHD6-13*01>1′_IGHJ4*01
2520
V1-13_IGLJ5*01
837
1442


2042
VH3-23_IGHD6-13*01>2′_IGHJ4*01
2521
V1-13_IGLJ5*01
837
1442


2043
VH3-23_IGHD6-13*01>2_IGHJ4*01_B
2522
V1-13_IGLJ5*01
837
1442


2044
VH3-23_IGHD6-19*01>1′_IGHJ4*01
2523
V1-13_IGLJ5*01
837
1442


2045
VH3-23_IGHD6-19*01>2′_IGHJ4*01
2524
V1-13_IGLJ5*01
837
1442


2046
VH3-23_IGHD6-19*01>2_IGHJ4*01_B
2525
V1-13_IGLJ5*01
837
1442


2047
VH3-23_IGHD6-25*01>1′_IGHJ4*01
2526
V1-13_IGLJ5*01
837
1442


2048
VH3-23_IGHD6-25*01>3′_IGHJ4*01
2527
V1-13_IGLJ5*01
837
1442


2049
VH3-23_IGHD7-27*01>1′_IGHJ4*01
2528
V1-13_IGLJ5*01
837
1442


2050
VH3-23_IGHD7-27*01>2′_IGHJ4*01
2529
V1-13_IGLJ5*01
837
1442


2051
VH3-23_IGHD6-6*01>2_IGHJ5*01
2578
V1-13_IGLJ5*01
837
1442


2052
VH3-23_IGHD6-13*01>1_IGHJ5*01
2579
V1-13_IGLJ5*01
837
1442


2053
VH3-23_IGHD6-13*01>2_IGHJ5*01
2580
V1-13_IGLJ5*01
837
1442


2054
VH3-23_IGHD6-19*01>1_IGHJ5*01
2581
V1-13_IGLJ5*01
837
1442


2055
VH3-23_IGHD6-19*01>2_IGHJ5*01
2582
V1-13_IGLJ5*01
837
1442


2056
VH3-23_IGHD6-25*01>1_IGHJ5*01
2583
V1-13_IGLJ5*01
837
1442


2057
VH3-23_IGHD6-25*01>2_IGHJ5*01
2584
V1-13_IGLJ5*01
837
1442


2058
VH3-23_IGHD7-27*01>1_IGHJ5*01
2585
V1-13_IGLJ5*01
837
1442


2059
VH3-23_IGHD7-27*01>3_IGHJ5*01
2586
V1-13_IGLJ5*01
837
1442


2060
VH3-23_IGHD6-13*01>1′_IGHJ5*01
2635
V1-13_IGLJ5*01
837
1442


2061
VH3-23_IGHD6-13*01>2′_IGHJ5*01
2636
V1-13_IGLJ5*01
837
1442


2062
VH3-23_IGHD6-13*01>3′_IGHJ5*01
2637
V1-13_IGLJ5*01
837
1442


2063
VH3-23_IGHD6-19*01>1′_IGHJ5*01
2638
V1-13_IGLJ5*01
837
1442


2064
VH3-23_IGHD6-19*01>2′_IGHJ5*01
2639
V1-13_IGLJ5*01
837
1442


2065
VH3-23_IGHD6-19*01>2_IGHJ5*01_B
2640
V1-13_IGLJ5*01
837
1442


2066
VH3-23_IGHD6-25*01>1′_IGHJ5*01
2641
V1-13_IGLJ5*01
837
1442


2067
VH3-23_IGHD6-25*01>3′_IGHJ5*01
2642
V1-13_IGLJ5*01
837
1442


2068
VH3-23_IGHD7-27*01>1′_IGHJ5*01
2643
V1-13_IGLJ5*01
837
1442


2069
VH3-23_IGHD7-27*01>2′_IGHJ5*01
2644
V1-13_IGLJ5*01
837
1442


2070
VH3-23_IGHD6-6*01>2_IGHJ6*01
2693
V1-13_IGLJ5*01
837
1442


2071
VH3-23_IGHD6-6*01>2_IGHJ1*01
2118
V1-16_IGLJ6*01
838
1443


2072
VH3-23_IGHD6-13*01>1_IGHJ1*01
2119
V1-16_IGLJ6*01
838
1443


2073
VH3-23_IGHD6-13*01>2_IGHJ1*01
2120
V1-16_IGLJ6*01
838
1443


2074
VH3-23_IGHD6-19*01>1_IGHJ1*01
2121
V1-16_IGLJ6*01
838
1443


2075
VH3-23_IGHD6-19*01>2_IGHJ1*01
2122
V1-16_IGLJ6*01
838
1443


2076
VH3-23_IGHD6-25*01>1_IGHJ1*01
2123
V1-16_IGLJ6*01
838
1443


2077
VH3-23_IGHD6-25*01>2_IGHJ1*01
2124
V1-16_IGLJ6*01
838
1443


2078
VH3-23_IGHD7-27*01>1_IGHJ1*01
2125
V1-16_IGLJ6*01
838
1443


2079
VH3-23_IGHD7-27*01>3_IGHJ1*01
2126
V1-16_IGLJ6*01
838
1443


2080
VH3-23_IGHD6-13*01>1′_IGHJ1*01
2175
V1-16_IGLJ6*01
838
1443


2081
VH3-23_IGHD6-13*01>2′_IGHJ1*01
2176
V1-16_IGLJ6*01
838
1443


2082
VH3-23_IGHD6-13*01>2_IGHJ1*01_B
2177
V1-16_IGLJ6*01
838
1443


2083
VH3-23_IGHD6-19*01>1′_IGHJ1*01
2178
V1-16_IGLJ6*01
838
1443


2084
VH3-23_IGHD6-19*01>2′_IGHJ1*01
2179
V1-16_IGLJ6*01
838
1443


2085
VH3-23_IGHD6-19*01>2_IGHJ1*01_B
2180
V1-16_IGLJ6*01
838
1443


2086
VH3-23_IGHD6-25*01>1′_IGHJ1*01
2181
V1-16_IGLJ6*01
838
1443


2087
VH3-23_IGHD6-25*01>3′_IGHJ1*01
2182
V1-16_IGLJ6*01
838
1443


2088
VH3-23_IGHD7-27*01>1′_IGHJ1*01_B
2183
V1-16_IGLJ6*01
838
1443


2089
VH3-23_IGHD7-27*01>2′_IGHJ1*01
2184
V1-16_IGLJ6*01
838
1443


2090
VH3-23_IGHD6-6*01>2_IGHJ2*01
2233
V1-16_IGLJ6*01
838
1443


2091
VH3-23_IGHD6-13*01>1_IGHJ2*01
2234
V1-16_IGLJ6*01
838
1443


2092
VH3-23_IGHD6-13*01>2_IGHJ2*01
2235
V1-16_IGLJ6*01
838
1443


2093
VH3-23_IGHD6-19*01>1_IGHJ2*01
2236
V1-16_IGLJ6*01
838
1443


2094
VH3-23_IGHD6-19*01>2_IGHJ2*01
2237
V1-16_IGLJ6*01
838
1443


2095
VH3-23_IGHD6-25*01>1_IGHJ2*01
2238
V1-16_IGLJ6*01
838
1443


2096
VH3-23_IGHD6-25*01>2_IGHJ2*01
2239
V1-16_IGLJ6*01
838
1443


2097
VH3-23_IGHD7-27*01>1_IGHJ2*01
2240
V1-16_IGLJ6*01
838
1443


2098
VH3-23_IGHD7-27*01>3_IGHJ2*01
2241
V1-16_IGLJ6*01
838
1443


2099
VH3-23_IGHD6-13*01>1′_IGHJ2*01
2290
V1-16_IGLJ6*01
838
1443


2100
VH3-23_IGHD6-13*01>2′_IGHJ2*01
2291
V1-16_IGLJ6*01
838
1443


2101
VH3-23_IGHD6-13*01>2_IGHJ2*01_B
2292
V1-16_IGLJ6*01
838
1443


2102
VH3-23_IGHD6-19*01>1′_IGHJ2*01
2293
V1-16_IGLJ6*01
838
1443


2103
VH3-23_IGHD6-19*01>2′_IGHJ2*01
2294
V1-16_IGLJ6*01
838
1443


2104
VH3-23_IGHD6-19*01>2_IGHJ2*01_B
2295
V1-16_IGLJ6*01
838
1443


2105
VH3-23_IGHD6-25*01>1′_IGHJ2*01
2296
V1-16_IGLJ6*01
838
1443


2106
VH3-23_IGHD6-25*01>3′_IGHJ2*01
2297
V1-16_IGLJ6*01
838
1443


2107
VH3-23_IGHD7-27*01>1′_IGHJ2*01
2298
V1-16_IGLJ6*01
838
1443


2108
VH3-23_IGHD7-27*01>2′_IGHJ2*01
2299
V1-16_IGLJ6*01
838
1443


2109
VH3-23_IGHD6-6*01>2_IGHJ3*01
2348
V1-16_IGLJ6*01
838
1443


2110
VH3-23_IGHD6-13*01>1_IGHJ3*01
2349
V1-16_IGLJ6*01
838
1443


2111
VH3-23_IGHD6-13*01>2_IGHJ3*01
2350
V1-16_IGLJ6*01
838
1443


2112
VH3-23_IGHD6-19*01>1_IGHJ3*01
2351
V1-16_IGLJ6*01
838
1443


2113
VH3-23_IGHD6-19*01>2_IGHJ3*01
2352
V1-16_IGLJ6*01
838
1443


2114
VH3-23_IGHD6-25*01>1_IGHJ3*01
2353
V1-16_IGLJ6*01
838
1443


2115
VH3-23_IGHD6-25*01>2_IGHJ3*01
2354
V1-16_IGLJ6*01
838
1443


2116
VH3-23_IGHD7-27*01>1_IGHJ3*01
2355
V1-16_IGLJ6*01
838
1443


2117
VH3-23_IGHD7-27*01>3_IGHJ3*01
2356
V1-16_IGLJ6*01
838
1443


2118
VH3-23_IGHD6-13*01>1′_IGHJ3*01
2405
V1-16_IGLJ6*01
838
1443


2119
VH3-23_IGHD6-13*01>2′_IGHJ3*01
2406
V1-16_IGLJ6*01
838
1443


2120
VH3-23_IGHD6-13*01>1_IGHJ6*01
2407
V1-16_IGLJ6*01
838
1443


2121
VH3-23_IGHD6-19*01>1′_IGHJ3*01
2408
V1-16_IGLJ6*01
838
1443


2122
VH3-23_IGHD6-19*01>2′_IGHJ3*01
2409
V1-16_IGLJ6*01
838
1443


2123
VH3-23_IGHD6-19*01>3′_IGHJ3*01
2410
V1-16_IGLJ6*01
838
1443


2124
VH3-23_IGHD6-25*01>1′_IGHJ3*01
2411
V1-16_IGLJ6*01
838
1443


2125
VH3-23_IGHD6-25*01>3′_IGHJ3*01
2412
V1-16_IGLJ6*01
838
1443


2126
VH3-23_IGHD7-27*01>1′_IGHJ3*01
2413
V1-16_IGLJ6*01
838
1443


2127
VH3-23_IGHD7-27*01>2′_IGHJ3*01
2414
V1-16_IGLJ6*01
838
1443


2128
VH3-23_IGHD6-6*01>2_IGHJ4*01
2463
V1-16_IGLJ6*01
838
1443


2129
VH3-23_IGHD6-13*01>1_IGHJ4*01
2464
V1-16_IGLJ6*01
838
1443


2130
VH3-23_IGHD6-13*01>2_IGHJ4*01
2465
V1-16_IGLJ6*01
838
1443


2131
VH3-23_IGHD6-19*01>1_IGHJ4*01
2466
V1-16_IGLJ6*01
838
1443


2132
VH3-23_IGHD6-19*01>2_IGHJ4*01
2467
V1-16_IGLJ6*01
838
1443


2133
VH3-23_IGHD6-25*01>1_IGHJ4*01
2468
V1-16_IGLJ6*01
838
1443


2134
VH3-23_IGHD6-25*01>2_IGHJ4*01
2469
V1-16_IGLJ6*01
838
1443


2135
VH3-23_IGHD7-27*01>1_IGHJ4*01
2470
V1-16_IGLJ6*01
838
1443


2136
VH3-23_IGHD7-27*01>3_IGHJ4*01
2471
V1-16_IGLJ6*01
838
1443


2137
VH3-23_IGHD6-13*01>1′_IGHJ4*01
2520
V1-16_IGLJ6*01
838
1443


2138
VH3-23_IGHD6-13*01>2′_IGHJ4*01
2521
V1-16_IGLJ6*01
838
1443


2139
VH3-23_IGHD6-13*01>2_IGHJ4*01_B
2522
V1-16_IGLJ6*01
838
1443


2140
VH3-23_IGHD6-19*01>1′_IGHJ4*01
2523
V1-16_IGLJ6*01
838
1443


2141
VH3-23_IGHD6-19*01>2′_IGHJ4*01
2524
V1-16_IGLJ6*01
838
1443


2142
VH3-23_IGHD6-19*01>2_IGHJ4*01_B
2525
V1-16_IGLJ6*01
838
1443


2143
VH3-23_IGHD6-25*01>1′_IGHJ4*01
2526
V1-16_IGLJ6*01
838
1443


2144
VH3-23_IGHD6-25*01>3′_IGHJ4*01
2527
V1-16_IGLJ6*01
838
1443


2145
VH3-23_IGHD7-27*01>1′_IGHJ4*01
2528
V1-16_IGLJ6*01
838
1443


2146
VH3-23_IGHD7-27*01>2′_IGHJ4*01
2529
V1-16_IGLJ6*01
838
1443


2147
VH3-23_IGHD6-6*01>2_IGHJ5*01
2578
V1-16_IGLJ6*01
838
1443


2148
VH3-23_IGHD6-13*01>1_IGHJ5*01
2579
V1-16_IGLJ6*01
838
1443


2149
VH3-23_IGHD6-13*01>2_IGHJ5*01
2580
V1-16_IGLJ6*01
838
1443


2150
VH3-23_IGHD6-19*01>1_IGHJ5*01
2581
V1-16_IGLJ6*01
838
1443


2151
VH3-23_IGHD6-19*01>2_IGHJ5*01
2582
V1-16_IGLJ6*01
838
1443


2152
VH3-23_IGHD6-25*01>1_IGHJ5*01
2583
V1-16_IGLJ6*01
838
1443


2153
VH3-23_IGHD6-25*01>2_IGHJ5*01
2584
V1-16_IGLJ6*01
838
1443


2154
VH3-23_IGHD7-27*01>1_IGHJ5*01
2585
V1-16_IGLJ6*01
838
1443


2155
VH3-23_IGHD7-27*01>3_IGHJ5*01
2586
V1-16_IGLJ6*01
838
1443


2156
VH3-23_IGHD6-13*01>1′_IGHJ5*01
2635
V1-16_IGLJ6*01
838
1443


2157
VH3-23_IGHD6-13*01>2′_IGHJ5*01
2636
V1-16_IGLJ6*01
838
1443


2158
VH3-23_IGHD6-13*01>3′_IGHJ5*01
2637
V1-16_IGLJ6*01
838
1443


2159
VH3-23_IGHD6-19*01>1′_IGHJ5*01
2638
V1-16_IGLJ6*01
838
1443


2160
VH3-23_IGHD6-19*01>2′_IGHJ5*01
2639
V1-16_IGLJ6*01
838
1443


2161
VH3-23_IGHD6-19*01>2_IGHJ5*01_B
2640
V1-16_IGLJ6*01
838
1443


2162
VH3-23_IGHD6-25*01>1′_IGHJ5*01
2641
V1-16_IGLJ6*01
838
1443


2163
VH3-23_IGHD6-25*01>3′_IGHJ5*01
2642
V1-16_IGLJ6*01
838
1443


2164
VH3-23_IGHD7-27*01>1′_IGHJ5*01
2643
V1-16_IGLJ6*01
838
1443


2165
VH3-23_IGHD7-27*01>2′_IGHJ5*01
2644
V1-16_IGLJ6*01
838
1443


2166
VH3-23_IGHD6-6*01>2_IGHJ6*01
2693
V1-16_IGLJ6*01
838
1443


2167
VH3-23_IGHD6-6*01>2_IGHJ1*01
2118
V1-2_IGLJ7*01
840
1445


2168
VH3-23_IGHD6-13*01>1_IGHJ1*01
2119
V1-2_IGLJ7*01
840
1445


2169
VH3-23_IGHD6-13*01>2_IGHJ1*01
2120
V1-2_IGLJ7*01
840
1445


2170
VH3-23_IGHD6-19*01>1_IGHJ1*01
2121
V1-2_IGLJ7*01
840
1445


2171
VH3-23_IGHD6-19*01>2_IGHJ1*01
2122
V1-2_IGLJ7*01
840
1445


2172
VH3-23_IGHD6-25*01>1_IGHJ1*01
2123
V1-2_IGLJ7*01
840
1445


2173
VH3-23_IGHD6-25*01>2_IGHJ1*01
2124
V1-2_IGLJ7*01
840
1445


2174
VH3-23_IGHD7-27*01>1_IGHJ1*01
2125
V1-2_IGLJ7*01
840
1445


2175
VH3-23_IGHD7-27*01>3_IGHJ1*01
2126
V1-2_IGLJ7*01
840
1445


2176
VH3-23_IGHD6-13*01>1′_IGHJ1*01
2175
V1-2_IGLJ7*01
840
1445


2177
VH3-23_IGHD6-13*01>2′_IGHJ1*01
2176
V1-2_IGLJ7*01
840
1445


2178
VH3-23_IGHD6-13*01>2_IGHJ1*01_B
2177
V1-2_IGLJ7*01
840
1445


2179
VH3-23_IGHD6-19*01>1′_IGHJ1*01
2178
V1-2_IGLJ7*01
840
1445


2180
VH3-23_IGHD6-19*01>2′_IGHJ1*01
2179
V1-2_IGLJ7*01
840
1445


2181
VH3-23_IGHD6-19*01>2_IGHJ1*01_B
2180
V1-2_IGLJ7*01
840
1445


2182
VH3-23_IGHD6-25*01>1′_IGHJ1*01
2181
V1-2_IGLJ7*01
840
1445


2183
VH3-23_IGHD6-25*01>3′_IGHJ1*01
2182
V1-2_IGLJ7*01
840
1445


2184
VH3-23_IGHD7-27*01>1′_IGHJ1*01_B
2183
V1-2_IGLJ7*01
840
1445


2185
VH3-23_IGHD7-27*01>2′_IGHJ1*01
2184
V1-2_IGLJ7*01
840
1445


2186
VH3-23_IGHD6-6*01>2_IGHJ2*01
2233
V1-2_IGLJ7*01
840
1445


2187
VH3-23_IGHD6-13*01>1_IGHJ2*01
2234
V1-2_IGLJ7*01
840
1445


2188
VH3-23_IGHD6-13*01>2_IGHJ2*01
2235
V1-2_IGLJ7*01
840
1445


2189
VH3-23_IGHD6-19*01>1_IGHJ2*01
2236
V1-2_IGLJ7*01
840
1445


2190
VH3-23_IGHD6-19*01>2_IGHJ2*01
2237
V1-2_IGLJ7*01
840
1445


2191
VH3-23_IGHD6-25*01>1_IGHJ2*01
2238
V1-2_IGLJ7*01
840
1445


2192
VH3-23_IGHD6-25*01>2_IGHJ2*01
2239
V1-2_IGLJ7*01
840
1445


2193
VH3-23_IGHD7-27*01>1_IGHJ2*01
2240
V1-2_IGLJ7*01
840
1445


2194
VH3-23_IGHD7-27*01>3_IGHJ2*01
2241
V1-2_IGLJ7*01
840
1445


2195
VH3-23_IGHD6-13*01>1′_IGHJ2*01
2290
V1-2_IGLJ7*01
840
1445


2196
VH3-23_IGHD6-13*01>2′_IGHJ2*01
2291
V1-2_IGLJ7*01
840
1445


2197
VH3-23_IGHD6-13*01>2_IGHJ2*01_B
2292
V1-2_IGLJ7*01
840
1445


2198
VH3-23_IGHD6-19*01>1′_IGHJ2*01
2293
V1-2_IGLJ7*01
840
1445


2199
VH3-23_IGHD6-19*01>2′_IGHJ2*01
2294
V1-2_IGLJ7*01
840
1445


2200
VH3-23_IGHD6-19*01>2_IGHJ2*01_B
2295
V1-2_IGLJ7*01
840
1445


2201
VH3-23_IGHD6-25*01>1′_IGHJ2*01
2296
V1-2_IGLJ7*01
840
1445


2202
VH3-23_IGHD6-25*01>3′_IGHJ2*01
2297
V1-2_IGLJ7*01
840
1445


2203
VH3-23_IGHD7-27*01>1′_IGHJ2*01
2298
V1-2_IGLJ7*01
840
1445


2204
VH3-23_IGHD7-27*01>2′_IGHJ2*01
2299
V1-2_IGLJ7*01
840
1445


2205
VH3-23_IGHD6-6*01>2_IGHJ3*01
2348
V1-2_IGLJ7*01
840
1445


2206
VH3-23_IGHD6-13*01>1_IGHJ3*01
2349
V1-2_IGLJ7*01
840
1445


2207
VH3-23_IGHD6-13*01>2_IGHJ3*01
2350
V1-2_IGLJ7*01
840
1445


2208
VH3-23_IGHD6-19*01>1_IGHJ3*01
2351
V1-2_IGLJ7*01
840
1445


2209
VH3-23_IGHD6-19*01>2_IGHJ3*01
2352
V1-2_IGLJ7*01
840
1445


2210
VH3-23_IGHD6-25*01>1_IGHJ3*01
2353
V1-2_IGLJ7*01
840
1445


2211
VH3-23_IGHD6-25*01>2_IGHJ3*01
2354
V1-2_IGLJ7*01
840
1445


2212
VH3-23_IGHD7-27*01>1_IGHJ3*01
2355
V1-2_IGLJ7*01
840
1445


2213
VH3-23_IGHD7-27*01>3_IGHJ3*01
2356
V1-2_IGLJ7*01
840
1445


2214
VH3-23_IGHD6-13*01>1′_IGHJ3*01
2405
V1-2_IGLJ7*01
840
1445


2215
VH3-23_IGHD6-13*01>2′_IGHJ3*01
2406
V1-2_IGLJ7*01
840
1445


2216
VH3-23_IGHD6-13*01>1_IGHJ6*01
2407
V1-2_IGLJ7*01
840
1445


2217
VH3-23_IGHD6-19*01>1′_IGHJ3*01
2408
V1-2_IGLJ7*01
840
1445


2218
VH3-23_IGHD6-19*01>2′_IGHJ3*01
2409
V1-2_IGLJ7*01
840
1445


2219
VH3-23_IGHD6-19*01>3′_IGHJ3*01
2410
V1-2_IGLJ7*01
840
1445


2220
VH3-23_IGHD6-25*01>1′_IGHJ3*01
2411
V1-2_IGLJ7*01
840
1445


2221
VH3-23_IGHD6-25*01>3′_IGHJ3*01
2412
V1-2_IGLJ7*01
840
1445


2222
VH3-23_IGHD7-27*01>1′_IGHJ3*01
2413
V1-2_IGLJ7*01
840
1445


2223
VH3-23_IGHD7-27*01>2′_IGHJ3*01
2414
V1-2_IGLJ7*01
840
1445


2224
VH3-23_IGHD6-6*01>2_IGHJ4*01
2463
V1-2_IGLJ7*01
840
1445


2225
VH3-23_IGHD6-13*01>1_IGHJ4*01
2464
V1-2_IGLJ7*01
840
1445


2226
VH3-23_IGHD6-13*01>2_IGHJ4*01
2465
V1-2_IGLJ7*01
840
1445


2227
VH3-23_IGHD6-19*01>1_IGHJ4*01
2466
V1-2_IGLJ7*01
840
1445


2228
VH3-23_IGHD6-19*01>2_IGHJ4*01
2467
V1-2_IGLJ7*01
840
1445


2229
VH3-23_IGHD6-25*01>1_IGHJ4*01
2468
V1-2_IGLJ7*01
840
1445


2230
VH3-23_IGHD6-25*01>2_IGHJ4*01
2469
V1-2_IGLJ7*01
840
1445


2231
VH3-23_IGHD7-27*01>1_IGHJ4*01
2470
V1-2_IGLJ7*01
840
1445


2232
VH3-23_IGHD7-27*01>3_IGHJ4*01
2471
V1-2_IGLJ7*01
840
1445


2233
VH3-23_IGHD6-13*01>1′_IGHJ4*01
2520
V1-2_IGLJ7*01
840
1445


2234
VH3-23_IGHD6-13*01>2′_IGHJ4*01
2521
V1-2_IGLJ7*01
840
1445


2235
VH3-23_IGHD6-13*01>2_IGHJ4*01_B
2522
V1-2_IGLJ7*01
840
1445


2236
VH3-23_IGHD6-19*01>1′_IGHJ4*01
2523
V1-2_IGLJ7*01
840
1445


2237
VH3-23_IGHD6-19*01>2′_IGHJ4*01
2524
V1-2_IGLJ7*01
840
1445


2238
VH3-23_IGHD6-19*01>2_IGHJ4*01_B
2525
V1-2_IGLJ7*01
840
1445


2239
VH3-23_IGHD6-25*01>1′_IGHJ4*01
2526
V1-2_IGLJ7*01
840
1445


2240
VH3-23_IGHD6-25*01>3′_IGHJ4*01
2527
V1-2_IGLJ7*01
840
1445


2241
VH3-23_IGHD7-27*01>1′_IGHJ4*01
2528
V1-2_IGLJ7*01
840
1445


2242
VH3-23_IGHD7-27*01>2′_IGHJ4*01
2529
V1-2_IGLJ7*01
840
1445


2243
VH3-23_IGHD6-6*01>2_IGHJ5*01
2578
V1-2_IGLJ7*01
840
1445


2244
VH3-23_IGHD6-13*01>1_IGHJ5*01
2579
V1-2_IGLJ7*01
840
1445


2245
VH3-23_IGHD6-13*01>2_IGHJ5*01
2580
V1-2_IGLJ7*01
840
1445


2246
VH3-23_IGHD6-19*01>1_IGHJ5*01
2581
V1-2_IGLJ7*01
840
1445


2247
VH3-23_IGHD6-19*01>2_IGHJ5*01
2582
V1-2_IGLJ7*01
840
1445


2248
VH3-23_IGHD6-25*01>1_IGHJ5*01
2583
V1-2_IGLJ7*01
840
1445


2249
VH3-23_IGHD6-25*01>2_IGHJ5*01
2584
V1-2_IGLJ7*01
840
1445


2250
VH3-23_IGHD7-27*01>1_IGHJ5*01
2585
V1-2_IGLJ7*01
840
1445


2251
VH3-23_IGHD7-27*01>3_IGHJ5*01
2586
V1-2_IGLJ7*01
840
1445


2252
VH3-23_IGHD6-13*01>1′_IGHJ5*01
2635
V1-2_IGLJ7*01
840
1445


2253
VH3-23_IGHD6-13*01>2′_IGHJ5*01
2636
V1-2_IGLJ7*01
840
1445


2254
VH3-23_IGHD6-13*01>3′_IGHJ5*01
2637
V1-2_IGLJ7*01
840
1445


2255
VH3-23_IGHD6-19*01>1′_IGHJ5*01
2638
V1-2_IGLJ7*01
840
1445


2256
VH3-23_IGHD6-19*01>2′_IGHJ5*01
2639
V1-2_IGLJ7*01
840
1445


2257
VH3-23_IGHD6-19*01>2_IGHJ5*01_B
2640
V1-2_IGLJ7*01
840
1445


2258
VH3-23_IGHD6-25*01>1′_IGHJ5*01
2641
V1-2_IGLJ7*01
840
1445


2259
VH3-23_IGHD6-25*01>3′_IGHJ5*01
2642
V1-2_IGLJ7*01
840
1445


2260
VH3-23_IGHD7-27*01>1′_IGHJ5*01
2643
V1-2_IGLJ7*01
840
1445


2261
VH3-23_IGHD7-27*01>2′_IGHJ5*01
2644
V1-2_IGLJ7*01
840
1445


2262
VH3-23_IGHD6-6*01>2_IGHJ6*01
2693
V1-2_IGLJ7*01
840
1445


2263
VH3-23_IGHD6-6*01>2_IGHJ1*01
2118
V1-20_IGLJ6*01
841
1446


2264
VH3-23_IGHD6-13*01>1_IGHJ1*01
2119
V1-20_IGLJ6*01
841
1446


2265
VH3-23_IGHD6-13*01>2_IGHJ1*01
2120
V1-20_IGLJ6*01
841
1446


2266
VH3-23_IGHD6-19*01>1_IGHJ1*01
2121
V1-20_IGLJ6*01
841
1446


2267
VH3-23_IGHD6-19*01>2_IGHJ1*01
2122
V1-20_IGLJ6*01
841
1446


2268
VH3-23_IGHD6-25*01>1_IGHJ1*01
2123
V1-20_IGLJ6*01
841
1446


2269
VH3-23_IGHD6-25*01>2_IGHJ1*01
2124
V1-20_IGLJ6*01
841
1446


2270
VH3-23_IGHD7-27*01>1_IGHJ1*01
2125
V1-20_IGLJ6*01
841
1446


2271
VH3-23_IGHD7-27*01>3_IGHJ1*01
2126
V1-20_IGLJ6*01
841
1446


2272
VH3-23_IGHD6-13*01>1′_IGHJ1*01
2175
V1-20_IGLJ6*01
841
1446


2273
VH3-23_IGHD6-13*01>2′_IGHJ1*01
2176
V1-20_IGLJ6*01
841
1446


2274
VH3-23_IGHD6-13*01>2_IGHJ1*01_B
2177
V1-20_IGLJ6*01
841
1446


2275
VH3-23_IGHD6-19*01>1′_IGHJ1*01
2178
V1-20_IGLJ6*01
841
1446


2276
VH3-23_IGHD6-19*01>2′_IGHJ1*01
2179
V1-20_IGLJ6*01
841
1446


2277
VH3-23_IGHD6-19*01>2_IGHJ1*01_B
2180
V1-20_IGLJ6*01
841
1446


2278
VH3-23_IGHD6-25*01>1′_IGHJ1*01
2181
V1-20_IGLJ6*01
841
1446


2279
VH3-23_IGHD6-25*01>3′_IGHJ1*01
2182
V1-20_IGLJ6*01
841
1446


2280
VH3-23_IGHD7-27*01>1′_IGHJ1*01_B
2183
V1-20_IGLJ6*01
841
1446


2281
VH3-23_IGHD7-27*01>2′_IGHJ1*01
2184
V1-20_IGLJ6*01
841
1446


2282
VH3-23_IGHD6-6*01>2_IGHJ2*01
2233
V1-20_IGLJ6*01
841
1446


2283
VH3-23_IGHD6-13*01>1_IGHJ2*01
2234
V1-20_IGLJ6*01
841
1446


2284
VH3-23_IGHD6-13*01>2_IGHJ2*01
2235
V1-20_IGLJ6*01
841
1446


2285
VH3-23_IGHD6-19*01>1_IGHJ2*01
2236
V1-20_IGLJ6*01
841
1446


2286
VH3-23_IGHD6-19*01>2_IGHJ2*01
2237
V1-20_IGLJ6*01
841
1446


2287
VH3-23_IGHD6-25*01>1_IGHJ2*01
2238
V1-20_IGLJ6*01
841
1446


2288
VH3-23_IGHD6-25*01>2_IGHJ2*01
2239
V1-20_IGLJ6*01
841
1446


2289
VH3-23_IGHD7-27*01>1_IGHJ2*01
2240
V1-20_IGLJ6*01
841
1446


2290
VH3-23_IGHD7-27*01>3_IGHJ2*01
2241
V1-20_IGLJ6*01
841
1446


2291
VH3-23_IGHD6-13*01>1′_IGHJ2*01
2290
V1-20_IGLJ6*01
841
1446


2292
VH3-23_IGHD6-13*01>2′_IGHJ2*01
2291
V1-20_IGLJ6*01
841
1446


2293
VH3-23_IGHD6-13*01>2_IGHJ2*01_B
2292
V1-20_IGLJ6*01
841
1446


2294
VH3-23_IGHD6-19*01>1′_IGHJ2*01
2293
V1-20_IGLJ6*01
841
1446


2295
VH3-23_IGHD6-19*01>2′_IGHJ2*01
2294
V1-20_IGLJ6*01
841
1446


2296
VH3-23_IGHD6-19*01>2_IGHJ2*01_B
2295
V1-20_IGLJ6*01
841
1446


2297
VH3-23_IGHD6-25*01>1′_IGHJ2*01
2296
V1-20_IGLJ6*01
841
1446


2298
VH3-23_IGHD6-25*01>3′_IGHJ2*01
2297
V1-20_IGLJ6*01
841
1446


2299
VH3-23_IGHD7-27*01>1′_IGHJ2*01
2298
V1-20_IGLJ6*01
841
1446


2300
VH3-23_IGHD7-27*01>2′_IGHJ2*01
2299
V1-20_IGLJ6*01
841
1446


2301
VH3-23_IGHD6-6*01>2_IGHJ3*01
2348
V1-20_IGLJ6*01
841
1446


2302
VH3-23_IGHD6-13*01>1_IGHJ3*01
2349
V1-20_IGLJ6*01
841
1446


2303
VH3-23_IGHD6-13*01>2_IGHJ3*01
2350
V1-20_IGLJ6*01
841
1446


2304
VH3-23_IGHD6-19*01>1_IGHJ3*01
2351
V1-20_IGLJ6*01
841
1446


2305
VH3-23_IGHD6-19*01>2_IGHJ3*01
2352
V1-20_IGLJ6*01
841
1446


2306
VH3-23_IGHD6-25*01>1_IGHJ3*01
2353
V1-20_IGLJ6*01
841
1446


2307
VH3-23_IGHD6-25*01>2_IGHJ3*01
2354
V1-20_IGLJ6*01
841
1446


2308
VH3-23_IGHD7-27*01>1_IGHJ3*01
2355
V1-20_IGLJ6*01
841
1446


2309
VH3-23_IGHD7-27*01>3_IGHJ3*01
2356
V1-20_IGLJ6*01
841
1446


2310
VH3-23_IGHD6-13*01>1′_IGHJ3*01
2405
V1-20_IGLJ6*01
841
1446


2311
VH3-23_IGHD6-13*01>2′_IGHJ3*01
2406
V1-20_IGLJ6*01
841
1446


2312
VH3-23_IGHD6-13*01>1_IGHJ6*01
2407
V1-20_IGLJ6*01
841
1446


2313
VH3-23_IGHD6-19*01>1′_IGHJ3*01
2408
V1-20_IGLJ6*01
841
1446


2314
VH3-23_IGHD6-19*01>2′_IGHJ3*01
2409
V1-20_IGLJ6*01
841
1446


2315
VH3-23_IGHD6-19*01>3′_IGHJ3*01
2410
V1-20_IGLJ6*01
841
1446


2316
VH3-23_IGHD6-25*01>1′_IGHJ3*01
2411
V1-20_IGLJ6*01
841
1446


2317
VH3-23_IGHD6-25*01>3′_IGHJ3*01
2412
V1-20_IGLJ6*01
841
1446


2318
VH3-23_IGHD7-27*01>1′_IGHJ3*01
2413
V1-20_IGLJ6*01
841
1446


2319
VH3-23_IGHD7-27*01>2′_IGHJ3*01
2414
V1-20_IGLJ6*01
841
1446


2320
VH3-23_IGHD6-6*01>2_IGHJ4*01
2463
V1-20_IGLJ6*01
841
1446


2321
VH3-23_IGHD6-13*01>1_IGHJ4*01
2464
V1-20_IGLJ6*01
841
1446


2322
VH3-23_IGHD6-13*01>2_IGHJ4*01
2465
V1-20_IGLJ6*01
841
1446


2323
VH3-23_IGHD6-19*01>1_IGHJ4*01
2466
V1-20_IGLJ6*01
841
1446


2324
VH3-23_IGHD6-19*01>2_IGHJ4*01
2467
V1-20_IGLJ6*01
841
1446


2325
VH3-23_IGHD6-25*01>1_IGHJ4*01
2468
V1-20_IGLJ6*01
841
1446


2326
VH3-23_IGHD6-25*01>2_IGHJ4*01
2469
V1-20_IGLJ6*01
841
1446


2327
VH3-23_IGHD7-27*01>1_IGHJ4*01
2470
V1-20_IGLJ6*01
841
1446


2328
VH3-23_IGHD7-27*01>3_IGHJ4*01
2471
V1-20_IGLJ6*01
841
1446


2329
VH3-23_IGHD6-13*01>1′_IGHJ4*01
2520
V1-20_IGLJ6*01
841
1446


2330
VH3-23_IGHD6-13*01>2′_IGHJ4*01
2521
V1-20_IGLJ6*01
841
1446


2331
VH3-23_IGHD6-13*01>2_IGHJ4*01_B
2522
V1-20_IGLJ6*01
841
1446


2332
VH3-23_IGHD6-19*01>1′_IGHJ4*01
2523
V1-20_IGLJ6*01
841
1446


2333
VH3-23_IGHD6-19*01>2′_IGHJ4*01
2524
V1-20_IGLJ6*01
841
1446


2334
VH3-23_IGHD6-19*01>2_IGHJ4*01_B
2525
V1-20_IGLJ6*01
841
1446


2335
VH3-23_IGHD6-25*01>1′_IGHJ4*01
2526
V1-20_IGLJ6*01
841
1446


2336
VH3-23_IGHD6-25*01>3′_IGHJ4*01
2527
V1-20_IGLJ6*01
841
1446


2337
VH3-23_IGHD7-27*01>1′_IGHJ4*01
2528
V1-20_IGLJ6*01
841
1446


2338
VH3-23_IGHD7-27*01>2′_IGHJ4*01
2529
V1-20_IGLJ6*01
841
1446


2339
VH3-23_IGHD6-6*01>2_IGHJ5*01
2578
V1-20_IGLJ6*01
841
1446


2340
VH3-23_IGHD6-13*01>1_IGHJ5*01
2579
V1-20_IGLJ6*01
841
1446


2341
VH3-23_IGHD6-13*01>2_IGHJ5*01
2580
V1-20_IGLJ6*01
841
1446


2342
VH3-23_IGHD6-19*01>1_IGHJ5*01
2581
V1-20_IGLJ6*01
841
1446


2343
VH3-23_IGHD6-19*01>2_IGHJ5*01
2582
V1-20_IGLJ6*01
841
1446


2344
VH3-23_IGHD6-25*01>1_IGHJ5*01
2583
V1-20_IGLJ6*01
841
1446


2345
VH3-23_IGHD6-25*01>2_IGHJ5*01
2584
V1-20_IGLJ6*01
841
1446


2346
VH3-23_IGHD7-27*01>1_IGHJ5*01
2585
V1-20_IGLJ6*01
841
1446


2347
VH3-23_IGHD7-27*01>3_IGHJ5*01
2586
V1-20_IGLJ6*01
841
1446


2348
VH3-23_IGHD6-13*01>1′_IGHJ5*01
2635
V1-20_IGLJ6*01
841
1446


2349
VH3-23_IGHD6-13*01>2′_IGHJ5*01
2636
V1-20_IGLJ6*01
841
1446


2350
VH3-23_IGHD6-13*01>3′_IGHJ5*01
2637
V1-20_IGLJ6*01
841
1446


2351
VH3-23_IGHD6-19*01>1′_IGHJ5*01
2638
V1-20_IGLJ6*01
841
1446


2352
VH3-23_IGHD6-19*01>2′_IGHJ5*01
2639
V1-20_IGLJ6*01
841
1446


2353
VH3-23_IGHD6-19*01>2_IGHJ5*01_B
2640
V1-20_IGLJ6*01
841
1446


2354
VH3-23_IGHD6-25*01>1′_IGHJ5*01
2641
V1-20_IGLJ6*01
841
1446


2355
VH3-23_IGHD6-25*01>3′_IGHJ5*01
2642
V1-20_IGLJ6*01
841
1446


2356
VH3-23_IGHD7-27*01>1′_IGHJ5*01
2643
V1-20_IGLJ6*01
841
1446


2357
VH3-23_IGHD7-27*01>2′_IGHJ5*01
2644
V1-20_IGLJ6*01
841
1446


2358
VH3-23_IGHD6-6*01>2_IGHJ6*01
2693
V1-20_IGLJ6*01
841
1446


2359
VH3-23_IGHD6-6*01>2_IGHJ1*01
2118
V1-3_IGLJ1*01
842
1447


2360
VH3-23_IGHD6-13*01>1_IGHJ1*01
2119
V1-3_IGLJ1*01
842
1447


2361
VH3-23_IGHD6-13*01>2_IGHJ1*01
2120
V1-3_IGLJ1*01
842
1447


2362
VH3-23_IGHD6-19*01>1_IGHJ1*01
2121
V1-3_IGLJ1*01
842
1447


2363
VH3-23_IGHD6-19*01>2_IGHJ1*01
2122
V1-3_IGLJ1*01
842
1447


2364
VH3-23_IGHD6-25*01>1_IGHJ1*01
2123
V1-3_IGLJ1*01
842
1447


2365
VH3-23_IGHD6-25*01>2_IGHJ1*01
2124
V1-3_IGLJ1*01
842
1447


2366
VH3-23_IGHD7-27*01>1_IGHJ1*01
2125
V1-3_IGLJ1*01
842
1447


2367
VH3-23_IGHD7-27*01>3_IGHJ1*01
2126
V1-3_IGLJ1*01
842
1447


2368
VH3-23_IGHD6-13*01>1′_IGHJ1*01
2175
V1-3_IGLJ1*01
842
1447


2369
VH3-23_IGHD6-13*01>2′_IGHJ1*01
2176
V1-3_IGLJ1*01
842
1447


2370
VH3-23_IGHD6-13*01>2_IGHJ1*01_B
2177
V1-3_IGLJ1*01
842
1447


2371
VH3-23_IGHD6-19*01>1′_IGHJ1*01
2178
V1-3_IGLJ1*01
842
1447


2372
VH3-23_IGHD6-19*01>2′_IGHJ1*01
2179
V1-3_IGLJ1*01
842
1447


2373
VH3-23_IGHD6-19*01>2_IGHJ1*01_B
2180
V1-3_IGLJ1*01
842
1447


2374
VH3-23_IGHD6-25*01>1′_IGHJ1*01
2181
V1-3_IGLJ1*01
842
1447


2375
VH3-23_IGHD6-25*01>3′_IGHJ1*01
2182
V1-3_IGLJ1*01
842
1447


2376
VH3-23_IGHD7-27*01>1′_IGHJ1*01_B
2183
V1-3_IGLJ1*01
842
1447


2377
VH3-23_IGHD7-27*01>2′_IGHJ1*01
2184
V1-3_IGLJ1*01
842
1447


2378
VH3-23_IGHD6-6*01>2_IGHJ2*01
2233
V1-3_IGLJ1*01
842
1447


2379
VH3-23_IGHD6-13*01>1_IGHJ2*01
2234
V1-3_IGLJ1*01
842
1447


2380
VH3-23_IGHD6-13*01>2_IGHJ2*01
2235
V1-3_IGLJ1*01
842
1447


2381
VH3-23_IGHD6-19*01>1_IGHJ2*01
2236
V1-3_IGLJ1*01
842
1447


2382
VH3-23_IGHD6-19*01>2_IGHJ2*01
2237
V1-3_IGLJ1*01
842
1447


2383
VH3-23_IGHD6-25*01>1_IGHJ2*01
2238
V1-3_IGLJ1*01
842
1447


2384
VH3-23_IGHD6-25*01>2_IGHJ2*01
2239
V1-3_IGLJ1*01
842
1447


2385
VH3-23_IGHD7-27*01>1_IGHJ2*01
2240
V1-3_IGLJ1*01
842
1447


2386
VH3-23_IGHD7-27*01>3_IGHJ2*01
2241
V1-3_IGLJ1*01
842
1447


2387
VH3-23_IGHD6-13*01>1′_IGHJ2*01
2290
V1-3_IGLJ1*01
842
1447


2388
VH3-23_IGHD6-13*01>2′_IGHJ2*01
2291
V1-3_IGLJ1*01
842
1447


2389
VH3-23_IGHD6-13*01>2_IGHJ2*01_B
2292
V1-3_IGLJ1*01
842
1447


2390
VH3-23_IGHD6-19*01>1′_IGHJ2*01
2293
V1-3_IGLJ1*01
842
1447


2391
VH3-23_IGHD6-19*01>2′_IGHJ2*01
2294
V1-3_IGLJ1*01
842
1447


2392
VH3-23_IGHD6-19*01>2_IGHJ2*01_B
2295
V1-3_IGLJ1*01
842
1447


2393
VH3-23_IGHD6-25*01>1′_IGHJ2*01
2296
V1-3_IGLJ1*01
842
1447


2394
VH3-23_IGHD6-25*01>3′_IGHJ2*01
2297
V1-3_IGLJ1*01
842
1447


2395
VH3-23_IGHD7-27*01>1′_IGHJ2*01
2298
V1-3_IGLJ1*01
842
1447


2396
VH3-23_IGHD7-27*01>2′_IGHJ2*01
2299
V1-3_IGLJ1*01
842
1447


2397
VH3-23_IGHD6-6*01>2_IGHJ3*01
2348
V1-3_IGLJ1*01
842
1447


2398
VH3-23_IGHD6-13*01>1_IGHJ3*01
2349
V1-3_IGLJ1*01
842
1447


2399
VH3-23_IGHD6-13*01>2_IGHJ3*01
2350
V1-3_IGLJ1*01
842
1447


2400
VH3-23_IGHD6-19*01>1_IGHJ3*01
2351
V1-3_IGLJ1*01
842
1447


2401
VH3-23_IGHD6-19*01>2_IGHJ3*01
2352
V1-3_IGLJ1*01
842
1447


2402
VH3-23_IGHD6-25*01>1_IGHJ3*01
2353
V1-3_IGLJ1*01
842
1447


2403
VH3-23_IGHD6-25*01>2_IGHJ3*01
2354
V1-3_IGLJ1*01
842
1447


2404
VH3-23_IGHD7-27*01>1_IGHJ3*01
2355
V1-3_IGLJ1*01
842
1447


2405
VH3-23_IGHD7-27*01>3_IGHJ3*01
2356
V1-3_IGLJ1*01
842
1447


2406
VH3-23_IGHD6-13*01>1′_IGHJ3*01
2405
V1-3_IGLJ1*01
842
1447


2407
VH3-23_IGHD6-13*01>2′_IGHJ3*01
2406
V1-3_IGLJ1*01
842
1447


2408
VH3-23_IGHD6-13*01>1_IGHJ6*01
2407
V1-3_IGLJ1*01
842
1447


2409
VH3-23_IGHD6-19*01>1′_IGHJ3*01
2408
V1-3_IGLJ1*01
842
1447


2410
VH3-23_IGHD6-19*01>2′_IGHJ3*01
2409
V1-3_IGLJ1*01
842
1447


2411
VH3-23_IGHD6-19*01>3′_IGHJ3*01
2410
V1-3_IGLJ1*01
842
1447


2412
VH3-23_IGHD6-25*01>1′_IGHJ3*01
2411
V1-3_IGLJ1*01
842
1447


2413
VH3-23_IGHD6-25*01>3′_IGHJ3*01
2412
V1-3_IGLJ1*01
842
1447


2414
VH3-23_IGHD7-27*01>1′_IGHJ3*01
2413
V1-3_IGLJ1*01
842
1447


2415
VH3-23_IGHD7-27*01>2′_IGHJ3*01
2414
V1-3_IGLJ1*01
842
1447


2416
VH3-23_IGHD6-6*01>2_IGHJ4*01
2463
V1-3_IGLJ1*01
842
1447


2417
VH3-23_IGHD6-13*01>1_IGHJ4*01
2464
V1-3_IGLJ1*01
842
1447


2418
VH3-23_IGHD6-13*01>2_IGHJ4*01
2465
V1-3_IGLJ1*01
842
1447


2419
VH3-23_IGHD6-19*01>1_IGHJ4*01
2466
V1-3_IGLJ1*01
842
1447


2420
VH3-23_IGHD6-19*01>2_IGHJ4*01
2467
V1-3_IGLJ1*01
842
1447


2421
VH3-23_IGHD6-25*01>1_IGHJ4*01
2468
V1-3_IGLJ1*01
842
1447


2422
VH3-23_IGHD6-25*01>2_IGHJ4*01
2469
V1-3_IGLJ1*01
842
1447


2423
VH3-23_IGHD7-27*01>1_IGHJ4*01
2470
V1-3_IGLJ1*01
842
1447


2424
VH3-23_IGHD7-27*01>3_IGHJ4*01
2471
V1-3_IGLJ1*01
842
1447


2425
VH3-23_IGHD6-13*01>1′_IGHJ4*01
2520
V1-3_IGLJ1*01
842
1447


2426
VH3-23_IGHD6-13*01>2′_IGHJ4*01
2521
V1-3_IGLJ1*01
842
1447


2427
VH3-23_IGHD6-13*01>2_IGHJ4*01_B
2522
V1-3_IGLJ1*01
842
1447


2428
VH3-23_IGHD6-19*01>1′_IGHJ4*01
2523
V1-3_IGLJ1*01
842
1447


2429
VH3-23_IGHD6-19*01>2′_IGHJ4*01
2524
V1-3_IGLJ1*01
842
1447


2430
VH3-23_IGHD6-19*01>2_IGHJ4*01_B
2525
V1-3_IGLJ1*01
842
1447


2431
VH3-23_IGHD6-25*01>1′_IGHJ4*01
2526
V1-3_IGLJ1*01
842
1447


2432
VH3-23_IGHD6-25*01>3′_IGHJ4*01
2527
V1-3_IGLJ1*01
842
1447


2433
VH3-23_IGHD7-27*01>1′_IGHJ4*01
2528
V1-3_IGLJ1*01
842
1447


2434
VH3-23_IGHD7-27*01>2′_IGHJ4*01
2529
V1-3_IGLJ1*01
842
1447


2435
VH3-23_IGHD6-6*01>2_IGHJ5*01
2578
V1-3_IGLJ1*01
842
1447


2436
VH3-23_IGHD6-13*01>1_IGHJ5*01
2579
V1-3_IGLJ1*01
842
1447


2437
VH3-23_IGHD6-13*01>2_IGHJ5*01
2580
V1-3_IGLJ1*01
842
1447


2438
VH3-23_IGHD6-19*01>1_IGHJ5*01
2581
V1-3_IGLJ1*01
842
1447


2439
VH3-23_IGHD6-19*01>2_IGHJ5*01
2582
V1-3_IGLJ1*01
842
1447


2440
VH3-23_IGHD6-25*01>1_IGHJ5*01
2583
V1-3_IGLJ1*01
842
1447


2441
VH3-23_IGHD6-25*01>2_IGHJ5*01
2584
V1-3_IGLJ1*01
842
1447


2442
VH3-23_IGHD7-27*01>1_IGHJ5*01
2585
V1-3_IGLJ1*01
842
1447


2443
VH3-23_IGHD7-27*01>3_IGHJ5*01
2586
V1-3_IGLJ1*01
842
1447


2444
VH3-23_IGHD6-13*01>1′_IGHJ5*01
2635
V1-3_IGLJ1*01
842
1447


2445
VH3-23_IGHD6-13*01>2′_IGHJ5*01
2636
V1-3_IGLJ1*01
842
1447


2446
VH3-23_IGHD6-13*01>3′_IGHJ5*01
2637
V1-3_IGLJ1*01
842
1447


2447
VH3-23_IGHD6-19*01>1′_IGHJ5*01
2638
V1-3_IGLJ1*01
842
1447


2448
VH3-23_IGHD6-19*01>2′_IGHJ5*01
2639
V1-3_IGLJ1*01
842
1447


2449
VH3-23_IGHD6-19*01>2_IGHJ5*01_B
2640
V1-3_IGLJ1*01
842
1447


2450
VH3-23_IGHD6-25*01>1′_IGHJ5*01
2641
V1-3_IGLJ1*01
842
1447


2451
VH3-23_IGHD6-25*01>3′_IGHJ5*01
2642
V1-3_IGLJ1*01
842
1447


2452
VH3-23_IGHD7-27*01>1′_IGHJ5*01
2643
V1-3_IGLJ1*01
842
1447


2453
VH3-23_IGHD7-27*01>2′_IGHJ5*01
2644
V1-3_IGLJ1*01
842
1447


2454
VH3-23_IGHD6-6*01>2_IGHJ6*01
2693
V1-3_IGLJ1*01
842
1447


2455
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
V2-13_IGLJ2*01
849
1454


2456
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
V2-13_IGLJ2*01
849
1454


2457
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
V2-13_IGLJ2*01
849
1454


2458
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
V2-13_IGLJ2*01
849
1454


2459
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
V2-13_IGLJ2*01
849
1454


2460
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
V2-13_IGLJ2*01
849
1454


2461
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
V2-13_IGLJ2*01
849
1454


2462
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
V2-13_IGLJ2*01
849
1454


2463
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
V2-13_IGLJ2*01
849
1454


2464
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
V2-13_IGLJ2*01
849
1454


2465
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
V2-13_IGLJ2*01
849
1454


2466
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
V2-13_IGLJ2*01
849
1454


2467
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
V2-13_IGLJ2*01
849
1454


2468
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
V2-13_IGLJ2*01
849
1454


2469
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
V2-13_IGLJ2*01
849
1454


2470
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
V2-13_IGLJ2*01
849
1454


2471
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
V2-13_IGLJ2*01
849
1454


2472
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
V2-13_IGLJ2*01
849
1454


2473
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
V2-13_IGLJ2*01
849
1454


2474
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
V2-13_IGLJ2*01
849
1454


2475
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
V2-13_IGLJ2*01
849
1454


2476
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
V2-13_IGLJ2*01
849
1454


2477
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
V2-13_IGLJ2*01
849
1454


2478
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
V2-13_IGLJ2*01
849
1454


2479
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
V2-13_IGLJ2*01
849
1454


2480
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
V2-13_IGLJ2*01
849
1454


2481
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
V2-13_IGLJ2*01
849
1454


2482
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
V2-13_IGLJ2*01
849
1454


2483
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
V2-13_IGLJ2*01
849
1454


2484
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
V2-13_IGLJ2*01
849
1454


2485
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
V2-13_IGLJ2*01
849
1454


2486
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
V2-13_IGLJ2*01
849
1454


2487
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
V2-13_IGLJ2*01
849
1454


2488
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
V2-13_IGLJ2*01
849
1454


2489
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
V2-13_IGLJ2*01
849
1454


2490
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
V2-13_IGLJ2*01
849
1454


2491
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
V2-13_IGLJ2*01
849
1454


2492
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
V2-13_IGLJ2*01
849
1454


2493
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
V2-13_IGLJ2*01
849
1454


2494
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
V2-13_IGLJ2*01
849
1454


2495
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
V2-13_IGLJ2*01
849
1454


2496
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
V2-13_IGLJ2*01
849
1454


2497
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
V2-13_IGLJ2*01
849
1454


2498
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
V2-13_IGLJ2*01
849
1454


2499
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
V2-13_IGLJ2*01
849
1454


2500
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
V2-13_IGLJ2*01
849
1454


2501
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
V2-13_IGLJ2*01
849
1454


2502
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
V2-13_IGLJ2*01
849
1454


2503
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
V2-13_IGLJ2*01
849
1454


2504
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
V2-13_IGLJ2*01
849
1454


2505
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
V2-13_IGLJ2*01
849
1454


2506
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
V2-13_IGLJ2*01
849
1454


2507
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
V2-13_IGLJ2*01
849
1454


2508
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
V2-13_IGLJ2*01
849
1454


2509
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
V2-13_IGLJ2*01
849
1454


2510
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
V2-13_IGLJ2*01
849
1454


2511
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
V2-13_IGLJ2*01
849
1454


2512
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
V2-13_IGLJ2*01
849
1454


2513
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
V2-13_IGLJ2*01
849
1454


2514
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
V2-13_IGLJ2*01
849
1454


2515
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
V2-13_IGLJ2*01
849
1454


2516
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
V2-13_IGLJ2*01
849
1454


2517
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
V2-13_IGLJ2*01
849
1454


2518
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
V2-13_IGLJ2*01
849
1454


2519
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
V2-13_IGLJ2*01
849
1454


2520
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
V2-13_IGLJ2*01
849
1454


2521
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
V2-13_IGLJ2*01
849
1454


2522
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
V2-13_IGLJ2*01
849
1454


2523
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
V2-13_IGLJ2*01
849
1454


2524
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
V2-13_IGLJ2*01
849
1454


2525
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
V2-13_IGLJ2*01
849
1454


2526
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
V2-13_IGLJ2*01
849
1454


2527
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
V2-13_IGLJ2*01
849
1454


2528
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
V2-13_IGLJ2*01
849
1454


2529
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
V2-13_IGLJ2*01
849
1454


2530
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
V2-13_IGLJ2*01
849
1454


2531
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
V2-13_IGLJ2*01
849
1454


2532
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
V2-13_IGLJ2*01
849
1454


2533
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
V2-13_IGLJ2*01
849
1454


2534
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
V2-13_IGLJ2*01
849
1454


2535
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
V2-13_IGLJ2*01
849
1454


2536
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
V2-13_IGLJ2*01
849
1454


2537
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
V2-13_IGLJ2*01
849
1454


2538
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
V2-13_IGLJ2*01
849
1454


2539
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
V2-13_IGLJ2*01
849
1454


2540
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
V2-13_IGLJ2*01
849
1454


2541
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
V2-13_IGLJ2*01
849
1454


2542
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
V2-13_IGLJ2*01
849
1454


2543
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
V2-13_IGLJ2*01
849
1454


2544
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
V2-13_IGLJ2*01
849
1454


2545
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
V2-13_IGLJ2*01
849
1454


2546
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
V2-13_IGLJ2*01
849
1454


2547
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
V2-13_IGLJ2*01
849
1454


2548
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
V2-13_IGLJ2*01
849
1454


2549
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
V2-13_IGLJ2*01
849
1454


2550
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
V2-13_IGLJ2*01
849
1454


2551
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
V2-14_IGLJ4*01
850
1455


2552
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
V2-14_IGLJ4*01
850
1455


2553
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
V2-14_IGLJ4*01
850
1455


2554
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
V2-14_IGLJ4*01
850
1455


2555
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
V2-14_IGLJ4*01
850
1455


2556
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
V2-14_IGLJ4*01
850
1455


2557
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
V2-14_IGLJ4*01
850
1455


2558
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
V2-14_IGLJ4*01
850
1455


2559
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
V2-14_IGLJ4*01
850
1455


2560
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
V2-14_IGLJ4*01
850
1455


2561
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
V2-14_IGLJ4*01
850
1455


2562
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
V2-14_IGLJ4*01
850
1455


2563
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
V2-14_IGLJ4*01
850
1455


2564
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
V2-14_IGLJ4*01
850
1455


2565
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
V2-14_IGLJ4*01
850
1455


2566
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
V2-14_IGLJ4*01
850
1455


2567
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
V2-14_IGLJ4*01
850
1455


2568
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
V2-14_IGLJ4*01
850
1455


2569
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
V2-14_IGLJ4*01
850
1455


2570
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
V2-14_IGLJ4*01
850
1455


2571
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
V2-14_IGLJ4*01
850
1455


2572
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
V2-14_IGLJ4*01
850
1455


2573
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
V2-14_IGLJ4*01
850
1455


2574
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
V2-14_IGLJ4*01
850
1455


2575
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
V2-14_IGLJ4*01
850
1455


2576
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
V2-14_IGLJ4*01
850
1455


2577
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
V2-14_IGLJ4*01
850
1455


2578
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
V2-14_IGLJ4*01
850
1455


2579
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
V2-14_IGLJ4*01
850
1455


2580
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
V2-14_IGLJ4*01
850
1455


2581
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
V2-14_IGLJ4*01
850
1455


2582
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
V2-14_IGLJ4*01
850
1455


2583
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
V2-14_IGLJ4*01
850
1455


2584
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
V2-14_IGLJ4*01
850
1455


2585
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
V2-14_IGLJ4*01
850
1455


2586
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
V2-14_IGLJ4*01
850
1455


2587
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
V2-14_IGLJ4*01
850
1455


2588
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
V2-14_IGLJ4*01
850
1455


2589
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
V2-14_IGLJ4*01
850
1455


2590
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
V2-14_IGLJ4*01
850
1455


2591
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
V2-14_IGLJ4*01
850
1455


2592
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
V2-14_IGLJ4*01
850
1455


2593
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
V2-14_IGLJ4*01
850
1455


2594
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
V2-14_IGLJ4*01
850
1455


2595
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
V2-14_IGLJ4*01
850
1455


2596
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
V2-14_IGLJ4*01
850
1455


2597
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
V2-14_IGLJ4*01
850
1455


2598
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
V2-14_IGLJ4*01
850
1455


2599
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
V2-14_IGLJ4*01
850
1455


2600
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
V2-14_IGLJ4*01
850
1455


2601
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
V2-14_IGLJ4*01
850
1455


2602
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
V2-14_IGLJ4*01
850
1455


2603
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
V2-14_IGLJ4*01
850
1455


2604
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
V2-14_IGLJ4*01
850
1455


2605
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
V2-14_IGLJ4*01
850
1455


2606
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
V2-14_IGLJ4*01
850
1455


2607
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
V2-14_IGLJ4*01
850
1455


2608
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
V2-14_IGLJ4*01
850
1455


2609
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
V2-14_IGLJ4*01
850
1455


2610
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
V2-14_IGLJ4*01
850
1455


2611
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
V2-14_IGLJ4*01
850
1455


2612
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
V2-14_IGLJ4*01
850
1455


2613
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
V2-14_IGLJ4*01
850
1455


2614
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
V2-14_IGLJ4*01
850
1455


2615
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
V2-14_IGLJ4*01
850
1455


2616
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
V2-14_IGLJ4*01
850
1455


2617
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
V2-14_IGLJ4*01
850
1455


2618
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
V2-14_IGLJ4*01
850
1455


2619
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
V2-14_IGLJ4*01
850
1455


2620
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
V2-14_IGLJ4*01
850
1455


2621
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
V2-14_IGLJ4*01
850
1455


2622
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
V2-14_IGLJ4*01
850
1455


2623
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
V2-14_IGLJ4*01
850
1455


2624
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
V2-14_IGLJ4*01
850
1455


2625
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
V2-14_IGLJ4*01
850
1455


2626
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
V2-14_IGLJ4*01
850
1455


2627
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
V2-14_IGLJ4*01
850
1455


2628
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
V2-14_IGLJ4*01
850
1455


2629
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
V2-14_IGLJ4*01
850
1455


2630
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
V2-14_IGLJ4*01
850
1455


2631
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
V2-14_IGLJ4*01
850
1455


2632
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
V2-14_IGLJ4*01
850
1455


2633
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
V2-14_IGLJ4*01
850
1455


2634
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
V2-14_IGLJ4*01
850
1455


2635
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
V2-14_IGLJ4*01
850
1455


2636
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
V2-14_IGLJ4*01
850
1455


2637
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
V2-14_IGLJ4*01
850
1455


2638
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
V2-14_IGLJ4*01
850
1455


2639
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
V2-14_IGLJ4*01
850
1455


2640
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
V2-14_IGLJ4*01
850
1455


2641
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
V2-14_IGLJ4*01
850
1455


2642
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
V2-14_IGLJ4*01
850
1455


2643
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
V2-14_IGLJ4*01
850
1455


2644
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
V2-14_IGLJ4*01
850
1455


2645
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
V2-14_IGLJ4*01
850
1455


2646
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
V2-14_IGLJ4*01
850
1455


2647
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
V2-15_IGLJ7*01
850
1455


2648
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
V2-15_IGLJ7*01
851
1456


2649
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
V2-15_IGLJ7*01
851
1456


2650
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
V2-15_IGLJ7*01
851
1456


2651
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
V2-15_IGLJ7*01
851
1456


2652
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
V2-15_IGLJ7*01
851
1456


2653
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
V2-15_IGLJ7*01
851
1456


2654
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
V2-15_IGLJ7*01
851
1456


2655
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
V2-15_IGLJ7*01
851
1456


2656
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
V2-15_IGLJ7*01
851
1456


2657
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
V2-15_IGLJ7*01
851
1456


2658
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
V2-15_IGLJ7*01
851
1456


2659
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
V2-15_IGLJ7*01
851
1456


2660
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
V2-15_IGLJ7*01
851
1456


2661
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
V2-15_IGLJ7*01
851
1456


2662
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
V2-15_IGLJ7*01
851
1456


2663
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
V2-15_IGLJ7*01
851
1456


2664
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
V2-15_IGLJ7*01
851
1456


2665
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
V2-15_IGLJ7*01
851
1456


2666
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
V2-15_IGLJ7*01
851
1456


2667
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
V2-15_IGLJ7*01
851
1456


2668
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
V2-15_IGLJ7*01
851
1456


2669
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
V2-15_IGLJ7*01
851
1456


2670
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
V2-15_IGLJ7*01
851
1456


2671
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
V2-15_IGLJ7*01
851
1456


2672
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
V2-15_IGLJ7*01
851
1456


2673
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
V2-15_IGLJ7*01
851
1456


2674
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
V2-15_IGLJ7*01
851
1456


2675
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
V2-15_IGLJ7*01
851
1456


2676
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
V2-15_IGLJ7*01
851
1456


2677
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
V2-15_IGLJ7*01
851
1456


2678
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
V2-15_IGLJ7*01
851
1456


2679
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
V2-15_IGLJ7*01
851
1456


2680
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
V2-15_IGLJ7*01
851
1456


2681
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
V2-15_IGLJ7*01
851
1456


2682
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
V2-15_IGLJ7*01
851
1456


2683
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
V2-15_IGLJ7*01
851
1456


2684
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
V2-15_IGLJ7*01
851
1456


2685
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
V2-15_IGLJ7*01
851
1456


2686
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
V2-15_IGLJ7*01
851
1456


2687
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
V2-15_IGLJ7*01
851
1456


2688
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
V2-15_IGLJ7*01
851
1456


2689
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
V2-15_IGLJ7*01
851
1456


2690
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
V2-15_IGLJ7*01
851
1456


2691
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
V2-15_IGLJ7*01
851
1456


2692
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
V2-15_IGLJ7*01
851
1456


2693
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
V2-15_IGLJ7*01
851
1456


2694
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
V2-15_IGLJ7*01
851
1456


2695
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
V2-15_IGLJ7*01
851
1456


2696
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
V2-15_IGLJ7*01
851
1456


2697
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
V2-15_IGLJ7*01
851
1456


2698
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
V2-15_IGLJ7*01
851
1456


2699
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
V2-15_IGLJ7*01
851
1456


2700
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
V2-15_IGLJ7*01
851
1456


2701
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
V2-15_IGLJ7*01
851
1456


2702
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
V2-15_IGLJ7*01
851
1456


2703
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
V2-15_IGLJ7*01
851
1456


2704
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
V2-15_IGLJ7*01
851
1456


2705
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
V2-15_IGLJ7*01
851
1456


2706
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
V2-15_IGLJ7*01
851
1456


2707
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
V2-15_IGLJ7*01
851
1456


2708
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
V2-15_IGLJ7*01
851
1456


2709
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
V2-15_IGLJ7*01
851
1456


2710
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
V2-15_IGLJ7*01
851
1456


2711
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
V2-15_IGLJ7*01
851
1456


2712
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
V2-15_IGLJ7*01
851
1456


2713
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
V2-15_IGLJ7*01
851
1456


2714
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
V2-15_IGLJ7*01
851
1456


2715
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
V2-15_IGLJ7*01
851
1456


2716
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
V2-15_IGLJ7*01
851
1456


2717
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
V2-15_IGLJ7*01
851
1456


2718
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
V2-15_IGLJ7*01
851
1456


2719
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
V2-15_IGLJ7*01
851
1456


2720
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
V2-15_IGLJ7*01
851
1456


2721
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
V2-15_IGLJ7*01
851
1456


2722
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
V2-15_IGLJ7*01
851
1456


2723
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
V2-15_IGLJ7*01
851
1456


2724
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
V2-15_IGLJ7*01
851
1456


2725
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
V2-15_IGLJ7*01
851
1456


2726
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
V2-15_IGLJ7*01
851
1456


2727
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
V2-15_IGLJ7*01
851
1456


2728
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
V2-15_IGLJ7*01
851
1456


2729
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
V2-15_IGLJ7*01
851
1456


2730
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
V2-15_IGLJ7*01
851
1456


2731
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
V2-15_IGLJ7*01
851
1456


2732
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
V2-15_IGLJ7*01
851
1456


2733
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
V2-15_IGLJ7*01
851
1456


2734
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
V2-15_IGLJ7*01
851
1456


2735
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
V2-15_IGLJ7*01
851
1456


2736
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
V2-15_IGLJ7*01
851
1456


2737
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
V2-15_IGLJ7*01
851
1456


2738
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
V2-15_IGLJ7*01
851
1456


2739
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
V2-15_IGLJ7*01
851
1456


2740
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
V2-15_IGLJ7*01
851
1456


2741
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
V2-15_IGLJ7*01
851
1456


2742
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
V2-15_IGLJ7*01
851
1456


2743
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
V2-17_IGLJ2*01
852
1457


2744
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
V2-17_IGLJ2*01
852
1457


2745
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
V2-17_IGLJ2*01
852
1457


2746
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
V2-17_IGLJ2*01
852
1457


2747
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
V2-17_IGLJ2*01
852
1457


2748
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
V2-17_IGLJ2*01
852
1457


2749
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
V2-17_IGLJ2*01
852
1457


2750
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
V2-17_IGLJ2*01
852
1457


2751
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
V2-17_IGLJ2*01
852
1457


2752
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
V2-17_IGLJ2*01
852
1457


2753
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
V2-17_IGLJ2*01
852
1457


2754
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
V2-17_IGLJ2*01
852
1457


2755
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
V2-17_IGLJ2*01
852
1457


2756
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
V2-17_IGLJ2*01
852
1457


2757
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
V2-17_IGLJ2*01
852
1457


2758
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
V2-17_IGLJ2*01
852
1457


2759
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
V2-17_IGLJ2*01
852
1457


2760
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
V2-17_IGLJ2*01
852
1457


2761
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
V2-17_IGLJ2*01
852
1457


2762
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
V2-17_IGLJ2*01
852
1457


2763
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
V2-17_IGLJ2*01
852
1457


2764
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
V2-17_IGLJ2*01
852
1457


2765
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
V2-17_IGLJ2*01
852
1457


2766
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
V2-17_IGLJ2*01
852
1457


2767
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
V2-17_IGLJ2*01
852
1457


2768
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
V2-17_IGLJ2*01
852
1457


2769
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
V2-17_IGLJ2*01
852
1457


2770
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
V2-17_IGLJ2*01
852
1457


2771
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
V2-17_IGLJ2*01
852
1457


2772
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
V2-17_IGLJ2*01
852
1457


2773
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
V2-17_IGLJ2*01
852
1457


2774
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
V2-17_IGLJ2*01
852
1457


2775
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
V2-17_IGLJ2*01
852
1457


2776
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
V2-17_IGLJ2*01
852
1457


2777
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
V2-17_IGLJ2*01
852
1457


2778
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
V2-17_IGLJ2*01
852
1457


2779
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
V2-17_IGLJ2*01
852
1457


2780
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
V2-17_IGLJ2*01
852
1457


2781
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
V2-17_IGLJ2*01
852
1457


2782
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
V2-17_IGLJ2*01
852
1457


2783
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
V2-17_IGLJ2*01
852
1457


2784
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
V2-17_IGLJ2*01
852
1457


2785
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
V2-17_IGLJ2*01
852
1457


2786
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
V2-17_IGLJ2*01
852
1457


2787
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
V2-17_IGLJ2*01
852
1457


2788
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
V2-17_IGLJ2*01
852
1457


2789
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
V2-17_IGLJ2*01
852
1457


2790
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
V2-17_IGLJ2*01
852
1457


2791
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
V2-17_IGLJ2*01
852
1457


2792
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
V2-17_IGLJ2*01
852
1457


2793
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
V2-17_IGLJ2*01
852
1457


2794
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
V2-17_IGLJ2*01
852
1457


2795
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
V2-17_IGLJ2*01
852
1457


2796
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
V2-17_IGLJ2*01
852
1457


2797
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
V2-17_IGLJ2*01
852
1457


2798
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
V2-17_IGLJ2*01
852
1457


2799
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
V2-17_IGLJ2*01
852
1457


2800
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
V2-17_IGLJ2*01
852
1457


2801
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
V2-17_IGLJ2*01
852
1457


2802
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
V2-17_IGLJ2*01
852
1457


2803
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
V2-17_IGLJ2*01
852
1457


2804
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
V2-17_IGLJ2*01
852
1457


2805
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
V2-17_IGLJ2*01
852
1457


2806
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
V2-17_IGLJ2*01
852
1457


2807
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
V2-17_IGLJ2*01
852
1457


2808
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
V2-17_IGLJ2*01
852
1457


2809
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
V2-17_IGLJ2*01
852
1457


2810
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
V2-17_IGLJ2*01
852
1457


2811
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
V2-17_IGLJ2*01
852
1457


2812
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
V2-17_IGLJ2*01
852
1457


2813
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
V2-17_IGLJ2*01
852
1457


2814
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
V2-17_IGLJ2*01
852
1457


2815
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
V2-17_IGLJ2*01
852
1457


2816
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
V2-17_IGLJ2*01
852
1457


2817
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
V2-17_IGLJ2*01
852
1457


2818
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
V2-17_IGLJ2*01
852
1457


2819
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
V2-17_IGLJ2*01
852
1457


2820
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
V2-17_IGLJ2*01
852
1457


2821
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
V2-17_IGLJ2*01
852
1457


2822
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
V2-17_IGLJ2*01
852
1457


2823
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
V2-17_IGLJ2*01
852
1457


2824
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
V2-17_IGLJ2*01
852
1457


2825
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
V2-17_IGLJ2*01
852
1457


2826
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
V2-17_IGLJ2*01
852
1457


2827
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
V2-17_IGLJ2*01
852
1457


2828
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
V2-17_IGLJ2*01
852
1457


2829
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
V2-17_IGLJ2*01
852
1457


2830
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
V2-17_IGLJ2*01
852
1457


2831
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
V2-17_IGLJ2*01
852
1457


2832
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
V2-17_IGLJ2*01
852
1457


2833
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
V2-17_IGLJ2*01
852
1457


2834
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
V2-17_IGLJ2*01
852
1457


2835
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
V2-17_IGLJ2*01
852
1457


2836
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
V2-17_IGLJ2*01
852
1457


2837
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
V2-17_IGLJ2*01
852
1457


2838
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
V2-17_IGLJ2*01
852
1457


2839
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
V2-6_IGLJ4*01
854
1459


2840
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
V2-6_IGLJ4*01
854
1459


2841
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
V2-6_IGLJ4*01
854
1459


2842
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
V2-6_IGLJ4*01
854
1459


2843
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
V2-6_IGLJ4*01
854
1459


2844
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
V2-6_IGLJ4*01
854
1459


2845
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
V2-6_IGLJ4*01
854
1459


2846
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
V2-6_IGLJ4*01
854
1459


2847
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
V2-6_IGLJ4*01
854
1459


2848
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
V2-6_IGLJ4*01
854
1459


2849
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
V2-6_IGLJ4*01
854
1459


2850
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
V2-6_IGLJ4*01
854
1459


2851
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
V2-6_IGLJ4*01
854
1459


2852
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
V2-6_IGLJ4*01
854
1459


2853
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
V2-6_IGLJ4*01
854
1459


2854
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
V2-6_IGLJ4*01
854
1459


2855
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
V2-6_IGLJ4*01
854
1459


2856
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
V2-6_IGLJ4*01
854
1459


2857
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
V2-6_IGLJ4*01
854
1459


2858
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
V2-6_IGLJ4*01
854
1459


2859
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
V2-6_IGLJ4*01
854
1459


2860
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
V2-6_IGLJ4*01
854
1459


2861
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
V2-6_IGLJ4*01
854
1459


2862
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
V2-6_IGLJ4*01
854
1459


2863
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
V2-6_IGLJ4*01
854
1459


2864
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
V2-6_IGLJ4*01
854
1459


2865
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
V2-6_IGLJ4*01
854
1459


2866
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
V2-6_IGLJ4*01
854
1459


2867
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
V2-6_IGLJ4*01
854
1459


2868
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
V2-6_IGLJ4*01
854
1459


2869
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
V2-6_IGLJ4*01
854
1459


2870
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
V2-6_IGLJ4*01
854
1459


2871
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
V2-6_IGLJ4*01
854
1459


2872
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
V2-6_IGLJ4*01
854
1459


2873
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
V2-6_IGLJ4*01
854
1459


2874
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
V2-6_IGLJ4*01
854
1459


2875
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
V2-6_IGLJ4*01
854
1459


2876
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
V2-6_IGLJ4*01
854
1459


2877
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
V2-6_IGLJ4*01
854
1459


2878
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
V2-6_IGLJ4*01
854
1459


2879
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
V2-6_IGLJ4*01
854
1459


2880
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
V2-6_IGLJ4*01
854
1459


2881
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
V2-6_IGLJ4*01
854
1459


2882
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
V2-6_IGLJ4*01
854
1459


2883
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
V2-6_IGLJ4*01
854
1459


2884
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
V2-6_IGLJ4*01
854
1459


2885
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
V2-6_IGLJ4*01
854
1459


2886
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
V2-6_IGLJ4*01
854
1459


2887
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
V2-6_IGLJ4*01
854
1459


2888
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
V2-6_IGLJ4*01
854
1459


2889
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
V2-6_IGLJ4*01
854
1459


2890
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
V2-6_IGLJ4*01
854
1459


2891
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
V2-6_IGLJ4*01
854
1459


2892
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
V2-6_IGLJ4*01
854
1459


2893
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
V2-6_IGLJ4*01
854
1459


2894
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
V2-6_IGLJ4*01
854
1459


2895
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
V2-6_IGLJ4*01
854
1459


2896
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
V2-6_IGLJ4*01
854
1459


2897
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
V2-6_IGLJ4*01
854
1459


2898
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
V2-6_IGLJ4*01
854
1459


2899
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
V2-6_IGLJ4*01
854
1459


2900
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
V2-6_IGLJ4*01
854
1459


2901
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
V2-6_IGLJ4*01
854
1459


2902
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
V2-6_IGLJ4*01
854
1459


2903
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
V2-6_IGLJ4*01
854
1459


2904
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
V2-6_IGLJ4*01
854
1459


2905
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
V2-6_IGLJ4*01
854
1459


2906
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
V2-6_IGLJ4*01
854
1459


2907
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
V2-6_IGLJ4*01
854
1459


2908
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
V2-6_IGLJ4*01
854
1459


2909
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
V2-6_IGLJ4*01
854
1459


2910
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
V2-6_IGLJ4*01
854
1459


2911
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
V2-6_IGLJ4*01
854
1459


2912
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
V2-6_IGLJ4*01
854
1459


2913
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
V2-6_IGLJ4*01
854
1459


2914
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
V2-6_IGLJ4*01
854
1459


2915
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
V2-6_IGLJ4*01
854
1459


2916
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
V2-6_IGLJ4*01
854
1459


2917
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
V2-6_IGLJ4*01
854
1459


2918
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
V2-6_IGLJ4*01
854
1459


2919
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
V2-6_IGLJ4*01
854
1459


2920
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
V2-6_IGLJ4*01
854
1459


2921
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
V2-6_IGLJ4*01
854
1459


2922
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
V2-6_IGLJ4*01
854
1459


2923
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
V2-6_IGLJ4*01
854
1459


2924
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
V2-6_IGLJ4*01
854
1459


2925
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
V2-6_IGLJ4*01
854
1459


2926
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
V2-6_IGLJ4*01
854
1459


2927
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
V2-6_IGLJ4*01
854
1459


2928
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
V2-6_IGLJ4*01
854
1459


2929
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
V2-6_IGLJ4*01
854
1459


2930
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
V2-6_IGLJ4*01
854
1459


2931
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
V2-6_IGLJ4*01
854
1459


2932
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
V2-6_IGLJ4*01
854
1459


2933
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
V2-6_IGLJ4*01
854
1459


2934
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
V2-6_IGLJ4*01
854
1459


2935
VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
V2-7_IGLJ2*01
855
1460


2936
VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
V2-7_IGLJ2*01
855
1460


2937
VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
V2-7_IGLJ2*01
855
1460


2938
VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
V2-7_IGLJ2*01
855
1460


2939
VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
V2-7_IGLJ2*01
855
1460


2940
VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
V2-7_IGLJ2*01
855
1460


2941
VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
V2-7_IGLJ2*01
855
1460


2942
VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
V2-7_IGLJ2*01
855
1460


2943
VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
V2-7_IGLJ2*01
855
1460


2944
VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
V2-7_IGLJ2*01
855
1460


2945
VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
V2-7_IGLJ2*01
855
1460


2946
VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
V2-7_IGLJ2*01
855
1460


2947
VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
V2-7_IGLJ2*01
855
1460


2948
VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
V2-7_IGLJ2*01
855
1460


2949
VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
V2-7_IGLJ2*01
855
1460


2950
VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
V2-7_IGLJ2*01
855
1460


2951
VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
V2-7_IGLJ2*01
855
1460


2952
VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
V2-7_IGLJ2*01
855
1460


2953
VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
V2-7_IGLJ2*01
855
1460


2954
VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
V2-7_IGLJ2*01
855
1460


2955
VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
V2-7_IGLJ2*01
855
1460


2956
VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
V2-7_IGLJ2*01
855
1460


2957
VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
V2-7_IGLJ2*01
855
1460


2958
VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
V2-7_IGLJ2*01
855
1460


2959
VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
V2-7_IGLJ2*01
855
1460


2960
VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
V2-7_IGLJ2*01
855
1460


2961
VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
V2-7_IGLJ2*01
855
1460


2962
VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
V2-7_IGLJ2*01
855
1460


2963
VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
V2-7_IGLJ2*01
855
1460


2964
VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01
2674
V2-7_IGLJ2*01
855
1460


2965
VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01
2675
V2-7_IGLJ2*01
855
1460


2966
VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01
2676
V2-7_IGLJ2*01
855
1460


2967
VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01
2677
V2-7_IGLJ2*01
855
1460


2968
VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
V2-7_IGLJ2*01
855
1460


2969
VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
V2-7_IGLJ2*01
855
1460


2970
VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
V2-7_IGLJ2*01
855
1460


2971
VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
V2-7_IGLJ2*01
855
1460


2972
VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01
2682
V2-7_IGLJ2*01
855
1460


2973
VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01
2683
V2-7_IGLJ2*01
855
1460


2974
VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01
2684
V2-7_IGLJ2*01
855
1460


2975
VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
V2-7_IGLJ2*01
855
1460


2976
VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
V2-7_IGLJ2*01
855
1460


2977
VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01
2687
V2-7_IGLJ2*01
855
1460


2978
VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01
2688
V2-7_IGLJ2*01
855
1460


2979
VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01
2689
V2-7_IGLJ2*01
855
1460


2980
VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
V2-7_IGLJ2*01
855
1460


2981
VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
V2-7_IGLJ2*01
855
1460


2982
VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
V2-7_IGLJ2*01
855
1460


2983
VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
V2-7_IGLJ2*01
855
1460


2984
VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
V2-7_IGLJ2*01
855
1460


2985
VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
V2-7_IGLJ2*01
855
1460


2986
VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
V2-7_IGLJ2*01
855
1460


2987
VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
V2-7_IGLJ2*01
855
1460


2988
VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
V2-7_IGLJ2*01
855
1460


2989
VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
V2-7_IGLJ2*01
855
1460


2990
VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
V2-7_IGLJ2*01
855
1460


2991
VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
V2-7_IGLJ2*01
855
1460


2992
VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
V2-7_IGLJ2*01
855
1460


2993
VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
V2-7_IGLJ2*01
855
1460


2994
VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
V2-7_IGLJ2*01
855
1460


2995
VH3-23_IGHD1-26*01>1_IGHJ6*01_B
2714
V2-7_IGLJ2*01
855
1460


2996
VH3-23_IGHD2-2*01>2_IGHJ6*01_B
2715
V2-7_IGLJ2*01
855
1460


2997
VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
V2-7_IGLJ2*01
855
1460


2998
VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
V2-7_IGLJ2*01
855
1460


2999
VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
V2-7_IGLJ2*01
855
1460


3000
VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
V2-7_IGLJ2*01
855
1460


3001
VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
V2-7_IGLJ2*01
855
1460


3002
VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
V2-7_IGLJ2*01
855
1460


3003
VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
V2-7_IGLJ2*01
855
1460


3004
VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
V2-7_IGLJ2*01
855
1460


3005
VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
V2-7_IGLJ2*01
855
1460


3006
VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
V2-7_IGLJ2*01
855
1460


3007
VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
V2-7_IGLJ2*01
855
1460


3008
VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
V2-7_IGLJ2*01
855
1460


3009
VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
V2-7_IGLJ2*01
855
1460


3010
VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
V2-7_IGLJ2*01
855
1460


3011
VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
V2-7_IGLJ2*01
855
1460


3012
VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01
2731
V2-7_IGLJ2*01
855
1460


3013
VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01
2732
V2-7_IGLJ2*01
855
1460


3014
VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01
2733
V2-7_IGLJ2*01
855
1460


3015
VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01
2734
V2-7_IGLJ2*01
855
1460


3016
VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
V2-7_IGLJ2*01
855
1460


3017
VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
V2-7_IGLJ2*01
855
1460


3018
VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
V2-7_IGLJ2*01
855
1460


3019
VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
V2-7_IGLJ2*01
855
1460


3020
VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01
2739
V2-7_IGLJ2*01
855
1460


3021
VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01
2740
V2-7_IGLJ2*01
855
1460


3022
VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
V2-7_IGLJ2*01
855
1460


3023
VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
V2-7_IGLJ2*01
855
1460


3024
VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01
2743
V2-7_IGLJ2*01
855
1460


3025
VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01
2744
V2-7_IGLJ2*01
855
1460


3026
VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
V2-7_IGLJ2*01
855
1460


3027
VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
V2-7_IGLJ2*01
855
1460


3028
VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
V2-7_IGLJ2*01
855
1460


3029
VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
V2-7_IGLJ2*01
855
1460


3030
VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
V2-7_IGLJ2*01
855
1460









4. Antibody Libraries
Provided herein are antibody libraries containing antibodies or portions thereof minimally containing a VH chain and a VL chain or a portion thereof containing a sufficient antigen binding site. The VH chain or portion thereof of the antibody members in the libraries provided herein are encoded by any of the nucleic acid members of the library set forth in Section E.1 above. The VL chain or portion thereof of the antibody members in the libraries provided herein are encoded by any of the nucleic acid members set forth in Section E.2 above. Thus, each antibody member of the library is derived in full or in part from germline segment sequences and/or are derived from modified sequences thereof. In some examples, the libraries are provided as addressed libraries.
In addition, each antibody in the library is productive and functional by virtue of containing a sufficient antigen binding site. The antibodies in the library are different, and when provided in an addressed format, each locus of the library contains a different antibody from all other loci in the library. Thus, the libraries provided herein exhibit high antibody diversity. The antibody libraries provided herein contain as few as 102 different members and typically contain about or 103, 104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 105 and more unique members, including about or 106, 107, 108, 109 and more unique members. The antibody libraries provided herein can be produced by the methods herein, whereby the natural recombination process and natural structural diversity of the antibody repertoire is mimicked.
Besides containing a VH chain and a VL chain, or a portion thereof containing a sufficient antigen-binding site, the resulting antibodies in the library provided herein can contain all or a portion of a constant region. For example, the antibodies can contain one or more of a CH1, CH2, CH3 or CL portion. Generally, the antibodies or portions thereof contain a CH1 portion. The resulting antibodies or portions thereof include, but are not limited to a full-length antibody, Fab, Fab′, F(ab′)2, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments. Exemplary addressed antibody libraries provided herein are Fab libraries.
The number of different members in the antibody libraries provided herein can be restricted such that each member of the library is selected based on sequence similarities or differences or based on shared characteristics (e.g., a V region family, CDR3 length or composition or other biochemical attribute). For example, members of the library can be selected so that individual encoding germline segments of the VL or VH chains of the resulting antibodies have shared characteristics (e.g. are of the same subgroup or gene family) or otherwise contain similar or different sequence identity. In another example, the members of the library can be selected based on the sequence diversity of the VH or VL chains of the resulting antibody members. The antibody members of the library can be selected to be diverse in the represented sequences or to be similar. Thus, in some instances, library members represent a group of highly diverse antibodies. In other instances, library members represent a group similar, non-diverse antibodies. For example, library members can have 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity of the VH or VL to all other members in the library. The choice of antibody library is a function of the application and is within the level of one of skill in the art.
In some examples, the addressed libraries provided herein are human naïve libraries. That means that every member of the library is derived completely from human germline segment sequences. For example, the VH chain of each antibody member is encoded by a nucleic acid molecule made up of a combination of a VH, DH and JH germline segment, for example, any set forth in Table 3 above, or a subset thereof, such that the resulting nucleic acid molecule has a sequence where the VH segment is 5′ to the DH segment which is 5′ to the JH segment. The VL chain of each antibody member is encoded by a combination of a Vκ and Jκ germline segments and/or Vλ and Jλ germline segments, for example, any set forth in Tables 3-4 above, or a subset thereof, such that the resulting nucleic acid molecule has a sequence where the VL segment (Vκ or Vλ) is 5′ to the JL segment (Jκ or Jλ). It is understood that the library is considered to be naïve and derived from germline even though, in practicing the method herein, the joint regions of the segments are altered to render the resulting encoding nucleic acid molecules in frame. Such alterations, however, are minor and variously include insertion or deletion generally of only a single nucleotide of a germline segment. In addition, other modification made to the recombined nucleic acid sequence by virtue of practice of the method herein, such as removal of stop codons and restriction enzyme site sequences, also result in naïve antibodies. Naïve antibody libraries provided herein can include the entire repertoire of naïve antibodies, or a subset thereof. For example, a naïve library provided herein can include 103, 104, 2×104, 3×104 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 105 and more unique members, including about or 106, 107 or more members.
In particular examples, the VH chain of members of the antibody library is encoded by a sequence of nucleotides made up entirely of a VH and a JH germline segment, and also any sequence of nucleotides between the VH and JH germline segment. The VH segment is 5′ to the random sequence of nucleotides which is 5′ to the JH segment in the encoding nucleic acid molecule. Thus, the resulting antibody members of the library contain a random sequence of amino acids in the region that includes the central portion of the CDRH3, which is largely responsible for the antigen specificity of the resulting antibody. The sequence of nucleotides can be any random sequence of nucleotides. In some instances, the sequence of nucleotides is a sequence that encodes a peptide mimetic against any desired target, for example, a cell surface receptor. Exemplary peptide mimetics are set forth in Table 16. In other examples, the random sequence of nucleotides is a DH germline segment, or modified form thereof. Generally, the sequence of nucleotides is or is about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more nucleotides in length.
Also provided herein are antibody libraries where the antibodies in the libraries are derived at least in part from modified germline segment sequences. In some examples, all encoding germline segments are modified, for example, by random mutation. In other examples, particular regions of germline segments are targeted for modification. For example, the modified germline segment sequences can include modifications, such as by amino acid mutation, of one or more CDR. In particular, resulting antibody members of the libraries provided herein can contain modification of CDR3, such as of CDRH3. Thus, the resulting members of the library can contain one or more amino acid replacement of a CDR as compared to a naïve antibody.
In some examples, the members of the antibody libraries provided herein can be directed toward a desired target or function by incorporation of sequences into the variable regions of a naive antibody that correspond to a polynucleotide target of interest. Thus, except for the incorporated sequence, which is added by replacement or insertion into the antibody sequence, the remaining antibody represents germline segment sequences. These incorporated sequences include, but are not limited to, peptides as well as portions of monoclonal antibodies.
For example, the antibody can contain a directed peptide that acts as a mimetic against a particular target. Generally, such antibodies act as agonists toward the target, but in some instances, can be antagonists. The peptides can be included in any region of the antibody, but generally are included in the variable region, and more generally in one or more of the CDR regions. In particular, directed peptides are included in the CDR3 region of the antibody. Each member antibody member of the library can be modified with the same directed peptide and/or with different directed peptides.
In related examples, the antibody can contain a sequence portion from a known monoclonal antibodies. The portion can include a portion corresponding to one or more CDRs of a known monoclonal antibody. In other cases, antibodies in the addressed library can contain entire variable region (for example, VL or VH) of a monoclonal antibody. For example, antibodies in the library can contain a VH or VL of a known monoclonal antibody, which can be combined with a VL or VH derived from germline segments as described herein. Exemplary of such an antibody library is one where one or more of the antibody members contain a VH or VL chain from any known monoclonal antibody set forth in Table 9 above.
The antibody libraries of provided herein can include members that represent one, some or all of the above examples.
In the libraries provided herein, the individual VH and VL chains or portions thereof can be the same of different from the VH and VL chains of other antibodies in the library. It is the combination of the VH and VL chain that renders each antibody in the library different, for example, each addressed antibody at a locus. Thus, for example, libraries provided herein can all contain a common VH chain, but can each contain a different VL chain. Thus, the resulting antibody members of the library each contain a different combination of VH and VL chain.
An antibody library provided herein can include a VH encoded from any of nucleic acid sequences SEQ ID NOS: 454-805 (each encoding a VH chain), and a VL chain encoded by any of nucleic acid molecule set forth in any of SEQ ID NOS: 806-815, 817, 819-834, and 836-867 (each encoding a VL chain), or sequences thereof that do not contain heterologous sequence for restriction sites at the 3′ and 5′ ends, and subsets thereof. An antibody library provided herein also can include a VH encoded from any of nucleic acid sequences SEQ ID NOS: 2070-2759 (each encoding a VH chain), and a VL chain encoded by any of nucleic acid molecule set forth in any of SEQ ID NOS: 806-815, 817, 819-834, and 836-867 (each encoding a VL chain), or sequences thereof not do not contain heterologous sequence for restriction sites at the 3′ and 5′ ends. Any combination of VH and VL from any of the above recombined nucleic acids provided herein can be paired to generate an antibody library. The components of the library, the size of the library and the type of library (e.g. Fab, full-length antibody, etc) all can be varied
Exemplary of such an antibody library provided herein incudes a VH and a VL encoded by a sequence of nucleic acids set forth in Table 17. An additional exemplary antibody library includes a subset of members set forth in Table 17, including a VH and a VL chain encoded from nucleic acid sequences set forth in Table 22 below. For example, in such an exemplary library any of the VH chain sequences set forth in any of SEQ ID NOS: 1475-1826 is paired with any of the VL light chain sequences set forth in SEQ ID NOS:1827-1838, 1840-1855, 1857-1888. The resulting library, when expressed with a CH as exemplified in Example 9, is a Fab library. By virtue of the pairing of VH and VL chain the resulting library can contain at or about 2.1.0×105 or more members, or a subset thereof. Each member pair is encoded by germline segments and represents a naïve antibody.
Exemplary of such an antibody library provided herein includes a VH and a VL encoded by a sequence of nucleic acids set forth in 17.A above.
5. Addressable Formats
The libraries provided herein are provided as addressed libraries such that each loci is a distinct address containing a different member of the library compared to all other loci of the library, and each can be identified by virtue of its address. For example, members at a loci can be identified by prior recording of their distinct location or by an identifiable tag. Where the library is a nucleic acid library, each loci of the library contains a nucleic acid molecule that is different from all other nucleic acid molecules at other loci in the library. Where the library is an antibody library, each loci of the library contains an antibody or portion thereof that is different from all other antibodies or portion thereof at other loci in the library.
The libraries provided herein can be presented in any addressable format known to one of skill in the art. Addressing can be effected by position on a surface or other physical locus or can be otherwise identifiable and/or sortable by other identifiers, such as a tag encoded with a bar code or other symbology, a chemical tag, an electronic, such RF tag, a color-coded tag or other such identifier. One of skill in the art is familiar with various addressable format. Exemplary of such formats is as an array. The library members can be provided on or in the array by immobilization or attachment, or can be provided in solution.
Any nucleic acid molecule or antibody provided herein can be provided in overlapping areas or at random locations on an array, such as a multiwell plate. Alternatively the library members can be provided in spatial array, such that each nucleic acid molecule or antibody is provided in a distinct region of the array which does not overlap with the any other region containing any other antibody or nucleic acid molecules. Any addressable array technology known in the art can be employed with the antibodies.
a. Multiwell Plate
Nucleic acid molecules or antibodies can be spatially arrayed in multiwell plates, such that each individual locus of the plate corresponds to one individual antibody. Multiwell plates can include, but are not limited to, 12-well, 24-well, 48-well, 96-well plates, 384-well plates, and 1536-well plates. In this instance, the identity of each member in each well is known. The members can be attached to the support or provided in solution. For example, one advantage of this technique is that antibodies are presented in solution and are therefore fully folded and functional thereby eliminating the loss of activity observed when proteins are immobilized on filters, chip surfaces or slides. In addition, in the case of antibodies, the antibodies can be screened for any desired activity, including but not limited to binding, cytotoxicity, differentiation or proliferation of cells, and alteration of gene expression. Since the identity of each antibody is known, information on structure activity relationships (SAR) is immediately available. Finally, pharmacokinetics and/or dose response experiments can be performed during screening or immediately following identification of a “hit” or lead compound.
In another instance, nucleic acid molecule and antibody members can be spatially arrayed in multiwell plates, such that each individual locus of the plate corresponds to a group of members. In this instance, the identity of every member within the group in any particular well is known. Groups of members can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more antibodies or nucleic acid molecules. For example, antibodies or nucleic acid molecules can be grouped randomly or by any desired characteristic, such as belonging to the same V-region family, containing similar CDR3 region amino acid composition or length, or any other biochemical attributes. For the case of antibodies, the group of addressed antibodies can be screened for any desired activity, including but not limited to binding, cytotoxicity, differentiation or proliferation of cells, and alteration of gene expression. Screening groups of libraries allows for screening a greater number of library members at any given time thereby covering a larger part of the available antibody diversity. Furthermore, following the identification of a “hit” or lead group, since the identity of every antibody within a group is known and the antibodies are all readily available individually, individual antibodies can be immediately screened to identify “active” library members within the group.
ii. Solid Support
Nucleic acid and antibody arrays include those in which members are immobilized on a solid support, such as in a microarray. For example, solid supports for attachment or immobilization include, but are not limited to, blotting on filters, on chip surfaces, or on cellulose and by attachment to slides using affinity tags. In some examples, cells expressing variant polypeptides can be naturally adsorbed to a bead, such that a population of beads contains a single cell per bead (Freeman et al. Biotechnol. Bioeng. (2004) 86:196-200). Following immobilization to a glass support, microcolonies can be grown and screened with a chromogenic or fluorogenic substrate. See, e.g., U.S. Pat. Nos. 6,372,483, 6,352,842, 6,346,416 and 6,242,266.
For example, it is contemplated herein that antibody members of the libraries can be immobilized on a chip. Exemplary of chips include those used for BIAcore. Determining the ability of the protein to bind to a target molecule can be accomplished, e.g., using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. Biacore is a method for measuring protein-protein interaction and binding affinity. The technology is based on surface plasmon resonance (SPR), an optical phenomenon that enables detection of unlabeled interactants in real time (Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268). The SPR-based biosensors can be used to monitor biomolecular interactions in real time to determine active concentration, screening and characterization in terms of both affinity and kinetics. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
The methodology relies on immobilization of ligands onto the surface of a sensor chip consisting of a glass substrate having a gold film covered by a monolayer of a long hydroxyalkyl thiol to which is covalently attached a thin layer of carboxymethylated dextran. The immobilization procedure is performed with the sensor chip in place in the instrument and is continuously monitored by the SPR detector. An unknown sample or ligate solution is introduced into the apparatus to contact the immobilized ligand. The interaction between ligand and ligate is observed directly by surface plasmon resonance techniques and the measurements recorded on a computer via a program such as Bialogue [Pharmacia].
6. Other Display Methods
The libraries also can be provided in other non-addressable formats. Exemplary of such other non-addressable formats include by display, in particular, any display format that facilitates screening of the members of the libraries for an activity or activities. Generally, such formats are used for the antibody libraries, but if desired can also be provided for the nucleic acid or vector libraries. Typically libraries are screened using a display technique, such that there is a physical link between individual molecules of the library (phenotype) and the genetic information encoding them (genotype). These methods include, but are not limited to, cell display, phage display, mRNA display, ribosome display and DNA display.
a. Cell Display
Antibody libraries for screening can be expressed on the surfaces of cells, including bacteria E. coli, yeast S. cerevisiae, and mammalian cells, by fusing them with a protein that is expressed on the surface of the cell. Cell display is a technology used to screen antibody libraries wherein immobilization of the target antigen is unnecessary. Instead a technique, such as fluorescence-activated cell sorting (FACS), can be used to identify desired antibodies. FACS permits the separation of subpopulations of cells on the basis of their light scatter properties as they pass through a laser beam. See e.g. United States Published Patent Application Nos. US 2003/0100023 and US 2003/0036092. Single chain antibodies can be expressed on the external surface of E. coli by fusing them to a protein previously shown to direct heterologous proteins to the bacterial surface (Francisco et al., (1993) Proc. Natl. Acad. Sci., USA, 90:10444-10448). Single chain and Fab antibodies can be displayed on the surface of a yeast cell, and homologous recombination in yeast can be exploited to generate libraries of transformants (see e.g. Kieke et al., (1997) Prot. Eng., 10:1303-1310; Weaver-Feldhaus et al., (2004) FEBS Lett., 564:24-34; and Swers et al., (2004) Nucleic Acids Res., 32:e36). Mammalian cell display has been utilized to screen scFv libraries as well as IgGs (Ho et al., (2005) J. Biol. Chem., 280:07-617).
b. Phage Display
Phage display is a widely used method for screening antibody libraries for their ability to bind to a particular antigen. Phage display is a cell based method in which proteins or peptides are expressed individually on the surface of phage as fusions to a coat protein, while the same phage particle carries the DNA encoding the protein or peptide (Smith, G. P. (1985) Science 228:1315-1317). Selection of the phage is achieved through a specific binding reaction involving recognition of the protein or peptide, enabling the particular phage to be isolated and cloned and the DNA for the protein or peptide to be recovered and propagated or expressed. Use of phage display is rapid and facile due to its reliance upon E. coli for amplification and propagation. Typical use involves panning phage libraries against an immobilized antigen.
c. mRNA Display and Ribosome Display
The use of mRNA display and ribosome display allow for totally in vitro construction of antibody libraries. mRNA display is a method of displaying proteins or peptides in which the nascent protein is caused to bind covalently to its mRNA through a puromycin link (Roberts et al. (1997) Proc. Natl. Acad. Sci, U.S.A. 64:12297-12302). Puromycin acts as a mimic of aminacyl tRNA, enters the ribosome A site, and the nascent protein is bound covalently to it by the peptidyl-transferase activity of the ribosome. Selection is carried out on these protein-mRNA fusions after dissociation of the ribosome. Alternatively, ribosome display is a method of displaying proteins or peptides in nascent form on the surface of ribosomes, such that a stable complex with the encoding mRNA is formed; the complexes are selected with a ligand for the protein or peptide and the genetic information is obtained by reverse transcription of the isolated mRNA (see e.g. U.S. Pat. Nos. 5,643,768 and 5,658,754). Selection techniques are similar to that of phage display wherein an ribosome display libraries are panned against an immobilized antigen.
d. DNA Display
In DNA display the DNA encoding the peptide is linked to the peptide. In non-covalent DNA display, the DNA-protein linkage is promoted by the recognition of the bacterial RepA protein as well as its own origin of replication sequence integrated into the template DNA (Odegrip et al. (2004) Proc. Natl. Acad. Sci, U.S.A. 101:2806-2810). Alternatively, a biotin-streptavidin interaction can be utilized. In covalent DNA display a bacteriophage P2 protein genetically fused to an antibody fragment binds to its own DNA sequence (Reiersen et al. (2005) Nucl. Acids Res. 33:e10). Alternatively, the DNA and the peptide can be compartmentalized, such as in an oil-in-water emulsion. Selection techniques are similar to that of phage display wherein DNA display libraries are panned against an immobilized antigen. See e.g. International Patent Publication No. WO 98/037186.
F. Methods of Production of Antibodies
Nucleic acid molecules and antibody members of the libraries provided herein can be made by any method known to one of skill in the art. Such procedures are routine and are well known to the skill artisan. They include routine molecular biology techniques including gene synthesis, PCR, ligation, cloning, transfection and purification techniques. A description of such procedures is provided below.
For example, nucleic acid sequences can be constructed using gene synthesis techniques as discussed herein above. Gene synthesis or routine molecular biology techniques also can be used to effect insertion, deletion, addition or replacement of nucleotides. For example, additional nucleotide sequences can be joined to a nucleic acid sequence. In one example linker sequences can be added, such as sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, a protein expression vector or a vector designed for the amplification of the antibody constant region coding DNA sequences. Furthermore, additional nucleotide sequences specifying functional DNA elements can be operatively linked to a recombined germline encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and leader peptide sequences designed to facilitate protein secretion. Additional nucleotide sequences such as sequences specifying protein binding regions also can be linked to nucleic acid sequences. Such regions include, but are not limited to, sequences to facilitate uptake of recombined antibodies or fragments thereof into specific target cells, or otherwise enhance the pharmacokinetics of the synthetic gene.
Nucleic acid sequences can be further engineered as described herein, such as by mutagenesis, to generate mutant antibodies. Mutagenesis can be effected entirely through gene synthesis. For example, nucleic acid molecules can be designed manually or in silico for synthesis to encode mutant antibodies. The benefit of using gene synthesis methods is that the mutations can be effected so that the resulting nucleic acid molecules are in-frame and are “productive” as discussed herein above. Other methods of synthesis exist where randomization can be achieved during the gene synthesis. For example, a protocol has been developed by which synthesis of an oligonucleotide is “doped” with non-native phosphoramidites, resulting in randomization of the gene section targeted for random mutagenesis (Wang and Hoover (1997) J. Bacteriol., 179:5812-9). This method allows control of position selection while retaining a random substitution rate. Alternatively, mutagenesis can be effected through other molecular biology techniques. Generally, site-directed mutagenesis strategies can be employed.
Other current methods can be used to create mutant antibody libraries from a template nucleic acid molecule or molecules, such as a germline recombined nucleic acid molecule encoding a naïve antibody. Such methods include, but are not limited to, error-prone polymerase chain reaction (Caldwell and Joyce (1992); Gram et al. (1992) Proc. Natl. Acad. Sci., 89:3576-80); cassette mutagenesis in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide (Stemmer and Morris (1992) Biotechniques, 13:214-20); Arkin and Youvan (1992) Proc. Natl. Acad. Sci., 89:7811-7815; Oliphant et al. (1986) Gene, 44:177-83; Hermes et al. (1990) Proc. Natl. Acad. Sci, 87:696-700); the use of mutator strains of hosts cells to add mutational frequency (Greener et al. (1997) Mol. Biotechnol., 7:189-95); DNA shuffling (Crameri et al. (1998) Nature, 391:288-291; U.S. Pat. Nos. 6,177,263; 5,965,408; Ostermeier et al. (1999) Nat. Biotechnol., 17:1205-1209); and other random mutagenesis methods.
1. Vectors
Provided herein are vectors that contain nucleic acid encoding the recombined antibodies or portions thereof. The nucleic acids encoding antibody polypeptides are typically cloned into a intermediate vector before transformation into prokaryotic or eukaryotic cells. Choice of vector can depend on the desired application. For example, after insertion of the nucleic acid, the vectors typically are used to transform host cells, for example, to amplify the recombined antibody genes for replication and/or expression thereof. In such examples, a vector suitable for high level expression is used. In other cases, a vector is chosen that is compatible with display of the expressed polypeptide on the surface of the cell.
Many expression vectors are available and known to those of skill in the art for the expression of recombined antibodies or portions thereof. The choice of an expression vector is influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vectors in the cells. Vectors also generally can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule (e.g. His tag, Flag tag). For purposes herein, vectors generally include sequences encoding the constant region. Thus, recombined antibodies or portions thereof also can be expressed as protein fusions. For example, a fusion can be generated to add additional functionality to a polypeptide. Examples of fusion proteins include, but are not limited to, fusions of a signal sequence, an epitope tag such as for localization, e.g. a his6 tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and/or membrane association.
For example, expression of the proteins can be controlled by any promoter/enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. Promoters which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310 (1981)), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the b-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. ScL USA 78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”: in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrara-Estrella et al., Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 9:2871 (1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 315:115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature 318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 45:485-495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 48:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 314:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 234:1372-1378 (1986)).
In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the antibody, or portion thereof, in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the germline antibody chain and signals required for efficient polyadenylation of the transcript, ribosome binding sites and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.
Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a germline antibody chain under the direction of the polyhedron promoter or other strong baculovirus promoter.
For purposes herein, vectors are provided that contain a sequence of nucleotides that encodes a constant region of an antibody operably linked to the nucleic acid sequence encoding the recombined variable region of the antibody. The vector can include the sequence for one or all of a CH1, CH2, CH3 or CH4 and/or CL. Generally, such as for expression of Fabs, the vector contains the sequence for a CH1 or CL. Exemplary of such vectors containing a heavy chain constant region gene (e.g. CH1) are plasmids A and D, described herein. Exemplary of such vectors containing a light chain constant region genes are plasmids C and E, described herein.
Exemplary expression vectors include any mammalian expression vector such as, for example, pCMV. For bacterial expression, such vectors include pBR322, pUC, pSKF, pET23D, and fusion vectors such as MBP, GST and LacZ. Exemplary of such a vector are bacterial expression vectors such as, for example, plasmid A, plasmid C, plasmid D and plasmid E, described herein. Other eukaryotic vectors, for example any containing regulatory elements from eukaryotic viruses can be used as eukaryotic expression vectors. These include, for example, SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Bar virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, baculovirus pDSCE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedron promoter, or other promoters shown effective for expression in eukaryotes.
Exemplary plasmid vectors for transformation of E. coli cells, include, for example, the ColE1 replication vectors described herein. Several features common to all these vectors include (a) a pBAD inducible promoter; (b) an AraC gene, which controls the pBAD promoter; (c) a synthetic ribosomal binding site (RBS) for efficient translation; (d) a ColE1 origin of replication, allowing for high copy expression; (e) a STII leader sequence, allowing for expressed proteins to be translocated to the periplasm; (f) a f1 origin of replication; and (g) a gene for conferring antibiotic resistance. Such plasmids include plasmid A (FIG. 3), plasmid C (FIG. 5), plasmid D (FIG. 4) and plasmid E (FIG. 6). Plasmid A and Plasmid D are utilized for expression of heavy chain antibody genes in as they contain a gene for the heavy chain constant region (CH1) operably linked to the inserted gene for the heavy chain variable region. The vectors contain Nhel and Ncol restriction sites to allow for cloning of the recombined antibody genes described herein. Both vectors contain a pUC origin of replication, a ColE1 type origin of replication, and an aminoglycoside phosphotransferase gene conferring kanamycin resistance. Plasmid A contains a (His)6 Tag and a Flag Tag for protein purification. Plasmid D contains both a (His)6 Tag and a Flag Tag, and an additional LPETG tag, which allows for covalent attachment of the resulting protein using a sortase. Plasmid C and Plasmid E are utilized for expression of light chain antibody genes in as they contain a gene for the light chain constant region (CL) operably linked to the inserted gene for the light chain variable region. Plasmid C is specific for kappa light chains and contains BseWI and NcoI restriction sites to allow for cloning of the recombined antibody genes described herein. Plasmid E is specific for lambda light chains and contains AcrII and NcoI restriction sites to allow for cloning of the recombined antibody genes described herein. Both vectors contain a 3.3 origin of replication, a ColE1 type origin of replication, and a gene conferring chloramphenicol resistance. The vectors described above are designed to be utilized in a dual vector system, in which a light chain vector and a heavy chain vector are co-transformed. Thus, they contain two different but compatible ColE1 origins of replication utilized, one for heavy chains and one light chain. This allows for efficient expression of both chains of the antibody when the vectors are co-transformed and expressed.
Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding an antibody chain. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.
2. Cells and Expression Systems
Cells containing the vectors also are provided. Generally, any cell type that can be engineered to express heterologous DNA and has a secretory pathway is suitable. Expression hosts include prokaryotic and eukaryotic organisms such as bacterial cells (e.g. E. coli), yeast cells, fungal cells, Archea, plant cells, insect cells and animal cells including human cells. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. Further, the choice of expression host is often related to the choice of vector and transcription and translation elements used. For example, the choice of expression host is often, but not always, dependent on the choice of precursor sequence utilized. For example, many heterologous signal sequences can only be expressed in a host cell of the same species (i.e., an insect cell signal sequence is optimally expressed in an insect cell). In contrast, other signal sequences can be used in heterologous hosts such as, for example, the human serum albumin (hHSA) signal sequence which works well in yeast, insect, or mammalian host cells and the tissue plasminogen activator pre/pro sequence which has been demonstrated to be functional in insect and mammalian cells (Tan et al., (2002) Protein Eng. 15:337). The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification. Thus, the vector system must be compatible with the host cell used.
Expression in eukaryotic hosts can include expression in yeasts such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as Drosophila cells and lepidopteran cells, plants and plant cells such as tobacco, corn, rice, algae, and lemna. Eukaryotic cells for expression also include mammalian cells lines such as Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. Eukaryotic expression hosts also include production in transgenic animals, for example, including production in serum, milk and eggs.
Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated. Generally, standard transfection methods are used to produce bacterial, mammalian, yeast, or insect cell lines that express large quantity of antibody chains, which is then purified using standard techniques (see e.g., Colley et al. (1989) J. Biol. Chem., 264:17619-17622; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed.), 1990). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison (1977) J. Bact. 132:349-351; Clark-Curtiss and Curtiss (1983) Methods in Enzymology, 101, 347-362). For example, any of the well-known procedures for introducing foreign nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any other the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell. Generally, for purposes herein, host cells are transfected with a first vector encoding at least a VH chain and a second vector encoding at least a VL chain. Thus, it is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least both genes into the host cell capable of expressing germline, or modified form thereof, antibody polypeptide.
Transformation of host cells with recombinant DNA molecules that incorporate the isolated recombined variable region gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene can be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. Generally, After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the germline chain, which is recovered from the culture using standard purification techniques identified below.
Antibodies and portions thereof can be produced using a high throughput approach by any methods known in the art for protein production including in vitro and in vivo methods such as, for example, the introduction of nucleic acid molecules encoding recombined antibodies or portions thereof into a host cell or host animal and expression from nucleic acid molecules encoding recombined antibodies in vitro. Prokaryotes, especially E. coli, provide a system for producing large amounts of recombined antibodies or portions thereof, and are particularly desired in applications of high-throughput expression and purification of proteins. Transformation of E. coli is a simple and rapid technique well known to those of skill in the art. E. coli host strains for high throughput expression include, but are not limited to, BL21 (EMD Biosciences) and LMG194 (ATCC). Exemplary of such an E. coli host strain is BL21. Vectors for high throughput expression include, but are not limited to, pBR322 and pUC vectors. Exemplary of such vectors are the vectors described herein, including plasmid A, plasmid C, plasmid D and plasmid E. Automation of expression and purification can facilitate high-throughput expression. For example, use of a PiccoloTM system, a fully automatic system that combines cell culture with automated harvesting, lysing and purification units, or other similar robotic system can be employed.
a. Prokaryotic Expression
Prokaryotes, especially E. coli, provide a system for producing large amounts of recombined antibodies or portions thereof. Transformation of E. coli is a simple and rapid technique well known to those of skill in the art. Expression vectors for E. coli can contain inducible promoters that are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated PL promoter.
Recombined antibodies or portions thereof can be expressed in the cytoplasmic environment of E. coli. The cytoplasm is a reducing environment and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreitol and -mercaptoethanol and denaturants (e.g., such as guanidine-HCl and urea) can be used to resolubilize the proteins. An exemplary alternative approach is the expression of recombined antibodies or fragments thereof in the periplasmic space of bacteria which provides an oxidizing environment and chaperonin-like and disulfide isomerases leading to the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. There are three major pathways to translocate expressed proteins into the periplasm, namely the Sec pathway, the SRP pathway and the TAT pathway. Examples of periplasmic-targeting leader sequences include the pelB leader from the pectate lyase gene, the StII leader sequence, and the DsbA leader sequence. An exemplary leader sequence is a DsbA leader sequence. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. The secretion of proteins allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and solubility. Typically, temperatures between 25° C. and 37° C. are used. Mutations also can be used to increase solubility of expressed proteins. Typically, bacteria produce aglycosylated proteins. Thus, if proteins require glycosylation for function, glycosylation can be added in vitro after purification from host cells.
b. Yeast
Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris are useful expression hosts for recombined antibodies or portions thereof. Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include AOX1, GAL1, GAL7, and GAL5 and metallothionein promoters such as CUP1. Expression vectors often include a selectable marker such as LEU2, TRP1, HIS3, and URA3 for selection and maintenance of the transformed DNA. Proteins expressed in yeast are often soluble. Co-expression with chaperonins such as Bip and protein disulfide isomerase can improve expression levels and solubility. Additionally, proteins expressed in yeast can be directed for secretion using secretion signal peptide fusions such as the yeast mating type alpha-factor secretion signal from Saccharomyces cerevisae and fusions with yeast cell surface proteins such as the Aga2p mating adhesion receptor or the Arxula adeninivorans glucoamylase. A protease cleavage site such as for the Kex-2 protease, can be engineered to remove the fused sequences from the expressed polypeptides as they exit the secretion pathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs.
c. Insects
Insect cells, particularly using baculovirus expression, are useful for expressing antibodies or portions thereof. Insect cells express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculovirus have a restrictive host range which improves the safety and reduces regulatory concerns of eukaryotic expression. Typical expression vectors use a promoter for high level expression such as the polyhedrin promoter and p10 promoter of baculovirus. Commonly used baculovirus systems include the baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from Spodoptera frugiperda and TN derived from Trichoplusia ni. For high-level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. To generate baculovirus recombinants capable of expressing human antibodies, a dual-expression transfer, such as pAcUW51 (PharMingen) is utilized. Mammalian secretion signals are accurately processed in insect cells and can be used to secrete the expressed protein into the culture medium
An alternative expression system in insect cells is the use of stably transformed cells. Cell lines such as Sf9 derived cells from Spodoptera frugiperda and TN derived cells from Trichoplusia ni can be used for expression. The baculovirus immediate early gene promoter IE1 can be used to induce consistent levels of expression. Typical expression vectors include the pIE1-3 and pI31-4 transfer vectors (Novagen). Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin.
d. Mammalian Cells
Mammalian expression systems can be used to express antibodies or portions thereof. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. Such vectors often include transcriptional promoter-enhancers for high-level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic protein, myosin light chain 2, and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase and thymidine kinase. Antibodies are typically produced using a NEOR/G418 system, a dihydrofolate reductase (DHFR) system or a glutamine synthetase (GS) system. The GS system uses joint expression vectors, such as pEE12/pEE6, to express both heavy chain and light chain. Fusion with cell surface signaling molecules such as TCR- and Fc RI- can direct expression of the proteins in an active state on the cell surface.
Many cell lines are available for mammalian expression including mouse, rat human, monkey, chicken and hamster cells. Exemplary cell lines include but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NS0 (nonsecreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of secreted proteins from the cell culture media. One such example is the serum free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42.)
e. Plants
Transgenic plant cells and plants can be used to express proteins such as any antibody or portion thereof described herein. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer into protoplasts, and with agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements and translational control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as Arabidopsis and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus CaMV 35S promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the maize ubiquitin-1 (ubi-1) promoter promoters. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce proteases or modified proteases (see for example, Mayfield et al. (2003) PNAS 100:438-442). Because plants have different glycosylation patterns than mammalian cells, this can influence the choice of protein produced in these hosts.
3. Purification
Antibodies and portions thereof are purified by any procedure known to one of skill in the art. The recombined germline antibodies can be purified to substantial purity using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ionic exchange chromatography or column chromatography. For example, antibodies can be purified by column chromatography. Exemplary of a method to purify antibodies is by using column chromatography, wherein a solid support column material is linked to Protein G, a cell surface-associated protein from Streptococcus, that binds immunoglobulins with high affinity. The antibodies can be purified to 60%, 70%, 80% purity and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity. Purity can be assessed by standard methods such as by SDS-PAGE and coomassie staining.
Methods for purification of recombined antibodies or portions thereof from host cells depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary further the proteins can be extracted and further purified using standard methods in the art.
When antibodies are expressed by transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the polypeptides can form insoluble aggregates. There are several protocols that are suitable for purification of polypeptide inclusion bodies known to one of skill in the art. Numerous variations will be apparent to those of skill in the art.
For example, in one method, the cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCL (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It can be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies can be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers are apparent to those of skill in the art.
Alternatively, antibodies can be purified from bacteria periplasm. Where the polypeptide is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art. For example, in one method, to isolate recombinant polypeptides from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO4 and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant polypeptides present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art. These methods include, but are not limited to, the following steps: solubility fractionation, size differential filtration, and column chromatography.
G. Applications and Uses of the Libraries
Provided herein are methods of using the antibody libraries provided herein for screening to identify or select an antibody or antibodies that alter or modulate (increase or decrease) an activity of a target. As discussed above, the antibody libraries provided herein contain members 1) that are each different from all other members in the library; and 2) that are each productive and functional. By virtue of these characteristics the libaries are both diverse and robust in their screening capabilties. For example, using the libraries provided herein, it is possible to screen a small library (e.g. containing 1000 or fewer members) and identify antibodies having desired functions or activities. This is exemplified in Example 13 where a library of only 960 members was used in a screening assay for binding to various targets, resulting in the identification of several high affinity (nanomolar affinity) antibodies.
In the method of screening herein, any desired activity can be assayed for, including but not limited to binding, cytotoxicity, differentiation or proliferation of cells, cell migration, apoptosis, angiogenesis and alteration of gene expression. For example, the resulting libraries can be screened for the discovery of agonist or antagonist antibodies against therapeutic targets, such as, for example, targets involved in cell proliferation and differentiation, cell migration, apoptosis and angiogeneis. Such targets include, but not limited to, growth factors, cytokines, lymphocytic antigens, other cellular activators and receptors thereof.
In other examples, the libraries can be screened using “blind”functional screens to measure an observable biological processes of interest without requiring specific knowledge of the target in advance. Any Hit that is identified in such screening assays, for example by virtue of a strong functional activity, can be further analyzed to identify the specific target. The “blind” assay approach can be applied to any observable biological outcome. For example, apoptosis of B-cells can be readily assayed, and a screen for increased B-cell apoptosis can yield antibody Hits that promote B-cell killing. Such antibodies can bind known B-cell surface-proteins like CD-20, or bind a novel target. In another example, the increased (or decreased) secretion of specific cytokines, for example interferon alpha, from a population of cells such as bone marrow cells can be assayed by ELISA, allowing screening for antibodies which modify the secretion of these proteins. No a priori knowledge of the mechanism that achieves the screened outcome is necessary, and the specific antibody target can later be pursued if the effect is strong. The blind functional assays can also be merged with high-content screening approaches to find antibodies that induce morphological changes in a certain cell type or promote cell differentiation in a progenitor cell population.
The methods of screening provided herein involve contacting each member of an antibody library provided herein with a target protein, peptide or antigen or cell expressing a target protein, peptide or antigen or simply a cell population and identifying antibody members that modulate (increase or decrease) the activity of the target protein or cell. For example, the members of the library can be contacted to identify antibodies that bind to the target protein. In another example, antibodies can be screened for modulation of a functional activity of a target. Functional assays permit identification of agonist and antagonist antibodies. It is contemplated herein that this screening assay is particularly useful against membrane-bound proteins, such as receptor targets and other signaling molecules.
The antibody libraries provided herein can be presented in a format that permits screening, such as, for example, as addressable libraries, including spatial arrays; and cell display libraries, including yeast display and mammalian display, phage display, mRNA display, including ribosome display, and DNA display. For example, screening is performed on addressable antibody libraries, such as a spatial arrayed library, whereby each antibody member at a locus (e.g. well) is separately contacted and screened against a target protein or proteins from all other members in the library. Thus, upon identification of a “HIT” the identity of the antibody member is immediately known from its location in the array without any requirement for further enrichment, amplification or sequencing thereof. Library screening can be high-throughput by screening hundreds to thousands or more antibodies in the same screening assay.
Any technique known to one of skill in the art can be used for antibody screening herein to identify antibody members having a desired activity. For a typical high-throughput screen, sets of microtiter plates are generated, whereby each well in the microplate contains a different library member. For example, plates can be created where every well of a plate contains an antibody containing the same antibody heavy chain, but each well contains a different light chain such that the paired antibody (heavy and light chain) in each well is different from the antibody in all other wells of the plate. All wells are then loaded with the constant components necessary for performing the screening assay (for example, target protein or cell expressing a target protein, buffer, assay reagent), incubated for the appropriate time, and assayed for a readout of choice. This method can be used for screening for activities such as binding or other functional assays where reaction with a single purified target molecule or cell(s) is sufficient to give a readout.
For example, in such methods, a cell-based assay can be performed to effect activation of the target (e.g. in the presence of a ligand thereof) and activation (such as by assessing signaling, differentiation, proliferation, chemotaxis, apoptosis or other downstream process) is assessed in the presence of the antibody. Generally, the performance of such functional assays require the presence of soluble protein. Hence, it is an advantage of the antibody libraries provided herein that they permit screening in a soluble format. For example, the antibodies can be addressed, for example, in wells of a multiwall plate. Each antibody at each locus is different. Thus, upon assessing activity the identity of a “Hit” can be immediately determined.
Following an initial round of screening, identified “HITS” can be further optimized by iterative screening and/or by directed evolution methods. For example, following identification of a “Hit” ‘focused’ libraries can be created using information gained from the primary screening libraries. Focused libraries represent substructures of the original primary library because they are created from germline segment components that are related to those of the “Hit.” They can allow one to refine the properties of a selected “Hit”, such as by increasing its affinity of binding or associated functional activity.
The identity of the antibody is known by virtue of its address. Once identified, the antibody Hits can be generated as a full-length IgG or as Fab, Fab, Fab-SH and F(ab)2 fragments. The antibody or fragments thereof can be created by traditional means, such as using recombinant techniques and enzymatic digestion. Antibodies or fragments thereof can be chimeric or humanized. The antibodies or fragments thereof can be used for diagnostic and therapeutic purposes as set forth below.
1. Binding Assays
The antibody libraries provided herein can be screened for their ability to bind a selected target by any method known to one of skill in the art. Exemplary target antigens are described below. Binding assays can be performed in solution, suspension or on a solid support. For example, target antigens can be immobilized to a solid support (e.g. a carbon or plastic surface or chip) and contacted with antibody. Unbound antibody or target protein can be washed away and bound complexes can then be detected. Binding assays can be performed under conditions to reduce nonspecific binding, such as by using a high ionic strength buffer (e.g. 0.3-0.4 M NaCl) with nonionic detergent (e.g. 0.1% Triton X-100 or Tween 20) and/or blocking proteins (e.g. bovine serum albumin or gelatin). Negative controls also can be including in such assays as a measure of background binding. Binding affinities can be determined using Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)), BIACore or other methods known to one of skill in the art.
Exemplary binding assays include, but are not limited to immunoassays such as competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, Meso Scale Discovery (MSD, Gaithersburg, Md.), immunoprecipitation assays, ELISPOT, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., (1986) Amer. Clin. Prod. Rev. 5:34-41).
Generally, binding is detected using a detectable moiety or label (e.g. an enzyme, a radionuclide, a fluorescent probe, electrochemiluminescent label, or a color dye) typically attached to the target or, if desired, directly to the antibody members in the library. Alternatively, binding can be detected by a further third reagent that itself is labeled or detectable. For example, detection of an antibody bound to a target protein can be achieved using a labeled capture molecule in a sandwich assay format. Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G also can be used as the label agent. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al., (1973) J. Immunol. 111:1401-1406; Akerstrom et al., (1985) J. Immunol. 135:2589-2542). The detction agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.
The choice of label or detectable group used in the assay is not critical, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. Generally, the choice depends on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions. One of skill in the art is familiar with labels and can identify a detectable label suitable for and compatible with the assay employed.
The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), chemiluminescent labels (luciferin and 2,3-dihydrophtahlazinediones, e.g., luminol), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). For a review of various labeling or signal producing systems that can be used, see e.g. U.S. Pat. No. 4,391,904.
Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels can be detected simply by observing the color associated with the label.
Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples containing the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
Alternatively, the antibody libraries provided herein can be screened for their ability to bind to cells, using whole cell panning, with or without subtractive panning. Screening can be done against live cells or against intact, mildly fixed target cells. Methods for whole cell panning have been described previously (see e.g. Siegel et al. (1997) J. Immunol. Methods 206:73-85 incorporated herein by reference). Other techniques for screening which can be applied include fluorescent activated cell sorting (FACs).
For high-throughput screening, assays can be multiplexed. Thus, the binding affinities of antibodies to a number of different target proteins can be determined at once. In one example, different target proteins can be separately labeled with different detectable moieities. For example, different antigens can be coupled to color-coded beads (Schwenk et al. (2007) Mol. Cell. Prot., 6:125-132). In another example, multi-spot plates can be used that permit assay multiplexing by absorption of of up to 100 proteins in a locus of the plate (e.g. using Multi-Array or Multi-Spot plates from Meso Scale Discovery; MSD, Gaithersburg, Md.). In such an example, addressed antibody libraries provided herein can be screened by addition of a different antibody member to each well of a multi-spot plate. The assay readily permits the screening of thousands of antibodies at once against numerous target proteins. This is exemplified herein in Example 13.
In the methods of screening herein, the binding affinity of the antibodies is determined to identify or select antibodies that have high affinity for a target protein. Typically, antibodies are selected or identified that have a binding affinity that is or is about 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, 10−12M or lower. Generally, antibodies are screened until antibodies are identified having nanomolar or sub-nanomolar binding affinity. A “Hit” identified in a first round of screening that does not have the desired binding affinity can be optimized by iterative screening and/or directed evolution methods and further screened for binding to a target antigen to identify an antibody that has a high binding affinity.
Any method known to one of skill in the art can be used to measure the binding affinity of an antibody. For example, the binding properties of an antibody can be assessed by performing a saturation binding assay, for example, a saturation ELISA, whereby binding to a target protein is assessed with increasing amounts of antibody. In such experiments, it is possible to assess whether the binding is dose-dependent and/or saturable. In addition, the binding affinity can be extrapolated from the 50% binding signal. Typically, apparent binding affinity is measured in terms of its association constant (Ka) or dissociation constant (Kd) and determined using Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980). For example, binding affinity to a target protein can be assessed in a competition binding assay in where increasing concentrations of unlabeled protein is added, such as by radioimmunoassay (RIA) or ELISA. Binding affinity also can be analyzed using BIAcore kinetic analysis. This involves analyzing the binding and dissociation of an antibody member from chips containing immobilized target proteins on their surface. The Biacore evaluation software generates the values of Ka and Kd by fitting the data to interaction models. It is understood that the binding affinity of an antibody can vary depending on the assay and conditions employed, although all assays for binding affinity provide a rough approximation. By performing various assays under various conditions it is possible to estimate the binding affinity of an antibody.
In addition, binding affinities can differ depending on the structure of an antibody. For example, generally a bivalent antibody, for example a bivalent F(ab′)2 fragment or full-length IgG, has a better binding affinity then a monovalent Fab antibody. Hence, it is understood that where a Fab has a specified binding affinity for a particular target, it is excepted that the binding affinity is even greater for a full-lenth IgG that is bivalent.
2. Functional Activity
The antibody libraries provided herein can be screened for their ability to modulate the functional activity of a target by any method known to one of skill in the art. Assays can be designed to identify antibodies capable of binding and/or modulating cell surface receptors. Such antibodies can either be agonists, mimicking the normal effects of receptor binding, or antagonists, inhibiting the normal effects of receptor binding. Of particular interest is the identification of agents which bind to the receptors and modulate intracellular signalling.
In some example, such assays are cell-based assays. Generally, assays are performed using cell lines known to express the target of interest. Such cells are known to one of skill in the art. For example, one can consult the ATCC Catalog (atcc.org) to identify cell lines. Also, if a particular cell type is desired, the means for obtaining such cells, and/or their instantly available source is known to those in the art. An analysis of the scientific literature can readily reveal appropriate choice of cells expressing any desired target. Table 18 lists exemplary cells lines that express targets of interest that can be screened in functional activities herein against antibody libraries provided herein.







TABLE 18







Cell lines expressing targets









Target
Cell Lines
References





GP IIb/IIIa
MEG-01 chronic myelogenous
Ogura et al. Establishment of a novel human megakaryoblastic leukemia cell



leukemia megakaryoblast cells
line, MEG- 01, with positive Philadelphia chromosome. Blood 66: 1384-1392,



(ATCC CRL-2021);
1985; Komatsu et al. Establishment and Characterization of a Human



UT-7 human leukemia cell ine
Leukemic Cell Line with Megakaryocytic Features: Dependency on




Granulocyte-Macrophage Colony-stimulating Factor, Interleukin 3, or




Erythropoietin for Growth and Survival. Cancer Research 51: 341-348 (1991)


GM-CSF-R
VA-ES-BJ epitheloid sarcoma
Int J Oncol 1995; 7: 51-56; Ali Habib et al. A urokinase-activated recombinant



cells (ATCC CRL-2138);
diphtheria toxin targeting the granulocyte-macrophage colony-stimulating factor



TF1-HaRas;
receptor is selectively cytotoxic to human acute myeloid leukemia blasts. Blood



TF1-vRaf;
104(7): 2143-2148 (2004); Kiser et al. Oncogene-dependent engraftment of



TF1-vSrc;
human myeloid leukemia cells in immunosuppressed mice. Leukemia



HL-60 (ATCC CCL-240);
15(5): 814-818(2001)



U-937 (ATCC CRL-1593.2);



ML-2


VEGFA
Human A673
Gerber et al. Complete inhibition of rhabdomyosarcoma xenograft growth and



rhabdomyosarcoma cells (ATCC
neovascularization requires blockade of both tumor and host vascular



CRL-1598);
endothelial growth factor. Cancer Res. 60(22): 6253-8 (2000); Presta et al.



Breast carcinoma MDA-MB-435
Humanization of an anti-vascular endothelial growth factor monoclonal



cells (ATCC); Bovine adrenal
antibody for the therapy of solid tumors and other disorders. Cancer Research,



cortex-derived capillary
57(20): 4593-4599 (1997)



endothelial cells


CD3
Jurkat E6.1 Human leukemic T
Buhler et al. A bispecific diabody directed against prostate-specific membrane



cell lymphoblast (Sigma Aldrich
antigen and CD3 induces T-cell mediated lysis of prostate cancer cells. Cancer



88042803)
Immunol Immunother. 57(1): 43-52 (2008)


EGFR
DiFi human colorectal carcinoma
Olive et al. Characterization of the DiFi rectal carcinoma cell line derived from a



cells;
familial adenomatous polyposis patient. In Vitro Cell Dev Biol. 29A(3 Pt 1): 239-



A431 cells (ATCC CRL-1555);
248 (1993); Wu et al. Apoptosis induced by an anti-epidermal growth factor



Caco-2 colorectal
receptor monoclonal antibody in a human colorectal carcinoma cell line and its



adenocarcinoma cells (ATCC
delay by insulin. Clin. Invest. 95(4): 1897-1905 (1995)



HTB-37);



HRT-18 colorectal



adenocarcinoma cells (ATCC



CCL-244);



HT-29 colorectal



adenocarcinoma cells (ATCC



HTB-38)


EPO receptor
A2780 ovarian cancer cells;
Jeong et al. Characterization of erythropoietin receptor and erythropoietin



UT-7 human leukemia cell ine
expression and function in human ovarian cancer cells. Int J Cancer.




122(2): 274-280 (2008); Elliott et al. Activation of the Erythropoietin (EPO)




Receptor by Bivalent Anti-EPO Receptor Antibodies. J Biol Chem.




271(40): 24691-24697 (1996)


Her2/Neu
BT-474 ductal carcinoma breast
Le et al. Roles of human epidermal growth factor receptor 2, c-jun NH2-


receptor
cancer cell (ATCC HTB-20);
terminal kinase, phosphoinositide 3-kinase, and p70 S6 kinase pathways in



SK-BR-3 adenocarcinoma
regulation of cyclin G2 expression in human breast cancer cells. Mol Cancer



breast cancer cell (ATCC
Ther. 6(11): 2843-2857 (2007)



HTB-30);



MDA-MB-453 metastatic



carcinoma cell line (ATCC



HTB-131)


cMet
H1993 lung adenocarcinoma
Ma et al. Functional expression and mutations of c-Met and its therapeutic



cells (ATCC CRL-5909);
inhibition with SU11274 and small interfering RNA in non-small cell lung



H1838 lung adenocarcinoma
cancer. Cancer Res. 65(4): 1479-1488 (2005);



cells (ATCC CRL-5899);
Ma et al. A selective small molecule c-MET Inhibitor, PHA665752, cooperates



SW 900 lung squamous cell
with rapamycin. Clin Cancer Res 11(6): 2312-2319 (2005)



carcinoma cells (ATCC HTB-59);



H358 lung bronchioalveolar



carcinoma cells (ATCC



CRL-5807);



SK-Lu-1 lung adenocarcinoma



cells (ATCC HTB-57);



H441 Non-small cell lung cancer



cells (ATCC HTB-174)


CD20
Ramos Burkitt's lymphoma B
Jazirehi et al. Rituximab (anti-CD20) selectively modifies Bcl-xL and apoptosis



cells (ATCC CRL-1596);
protease activating factor-1 (Apaf-1) expression and sensitizes human non-



Raji Burkitt's lymphoma B
Hodgkin's lymphoma B cell lines to paclitaxel-induced apoptosis. Mol Cancer



cells (ATCC CCL-86):
Ther. 2(11): 1183-1193 (2003)



Daudi Burkitt's lymphoma B



cells (ATCC CCL-213);



2F7 Burkitt's lymphoma B



cells









In addition, cells lines expressing a target of interest can be generated by transient or stable transfection with an expression vector expressing a target of interest. Methods of transfection and expression are known to those of skill in the art (see e.g., Kaufman R. J. (1990) Methods in Enzymology 185:537-566; Kaufman et al. (1990) Methods in Enzymology 185:537-566). In addition, any primary cell or cell line can be assessed for expression of a particular target (e.g. cell surface marker). Cell surface markers can be assayed using fluorescently labeled antibodies and FACS. Suitable cell lines include A549 (lung), HeLa , Jurkat, BJAB, Colo205, H1299, MCF7, MDA-MB-231, PC3, HUMEC, HUVEC, and PrEC.
Any suitable functional effect can be measured, as described herein. For example, cellular morphology (e.g., cell volume, nuclear volume, cell perimeter, and nuclear perimeter), ligand binding, substrate binding, nuclease activity, apoptosis, chemotaxis or cell migrations, cell surface marker expression, cellular proliferation, GFP positivity and dye dilution assays (e.g., cell tracker assays with dyes that bind to cell membranes), DNA synthesis assays (e.g., 3H-thymidine and fluorescent DNA-binding dyes such as BrdU or Hoechst dye with FACS analysis) and nuclear foci assays, are all suitable assays to identify potential modulators using a cell based system. Other functional activities that can be measured include, but are not limited to, ligand binding, substrate binding, endonuclease and/or exonuclease activity, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism, changes related to cellular proliferation, cell surface marker expression, DNA synthesis, marker and dye dilution assays (e.g., GFP and cell tracker assays), contact inhibition, tumor growth in nude mice, and others.
For example, antibodies or portions thereof in the libraries provided herein can be assessed for their modulation of one or more phenotypes of a cell known to express a target protein. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to screen antibody libraries. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).
Cells determined to be appropriate for a particular phenotypic assay (i.e., A549, HeLa, Jurkat, BJAB, Colo205, H1299, MCF7, MDA-MB-231, PC3, HUMEC, HUVEC, and PrEC and any others known to express the target of interest) are treated with antibodies as well as control compounds. If necessary, a ligand for the receptor target is included so that activation of the receptor is effected. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
The assays can be performed to assess the direct effects of an antibody on a target protein. For example, if the target protein is a cell surface receptor, an antibody can be added to assess whether the target protein directly modulates, such as by stimulation, the activity or function of the receptor. In such instances, the antibody is deemed an agonist antibody. In other examples, if the target protein is a cell surface receptor, the activity of the receptor can be stimulated in the presence of a ligand or other stimulating agent in the presence or absence of the antibody to determine if the antibody modulates (e.g. inhibits) the actions of the antibody. For example, the antibody can act by blocking the ability of the ligand to interact with the receptor and/or otherwise induce a negative stimulatory signal. In such instances, the antibody is deemed to be an antagonist of the receptor. Thus, the methods of screening herein by functional activity permits identification of agonist and antagonist antibodies.
a. Differentiation
Cellular differentiation can be analyzed using any assay that allows a detection of a physical, chemical or phenotypic change. Various assays are used to quantitatively determine cellular proliferation and activation in response to an external stimuli. Cell proliferation assays are used to quantitatively determine cellular proliferation by incorporating a reagent into the DNA of newly synthesized cells upon cell division. Such reagents include, but are not limited to 3H-thymidine, 5-bromo-2-deoxyuridine (BrdU) and fluorescent Hoechst dyes. Cell viability assays are used to determine the number of healthy cells in a sample by staining cells with a dye and measuring how many cells uptake the dye based on the fact that living cells will exclude the dye. Such dyes include but are not limited to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT), and 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1). Uptake of the reagent is measured either colorimetrically using a spectrophotometer or by measuring radiation with a scintillation counter. Details of these methods are well-known to one skilled in the art. For example, Example 12 exemplifies an MTT proliferation assay to assess cell proliferation.
Fluorescent dyes are commonly used for the detection of live cells and key functional activities in a variety of cell-based assays. There are several non-radioactive, fluorescence-based assays that are not dependent on cellular metabolism. The fluorescent dye binds nucleic acids and the fluorescence can then be measured quantitatively or qualitatively. Such dyes include, but are not limited to, propidium iodide and Hoechst 33342. The cell number can then be quantitated based on the fluorescence. DNA content can also be quantitated using the tools available in the imaging instruments. Details of these methods are well known to one skilled in the art.
The degree of invasiveness into Matrigel or some other extracellular matrix constituent can be used as an assay to identify antibodies that are capable of inhibiting abnormal cell proliferation and tumor growth. Tumor cells exhibit a good correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Therefore, antibodies can be identified by measuring changes in the level of invasiveness between the host cells before and after the introduction of potential modulators.
Briefly, the level of invasion of host cells can be measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 125I and counting the radioactivity on the distal side of the filter or bottom of the dish. (see, e.g., Freshney, Culture of Animal Cells a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York (1994), herein incorporated by reference).
b. Alteration of Gene Expression
Detection of binding and/or modulation of a target by an antibody can be accomplished by detecting a biological response, such as, for example, measuring Ca2+ ion flux, cAMP, IP3, PIP3 or transcription of reporter genes. Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell using a reporter gene assay) after treatment is also used as an indicator of the efficacy or potency of the antibody. Hallmark genes, or those genes suspected to be associated with a signal transduction pathway are measured in both treated and untreated cells.
Assays can be performed that measure the activation of a reporter gene. Suitable reporter genes include endogenous genes as well as exogenous genes that are introduced into a cell by any of the standard methods familiar to the skilled artisan, such as transfection, electroporation, lipofection and viral infection. For example, cells expressing a recombinant receptor can be transfected with a reporter gene (e.g., chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase, -galactosidase and alkaline phosphatase) operably linked to a response element. The cells are then incubated with antibodies and the expression of the reporter gene is compared to expression in control cells that do not express the recombinant receptor but that are essentially identical in other respects. A statistically significant change in reporter gene expression in the receptor-expressing cells is indicative of a test compound that interacts with the receptor. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as red or green fluorescent protein (see, e.g., Mistili & Spector, (1997) Nature Biotechnology 15:961-964).
The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art. The use of a reporter gene assay using luciferase to measure activiation of STAT5 directly or by induction of cyclin-D promoter is exemplified in Example 12.
c. Cytotoxicity Activity
Antibodies can be screened for their ability to directly induce apoptosis or programmed cell death or to indirectly induce apoptosis by blocking growth factor receptors, thereby effectively arresting proliferation. Antibodies also bind complement, leading to direct cell toxicity, known as complement dependent cytotoxicity (CDC). Thus, assays can be performed to assess complement-dependent cytotoxicity.
A variety of assays to assess apoptosis are known to one of skill in the art. For example, apoptosis assays include those that assay for the activation of a caspase, which are enzymes involved in apoptosis. Caspase assays are based on teh measurement of zymogen processing to an active enzyme and proteolytic activity. A number of commercial kits and reagents are available to assess apoptosis based on caspase function including, but not limited to, PhiPhiLux (Oncolmmunin, Inc.), Caspase 3 activity assay (Roche Applied science), Homogenous Caspase assay (Roche Applied Science), Caspase-Glo Assays (Promega), Apo-ONE Homogeneous Caspase-3/7 Assay (Promega), CaspACE Assay System Colorimetric or Fluormetric (Promega), EnzChek Caspase-3 Assay Kit (Invitrogen), Imag-iT LIVE green Caspase-3 and 7 Detection Kit (Invitrogen), Active Caspase-3 Detection Kits (Stratagene), Caspase-mediated Apoptosis Products (BioVision) and CasPASE Apoptosis Assay Kit (Genotech). Example 11 exemplifies a assaying for apoptosis using a caspase assay.
Assays for apoptosis include TUNEL and DNA fragmentation assays that measure the activation of nucleases and subsequent cleavage of DNA into 180 to 200 base pair increments. Such assays and kits are commercially available and include, but are not limited to, Apoptotic DNA Ladder Kit (Roche Applied Science), Cellular DNA Fragmentation ELISA (Roche Applied Science), Cell Death Detection ELISAPLUS (Roche Applied Science), In Situ Cell Death Detection Kit (Roche Applied Science), DeadEnd Fluorometirc or Colorimetric TUNEL System (Promega), APO-BrdU TUNEL Assay Kit (Invitrogen), and TUNEL Apoptosis Detection Kit (Upstate).
Other assays to assess apoptosis include, for example, cell permeability assays that evaluate the loss of membrane integrity. For example, to determine whether the antibody is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue, or 7-aminoactinomycin D (7AAD) can be assessed relative to untreated cells. In addition, commercial kits such as APOPercentage Assay (Biocolor Assays) can be used to measure apoptosis. Annexin V assays also can be employed. Annexin V binds to phosphatidylserine, which is normally found on the inner surface of the cytoplasmic membrane. During apoptosis, phosphatidylserine is translocated to the outer surface and can be detected by Annexin V. For example, standard binding assays using a fluorescent labeled Annexin V can be used (e.g. Annexin V, Alex Fluor 350 Conjugate from Invitrogen). Apoptosis also can be measured by assessing the presence of other markers of apoptosis, assessing protein cleavage, and/or by mitochondrial and ATP/ADP assays. Such assays are routine and known to one of skill in the art.
For example, apoptosis analysis can be used as an assay to identify functional antibodies using cell lines, such as RKO or HCT116, or other cells expressing a target protein of interest. The cells can be co-transfected with a construct containing a marker gene, such as a gene that encodes green fluorescent protein, or a cell tracker dye. The apoptotic change can be determined using methods known in the art, such as DAPI staining and TUNEL assay using fluorescent microscope. For TUNEL assay, commercially available kit can be used (e.g., Fluorescein FragEL DNA Fragmentation Detection Kit (Oncogene Research Products, Cat.#QIA39) and Tetramethyl-rhodamine-5-dUTP (Roche, Cat. #1534 378)). Cells contacted with an antibody can exhibit, e.g., an increased apoptosis compared to control.
Cell death in vitro can be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death can be performed using heat inactivated serum (i.e. in the absence of complement) and in the absence of immune effector cells.
3. Targets
In the screening methods provided herein, the antibodies are screened for any activity, such as binding or other functional activity, against a target. The activity can be an agonist or antagonist activity of the target. The screening assays can be designed based on any contemplated target. Exemplary of such targets include membrane-bound proteins, receptors and ligands thereof; ion channels; G-protein coupled receptors; novel epitopes; and non-protein antigens. Any activity can be assessed and is a function of the target of interest. One of skill in the art is familiar with the activities of various targets, and can choose a screening assay based on such known activities. Many of these activities for exemplary targets are exemplified herein below. Binding activity can be assessed for all targets.
Membrane-Bound Proteins, Receptors and Ligands Thereof
Exemplary targets are membrane-bound proteins, receptors and ligands thereof that can play important roles in, among other things, the formation, differentiation and maintenance of multicellular organisms. Identifying antibodies that interfere with these functions is contemplated. For example, the fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment. Thus, identification of antibodies against any one or more of such targets (receptor or ligand) can modulate a pathway important in a disease, thereby modulating the disease and ameliorating symptoms thereof.
Such membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like cadherins and integrins, and ligands of any such receptors. Exemplary of such targets include, membrane bound receptors, such as cell surface receptors, including, but are not limited to, a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 4), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-b3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (recetpr for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d'origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epimermal growth factor homology domains receptor), CSF1R (colngly stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-R, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R and EPO-R. Other targets include membrane-bound proteins such as selected from among a cadherin, integrin, CD52 or CD44. Exemplary ligands that can be targets of the screening methods herein, include, but are not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO.
One of skill in the art is familiar with the activity and functions of various target proteins. Thus, screening assays (e.g. binding or functional assays), such as any described above, for example, binding, proliferation or apoptosis assays, can be chosen to screen antibody libraries herein. Table 18A provides a summary of the activities and known function of exemplary target proteins, and exemplary assays that can be employed to screen such target proteins. In another example, antibodies or fragments thereof can be screened for binding affinity for a target protein. Binding can be to a known epitope in a target protein or to a novel epitope, which can be identified as described herein below. Table 18B provides examples of known epitopes for exemplary target proteins recognized by therapeutic antibodies. The sections that follow also further exemplify the activity and function of target proteins, including exemplary screening assays and antibodies identified thereby. It is understood that similar assays can be employed against any target protein of interest to identify antibodies that exhibit high affinity to the target protein and/or otherwise modulate the activity of a target protein.







TABLE 18A







TARGET PROTEINS















Existing


Target
SEQ ID NO
Screening Assay
Functions/Activity
Antibody Drugs





CD20
2011
Apoptosis of B cell lymphoma cell lines
B-cell lymphoma
Rituxan




expressing CD20 on the surface


HER2/Neu
1999
Binding to HER2/Neu receptor and
Breast cancer
Herceptin


receptor

inhibition of cancer cell growth


VEGFA
2012
Binding
Colorectal cancer,
Avastin,





non-small lung cancer.
Lucentis





Neovascular age-





related macular





degeneration


EGFR
2000
Binding
Colorectal, head and
Erbitux,





neck, metastatic cancers
Vectibix


CD52
2013
Binding to CD52 on T and B cells
B-cell chronic
Campath





lymphocytic leukemia


TNFα
2014
Binding to TNFα blocks its interaction
Rheumatoid arthritis
Humira,




with p55 and p75 receptors, reducing the

Remicade




level of inflammation markers, CRP, ESR, IL6


CD25
2015
Binding to CD25 (IL2 receptor) inhibits
Prophylaxis against
Simulect,




lymphocyte activation
allograft rejection
Zenapax


CD3
2016 (delta)
Binding blocks T cell function
Allograft rejection,
Orthoclone



2017 (gamma)

acute renal and



2018 (zeta)

hepatic



2019 (epsilon)


IgE
2020 (constant)
Binding to high affinity IgE reduces
Asthma
Xolair



2021 (FcER1)
activation of mast cells, basophils and



2022 (FcERII)
release of inflammatory mediators


IIb/IIIa
2023 (IIIa)
Binding to the receptor inhibits platelet
Prevention of
ReoPro


integrin
2024 (IIb)
aggregation
cardiac


receptor


ischaemia


EPO-R
2009
EPO binding activates STAT and
Anaemia
Epogen




stimulates erythropoiesis


G-CSF
2025
G-CSF binding stimulates neutrophill
Neutropaenia
Neupogen,


receptor

proliferation

Neulasta


GM-CSF
2026
GM-CSF binding stiumulates proliferation
Leukopaenia
Lukine


receptor

and differentiation of neutrophils,




eosinophils and monocytes
















TABLE 18B







Epitopes













SEQ ID


TARGET
ANTIBODY
AMINO ACID SEQUENCE
NO





DLL4
YW26.82
ECIPHNGCRHGTCSTPWQCTCDEGWGGLFCD (252-282)
2029


 


EpoR
Mab 71 and Mab
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (49-78
2030



73




 


ErbB2
7C2 and 7F3
STQVCTGTDMKLRLPASPETHLDMLRHLYQGC (22-53)
2031


 



4D5
LPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVA
2032




RCPSGVKPDLSYMPIWKFPDEEGACQP (561-625)



 



3H4
VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFG
2033




PEADQCVACAHYKDPPFCVAR (541-599)



 


Her2/Neu
Herceptin
PEADQ(557-561) + DPPF(570-573) +
2034




KFPDEEGACQP(593-603)



 



C24
Q156 + H245 + YF(252, 257) + T268 +
2035



(Pertuzumab)
DVGSCTPLH(285-290, 294-296) + K311



 


EGFR
IMC-C225
R353 + Q384 + QHFVS(408-409, 412, 417-
2036



(Erbitux)
418) + ISK(438, 440, 443) + KIISN(465-





468, 473)



 



EGFR antibody
KK(293-294) + G + NYVVTD(298-303)
2037



[1i8k]




 


VEGFA
Fab12 (Avastin)
[fy(17, 21)] + [yk(45, 48) +
2038




qiMRIkhqGQhiGEM(79-84, 86-94)]



 



G6
[FMYQY(17, 18, 21, 22, 25) + Del(63, 64,
2039




66) + cp(104, 106)] + [k48 + mIHqgQhI(81,





83, 86-91)]



 



B20-4
[kFMDYqRYCH(16-19, 21-23, 25-27) +
2040




cndl(61-63, 66) + nkec(100, 101, 103,





104)] + [e30 + k48 + qmiQi(79, 81, 83,





89, 91)]



 



Fab 12 variant
Y(71) + K(74) + QIMRIK(105-110) +
2041



Y0317
HQGQHIGEM(112-120) + F(43) + Y(47)



 



Fab 12 variant
F(43) + Y(47) + Y(71) + K(74) +
2042



Y0317
QIMRIK(105-110) + HQGQHIGEM(112-120)



 


CD52
Campath
G + TSSPSA(32-37) + D
2043


 


CD20
C2h7
EPANPSEK 168-175
2044


 



C2b8 (Rituxan)
I(164) + NCEPANPSE(166-174)
2045


 



C2b8 (Rituxan)
IYNCEPANP(164-172) + K(175)
2046


 


CD40
5c8
EASS(129-132) + EKGYY(142-146) +
2047




C178 + C218 + SQVSHG(245-250)



 


CD41 (GPIIb)
10E5
RNVGSQ(77-82) + N149 + N158 +
2048




SRLWH(206, 208, 213-215)



 


TPO
TN1
ETKAQ(57-61) + RGALQSLLGTQLPPQ(98,
2049




102-115)









i. Notch and Notch Ligands
a) Notch Proteins
The Notch proteins (Notch1, set forth in SEQ ID NO:2002; Notch 2, set forth in SEQ ID NO:2003; Notch 3, set forth in SEQ ID NO: 2004; and Notch 4 set forth in SEQ ID NO:2005) are single-pass transmembrane receptor proteins that play a crucial role in cell-to-cell communication. Cell surface receptors in the Notch family are expressed on numerous types of cells, including many types of stem cells and undifferentiated progenitor cells, either in the embryo or in self-renewing tissues after birth, see e.g. Artavanis-Tsakonas, et al., (1995) Science 268:225-32. For example, human primary macophages express all Notch receptors, with Notch 3 being selectively increased during macrophage differentiation (see e.g., Fung et al. (2007) Circulation, 115: 2948-2956). Notch 4 also is expressed specifically in endothelial cells and plays a role in angiogenesis. Notch also is expressed on lymphocytes where Notch signaling participates in lymphocyte development, maturation, activation and transformation.
There are five Notch ligands, designated Delta Like-1 (DLL-1), Delta Like-3 (DLL-3), Delta Like-4 (DLL-4), Jagged-1 and Jagged-2. When Notch is activated by a ligand, its intracellular domain is proteolytically cleaved and transported to the nucleus, along with CSL (CBF-1/Su(H)/Lag-1/RBP-JK) transcription factor to activate transcription of downstream effectors. The resulting effector can repress the transcriptional activity of other genes encoding transcription factors for entry into terminal differentiation.
The Notch Signaling Pathway (NSP) is involved in many cellular processes, such as differentiation, cell fate decisions, maintenance of stem cells, cell motility, proliferation, and apoptosis in various cell types during development and tissue homeostasis. Notch signalling is dysregulated in many cancers, and faulty Notch signalling is implicated in many diseases including T-ALL (T-cell acute lymphoblastic leukemia), CADASIL, (Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy), Multiple Sclerosis (MS), Tetralogy of Fallot, Alagille Syndrome, multiple myeloma and other disease states thereby making Notch an important target in protein therapeuics (see e.g. U.S. Pat. No. 6,083,904).
Antibody libraries provided herein can be screened for modulation of an acitivity by assaying for binding to a Notch receptor and/or assaying for a functional activity, for example, proliferation, cell motility or apoptosis of cells known to express a Notch receptor. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to Notch-1. Assays also include signal transduction assays such as by using an RBP-J/CBF-1 luciferase reporter assay (Fung et al. (2007) Circulation, 115: 2948-2956.) Assays can be performed in the presence or absence of a Notch ligand, for example, by coincubation of cells that express DLL4 (see e.g., Fung et al. (2007) Circulation, 115: 2948-2956) or by immobilization of a ligand (see e.g., Lefort et al. (2003) Experimental Hematology, 34:1720-1729). For example, as described in Fung et al., cells can be stably transfected with a construct expressing DLL4, and can be overlaid on human primary macrophages that express Notch and assayed in the presence of antibody library members.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of a Notch receptor (Notch 1, Notch 2, Notch 3 and/or Notch 4) can be used for the treatment or prevention of a disease associated with expression and/or activity of Notch. The antibodies can be used as agonist antibodies or antagonist antibodies in such treatments. For example, agonist antibodies to Notch can be used to inhibit or decrease cancer cell growth or proliferation, such as in the treatment of various cancers including, but not limited to, prostate disorders and leukemia (see e.g., U.S. Pat. No. 6,689,744).
Antagonist antibodies provided herein also can be used in the treatment of a T-cell acute lymphoblastic leukemia; lymphoma; liver disease involving aberrant bascularization; diabetes; ovarian cancer; diseases involving vascular cell fate; rheumatoid arthritis; pancreatic cancer; non-small cell lung carcinoma; plasma cell neoplasmas such as multiple myeloma, plasma cell leukemia and extramedullary plasmacytoma; and neuroblastoma; and in treatment of plasma cell disorders such as multiple myeloma; angiogenesis; cancers such as sarcomas and carcinomas including but not limited to, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, prostate cancer, liver cancer, ovarian cancer, head and neck cancer, skin cancer, brain cancer or blood cancer (see e.g., US20080226621, WO2008/091641, WO2005/054434). Notch signaling is associated in the development of skin, blood vessels and fat, and activated receptor can transform mammary epithelium. Hence, antagonist antibodies to Notch can be used in the treatment of breast tumors (see e.g. US20080206753).
Provided herein are antibodies that modulate the activity of Notch-1 and therefore can be used in the treatment of diseases or conditions associated with expression or activity of Notch-1. Such antibodies include those that have a VH chain and a VL chain encoded by a sequence of nucleotides compiled from germline segments, or any antibodies optimized therefrom. Exemplary of such antibodies are Fab antibodies. The antibodies further can contain a constant region. The antibodies include those that have a binding affinity for Notch-1 that is or is about 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, 1012M, or lower, in particulary any that have a nanomolar or sub-nanomolar binding affinity.
Anti-Notch-1 antibodies provided herein include antibodies having at least one CDR that is a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, a CDRH1 is GYTFTSYYMH (amino acids 26-35 of SEQ ID NO:1512); a CDRH2 is IINPSGGSTSYAQKFQG (amino acids 50-66 of SEQ ID NO:1512); a CDRH3 is EGYSSSWYDYFDY (amino acids 99-111 of SEQ ID NO:1512); a CDRH3 is EYYYGSGSYYNDYFDY (amino acids 99-114 of SEQ ID NO:1509); a CDRL1 is RASQSVSSNLA (amino acids 24-34 of SEQ ID NO:1843); a CDRL1 is RASQSVSSSYLA (amino acids 24-35 of SEQ ID NO:1833); a CDRL1 is RASQSISSWLA (amino acids 24-34 of SEQ ID NO:1841); a CDRL2 is GASTRAT (amino acids 50-56 of SEQ ID NO:1843); a CDRL2 is GASSRAT (amino acids 51-57 of SEQ ID NO:1833); a CDRL2 is DASSLES (amino acids 50-56 of SEQ ID NO:1841); a CDRL3 is QQYNNWPPWT (amino acids 8-98 of SEQ ID NO:1843); a CDRL3 is QQYGSSPPWT (amino acids 90-99 of SEQ ID NO:1833); and a CDRL3 is QQYNSYSPWT (amino acids 89-98 of SEQ ID NO:1841). Also provided herein is a CDR that exhibits 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the above CDRs.
For example, antibodies that modulate an activity of Notch-1 include any that contain a VH chain encoded by a sequence of nucleotides containing germline components compiled from a VH germline segment that is an IGHV1 (e.g. any set forth in any of SEQ ID NOS:1-43); a DH germline segment that is an IGHD6 (e.g. any set forth in SEQ ID NOS: 268-271) or an IGHD3 (e.g. any set forth in SEQ ID NOS: 252-259); and a JH germline segment that is an IGHJ4 (e.g. set forth in SEQ ID NO: 278 or 279). Such antibodies also include any that contain a VL chain encoded by a sequence of nucleotides containing germline components compiled from a V germline segment that is an IGKV1 (set forth in any of SEQ ID NOS: 286-316) or an IGKV3 (e.g. any set forth in any of SEQ ID NOS:332-350); and a J germline segment that is an IGKJ1 (e.g. set forth in SEQ ID NO: 356). Such antibodies also include any that are encoded by a sequence of nucleotides containing germline segments that are variants of any of the above germline segments, for example due to conservative mutations or other mutations, so long as the resulting antibody is a functional and productive antibody and binds to Notch-1 and/or modulates a functional activity.
Exemplary of antibodies against Notch-1 include an antibody where the VH chain is encoded by a sequence of nucleotides compiled from a VH germline segment that is a IGHV1-46 (e.g. an IGHV1-46*01, IGHV1-46*02, or an IGHV1-46*03); a DH germline segment that is an IGHD3-10 (e.g. IGHD3-10*01, IGHD3-10*02) or an IGHD6-13 (e.g. IGHD6-13*01); and a JH germline segment that is an IGHJ4 (e.g. IGHJ4*01, IGHJ4*02, IGHJ4*03). The VL chain is encoded by a sequence of nucleotides compiled from a V germline segment that is an IGKV3-15 (e.g. IGKV3-15*01), IGKV3-20 (e.g. IGKV3-20*01, IGKV3-20*02) or an IGKV1-5 (e.g. IGKV1-5*01, IGKV1-5*02, IGKV1-5*03); and a J germline segment that is a IGKJ1*01. Exemplary antibodies provided herein that modulate an activity of Notch-1 are set forth in Table 18C.







TABLE 18C







Anti-Notch-1 Antibodies











SEQ ID NO

SEQ ID NO












Heavy Chain Germline
nucleo-
Amino
Light Chain Germline
nucleo-
Amino


Segments
tide
acid
Segments
tide
acid





IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-15*01; IGKJ1*01
1427
1843


IGHJ4*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-20*01; IGKJ1*01
1417
1833


IGHJ4*01


IGHV1-46*01; IGHD3-10*01;
1093
1509
IGKV1-5*01; IGKJ1*01
1425
1841


IGHJ4*01









b) DLL4
DLL4 (set forth in SEQ ID NO:2010) is a transmembrane protein ligand for Notch transmembrane receptors. It is expressed widely in a variety of tissues, but its expression is predominantly localized to the vasculature. DLL4 activiates Notch-1 and Notch-4 receptors. It is required for normal vascular development and is expressed on tumor vessels. It is upregulated in blood vessels during tumor angiogenesis and expression is dependent on VEGF signaling. DLL4 expression on angiogenic endothelial cells acts as a negative regulator of vascular growth by acting to allow angiogenesis to productively proceed (Ridgway et al. (2006) Nature, 444:1083; Noguera-Troise et al. (2006) Nature, 444:1032). It acts to inhibit endothelial cell proliferation. Blockage of DLL4, however, is associated with increased angiogenesis characterized by sprouting and branching of blood vessels, but a decrease in vessel function, thereby resulting in decreased tumor growth (Ridgway et al. (2006) Nature, 444:1083; Noguera-Troise et al. (2006) Nature, 444:1032). Thus, DLL4 function is associated with an uncoupling of tumor growth from tumor vascular density. DLL4 also is expressed on activated macrophages exposed to proinflammatory stimuli such as lipopolysaccharide, interleukin-1, Toll-like receptor 4 ligands and other proinflammatory stimuli and it's signaling through the Notch pathway plays a role in inflammatory states characterized by macrophage activation (Fung et al. (2007) Circulation, 115: 2948-2956).
Antibody libraries provided herein can be screened for modulation of an acitivity by assaying for binding to DLL4 and/or assaying for a functional activity. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to DLL4. Assays also include binding assasys to assess the inhibition of DLL4-Notch interaction in the presence of an anti-DLL4 antibody. Such assays can be used to identify anagonist antibodies. This is exemplified in Example 17. Assays for functional activity include those that assess activation of Notch signaling by DLL4 by assaying for signal transduction and/or down stream functional activities such as are described above. Activiation of Notch can be achieved, for example, by coincubation with cells that express DLL4 and/or immobilization of DLL4, and assays performed in the presence of antibody members. In such assays, for example, the effects of antibodies on endothelial cell proliferation (e.g. HUVECs) induced by DLL4 can be assessed (see e.g., Ridgway et al. (2006) Nature, 444:1083). In some examples, antibodies can be used to assess effects on cell differentiation of a cell expressing Notch. The cells can be co-cultured with cells expressing a ligand for Notch, for example, DLL4 or Jag1. To identify antibodies that promote differentiation (i.e. interfere with Notch activation), antibodies can be added to the assay. An exemplary assay is set forth in Example 18.
Hence, the antibodies identified from the libraries provided herein or the antibodies provided herein bind (such as specifically bind) DLL4, and in some embodiments, can modulate one or more aspects of DLL4-associated effects, including but not limited to any one or more of reduction or blocking of Notch receptor activation, reduction or blocking of Notch receptor downstream molecular signaling, disruption or blocking of Notch receptor binding to DLL4, and/or promotion of endothelial cell proliferation, and/or inhibition of endothelial cell differentiation, and/or inhibition of arterial differentiation, and/or inhibition of tumor vascular perfusion, and/or treatment and/or prevention of a tumor, cell proliferative disorder or a cancer; and/or treatment or prevention of a disorder associated with DLL4 expression and/or activity and/or treatment or prevention of a disorder associated with Notch receptor expression and/or activity. In some embodiments, the antibody specifically binds to DLL4. In some embodiments, the antibody specifically binds to the DLL4 extracellular domain (ECD). In some embodiments, an antibody reduces, inhibits, and/or blocks DLL4 activity in vivo and/or in vitro. In some embodiments, the antibody competes for binding with DLL4-ligand (reduces and/or blocks Notch receptor binding to DLL4).
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of DLL4 can be used for the treatment or prevention of disease states associated with expression and/or activity of DLL4, such as increased expression and/or activity or undesired expression and/or activity (see e.g., U.S. Published Application Serial No. US20080175847 and International Published PCT Appl. No. WO2008060705, WO2008091222). Treatment includes neoplastic and non-neoplastic disorders. For example, the antibodies or portions thereof can be used to treat a tumor, a cancer (e.g. colon cancer, lung cancer or breast cancer) and/or a cell proliferative disorder and/or conditions associated with angiogenesis (e.g. intraocular neovascular disease). In particular, the antibodies or portions thereof can be used in combination with anti-VEGF therapies and/or in treatments that are resistant to anti-VEGF treatment.
Angiogenesis is implicated in the pathogenesis of a variety of disorders. These include solid tumors and metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular diseases such as proliferative retinopathies, e.g., diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis. Folkman et al., J. Biol. Chem. 267:10931-34 (1992); Klagsbrun et al., Annu. Rev. Physiol. 53:217-39 (1991); and Garner A., “Vascular diseases,” In: Pathobiology of Ocular Disease. A Dynamic Approach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710.
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment for the growth and metastasis of the tumor. Folkman et al., Nature 339:58 (1989). The neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. A tumor usually begins as a single aberrant cell which can proliferate only to a size of a few cubic millimeters due to the distance from available capillary beds, and it can stay ‘dormant’ without further growth and dissemination for a long period of time. Some tumor cells then switch to the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow for continued growth of the primary tumor, but also for the dissemination and recolonization of metastatic tumor cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors. Weidner et al., N. Engl. J. Med. 324:1-6 (1991); Horak et al., Lancet 340:1120-24 (1992); Macchiarini et al., Lancet 340:145-46 (1992). The precise mechanisms that control the angiogenic switch is not well understood, but it is believed that neovascularization of tumor mass results from the net balance of a multitude of angiogenesis stimulators and inhibitors (Folkman, Nat. Med. 1(1):27-31 (1995)).
In addition, antibodies or portions thereof can be used to treat non-neoplastic disorders including, but not limited to, undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, edema from myocardial infarction, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal graft rejection, retinal/choroidal neovascularization, neovascularization of the angle (rubeosis), ocular neovascular disease, vascular restenosis, arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, acute lung injury/ARDS, sepsis, primary pulmonary hypertension, malignant pulmonary effusions, cerebral edema (e.g., associated with acute stroke/closed head injury/trauma), synovial inflammation, pannus formation in RA, myositis ossificans, hypertropic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartment syndrome, burns, bowel disease), uterine fibroids, premature labor, chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, nephrotic syndrome, undesired or aberrant tissue mass growth (non-cancer), obesity, adipose tissue mass growth, hemophilic joints, hypertrophic scars, inhibition of hair growth, Osier-Weber syndrome, pyogenic granuloma retrolental fibroplasias, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.
Provided herein are antibodies that modulate the activity of DLL4 and therefore can be used in the treatment of diseases or conditions associated with expression or activity of DLL4. Such antibodies include those that have a VH chain and a VL chain encoded by a sequence of nucleotides compiled from germline segments, or any antibodies optimized therefrom. Exemplary of such antibodies are Fab antibodies. The antibodies further can contain a constant region. The antibodies include those that have a binding affinity for DLL4 that is or is about 10−6 M, 10−7M, 10−8 M, 10−9M, 10−10M, 10−11M, 10−12 M or lower, in particulary any that have a nanomolar or sub-nanomolar binding affinity.
Anti-DLL4 antibodies provided herein include antibodies having at least one CDR that is a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, a CDRH1 is GYTFTSYYMH (amino acids 26-35 of SEQ ID NO:1513); a CDRH1 is GYSFTSYWIG (amino acids 26-35 of SEQ ID NO:1803); a CDRH1 is GDSVSSNSAAWN (amino acids 26-37 of SEQ ID NO:1812); a CDRH1 is GGSFSGYYWS (amino acids 26-35 of SEQ ID NO:1779); a CDRH1 is GYTFTSYAMH (amino acids 26-35 of SEQ ID NO:1494); a CDRH1 is GYTFTSYDIN (amino acids 26-35 of SEQ ID NO:1537); a CDRH1 is GGSISSGGYYWS (amino acids 26-37 of SEQ ID NO:1761); a CDRH2 is IINPSGGSTSYAQKFQG (amino acids 50-66 of SEQ ID NO:1513); a CDRH2 is IIYPGDSDTRYSPSFQG (amino acids 50-66 of SEQ ID NO:1803); a CDRH2 is RTYYRSKWYNDYAVSVKS (amino acids 52-69 of SEQ ID NO:1812); a CDRH2 is EINHSGSTNYNPSLKS (amino acids 50-65 of SEQ ID NO:1779); a CDRH2 is WSNAGNGNTKYSQEFQG (amino acids 50-66 of SEQ ID NO:1494); a CDRH2 is WMNPNSGNTGYAQKFQG (amino acids 50-66 of SEQ ID NO:1537); a CDRH2 is (amino acids 52-67 of SEQ ID NO:1761); a CDRH3 is EEYSSSSAEYKQH (amino acids 99-111 of SEQ ID NO:1513); a CDRH3 is RGYSYGYDYFDY (amino acids 99-110 of SEQ ID NO:1803); a CDRH3 is EYYDFWSGYYTDYFD (amino acids 102-117 of SEQ ID NO:1812); a CDRH3 is EGYSSSWYDYFDY (amino acids 99-111 of SEQ ID NO:1512); a CDRH3 is ANWGDYFDY (amino acids 89-106 of SEQ ID NO:1779); a CDRH3 is ANWGYWYFDL (amino acids 99-108 of SEQ ID NO:1514); a CDRH3 is DDYGGNSDYFDY (amino acids 99-110 of SEQ ID NO:1494); a CDRH3 is EGYCSGGSCYSYWYFDL (amino acids 99-115 of SEQ ID NO:1508); a CDRH3 is EYYYGSGSYYNDYFDY (amino acids 99-114 of SEQ ID NO:1509); a CDRH3 is GGYCSSTSCYADYYYYYGMDV (amino acids 99-119 of SEQ ID NO:1537); a CDRH3 is EGYCSGGSCYSYWYFDL (amino acids 100-116 of SEQ ID NO:1761); a CDRL1 is RASQSVSSYLA (amino acids 24-34 of SEQ ID NO:1850); a CDRL1 is GLSSGSVSTSYYPS (amino acids 23-36 of SEQ ID NO:1881); a CDRL1 is TLRSGINLGSYRIF (amino acids 23-36 of SEQ ID NO:1884); a CDRL1 is RASQSVSSNLA (amino acids 24-34 of SEQ ID NO:1843); a CDRL1 isRASQGISSWLA (amino acids 24-34 of SEQ ID NO:1849); a CDRL1 is RASQSVSSSYLA (amino acids 24-35 of SEQ ID NO:1833); a CDRL1 is RASQSISSWLA (amino acids 24-34 of SEQ ID NO:1841); a CDRL1 is RSSQSLLDSDDGNTYLD (amino acids 24-40 of SEQ ID NO:1853); a CDRL1 is TGTSSDVGGYNYVS (amino acids 23-36 of SEQ ID NO:1864); a CDRL1 is TLSSDLSVGGKNMF (amino acids 23-36 of SEQ ID NO:1886); a CDRL2 is DASNRAT (amino acids 50-56 of SEQ ID NO:1850); a CDRL2 is STNTRSS (amino acids 52-58 of SEQ ID NO:1881); a CDRL2 is YYSDSSK (amino acids 52-58 of SEQ ID NO:1884); a CDRL2 is GASTRAT (amino acids 50-56 of SEQ ID NO:1843); a CDRL2 is AASSLQS (amino acids 50-56 of SEQ ID NO:1849); a CDRL2 is GASSRAT (amino acids 51-57 of SEQ ID NO:1833); a CDRL2 is DASSLES (amino acids 50-56 of SEQ ID NO:1841); a CDRL2 is TLSYRAS (amino acids 56-62 of SEQ ID NO:1853); a CDRL2 is EVSNRPS (amino acids 52-58 of SEQ ID NO:1864); a CDRL2 is HYSDSDK (amino acids 52-58 of SEQ ID NO:1886); a CDRL3 is QQRSNWPPWT (amino acids 89-98 of SEQ ID NO:1850); a CDRL3 is VLYMGSGISYV (amino acids 91-101 of SEQ ID NO:1881); a CDRL3 is MIWHSSASFV (amino acids 97-106 of SEQ ID NO:1884); a CDRL3 is QQYNNWPPWT (amino acids 89-98 of SEQ ID NO:1843); a CDRL3 is QQANSFPPWT (amino acids 89-98 of SEQ ID NO:1849); a CDRL3 is QQYGSSPPWT (amino acids 90-99 of SEQ ID NO:1833); a CDRL3 is QQYNSYSPWT (amino acids 89-98 of SEQ ID NO:1841); a CDRL3 is MQRIEFPSWT (amino acids 95-104 of SEQ ID NO:1853); a CDRL3 is SSYTSSSTLFV (amino acids 91-101 of SEQ ID NO:1864); and a CDRL3 is QVYESSANFV (amino acids 89-98 of SEQ ID NO:1886). Also provided herein is a CDR that exhibits 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the above CDRs.
For example, antibodies that modulate an activity of DLL4 include any that contain a VH chain encoded by a sequence of nucleotides containing germline components compiled from a VH germline segment that is an IGHV1 (e.g. any set forth in any of SEQ ID NOS:10-43), an IGHV4 (e.g. any set forth in SEQ ID NOS: 153-224), an IGHV5 (e.g. any set forth in SEQ ID NOS: 225-232) or an IGHV6 (e.g., any set forth in any of SEQ ID NOS: 233 or 234); a DH germline segment that is an IGHD6 (e.g. any set forth in SEQ ID NOS: 268-271), an IGHD5 (e.g. any set forth in any of SEQ ID NOS: 264-267); an IGHD4 (e.g. any set forth in SEQ ID NOS: 260-263); an IGHD2 (e.g. any set forth in SEQ ID NOS: 244-251), an IGHD3 (e.g. any set forth in any of SEQ ID NOS: 252-259) an IGHD6 (e.g. any set forth in SEQ ID NO: 268-271), or an IGHD7 (e.g. set forth in SEQ ID NO:272); and a JH germline segment that is an IGHJ1 (e.g., set forth in SEQ ID NO:273), an IGHJ2 (set forth in SEQ ID NO:274), an IGHJ4 (e.g. any set forth in any of SEQ ID NOS: 277-279), or an IGHJ6 (e.g. any set forth in SEQ ID NOS: 282-285). Such antibodies also include any that contain a VL chain encoded by a sequence of nucleotides containing germline components compiled from a V germline segment that is an IGKV1 (e.g. any set forth in any of SEQ ID NOS: 286-316), an IGKV2 (e.g. any set forth in SEQ ID NOS: 317-331), or an IGKV3 (e.g. any set forth in any of SEQ ID NOS:332-350) and a J germline segment that is an IGKJ1 (e.g. set forth in SEQ ID NO: 356); or from a V germline segment that is an IGLV2 (e.g. any set forth in any of SEQ ID NOS:380-399), IGLV8 (e.g. any set forth in any of SEQ ID NOS: 436-438), IGLV11 (e.g. any set forth in any of SEQ ID NO: 379), or a IGLV5 (e.g. any set forth in any of SEQ ID NOS: 424-431) and a J germline segment that is a IGLJ1 (e.g. set forth in SEQ ID NO:442) or an IGLJ4 (e.g. set forth in SEQ ID NO:446). Such antibodies also include any that are encoded by a sequence of nucleotides containing germline segments that are variants of any of the above germline segments, for example due to conservative mutations or other nucleotide mutations, so long as the resulting antibody is a functional and productive antibody and binds to DLL4 and/or modulates a functional activity.
Exemplary of antibodies against DLL4 include an antibody where the VH chain is encoded by a sequence of nucleotides compiled from a VH germline segment that is an IGHV1-3 (e.g. IGHV1-3*01, IGHV1-3*02), an IGHV1-8*01, an IGHV1-46 (e.g. an IGHV1-46*01, IGHV1-46*02 or an IGHV1-46*03), an IGHV4-31 (e.g. IGHV4-31*01, IGHV4-31*02, IGHV4-31*03, IGHV4-31*04, IGHV4-31*05, IGHV4-31*06, IGHV4-31*07, IGHV4-31*08, IGHV4-31*09, IGHV4-31*10), an IGHV4-34 (e.g. IGHV4-34*01, IGHV4-34*02, IGHV4-34*03, IGHV4-34*04, IGHV4-34*05, IGHV4-34*06, IGHV4-34*07, IGHV4-34*08, IGHV4-34*09, IGHV4-34*10, IGHV4-34*11, IGHV4-34*12, IGHV4-34*13), an IGHV5-51 (e.g., an IGHV1-5-51*01, IGHV1-5-51*02, IGHV1-5-51*03, IGHV1-5-51*04 or IGHV1-5-51*05) or is an IGHV6-1 (e.g. an IGHV6-1*01 or an IGHV6-1*02); a DH germline segment that is an IGHD2-2 (e.g. IGHD2-2*01, IGHD2-2*02), an IGHD2-15*01, an IGHD4-23*01, an IGHV6-6 (e.g. IGHD6-6*01), an IGHD6-13*01, an IGHD5-18 (e.g. an IGHD5-18*01), an IGHD3-3 (e.g. an IGHD3-3*01 or IGHD3-3*02), an IGHD3-10 (e.g. IGHD3-10*01, IGHD3-10*02), or an IGHD7-27*01; and a JH germline segment that is a IGHJ1*01, IGHJ2*01, IGHJ4*01, IGHJ4*02, IGHJ4*03, or an IGHJ6 (e.g. IGHJ6*01, IGHJ6*02, IGHJ6*03, IGHJ6*04). The VL chain is encoded by a sequence of nucleotides compiled from a V germline segment that is an IGKV1-5 (e.g. IGKV1-5*01, IGKV1-5*02, IGKV1-5*03), an IGKV1-12 (e.g IGKV1-12*01, IGKV1-12*02), an IGKV2-D-40*01, an IGKV3-11 (e.g. IGKV3-11*01 or IGKV3-11*02), an IGKV3-15*01, an IGKV3-20 (e.g. IGKV3-20*01, IGKV3-20*02) and a J germline segment that is a IGKJ1*01; or is compiled from a V germline segment that is an IGLV2-14 (e.g. IGLV2-14*01, IGLV2-14*02, IGLV2-14*03, IGLV2-14*04), an IGLV8-61 (e.g. IGLV8-61*01, IGLV8-61*02 or IGLV8-61*03), an IGLV5 (e.g. IGLV5-48*01), or an IGLV11-55*01 and a J germline segment that is a IGLJ1*01 or IGLJ4*01. Exemplary antibodies provided herein that modulate an activity of DLL4 are set forth in Table 18D.







TABLE 18D







Anti-DLL4 Antibodies











SEQ ID NO

SEQ ID NO












Heavy Chain Germline
nucleo-
Amino
Light Chain Germline
nucleo-
Amino


Segments
tide
acid
Segments
tide
acid





IGHV1-46*01; IGHD6-6*01;
1097
1513
IGKV3-11*01; IGKJ1*01
1434
1850


IGHJ1*01


IGHV5-51*03; IGHD5-18*01>3;
1387
1803
IGLV8-61*01; IGLJ1*01
1465
1881


IGHJ4*01


IGHV6-1*01; IGHD3-3*01;
1396
1812
IGLV5-48*01; IGLJ4*01
1468
1884


IGHJ4*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-15*01; IGKJ1*01
1427
1843


IGHJ4*01


IGHV4-34*01; IGHD7-27*01;
1363
1779
IGKV1-12*01; IGKJ1*01
1433
1849


IGHJ4*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-20*01; IGKJ1*01
1417
1833


IGHJ4*01


IGHV1-3*02; IGHD4-23*01;
1078
1494
IGKV1-5*01; IGKJ1*01
1425
1841


IGHJ4*01


IGHV1-46*01; IGHD2-15*01;
1092
1508
IGKV1-5*01; IGKJ1*01
1425
1841


IGHJ2*01


IGHV1-46*01; IGHD3-10*01;
1093
1509
IGKV1-5*01; IGKJ1*01
1425
1841


IGHJ4*01


IGHV1-8*01; IGHD2-2*01;
1121
1537
IGKV1-5*01; IGKJ1*01
1425
1841


IGHJ6*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV2D-40*01; IGKJ1*01
1437
1853


IGHJ4*01


IGHV4-34*01; IGHD7-27*01;
1363
1779
IGLV2-14*01; IGLJ4*01
1448
1864


IGHJ4*01


IGHV4-31*02; IGHD2-15*01;
1345
1761
IGLV2-14*01; IGLJ4*01
1448
1864


IGHJ2*01


IGHV4-34*01; IGHD7-27*01;
1363
1779
IGLV11-55*01; IGLJ4*01
1470
1886


IGHJ4*01









Anti-DLL4 antibodies provided herein include antibodies that are optimized compared to any of the identified anti-DLL4 germline Hits. The antibodies include one or more mutations in the VH chain and/or one or more mutations in the VL chain compared to an identified germline Hit. For example, the antibodies can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid replacements compared to the corresponding antibody germline Hit. The mutations can be in the VH chain, for example, in any one or more of the amino acid residues of a VH, DH or JH region. Alternatively, or in addition, the mutations can be in the VL chain, for example, in any one or more of the amino acid residues of the VL or JL region. Optimized antibodies containing one or more mutations exhibit improved activity compared to the parent antibody (e.g. germline Hit not containing the modifications). The antibodies are optimized to exhibit an improved functional activity, either agonistic or antagonistic, against the DLL4 target protein. In other examples, the antibodies are optimized to exhibit an improved binding affinity for DLL4. Generally, an activity or binding affinity is increased by at or about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more compared to an activity or binding affininity of the parent antibody (e.g. germline Hit not containing the modification(s)). For example, as described in the Examples, optimized anti-DLL4 antibodies provided herein exhibit a binding affinity that is improved by at least 100-fold compared to the parent antibody. Such antibodies exhibit a nanomolar binding affinity.
For example, provided herein are optimized anti-DLL4 antibodies that contain one or more mutations in the VH chain of an anti-DLL4 Hit, for example, any set forth in any of SEQ ID NOS: 1494, 1508-1509, 1512-1513, 1537, 1761, 1779, 1803 and 1812. In one example, the one or more mutations include mutations in the DH region. For example, an anti-DLL4 antibody provided herein can include a VH chain containing a germline segment compiled from a variant of a IGHD5-18*01 that encodes a DH region that contains a mutation(s) at position G1 and/or G5 corresponding to amino acid residues set forth in SEQ ID NO:3736 (also corresponding to amino acid residues G100 and/or G104 set forth in the VH chain of the anti-DLL4 Hit set forth in SEQ ID NO: 1803). The mutation can be to any other amino acid residue, in particular, the mutation is a lysine (K), Arginine (R), threonine (T). Exemplary of such mutations are G1K, G1R, and/or G5T corresponding to amino acid replacements in the DH region set forth in SEQ ID NO:3736 (also corresponding to amino acid replacements G100K, G100R, and/or G104T in the VH chain of the anti-DLL4 Hit set forth in SEQ ID NO:1803). In another example, an anti-DLL4 antibody provided herein can include a VH chain containing germline segments compiled from a variant of IGHD6-6*01 that encodes a DH region that contains a mutations(s) at positions S3, S4 and/or S5 corresponding to amino acid residues set forth in SEQ ID NO:3737 (also corresponding to amino acid residues S102, S103 and/or S104 in the VH chain of the anti-DLL4 Hit set forth in SEQ ID NO: 1513. The mutation can be to any other amino acid residue, in particular, the mutation is a alanine (A), proline (P) or phenylalanine (F). Exemplary of such mutations are S3A, S4P, S5F and/or S5A corresponding to amino acid replacements in the DH region set forth in SEQ ID NO:3737 (also corresponding to amino acid replacements S102A, S103P, S104F and S104A in the VH chain of the anti-DLL4 Hit set forth in SEQ ID NO:1513. Table 18E lists exemplary anti-DLL4 antibody variants containing one or more mutations in the DH region of the VH chain of anti-DLL4 Hits.
Also provided herein are optimized anti-DLL4 antibodies containing one or more mutations in the JH region of the VH chain of an anti-DLL4 hit. For example, an anti-DLL4 antibody provided herein can include a VH chain containing a germline segment compiled from a variant of an IGHJ1*01 that encodes a JH region that contains a mutation at position H6 corresponding to amino acid residue set forth in SEQ ID NO:3738 (also corresponding to amino acid residue H111 in the VH chain of the anti-DLL4 Hit set forth in SEQ ID NO:1513). The mutation can be to any other amino acid residue, in particular, the mutation is a phenylalanine (F) or a tyrosine (Y). Exemplary of such mutations are H6F and H6Y corresponding to amino acid replacements in the JH region set forth in SEQ ID NO:3738 (also corresponding to amino acid replacements H111F and H111Y in the JH region of the VH chain of the anti-DLL4 Hit set forth in SEQ ID NO:1513). Table 18E lists exemplary anti-DLL4 antibody variants containing one or more mutations in the JH region of the VH chain of anti-DLL4 Hits.
Optimized anti-DLL4 antibodies provided herein also can contain one or more amino acid mutations in the VL chain. For example, provided herein are optimized anti-DLL4 antibodies containing one or more mutation in the V region of the VL chain of an anti-DLL4 hit. For example, an anti-DLL4 antibody provided herein can include a VL chain containing a germline segment compiled from a variant of an IGKV3-11 (e.g. IGKV3-11*01 or IGKV3-11*02) that encodes a V region that contains a mutation at position S28, S30 and/or S31 corresponding to amino acid residues set forth in SEQ ID NO:3739 (also corresponding to amino acid resdues S28, S30 and/or S31 in the VL chain of the anti-DLL4 Hit set forth in SEQ ID NO:1850). The mutation can be to any other amino acid residue, in particular, the mutation is a proline (P), asparagine (N), or lysine (K). Exemplary of such mutations are S28P, S30N and/or S31K corresponding to amino acid replacements in the V region set forth in SEQ ID NO:3739 (also corresponding to amino acid replacements S28P, S30N and/or S31K in the V region of the VL chain of the anti-DLL4 Hit set forth in SEQ ID NO:1850). Table 18E lists exemplary anti-DLL4 antibody variants containing one or more mutations in the V region of the VL chain of anti-DLL4 Hits.







TABLE 18E







Anti-DLL4 antibody variants










Parent anti-
SEQ ID NO
Parent anti
SEQ ID NO














DLL4 VH chain
Muta-
Nucleo-
Amino
DLL4 VL chain
Muta-
Nucleo-
Amino


Hit
tion(s)
tide
acid
Hit
tion(s)
tide
acid





IGHV5-51*03;
G100K
3741
3720
IGLV8-61*01;
n/a
1465
1881


IGHD5-18*01>3;



IGLJ1*01


IGHJ4*01


IGHV5-51*03;
G100R
3742
3721
IGLV8-61*01;
n/a
1465
1881


IGHD5-18*01>3;



IGLJ1*01


IGHJ4*01


IGHV5-51*03;
G104T
3745
3724
IGLV8-61*01;
n/a
1465
1881


IGHD5-18*01>3;



IGLJ1*01


IGHJ4*01


IGHV5-51*03;
G100K/
3749
3728
IGLV8-61*01;
n/a
1465
1881


IGHD5-18*01>3;
G104T


IGLJ1*01


IGHJ4*01


IGHV1-46*01;
S104F
3743
3722
IGKV3-11*01;
n/a
1434
1850


IGHD6-6*01;



IGKJ1*01


IGHJ1*01


IGHV1-46*01;
S104A
3744
3723
IGKV3-11*01;
n/a
1434
1850


IGHD6-6*01;



IGKJ1*01


IGHJ1*01


IGHV1-46*01;
S103P
3746
3725
IGKV3-11*01;
n/a
1434
1850


IGHD6-6*01;



IGKJ1*01


IGHJ1*01


IGHV1-46*01;
S102A
3747
3726
IGKV3-11*01;
n/a
1434
1850


IGHD6-6*01;



IGKJ1*01


IGHJ1*01


IGHV1-46*01;
S102A/
3748
3727
IGKV3-11*01;
n/a
1434
1850


IGHD6-6*01;
S103P/


IGKJ1*01


IGHJ1*01
S104F


IGHV1-46*01;
S102A/
3750
3729
IGKV3-11*01;
n/a
1434
1850


IGHD6-6*01;
S103P/


IGKJ1*01


IGHJ1*01
S104F/



H111F


IGHV1-46*01;
S102A/
3751
3730
IGKV3-11*01;
n/a
1434
1850


IGHD6-6*01;
S103P/


IGKJ1*01


IGHJ1*01
S104F/



H111Y


IGHV1-46*01;
S102A/
3752
3731
IGKV3-11*01;
n/a
1434
1850


IGHD6-6*01;
S103P/


IGKJ1*01


IGHJ1*01
S104Y/



H111Y


IGHV1-46*01;
S102A/
3748
3727
IGKV3-11*01;
S28P
3753
3735


IGHD6-6*01;
S103P/


IGKJ1*01


IGHJ1*01
S104F


IGHV1-46*01;
S102A/
3748
3727
IGKV3-11*01;
S30N
3754
3733


IGHD6-6*01;
S103P/


IGKJ1*01


IGHJ1*01
S104F


IGHV1-46*01;
S102A/
3748
3727
IGKV3-11*01;
S31K
3755
3734


IGHD6-6*01;
S103P/


IGKJ1*01


IGHJ1*01
S104F









ii. ErbB Family
Group I receptor tyrosine kinases, including EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4), are widely expressed in epithelial, mesenchymal, and neuronal tissues and play fundamental roles in proliferation and differentiation. They are activated by a family of ligands that variously bind to the receptors. For example, epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha) and amphiregulin bind to ErbB1, but not to other receptors. The neuregulins bind to ErbB3 and ErbB4. Finally, b-cellulin (BTC), heparin-binding EGF and epiregulin bind to ErbB1 and ErbB4. ErbB2 has no characterized ligand, but can be activated by homodimerization in trans by heterodimerization with another ErbB family member.
a) Epidermal Growth Factor Receptor (EGFR)
The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans; set forth in SEQ ID NO:2000) is a cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. Upon binding by the ligand epidermal growth factor (EGF), EGFR dimerizes stimulating its intrinsic intracellular protein-tyrosine kinase activity. This autophosphorylation elicits downstream activation and signaling by several other proteins that associate with the phosphorylated tyrosines through their own phosphotyrosine-binding SH2 domains. These downstream signaling proteins initiate several signal transduction cascades, including MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation. Such proteins modulate phenotypes such as cell migration, adhesion, and proliferation. Therefore, mutations affecting EGFR expression or activity can result in cancer
Upregulation of EGFR is associated with poor cancer prognosis. ERBITUX® (cetuximab) is a chimeric monoclonal antibody approved for the treatment of colorectal and/or head and neck cancers. ERBITUX® binds the EGFR and thereby prevents intracellular signaling associated with DNA synthesis and cell proliferation. VECTIBIX® (panitumumab) is a fully human monoclonal antibody approved for the treatment of EGFR-expressing, metastatic colorectal carcinoma. Both ERBITUX® and VECTIBIX® can be used alone or in conjuction with a chemotherapeutic agent.
Antibody libraries provided herein can be screened for modulation of an acitivity by assaying for binding to EGFR and/or assaying for a functional activity, for example, signal transduction, cell migration, adhesion, and proliferation. Functional assays can be performed in the presence or absence of the EGF ligand. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to EGFR.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of EGFR can be used for the treatment or prevention of disease states associated with expression and/or activity of EGFR. For example, the antibodies or portions thereof can be used in the treatment of cancers including, but not limited to, glioblastoma, head and neck cancer, pancreatic cancer, colorectal cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retina cancer, skin cancer, liver cancer, genital-urinary cancer, bladder cancer, or a lung cancer such as a lun adenocarcinoma, lung squamous cell carcinoma or non-small cell lung cancer.
Provided herein are antibodies that modulate the activity of EGFR and therefore can be used in the treatment of diseases or conditions associated with expression or activity of EGFR. Such antibodies include those that have a VH chain and a VL chain encoded by a sequence of nucleotides compiled from germline segments, or any antibodies optimized therefrom. Exemplary of such antibodies are Fab antibodies. The antibodies further can contain a constant region. The antibodies include those that have a binding affinity for EGFR that is or is about 10−6 M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, 10−12 M or lower, in particular any that have a nanomolar or sub-nanomolar binding affinity.
Anti-EGFR antibodies provided herein include antibodies having at least one CDR that is a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, a CDRH1 is GYTFTSYYMH (amino acids 26-35 of SEQ ID NO:1508); a CDRH2 is IINPSGGSTSYAQKFQG (amino acids 50-66 of SEQ ID NO:1508); a CDRH3 is EGYCSGGSCYSYWYFDL (amino acids 99-115 of SEQ ID NO:1508); a CDRH3 is EGYSSSWYDYFDY (amino acids 99-111 of SEQ ID NO:1512); a CDRL1 is RASQSVSSNLA (amino acids 24-34 of SEQ ID NO:1843); a CDRL1 is RASQSISSWLA (amino acids 24-34 of SEQ ID NO:1841); a CDRL2 is GASTRAT (amino acids 50-56 of SEQ ID NO:1843); a CDRL2 is DASSLES (amino acids 50-56 of SEQ ID NO:1841); a CDRL3 is QQYNNWPPWT (amino acids 89-98 of SEQ ID NO:1843); and a CDRL3 is QQYNSYSPWT (amino acids 89-98 of SEQ ID NO:1841). Also provided herein is a CDR that exhibits 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the above CDRs.
For example, antibodies that modulate an activity of EGFR include any that contain a VH chain encoded by a sequence of nucleotides containing germline components compiled from a VH germline segment that is an IGHV1 (e.g. any set forth in any of SEQ ID NOS:1-43); a DH germline segment that is an IGHD6 (e.g. any set forth in SEQ ID NOS: 268-271) or an IGHD2 (e.g. any set forth in SEQ ID NOS: 244-251); and a JH germline segment that is an IGHJ2 (e.g., set forth in SEQ ID NO:274) or an IGHJ4 (e.g. set forth in SEQ ID NO: 278 or 279). Such antibodies also include any that contain a VL chain encoded by a sequence of nucleotides containing germline components compiled from a V germline segment that is an IGKV1 (set forth in any of SEQ ID NOS: 286-316) or an IGKV3 (e.g. any set forth in any of SEQ ID NOS:332-350); and a J germline segment that is an IGKJ1 (e.g. set forth in SEQ ID NO: 356). Such antibodies also include any that are encoded by a sequence of nucleotides containing germline segments that are variants of any of the above germline segments, for example due to conservative mutations or other mutations, so long as the resulting antibody is a functional and productive antibody and binds to EGFR and/or modulates a functional activity.
Exemplary of antibodies against EGFR include an antibody where the VH chain is encoded by a sequence of nucleotides compiled from a VH germline segment that is a IGHV1-46 (e.g. an IGHV1-46*01, IGHV1-46*01, or an IGHV1-46*03); a DH germline segment that is an IGHD2-15 (e.g. IGHD2-15*01) or an IGHD6-13 (e.g. IGHD6-13*01); and a JH germline segment that is an IGHJ2*01 or an IGHJ4 (e.g. IGHJ4*01, IGHJ4*02, IGHJ4*03). The VL chain is encoded by a sequence of nucleotides compiled from a V germline segment that is an IGKV1-5 (e.g. IGKV1-5*01, IGKV1-5*02, IGKV1-5*03) or an IGKV3-15(e.g. IGHV3-15*01); and a J germline segment that is a IGKJ1*01. Exemplary antibodies provided herein that modulate an activity of EGFR are set forth in Table 18F.







TABLE 18F







Anti-EGFR Antibodies











SEQ ID NO

SEQ ID NO












Heavy Chain Germline
nucleo-
Amino
Light Chain Germline
nucleo-
Amino


Segments
tide
acid
Segments
tide
acid





IGHV1-46*01; IGHD2-15*01;
1092
1508
IGKV3-15*01; IGKJ1*01
1427
1841


IGHJ2*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-15*01; IGKJ1*01
1427
1841


IGHJ4*01


IGHV1-46*01; IGHD2-15*01;
1092
1508
IGKV1-5*01; IGKJ1*01
1425
1843


IGHJ2*01









b) Human Epidermal Growth Factor Receptor 2 (HER2/neu)
HER2/neu (ErbB-2; set forth in SEQ ID NO: 1999) is a cell membrane surface-bound receptor tyrosine kinase normally involved in the signal transduction pathways leading to cell growth and differentiation. ErbB-2 is thought to be an orphan receptor, with none of the EGF family of ligands able to activate it. However, ErbB receptors dimerize on ligand binding, and ErbB-2 is the preferential dimerization partner of other members of the ErbB family. ErbB2 activation leads to kinase activation and cell proliferation. ErbB-2 is notable for its role in the pathogenesis of breast cancer and therefore as a target for treatment. In fact, ErbB-2 protein overexpression is observed in 25-30% of primary breast cancers. HERCEPTIN® (trastuzumab) is a recombinant DNA-derived humanized monoclonal antibody, used to treat breast cancer, that selectively binds to the extracellular domain of ErbB-2 Thus, ErbB-2is an attractive target for additional protein therapeutics. See e.g. Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89:4285-4289; and U.S. Pat. No. 5,725,856.
Antibody libraries provided herein can be screened for modulation of an acitivity by assaying for binding to ErbB-2 and/or assaying for a functional activity, for example, signal transduction, cell migration, adhesion, and proliferation. For example, cells that are known to express ErbB-2 can be assessed for proliferation in the presence or absence of an antibody or portion thereof. Alternatively, reporter system assays can be constructed, whereby the expression of a reporter protein, such as luciferase, is dependent on the activiation of ErbB2 (see e.g. Ueda et al. (2004) J Biol. Chem., 279:24505-24513). Assays can be performed in the presence of EGF, TGF or other ErbB lignads that are ligands for ErbB-2 binding partners. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to ErbB-2.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of ErbB-2 can be used for the treatment or prevention of disease states associated with expression and/or activity of ErbB-2. For example, the antibodies or portions thereof can be used in the treatment of proliferative diseases such as cancers, including, but not limited to, breast cancer, ovarian cancer, colon cancer, lung cancer and prostate cancer.
Provided herein are antibodies that modulate the activity of ErbB-2 and therefore can be used in the treatment of diseases or conditions associated with expression or activity of ErbB-2. Such antibodies include those that have a VH chain and a VL chain encoded by a sequence of nucleotides compiled from germline segments, or any antibodies optimized therefrom. Exemplary of such antibodies are Fab antibodies. The antibodies further can contain a constant region. The antibodies include those that have a binding affinity for ErbB-2 that is or is about 10−6 M, 10−7 M, 10−8M, 10−9 M, 10−10 M, 10−11 M, 10−12 M or lower, in particular any that have a nanomolar or sub-nanomolar binding affinity.
Anti-ErbB-2 antibodies provided herein include antibodies having at least one CDR that is a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, a CDRH1 is GGSISSGGYYWS (amino acids 26-37 of SEQ ID NO:1760); a CDRH1 is GYTFTSYYMH (amino acids 26-35 of SEQ ID NO:1512); a CDRH1 is GFSLSTSGVGVG (amino acids 26-37 of SEQ ID NO:1559); a CDRH1 is GGTFSSYAIS (amino acids 26-35 of SEQ ID NO:1522); a CDRH2 is YIYYSGSTYYNPSLKS (amino acids 52-67 of SEQ ID NO:1760); a CDRH2 is IINPSGGSTSYAQKFQG (amino acids 50-66 of SEQ ID NO:1512); a CDRH2 is LIYWNDDKRYSPSLKS (amino acids 52-67 of SEQ ID NO:1559); a CDRH2 is GIIPIFGTANYAQKFQG (amino acids 50-66 of SEQ ID NO:1522); a CDRH3 is EGYSSSWYDYFDY (amino acids 100-112 of SEQ ID NO:1760); a CDRH3 is GYSGSYYYWYFDL (amino acids 99-111 of SEQ ID NO:1512); a CDRH3 is EEYSSSSAEYKQH (amino acids 99-111 of SEQ ID NO:1513); a CDRH3 is RPNWGYWYFDL (amino acids 100-110 of SEQ ID NO:1559); a CDRH3 is GYNWNDDYYYYYGMDV (amino acids 99-114 of SEQ ID NO:1522); a CDRL1 is RASQSVSSSYLA (amino acids 24-35 of SEQ ID NO:1833); a CDRL1 is KSSQSVLYSSNNKNYLA (amino acids 24-40 of SEQ ID NO:1838); a CDRL1 is RASQSVSSNLA (amino acids 24-34 of SEQ ID NO:1843); a CDRL1 is RSSQSLVYSDGNTYLN (amino acids 24-39 of SEQ ID NO:1828); a CDRL2 is GASSRAT (amino acids 51-57 of SEQ ID NO:1833); a CDRL2 is WASTRES (amino acids 56-62 of SEQ ID NO:1838); a CDRL2 is GASTRAT (amino acids 50-56 of SEQ ID NO:1843); a CDRL2 is KVSNDRS (amino acids 55-61 of SEQ ID NO:1828); a CDRL3 is QQYGSSPPWT (amino acids 90-99 of SEQ ID NO:1833); a CDRL3 is QQYYSTPPWT (amino acids 95-104 of SEQ ID NO:1838); a CDRL3 is QQYNNWPPWT (amino acids 89-98 of SEQ ID NO:1843); and a CDRL3 is MQGTHWPPWT (amino acids 94-103 of SEQ ID NO:1828). Also provided herein is a CDR that exhibits 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the above CDRs.
For example, antibodies that modulate an activity of ErbB-2 include any that contain a VH chain encoded by a sequence of nucleotides containing germline components compiled from a VH germline segment that is an IGHV4 (e.g. any set forth in any of SEQ ID NOS:153-224), an IGHV1 (e.g. any set forth in SEQ ID NOS: 10-43), or an IGHV2 (e.g. any set forth in SEQ ID NOS:44-67); a DH germline segment that is an IGHD6 (e.g. any set forth in SEQ ID NOS: 268-271); an IGHD1 (e.g. any set forth in any of SEQ ID NOS: 239-243), or an IGHD7 (e.g. set forth in SEQ ID NO:272); and a JH germline segment that is an IGHJ1 (e.g., set forth in SEQ ID NO:273), an IGHJ2 (e.g. set forth in SEQ ID NO:274), an IGHJ4 (e.g. set forth in any of SEQ ID NOS: 277-279), or an IGHJ6 (e.g. set forth in any of SEQ ID NOS:282-285). Such antibodies also include any that contain a VL chain encoded by a sequence of nucleotides containing germline components compiled from a V germline segment that is a IGKV3 (e.g. any set forth in any of SEQ ID NOS:332-350), a IGKV4 (e.g. set forth in SEQ ID NO:351), or an IGKV2 (e.g. any set forth in SEQ ID NOS:317-331); and a J germline segment that is an IGKJ1 (e.g. set forth in SEQ ID NO: 356). Such antibodies also include any that are encoded by a sequence of nucleotides containing germline segments that are variants of any of the above germline segments, for example due to conservative mutations or other mutations, so long as the resulting antibody is a functional and productive antibody and binds to ErbB-2 and/or modulates a functional activity.
Exemplary of antibodies against ErbB-2 include an antibody where the VH chain is encoded by a sequence of nucleotides compiled from a VH germline segment that is a IGHV4-31 (e.g. an IGHV4-31*01, IGHV4-31*02, IGHV4-31*03, IGHV4-31*04, IGHV4-31*05, IGHV4-31*06, IGHV4-31*07, IGHV4-31*08, IGHV4-31*09, IGHV4-31*10), an IGHV1-46 (e.g.,IGHV1-46*01, IGHV1-46*02, IGHV1-46*03) an IGHV2-5 (e.g. IGHV2-5*01, IGHV2-5*02; IGHV2-5*03, IGHV2-5*04, IGHV2-5*05, IGHV2-5*06, IGHV2-5*07, IGHV2-5*08, IGHV2-5*09, IGHV2-5*10) IGHV1-69 (e.g. IGHV1-69*01, IGHV1-69*02, IGHV1-69*03, IGHV1-69*04, IGHV1-69*05, IGHV1-69*06, IGHV1-69*07, IGHV1-69*08, IGHV1-69*09, IGHV1-69*10, IGHV1-69*11, IGHV1-69*12, IGHV1-69*13); a DH germline segment that is a IGHD6-6 (e.g. IGHD6-6*01), an IGHD6-13 (e.g. IGHD6-13*01), an IGHD1-26 (e.g. IGHD1-26*01), an IGHD7-27*01, or an IGHD1-1*01; and a JH germline segment that is an IGHJ1*01, an IGHJ2*01, an IGHJ4 (e.g. IGHJ4*01, IGHJ4*02, IGHJ4*03) or an IGHJ6 (e.g. IGHJ6*01, IGHJ6*02, IGHJ6*03, IGHJ6*04). The VL chain is encoded by a sequence of nucleotides compiled from a V germline segment that is a IGKV3-20 (e.g. IGHV3-20*01 or IGHV3-20*02), a IGKV4-1 (e.g. IGKV4-1*01), an IGKV3-15*01, or an IGKV2-30*01; and a J germline segment that is a IGKJ1*01. Exemplary antibodies provided herein that modulate an activity of ErbB-2 are set forth in Table 18G.







TABLE 18G







Anti-ErbB2 Antibodies











SEQ ID NO

SEQ ID NO












Heavy Chain Germline
nucleo-
Amino
Light Chain Germline
nucleo-
Amino


Segments
tide
acid
Segments
tide
acid





IGHV4-31*02; IGHD1-26*01;
1344
1760
IGKV3-20*01; IGKJ1*01
1417
1833


IGHJ2*01


IGHV1-46*01, IGHD6-6*01
1097
1513
IGKV4-1*01; IGKJ1*01
1422
1838


and IGHJ1*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV4-1*01; IGKJ1*01
1422
1838


IGHJ4*01


IGHV2-5*01; IGHD7-27*01;
1143
1559
IGKV3-15*01; IGKJ1*01
1427
1843


IGHJ2*01


IGHV1-69*06; IGHD1-1*01;
1106
1522
IGKV2-30*01; IGKJ1*01
1412
1828


IGHJ6*01









iii. IGF-R1 (Insulin-Like Growth Factor 1 Receptor)
Insulin-like Growth Factor 1 Receptor (IGF-R1; set forth in SEQ ID NO:2007) is a transmembrane receptor activated by Insulin-like Growth Factor 1 (IGF-1) and Insulin-like Growth Factor 2 (IFG-2). Overexpression of Insulin-like Growth Factor Receptor-I has been demonstrated in several cancer cell lines and tumor tissues. IGFR1 is overexpressed in 40% of all breast cancer cell lines (Pandini, et al., (1999) Cancer Res. 5: 1935) and in 15% of lung cancer cell lines. In breast cancer tumor tissue, IGFR1 is overexpressed 6-14 fold and IGFR1 exhibits 2-4 fold higher kinase activity as compared to normal tissue (Webster, et al., (1996) Cancer Res. 56: 2781 and Pekonen, et al., (1998) Cancer Res. 48: 1343). Moreover, colorectal cancer tissue has been reported to exhibit strongly elevated IGFR1 levels (Weber et al., Cancer 95 (10): 2086-95 (2002)). Analysis of primary cervical cancer cell cultures and cervical cancer cell lines revealed 3-and 5-fold overexpression of IGFR1, respectively, as compared to normal ectocervical cells (Steller, et al., (1996) Cancer Res, 56: 1762). Expression of IGFR1 in synovial sarcoma cells also correlated with an aggressive phenotype (i.e., metastasis and high rate of proliferation; Xie, et al., (1999) Cancer Res. 59: 3588).
Activation of IGF-R1 causes survival and proliferation of mitosis-competent cells and growth in tissues such as skeletal muscle and cardiac muscle. The IGF-1 receptor is implicated in several cancers, most notably breast cancer. IGF-R1 can serve to increase the metastatic potential of the tumor by inferring the ability of the tumor to promote vascularization. In addition, IGF-R1's anti-apoptotic properties allow cancer cells to evade the cytotoxic properties of chemotherapeutic drugs or radiation. Crosstalk can occur between IGF-R1 and EGFR allowing EGFR signaling to resume, even in the presence of EGFR inhibitors. Inhibition of IGF-1R mediated signaling has been shown to reduce tumor growth rate, increase apoptosis, and increase killing of tumors by chemotherapy and other molecular target therapies.
Experimental approaches undertaken to inhibit IGF-1R function in tumors have provided encouraging but limited success, and their effectiveness in treating cancer is yet to be determined in the clinic. The ability of an antibody to inhibit IGF-R1 function was first demonstrated with a mouse monoclonal antibody (-IR3) targeting an unknown epitope in the subunit of IGF-1R (Kull et al., (1983) J. Biol. Chem. 258:6561-66). Subsequently other antibodies developed to the subunit of IGF-1R have been shown to inhibit IGF-R1 function to varying degrees in different experimental cancer models. There remains a need for IGF-1R antibodies with different or improved binding, efficacy, and safety characteristics for the treatment of various neoplastic diseases including cancer and metastases thereof.
Antibody libraries provided herein can be screened for modulation of an acitivity by assaying for binding to IGF-R1 and/or assaying for a functional activity, for example, cell proliferation. Assays can be peformed in the presence of IGF-1 or IGF-2. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to IGF-R1.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of IGF-R1 can be used for the treatment or prevention of disease states associated with expression and/or activity of IGF-R1. For example, the antibodies or portions thereof can be used in the treatment of rheumatoid arthritis, Grave's disease, multiple sclerosis, systemin lupus erythematosus, Hashimoto's Thyroiditis, Myasthenia Gravis, auto-immune thyroiditis, Bechet's disease, acromegaly, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical canner, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumors, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels or inappropriate microvascular proliferation.
iv. C-Met
C-Met (or hepatocyte growth factor receptor, HGFR; set forth in SEQ ID NO: 2001) is a membrane receptor found in cells of epithelial origin, including stem cells and progenitor cells. Upon stimulation of c-Met by its ligand, hepatocyte growth factor (HGF), c-Met induces several biological responses that trigger invasive growth, including mitogenesis, motogenesis and morphogenesis. C-Met also is expressed in tumor cell lines and in various human solid tumors. Abnormal c-Met activation in cancer cells correlates with poor prognosis and triggers tumor growth, angiogenesis and metastasis. C-Met engages multiple oncogenic signal transduction pathways, including RAS, which leads to morphogenesis; PI3K, which is associated with cell motility; STAT, which induces branching morphogenesis; and beta catenin, which participates in transcriptional regulation of numerous genes. HGF, through c-Met, has also been shown to be a mitogen for certain cell types, including melanocytes, renal tubular cells, keratinocytes, certain endothelial cells and cells of epithelial origin [Matsumoto et al., Biochem. Biophys. Res. Commun. 176: 45-51 (1991); Igawa et al., Biochem. Biophys. Res. Commun. 174: 831-838 (1991); Han et al., Biochem., 30: 9768-9780 (1991); Rubin et al., Proc. Natl. Acad. Sci. USA, 88: 415-419 (1991)].
Several cancer therapies involve interference of c-Met signaling. These therapies include kinase inhibitors, that prevent ATP from binding to c-Met preventing transphosphorylation; HGF inhibitors, that prevent HGF activation of c-Met; decoy MET inhibitors, that prevent ligand binding and homodimerization; and immunotherapy, including passive immunotherapy which activates CDC or ADCC and active immunotherapy with cytokines triggering non-specific stiulation of immune cells. In view of the important role that this pathway plays in the etiology of various pathological conditions, however, it is clear that there continues to be a need for agents that have clinical attributes that are optimal for development as therapeutic agents.
Antibody libraries provided herein can be screened for modulation of an acitivity by assaying for binding to c-Met and/or assaying for a functional activity, for example, cell proliferation or cell signaling. Assays can be performed in the presence of HGF. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to c-Met.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of c-Met can be used for the treatment or prevention of disease states associated with expression and/or activity of c-Met. For example, the antibodies or portions thereof can be used in the treatment of lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter (e. g., renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central nervous system (e.g., primary CNS lymphoma, spinal axis tumors, brain stem gliomas or pituitary adenomas).
Anti-c-Met antibodies provided herein include antibodies having at least one CDR that is a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, a CDRH1 is GFTFSSYAMS (amino acids 26-35 of SEQ ID NO:3353); a CDRH2 is SISGSGGSTYYADSVKG (amino acids 50-66 of SEQ ID NO:3353); a CDRH3 is EHIVVVIAISYYYYYYGMDV (amino acids 99-118 of SEQ ID NO:3353); a CDRH3 is EDIVVVPAAMSYYYYYYGMDV (amino acids 99-119 of SEQ ID NO:3347); a CDRH3 is EDIVLMVYAISYYYYYYGMDV (amino acids 99-119 of SEQ ID NO:3349); a CDRH3 is EDIVVVVAATSYYYYYYGMDV (amino acids 99-119 of SEQ ID NO:3351); a CDRL1 is QGDSLRSYYAS (amino acids 22-33 of SEQ ID NO:1870); a CDRL2 is GKNNRPS (amino acids 49-55 of SEQ ID NO:1870); and a CDRL3 is NSRDSSGNHLW (amino acids 88-99 of SEQ ID NO:1870). Also provided herein is a CDR that exhibits 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the above CDRs.
For example, antibodies that modulate an activity of c-Met include any that contain a VH chain encoded by a sequence of nucleotides containing germline components compiled from a VH germline segment that is an IGHV3 (e.g. any set forth in SEQ ID NOS: 68-152); a DH germline segment that is an IGHD2 (e.g., any set forth in any of SEQ ID NOS:244-251); and a JH germline segment that is an IGHJ6 (e.g. any set forth in SEQ ID NOS: 282-285), or is a modified form of a JH germline segment, for example, set forth in SEQ ID NO: 3455. Such antibodies also include any that contain a VL chain encoded by a sequence of nucleotides containing germline components compiled from a V germline segment that is an IGLV3 (e.g. set forth in SEQ ID NO: 400-417); and a J germline segment that is an IGLJ2 (e.g. set forth in SEQ ID NO: 443). Such antibodies also include any that are encoded by a sequence of nucleotides containing germline segments that are variants of any of the above germline segments, for example due to conservative mutations or other nucleotide mutations, so long as the resulting antibody is a functional and productive antibody and binds to c-Met and/or modulates a functional activity.
Exemplary of antibodies against c-Met include an antibody where the VH chain is encoded by a sequence of nucleotides compiled from a VH germline segment that is an IGHV3-23 (e.g. IGHV3-23*01, IGHV3-23*02, IGHV3-23*03, IGHV3-23*04 or IGHV3-23*05); a DH germline segment that is an IGHD2-15*01, an IGHD2-2 (e.g. IGHD2-2*01, IGHD2-2*02, or IGHD2-2*03), an IGHD2-8 (e.g. IGHD2-8*01 or IGHD2-8*02), or an IGHD2-21 (e.g. IGHD2-21*01 or IGHD2-21*02); and a JH germline segment that is an IGHJ6 (e.g. IGHJ6*01, IGHJ6*02, IGHJ6*03, IGHJ6*04), or any modified form thereof, for example, set forth in SEQ ID NOS: 3455. The VL chain is encoded by a sequence of nucleotides compiled from a a V germline segment that is an IGLV3-19*01; and a J germline segment that is an ILGJ2*01. Exemplary antibodies provided herein that modulate an activity of EpoR are set forth in Table 18H.







TABLE 18H







Anti-HGFR Antibodies











SEQ ID NO

SEQ ID NO












Heavy Chain Germline
nucleo-
Amino
Light Chain Germline
nucleo-
Amino


Segments
tide
acid
Segments
tide
acid





IGHV3-23*01; IGHD2-21*01>3;
2663
3353
IGLV3-19*01; IGLJ2*01
1454
1870


IGHJ6*01


IGHV3-23*01; IGHD2-2*01>3;
2657
3347
IGLV3-19*01; IGLJ2*01
1454
1870


IGHJ6*01


IGHV3-23*01; IGHD2-8*01>3;
2659
3349
IGLV3-19*01; IGLJ2*01
1454
1870


IGHJ6*01


IGHV3-23*01; IGHD2-15*01>3;
2661
3351
IGLV3-19*01; IGLJ2*01
1454
1870


IGHJ6*01









v. CD20—B-Lymphocyte Antigen
CD20 (human B-lymphocyte-restricted differentiation antigen, Bp35; set forth in SEQ ID NO: 2011), is a hydrophobic transmembrane protein located on pre-B and mature B lymphocytes. CD20 is found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs and is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present on both normal B cells as well as malignant B cells. In particular, CD20 is expressed on greater than 90% of B cell non-Hodgkin's lymphomas (NHL) (Anderson et al. (1984) Blood 63(6):1424-1433), but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissue (Tedder et al. (1985) J. Immunol. 135(2):973-979). CD20 also is expressed on tumor cells, e.g. NHL.
CD20 regulates an early step in the activation process for cell cycle initiation and differentiation. CD20 functions as an ion channel and operates as a store of calcium facilitating entry of extracellular calcium following BCR-induced emptying of intracellular stores (see e.g., Teeling et al. (2006) J Immunol., 177:362-371). Due to the expression of CD20 on almost all B-cells, but not stem cells, it is a target for antigenic modulation via mAb induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. In addition, when engaged by antibody, CD20 initiates signaling that can control growth and triggering cell death in tumors (Teeling et al. (2006) J Immunol., 177:362-371).
CD20 has been validated as a cancer cell target by RITUXAN® (Rituximab), a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes (see e.g. U.S. Pat. No. 5,736,137). RITUXAN® has been shown to induce B-cell lysis through apoptosis, complement-dependent cytotoxicity (CDC) and ADCC.
Antibody libraries provided herein can be screened for modulation of an acitivity by assaying for binding to CD20 and/or assaying for a functional activity, for example, complement-dependent cell-mediate cytotoxicity and killing of cells and apoptosis assays of cells expressing CD20. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to CD20. Example 11 exemplifies a lymphoma apoptosis assay for assessing the function of cross-linked Fab antibodies.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of CD20 can be used for the treatment or prevention of disease states associated with expression and/or activity of CD20. For example, the antibodies or portions thereof can be used in the treatment of lymphomas, autoimmune diseases and transplant rejections (e.g, to prevent rejection of organ and tissue grafts by suppressing autoimmune responses.) Lymphomas include, but are not limited to, non-Hodgkin's lymphomas (high-grade lymphomas, intermediate grade lymphomas, and low grade lymphomas), Hodgkin's disease, acute lymphoblastic leukemias, myelomas, chronic lymphocytic leukemias, and myeloblastic leukemias. Autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, Crohn's disease, psoriasis, autoimmune thrombocytopenic purpura, multiple sclerosis, ankylosing spondylitis, myasthenia gravis, and pemphigus vulgaris.
vii. Erythropoietin Receptor (Epo-R)
Erythropoietin (Epo; set forth in SEQ ID NO: 2009) is a glycoprotein hormone that induces proliferation and differentiation of erythroid progenitor cells. Epo is responsible for promoting the growth, differentiation and survival of erythroid progenitors, which give rise to mature red blood cells. In response to changes in the level of oxygen in the blood and tissues, erythropoietin appears to stimulate both proliferation and differentiation of immature erythroblasts. It also functions as a growth factor, stimulating the mitotic activity of erythroid progenitor cells, such as erythrocyte burst forming and colony-forming units. It also acts as a differentiation factor, triggering transformation of an erythrocyte colony-forming-unit into a proerythroblast (See Erslev, A., New Eng. J. Med., 316:101-103 (1987)).
The activity of Epo is mediated through the binding and activation of a cell surface receptor referred to as the erythropoietin receptor (EpoR). In the absence of ligands the Epo receptor exists in a preformed dimer. The binding of Epo to its receptor causes a conformational change such that the cytoplasmic domains are placed in close proximity. While not completely understood, it is believed that this “dimerization” plays a role in the activation of the receptor. The activation of the Epo receptor results in a number of biological effects. Some of these activities include stimulation of proliferation, stimulation of differentiation and inhibition of apoptosis (See U.S. Pat. No. 6,319,499, Liboi et al., PNAS USA, 90:11351 (1993), Koury, Science, 248:378 (1990)). Defects in the erythropoietin receptor can produce erythroleukemia and familial erythrocytosis.
Erythropoietin is an important pharmaceutical for use in a variety of therapies where stimulation of red blood cell proliferation (RBC) is desired. Epogen® (epoetin alfa) is a recombinant erythropoietin used to stimulate red blood cell proliferation and thereby treat anemia (see e.g. U.S. Pat. Nos. 4,703,008 and 5,955,422).
Antibody libraries provided herein can be screened for modulation of an acitivity by assaying for binding to EpoR and/or assaying for a functional activity, for example, proliferation, apoptosis or cell signaling. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to EpoR. Example 12 and 18 exemplify assays to assess modulation of proliferation or apoptosis induced by EpoR. Hence, such assays can be used to identify agonist antibodies.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of EpoR can be used for the treatment or prevention of disease states associated with expression and/or activity of EpoR. For example, the antibodies or portions thereof can be used in the treatment of disorders characterized by low red blood cell levels and/or decreased hemoglobin levels (e.g. anemia). In addition, such antibodies or portions thereof can be used for treating disorders characterized by decreased or subnormal levels of oxygen in the blood or tissue, such as, for example, hypoxemia or chronic tissue hypoxia and/or diseases characterized by inadequate blood circulation or reduced blood flow. Antibodies or antigen-binding portions thereof also can be useful in promoting wound healing or for protecting against neural cell and/or tissue damage, resulting from brain/spinal cord injury, stroke and the like. Non-limiting examples of conditions that can be treatable by the antibodies include anemia, such as chemotherapy-induced anemia, cancer associated anemia, anemia of chronic disease, HIV-associated anemia, bone marrow transplant-associated anemia and the like, heart failure, ischemic heart disease and renal failure.
Provided herein are antibodies that modulate the activity of EpoR and therefore can be used in the treatment of diseases or conditions associated with expression or activity of EpoR. Such antibodies include those that have a VH chain and a VL chain encoded by a sequence of nucleotides compiled from germline segments, or any antibodies optimized therefrom. Exemplary of such antibodies are Fab antibodies. The antibodies further can contain a constant region. The antibodies include those that have a binding affinity for EpoR that is or is about 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, 10−12M or lower, in particular any that have a nanomolar or sub-nanomolar binding affinity.
Anti-EpoR antibodies provided herein include antibodies having at least one CDR that is a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, a CDRH1 is GYTFTSYYMH (amino acids 26-35 of SEQ ID NO:1509); a CDRH1 is SGYSISSSNWWG (amino acids $26-37 of SEQ ID NO:1759); a CDRH1 is GGSISSGGYYWS (amino acids 26-37 of SEQ ID NO:1769); a CDRH1 is GFTFSSYAMS (amino acids 26-35 of SEQ ID NO:3359); a CDRH2 is IINPSGGSTSYAQKFQG (amino acids 50-66 of SEQ ID NO:1509); a CDRH2 is YIYYSGSTYYNPSLKS (amino acids 51-66 of SEQ ID NO:1759); a CDRH2 is YIYYSGSTYYNPSLKS (amino acids 52-67 of SEQ ID NO:1769); a CDRH2 is SISGSGGSTYYADSVKG (amino acids 50-66 of SEQ ID NO:3359); a CDRH3 is EYYYGSGSYYNDYFDY (amino acids 99-114 of SEQ ID NO:1509); a CDRH3 is EGYSSSWYDYFDY (amino acids 99-111 of SEQ ID NO:1512); a CDRH3 is TNWGAEYFQH (amino acids 99-108 of SEQ ID NO:1759); a CDRH3 is ANWGDNWFDS (amino acids 100-109 of SEQ ID NO:1769); a CDRH3 is ANWGYWYFDL (amino acids 99-108 of SEQ ID NO:1514); a CDRH3 is EGYCSGGSCYSYWYFDL (amino acids 99-115 of SEQ ID NO:1508); a CDRH3 is GITMVRGVIISYYYYYYGMDV (amino acids 99-119 of SEQ ID NO:3359); a CDRL1 is RASQSVSSSYLA (amino acids 24-35 of SEQ ID NO:1833); a CDRL1 is KSSQSVLYSSNNKNYLA (amino acids 24-40 of SEQ ID NO:1838); a CDRL1 is RASQSVSSNLA (amino acids 24-34 of SEQ ID NO:1843); a CDRL1 is RASQSISSWLA (amino acids 24-34 of SEQ ID NO:1841); a CDRL1 is RSSQSLLDSDDGNTYLD (amino acids 24-40 of SEQ ID NO:1853); a CDRL1 is RASQSISSYLN (amino acids 24-34 of SEQ ID NO:1854); a CDRL1 is QGDSLRSYYAS (amino acids 23-33 of SEQ ID NO:1870); a CDRL2 is GASSRAT (amino acids 51-57 of SEQ ID NO:1833); a CDRL2 is WASTRES (amino acids 56-62 of SEQ ID NO:1838); a CDRL2 is GASTRAT (amino acids 50-56 of SEQ ID NO:1843); a CDRL2 is DASSLES (amino acids 50-56 of SEQ ID NO:1841); a CDRL2 is TLSYRAS (amino acids 56-62 of SEQ ID NO:1853); a CDRL2 is AASSLQS (amino acids 50-56 of SEQ ID NO:1854); a CDRL2 is GKNNRPS (amino acids 49-55 of SEQ ID NO:1870); a CDRL3 is QQYGSSPPWT (amino acids 90-99 of SEQ ID NO:1833); a CDRL3 is QQYYSTPPWT (amino acids 95-104 of SEQ ID NO:1838); a CDRL3 is QQYNNWPPWT (amino acids 89-98 of SEQ ID NO:1843); a CDRL3 is QQYNSYSPWT (amino acids 89-98 of SEQ ID NO:1841); a CDRL3 is MQRIEFPSWT (amino acids 95-104 of SEQ ID NO:1853); a CDRL3 is QQSYSTPPWT (amino acids 89-98 of SEQ ID NO:1854); and a CDRL3 is NSRDSSGNHLW(amino acids 88-99 of SEQ ID NO:1870). Also provided herein is a CDR that exhibits 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the above CDRs.
For example, antibodies that modulate an activity of EpoR include any that contain a VH chain encoded by a sequence of nucleotides containing germline components compiled from a VH germline segment that is an IGHV1 (e.g. any set forth in SEQ ID NOS: 10-43), IGHV3 (e.g. any set forth in SEQ ID NOS: 68-152), or an IGHV4 (e.g. any set forth in SEQ ID NOS: 153-224); a DH germline segment that is an IGHD6 (e.g. any set forth in SEQ ID NOS: 268-271), an IGHD3 (e.g. any set forth in any of SEQ ID NOS: 252-259), an IGHD7 (e.g. any set forth in SEQ ID NO: 272); an IGHD2 (e.g., any set forth in any of SEQ ID NOS:244-251); and a JH germline segment that is an IGHJ1 (e.g., set forth in SEQ ID NO:273), IGHJ4 (e.g. set forth in SEQ ID NO:277-279), an IGHJ5 (e.g. set forth in SEQ ID NOS: 280 or 281), an IGHJ2 (e.g. set forth in SEQ ID NOS: 274), or an IGHJ6 (e.g. any set forth in SEQ ID NOS: 282-285), or is a modified form of a JH germline segment, for example, set forth in any of SEQ ID NOS: 3450-3455. Such antibodies also include any that contain a VL chain encoded by a sequence of nucleotides containing germline components compiled from a V germline segment that is an IGKV4 (e.g. set forth in SEQ ID NO:351), an IGKV3 (e.g. set forth in SEQ ID NOS:332-350); an IGKV1 (e.g. set forth in SEQ ID NOS:286-316 and 868); an IGKV2 (e.g. set forth in SEQ ID NOS:317-331); a J germline segment that is an IGKJ1 (e.g. set forth in SEQ ID NO: 356); a V germline segment that is an IGLV3 (e.g. set forth in SEQ ID NO: 400-417); and a J germline segment that is an IGLJ2 (e.g. set forth in SEQ ID NO: 443). Such antibodies also include any that are encoded by a sequence of nucleotides containing germline segments that are variants of any of the above germline segments, for example due to conservative mutations or other nucleotide mutations, so long as the resulting antibody is a functional and productive antibody and binds to EpoR and/or modulates a functional activity.
Exemplary of antibodies against EpoR include an antibody where the VH chain is encoded by a sequence of nucleotides compiled from a VH germline segment that is a IGHV1-46 (e.g.,IGHV1-46*01, IGHV1-46*02, IGHV1-46*03), an IGHV3-23 (e.g. IGHV3-23*01, IGHV3-23*02, IGHV3-23*03, IGHV3-23*04, IGHV3-23*05), an IGHV4-28 (e.g. IGHV4-28*01, IGHV4-28*02, IGHV4-28*03, IGHV4-28*04, IGHV4-28*05), or an IGHV4-31 (e.g. IGHV4-31*01, IGHV4-31*02, IGHV4-31*03; IGHV4-31*04, IGHV4-31*05, IGHV4-31*06, IGHV4-31*07, IGHV4-31*08, IGHV4-31*09, IGHV4-31*10); a DH germline segment that is a IGHD6-6 (e.g. IGHD6-6*01), an IGHD6-13 (e.g. IGHD6-13*01), an IGHD3-10 (e.g. IGHD3-10*01 or IGHD3-10*02), an IGHD7-27*01, an IGHD2-15*01, or an IGHD6-13*01; and a JH germline segment that is an IGHJ1*01, an IGHJ4 (e.g. IGHJ4*01, IGHJ4*02 or IGHJ4*03), an IGHJ5 (e.g. IGHJ5*01, IGHJ5*02), an IGHJ6 (e.g. IGHJ6*01, IGHJ6*02, IGHJ6*03, IGHJ6*04), or an IGHJ2*01, or any modified form thereof, for example, set forth in SEQ ID NOS: 3455. The VL chain is encoded by a sequence of nucleotides compiled from a V germline segment that is an IGKV4-1 (e.g. IGKV4-1*01), an IGKV3-15*01, an IGKV3-20 (e.g. IGKV3*01, IGKV3*02), an IGKV1-5 (e.g. IGKV1-5*01, IGKV1-5*02, IGKV1-5*03), an IGKV1-39*01, or an IGKV2D-40*01; a J germline segment that is a IGKJ1*01; a V germline segment that is an IGLV3-19*01; and a J germline segment that is an ILGJ2*01. Exemplary antibodies provided herein that modulate an activity of EpoR are set forth in Table 18I.







TABLE 18I







Anti-EpoR Antibodies











SEQ ID NO

SEQ ID NO












Heavy Chain Germline
nucleo-
Amino
Light Chain Germline
nucleo-
Amino


Segments
tide
acid
Segments
tide
acid





IGHV1-46*01; IGHD3-10*01;
1093
1509
IGKV4-1*01; IGKJ1*01
1422
1838


IGHJ4*01


IGHV1-46*01; IGHD6-6*01;
1097
1513
IGKV4-1*01; IGKJ1*01
1422
1838


IGHJ1*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV4-1*01; IGKJ1*01
1422
1838


IGHJ4*01


IGHV4-28*01; IGHD7-27*01;
1343
1759
IGKV3-15*01; IGKJ1*01
1427
1843


IGHJ1*01


IGHV4-31*02; IGHD7-27*01;
1353
1769
IGKV3-15*01; IGKJ1*01
1427
1843


IGHJ5*01


IGHV1-46*01; IGHD7-27*01;
1098
1514
IGKV3-20*01; IGKJ1*01
1417
1833


IGHJ2*01


IGHV1-46*01; IGHD2-15*01;
1092
1508
IGKV3-15*01; IGKJ1*01
1427
1843


IGHJ2*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-15*01; IGKJ1*01
1427
1843


IGHJ4*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-20*01; IGKJ1*01
1417
1833


IGHJ4*01


IGHV1-46*01; IGHD2-15*01;
1092
1508
IGKV1-5*01; IGKJ1*01
1425
1841


IGHJ2*01


IGHV1-46*01; IGHD3-10*01;
1093
1509
IGKV2D-40*01; IGKJ1*01
1437
1853


IGHJ4*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV2D-40*01; IGKJ1*01
1437
1853


IGHJ4*01


IGHV3-23*01; IGHD3-10*01>3;
2669
3359
IGKV1-39*01; IGKJ1*01
1438
1854


IGHJ6*01


IGHV3-23*01; IGHD3-10*01>3;
2669
3359
IGLV3-19*01; IGLJ2*01
1454
1870


IGHJ6*01









viii. Cadherins
Cadherins are a class of calcium dependent, type-1 transmembrane glycoproteins that are involved in cell adhesion, helping to ensure that cells within tissues are bound together. There are multiple classes of cadherin molecules, including but not limited to N-cadherin, E-cadherin and P-cadherin. Members of this family exhibit calcium-dependent homophilic interactions and are responsible for the selective cell-cell recognition and adhesion, which is necessary for allocating different cell types to their proper places during organ development. Cadherins also play an important role in maintaining the integrity of multicellular structures. During embryonic morphogenesis the expression of diverse members of the cadherin family is spatially and temporally regulated facilitating the orderly assembly of various cell types into functional structures. Cadherins are considered to play a significant role in the cellular connections of cancer and metastatic cells.
a) P-Cadherin (P-cad/CDH3)
P-cadherin (P-cad; set forth in SEQ ID NO: 2008) is a single-span type-1 transmembrane glycoprotein found in the placenta that is homologous to E-cadherin (epithelial cadherin, E-cad). Both P-cad and E-cad interact with the cytoskeleton by alpha-catenin. Like other cadherins, p-cadherin plays a role in epithelial cell-cell adhesion. Other major roles include the determination of cell phenotypes and involvement in cell dynamics, including migration and the dissemination of tumor cells. The expression of P-cad in epithelial tissues appears to identify cell populations with proliferative activity, and its expression decreases as cells differentiate.
Expression of P-cadherin, a calcium-dependent cellular adhesion protein, has been reported in poorly differentiated and invasive bladder carcinoma cells. Such bladder carcinoma cells exhibit reduced E-cadherin expression. (Mialhe, A. et al., J. Urol. 164:826 (2000)). Down-regulation of E-cadherin and P-cadherin has also been associated with cultured neoplastic prostate cells. (Wang, J. et al., Urol. Res. 5:308 (2000)). The development of human colorectal cancer has been attributed, at least in part, to a decrease in cellular levels of the E-cadherin/catenin complex. (Debruyne, P. et al., Acta Gastroenterol. Belg. 62(4):393 (1999)). Aberrant up-regulation of P-cadherin was recently reported to be associated with proliferative cell phenotypes that can be related to neoplastic transformation of tissues of the gastrointestinal tract, particularly metaplastic and adenomatous polyps. (Sanders, D. S., et al., J. Pathol. 190(5):526 (2000)). Hence, certain cancer types, particularly some digestive cancer types, e.g., colon cancer, are characterized by the upregulation and the overexpression of P-cadherin relative to normal cells. P-cadherin is a valid target for cancer diagnosis, prophylaxis or therapy (see e.g. United States Published Patent Application No. 2003/0194406 and International Published Patent Application No. WO 02/097395).
Antibody libraries provided herein can be screened for modulation of an activity by assaying for binding to pCad and/or assaying for a functional activity. Assays for functional activity, include but are not limited to, assays that assess proliferation and/or adhesion of tumor cells, ADCC or CDC activity, anti-apoptotic assays, or cell cycle checkpoint assays. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to pCad. Example 20 exemplifies an assay to assess effects of antibodies on cell to cell adhesion induced by p-cadherin. For example, an antibody that inhibits p-cadherin function can be identified based on the failure of cells to clump in the presence of the antibody.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of pCad can be used for the treatment or prevention of disease states associated with expression and/or activity of pCad. For example, the antibodies or portions thereof can be used in the treatment of digestive cancers such as colon cancer, stomach cancer and liver cancer, and other cancers, such as lung cancer and breast cancer.
Provided herein are antibodies that modulate the activity of p-cadherin and therefore can be used in the treatment of diseases or conditions associated with expression or activity of p-cadherin. Such antibodies include those that have a VH chain and a VL chain encoded by a sequence of nucleotides compiled from germline segments, or any antibodies optimized therefrom. Exemplary of such antibodies are Fab antibodies. The antibodies further can contain a constant region. The antibodies include those that have a binding affinity for p-cadherin that is or is about 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, 10−12 M or lower, in particular any that have a nanomolar or sub-nanomolar binding affinity.
Anti-p-cadherin antibodies provided herein include antibodies having at least one CDR that is a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, a CDRH1 is GYTFTSYYMH (amino acids 26-35 of SEQ ID NO:1512); a CDRH1 is GFTFSSYAMS (amino acids 26-35 of SEQ ID NO:3559); a CDRH2 is IINPSGGSTSYAQKFQG (amino acids 50-66 of SEQ ID NO:1512); a CDRH2 is SISGSGGSTYYADSVKG (amino acids 50-66 of SEQ ID NO:3359); a CDRH3 is EGYSSSWYDYFDY (amino acids 99-111 of SEQ ID NO:1512); a CDRH3 is ANWGYWYFDL (amino acids 99-108 of SEQ ID NO:1514); a CDRH3 is EYYYGSGSYYNDYFDY (amino acids 99-114 of 1509); a CDRH3 is GITMVRGVIISYYYYYYGMDV (amino acids 99-119 of SEQ ID NO:3359); a CDRH3 is VIITPRTIVISYAFDV (amino acids 99-114 of SEQ ID NO:3071; a CDRL1 is RASQSVSSNLA (amino acids 24-35 of SEQ ID NO:1843); a CDRL1 is RASQSVSSSYLA (amino acids 24-35 of SEQ ID NO:1833); a CDRL1 is RASQSISSWLA (amino acids 24-34 of SEQ ID NO:1841); a CDRL1 is RASQSISSYLN (amino acids 24-34 of SEQ ID NO:1854); a CDRL1 is QGDSLRSYYAS (amino acids 23-33 of SEQ ID NO:1870; a CDRL2 is GASTRAT (amino acids 50-56 of SEQ ID NO:1843); a CDRL2 is GASSRAT (amino acids 51-57 of SEQ ID NO:1833); a CDRL2 is DASSLES (amino acids 50-56 of SEQ ID NO:1841); a CDRL2 is AASSLQS (amino acids 50-56 of SEQ ID NO:1854); a CDRL2 is GKNNRPS (amino acids 49-55 of SEQ ID NO:1870); a CDRL3 is QQYNNWPPWT (amino acids 89-98 of SEQ ID NO:1843); a CDRL3 is QQYGSSPPWT (amino acids 90-99 of SEQ ID NO:1833); a CDRL3 is QQYNSYSPWT (amino acids 89-98 of SEQ ID NO:1841); a CDRL3 is QQSYSTPPWT (amino acids 89-98 of SEQ ID NO:1854); and a CDRL3 is NSRDSSGNHLW (amino acids 88-99 of SEQ ID NO:1870). Also provided herein is a CDR that exhibits 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the above CDRs.
For example, antibodies that modulate an activity of p-cadherin include any that contain a VH chain encoded by a sequence of nucleotides containing germline components compiled from a VH germline segment that is an IGHV1 (e.g. any set forth in any of SEQ ID NOS:1-43) or an IGHV3 (e.g. any set forth in any of SEQ ID NOS:68-152); a DH germline segment that is an IGHD3 (e.g. any set forth in SEQ ID NOS:252-259), an IGHD6 (e.g. any set forth in SEQ ID NOS: 268-271) or an IGHD7 (e.g. set forth in SEQ ID NO: 272); and a JH germline segment that is an IGHJ2 (e.g. set forth in SEQ ID NO:274), IGHJ3 (e.g. set forth in SEQ ID NO:275 or 276), is an IGHJ4 (e.g. set forth in SEQ ID NO: 278 or 279), an IGHJ6 (e.g. set forth in any of SEQ ID NOS: 282-285), or is a modified form of a JH germline segment, for example, set forth in any of SEQ ID NOS: 3450-3455. Such antibodies also include any that contain a VL chain encoded by a sequence of nucleotides containing germline components compiled from a V germline segment that is an IGKV1 (e.g. any set forth in SEQ ID NOS: 286-316), or an IGKV3 (e.g. any set forth in any of SEQ ID NOS:332-350); a J germline segment that is an IGKJ1 (e.g. set forth in SEQ ID NO: 356); a V germline segment that is an IGLV3 (e.g. set forth in SEQ ID NO: 400-417); and a J germline segment that is an IGLJ2 (e.g. set forth in SEQ ID NO: 443). Such antibodies also include any that are encoded by a sequence of nucleotides containing germline segments that are variants of any of the above germline segments, for example due to conservative mutations or other mutations, so long as the resulting antibody is a functional and productive antibody and binds to p-cadherin and/or modulates a functional activity.
Exemplary of antibodies against p-cadherin include an antibody where the VH chain is encoded by a sequence of nucleotides compiled from a VH germline segment that is a IGHV1-46 (e.g. an IGHV1-46*01, IGHV1-46*01, or an IGHV1-46*03) or an IGHV3-23 (e.g. IGHV3-23*01, IGHV3-23*02, IGHV3-3*03, IGHV3-23*04, IGHV3-23*05); a DH germline segment that is an IGHD3-10 (e.g. IGHD3-10*01, IGHD3-10*02) or an IGHD6-13 (e.g. IGHD6-13*01) or an IGHD7-27*01; and a JH germline segment that is an IGHJ3 (e.g. IGHJ3*01, IGHJ3*02), IGHJ4 (e.g. IGHJ4*01, IGHJ4*02, IGHJ4*03), an IGHJ6 (e.g. IGHJ6*01, IGHJ6*02, IGHJ6*03, IGHJ6*04), or an IGHJ2*01, or any modified form thereof, for example, set forth in SEQ ID NO: 3452 and 3455 and encoding a JH region set forth in SEQ ID NO:3458 and 3461, respectively. The VL chain is encoded by a sequence of nucleotides compiled from a V germline segment that is an IGKV1-5 (e.g. IGKV1-5*01, IGKV1-5*02, IGKV1-5*03), IGKV1-39*01, an IGKV3-15 (e.g. IGKV3-15*01), IGKV3-20 (e.g. IGKV3-20*01, IGKV3-20*02) or an IGKV3-11 (e.g. IGKV3-11*01, IGKV3-11*02); a J germline segment that is a IGKJ1*01; a V germline segment that is an IGLV3-19*01; and a J germline segment that is an ILGJ2*01. Exemplary antibodies provided herein that modulate an activity of p-cadherin are set forth in Table 18J.







TABLE 18J







Anti-p-cadherin Antibodies










Heavy Chain Germline
SEQ ID NO

SEQ ID NO












Segments or Modified
nucleo-
Amino
Light Chain Germline
nucleo-
Amino


forms thereof
tide
acid
Segments
tide
acid





IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-15*01; IGKJ1*01
1427
1843


IGHJ4*01


IGHV1-46*01; IGHD6-13*01;
1096
1512
IGKV3-20*01; IGKJ1*01
1417
1833


IGHJ4*01


IGHV1-46*01; IGHD7-27*01;
1098
1514
IGKV3-11*01; IGKJ1*01
1434
1850


IGHJ2*01


IGHV1-46*01; IGHD3-10*01;
1093
1509
IGKV1-5*01; IGKJ1*01
1425
1841


IGHJ4*01


IGHV3-23*01; IGHD3-10*01>3;
2669
3359
IGKV1-39*01; IGKJ1*01
1438
1854


IGHJ6*01


IGHV3-23*01; IGHD3-10*01>1′;
2381
3071
IGKV1-39*01; IGKJ1*01
1438
1854


IGHJ3*01


IGHV3-23*01; IGHD3-10*01>3;
2669
3359
IGLV3-19*01; IGLJ2*01
1454
1870


IGHJ6*01









ix. CD44
CD44 (set forth in SEQ ID NO: 2006) is a cell-surface integral membrane glycoprotein involved in cell-to-cell interactions, cell adhesion and migration. Transcripts of the CD44 gene undergo complex alternative splicing resulting in many distinct functional isoform variants. CD44 is a protein which is expressed in several different isoforms on the surface of a wide variety of cell types. The smallest isoform, standard CD44 (CD44s), which is expressed by a variety of different cells, is thought to mediate cell attachment to extracellular matrix components and can transmit a co-stimulus in lymphocyte and monocyte activation. In contrast, expression of splice variants of CD44 which contain the domain v6 (CD44v6) in the extracellular region, is restricted to a subset of epithelia.
CD44 participates in a wide variety of cellular functions, including lymphocyte activation, recirculation and homing, hematopoiesis and tumor metastasis. CD44 is a receptor for hyaluronic acid and can also interact with other ligands such as E-selectin and L-selectin and other ligands such as osteopontin, collagens and matrix metalloproteinases (MMPs). MMPs degrade proteins that keep vessel walls solid allowing endothelial cells to escape into the interstitial matrix causing sprouting angiogenesis. Inhibition of MMPs prevents formation of new capillaries. Interactions of CD44 with hyaluronan mediates cell adhesion interactions. Treatments that disrupt such interactions can be used in the treatment of a number of pathologies.
Some CD44 variations are cell surface markers for breast and prostate cancer cells. Overexpression of CD44 has been linked to the growth and spread of a range of different types of malignancies, particularly lymphomas. CD44v6, as well as other variant exons (CD44v3, CD44v5, CD44v7/v8, CD44v10) has been shown to be a tumor-associated antigen with a favorable expression pattern in human tumors and normal tissues (Heider K H et al. Eur. J. Cancer 31A:2385-2391, 1995; Heider K H et al. Cancer Immunology Immunotherapy 43:245-253, 1996; Dall et al., 1996; Beham-Schmid et al., 1998; Tempfer et al., 1998; Wagner et al., 1998) and has been subject to antibody-based diagnostic and therapeutic approaches, in particular radioimmunotherapy (RIT) of tumors (Stroomer J W et al. Clin Cancer Res 6(8):3046-55, 2000, WO 95/33771, WO 97/21104). CD44 has been the target of the development of anti-cancer therapeutics (see e.g. U.S. Pat. No. 5,990,299).
Antibody libraries provided herein can be screened for modulation of an activity by assaying for binding to CD44 and/or assaying for a functional activity. Example 13 exemplifies a binding assay to screen antibody libraries provided herein to select or identify antibodies that bind to pCad.
The antibodies or portions thereof identified from the libraries provided herein that modulate an activity of CD44 can be used for the treatment or prevention of disease states associated with expression and/or activity of CD44 or a variant thereof. For example, the antibodies or portions thereof can be used in the treatment of a cancer that is a head and neck squamous cell carcinoma (SCC), esophagus SCC, lung SCC, skin SCC, breast adenocarcinoma (AC), lung AC, cervix SCC, pancreas AC, colon AC, or stomach AC. The antibodies or portions thereof also can be used in the treatment of rheumatoid arthritis.
4. Iterative Screening
Upon identification of a “Hit” the methods described in Section C can be repeated to generate a further library for use in the screening methods herein. By virtue of the fact that antibodies are provided in an addressable format, identification of a “Hit” permits instant identification and assessment of the “structure/activity” relationship. For example, upon identification of a “Hit” the component germline segments of the encoded antibody can be immediately envisaged. A subsequent library can be generated that includes antibodies encoded by nucleic acid molecules derived from germline segments “related” to the identified “Hit”. Typically, the germline segments are related by sequence similarity, but can also be related by some other shared characteristic such as a CDR, directed peptide, or other biochemical attribute.
Generally, for the generation of a further antibody library in the iterative screening method, all gene families of the germline segments contained in a “Hit” are identified. All VH, DH and JH gene family segments thereof are combined in all possible permutations, or a subset thereof as desired, to generate a recombined nucleic acid molecule encoding a VH chain. All Vκ and Jκ, or Vλ and Jλ gene family segments thereof are combined in all possible permutation, or a subset thereof as desired, to generate a recombined nucleic acid molecule encoding a VL chain. Vectors and cells expressing the various paired VH and VL members, in all permutations, are generated, proteins expressed, and antibody members purified to generate a further antibody library. The further antibody library can be provided in an addressed format. The further library can be used in screening assays as described herein above to optimize the “Hit”.
In addition, it is contemplated herein that because libraries can be created whereby the identify of each member is known, a library might exist already containing the exact desired “related” library members. Thus, those members can be collected to create a sub-library of related “Hits” for screening without having to reperform the method herein.
It also is contemplated that the “Hits” or any subsequent members of a further library can be mutagenized, such as by directed evolution, to further optimize the “Hit”.
5. Directed Evolution
Antibody “Hits” identified by the screening methods herein can be further modified, e.g., by mutagenesis, to provide a library of modified antibodies. The modified antibodies are then evaluated to identify one or more antibodies which have altered functional properties (e.g., improved binding, improved stability, lengthened stability in vivo, and/or improved modulation of a functional activity). A further library of modified “Hits” can be generated to select or screen for an improved activity against a target protein. The library can be screened in an addressable format or other display format as described herein above. For example, higher affinity binding proteins can be identified from the second library, e.g., by using higher stringency or more competitive binding and washing conditions. Other screening techniques can also be used.
Various approaches have been used to create mutated antibody repertoires, including chain shuffling (Marks et al. (1992) Bio/Technology 10:779 and Clackson et al. (1991) Nature 352:624), error prone PCR (Hawkins et al. (1992) J. Mol. Biol. 226:889; and Leung et al. (1989) Technique 1:11-15), use of E. coli mutator strains (Low et al. (1996) J. Mol. Biol. 260,359), recombination (see, e.g., United States Publication No. 2004-0005709), DNA shuffling using random cleavage (Stemmer (1994) Nature 389-391; termed “nucleic acid shuffling”), RACHITT® (Coco et al. (2001) Nature Biotech. 19:354), or approaches more specifically directed to the complementarity determining regions (CDRs) of the antibody molecule, like CDR walking (Barbas et al. (1994) Proc. Natl. Acad. Sci. USA 91:3809 and Yang et al. (1995) J. Mol. Biol. 254:392), site-directed mutagenesis (Zoller et al. (1987) Nucl Acids Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) Methods Enzymol. 208:564-586) and incorporation of degenerate oligonucleotides (Griffiths et al. (1994) EMBO J 13:3245).
In some implementations, the mutagenesis is targeted to regions known or likely to be at the binding interface. For example, mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particular within ten, five, or three amino acids of a CDR junction. Additionally, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make step-wise improvements.
a. Random Mutagenesis
Antibodies bind to antigens via residues in their CDRs. Consequently, mutagenesis of CDRs is widely used to improve the affinity of Fab and Fv fragments of antibodies. Random mutagenesis methods include, for example, use of E. coli XL1red, UV irradiation, chemical modification such as by deamination, alkylation, or base analog mutagens, or PCR methods such as DNA shuffling, cassette mutagenesis, site-directed random mutagenesis, error prone PCR (see e.g. U.S. Application No.: 2006-0115874) or the use of commercially available random mutagenesis kits such as, for example, GeneMorph PCR-based random mutagenesis kits (Stratagene) or Diversify random mutagenesis kits (Clontech). The Diversify random mutagenesis kit allows the selection of a desired mutation rate for a given DNA sequence (from 2 to 8 mutations/1000 base pairs) by varying the amounts of manganese (Mn2+) and dGTP in the reaction mixture. Raising manganese levels initially increases the mutation rate, with a further mutation rate increase provided by increased concentration of dGTP. Even higher rates of mutation can be achieved by performing additional rounds of PCR. All of these approaches involve the construction of expression libraries of antibodies with mutations in the CDRs and selection for better binders. Any of a variety of general approaches for directed protein evolution based on mutagenesis can be employed. Any of these, alone or in combination can be used to modify a lead antibody to achieve a desired property. Any method known in the art can be used to modify or alter a lead antibody.
i. Saturation Mutagenesis
In one exemplary embodiment, a saturation mutagenesis technique is used in which the CDR residue(s) are mutated to each of the 20 possible amino acids (see e.g., the Kunkle method, Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Media Pa. and U.S. Pat. No. 6,562,594). In such a technique, a degenerate mutagenic oligonucleotide primer can be synthesized which contains randomization of nucleotides at the desired codon(s) encoding the selected amino acid(s). Exemplary randomization schemes include NNS- or NNK-randomization, where N represents any nucleotide, S represents guanine or cytosine and K represents guanine or thymine. The degenerate mutagenic primer is annealed to the single stranded DNA template and DNA polymerase is added to synthesize the complementary strand of the template. After ligation, the double stranded DNA template is transformed into E. coli for amplification. Alternatively, single amino acid changes are made using standard, commercially available site-directed mutagenesis kits such as QuikChange (Stratagene). In another embodiment, any method commonly known in the art for site specific amino acid mutation can be used.
ii. Error Prone PCR
Error prone PCR can introduce random mutations into nucleic acid sequences (See, e.g., Hawkins et al. (1992) J. Mol. Biol. 226(3):889-96). Briefly, PCR is run under conditions which compromise the fidelity of replication, thus introducing random mutations in sequences as those skilled in the art can accomplish. After generation of such random mutants, they can be placed into genetic display formats, panned and thus evaluated for activity.
iii. Cell Lines
A mutator strain of bacteria, such as E. coli mutD5 or Epicurian Cole XL1-Red Competent cells (Stratagene, La Jolla, Calif.), can be used during plasmid replication to generate a repertoire of antibodies that include single nucleotide point mutations. The libraries of random mutants are then screened for biological activity using a genetic display format.
iv. DNA Shuffling/Antibody Chain Shuffling
DNA shuffling can be employed to modulate the activities of lead antibodies. In DNA shuffling, variant DNA molecules are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations (see e.g. Stemmer, (1994) Nature 370:389-391; Stemmer (1994) Proc. Natl Acad. Sci. USA 91:10747-10751; U.S. Pat. Nos. 5,605,793, 5,811,238, and 5,830,721; and International Publication Nos. WO 95/022625 and WO 97/20078).
This technique can be modified by using a family of parent DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid “evolution” of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
In antibody chain shuffling, one selected VH sequence is paired with variety of VL sequences to create new VH/VL partners (see e.g. Kang et al. (1991) Proc. Natl. Acad. Sci. USA 88:11120 11123; Collet et al. (1992) Proc. Natl. Acad. Sci. USA 89:10026-10030; and Marks et al. (1992) Bio/Technology 10:779)
v. CDR Walking
In another example, the generation or selection of higher affinity antibodies can be carried out by CDR walking mutagenesis, which mimics the tertiary immune selection process. For example, saturation mutagenesis of the CDR's of an antibody can be used to generate one or more libraries of antibody fragments which are displayed on the surface of filamentous bacteriophage followed by the subsequent selection of the relevant antibody using immobilized antigen. Sequential and parallel optimization strategies can be used to then select the higher affinity antibody (see e.g. Barbas et al. (1994) Proc. Natl. Acad. Sci. USA 91:3809-3813; and Yang et al. (1995) J. Mol. Biol 254(3):392-403).
vi. Framework Stabilization
A mutation can be made in a framework region or constant domain to increase the half-life of the antibody. A mutation in a framework region or constant domain also can be made to alter the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molecule, or to alter such properties as complement fixation, FcR binding and antibody-dependent cell-mediated cytotoxicity (ADCC). A single antibody can have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant domain. See, e.g., PCT Publication No. WO 00/09560; Ewert et al. (2003) Biochemistry 42(6):1517-1528; Ewert et al. (2004) Methods 23(2):184-99; and Knappik et al. (1995) Protein Engineering 8:81-89.
6. Epitope Mapping
Once a “Hit” is identified its binding epitope can be determined. Hence, antibody libraries provided herein can be used to identify novel epitopes, including but not limited to, previously unidentified epitopes on known antigens or novel epitopes of unknown antigens, for example, those presented by a carcinoma cell line. Methods of epitope mapping are known to one skill in the art (see e.g., Olwyn M. R. Westwood and Frank C. Hay. Epitope Mapping. Oxford University Press, 2001; Sigma Catalog #Z373990 Epitope Mapping Protocols: Methods in Molecular Biology, Vol. 66). Methods for mapping of epitopes recognized by an antibody include, but are not limited to, binding assays such as by using BIAcore or ELISA (Reineke et al. 1999), ELISPOT, prediction software, combinatorial synthesis of peptide libraries onto a planar support (i.e. protein chip) followed by exposure of the surface to the antibody, phage display of a library of peptides derived from the sequence of the protein antigen, methods using mass spectrometry methods such as methods using MALDI or amide H/D exchange (see e.g. Baerga-Ortiz et al. (2002) Protein Science, 11:1300-1308) and surface plasma resonance.
7. In Vivo Assays of Identified Hits
Once a “Hit” is identified against a target of interest, it can be assessed in vivo assays associated with aberrant activity of the target. In general, the method involves administering an antibody to a subject, generally a non-human animal model for a disease or condition and determining the effect of the antibody on the on the disease or condition of the model animal. In vivo assays include controls, where suitable controls include a sample in the absence of the antibody. Generally a plurality of assay mixtures is run in parallel with different antibody concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection. An antibody of interest is one that modulates, i.e., reduces or increases a symptom of the animal model disease or condition by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or more, when compared to a control in the absence of the antibody. In general, an antibody of interest will cause a subject animal to be more similar to an equivalent animal that is not suffering from the disease or condition.
Non-human animals models include those induced to have a disease such as by injection with disease and/or phenotype-inducing substances prior to administration of the antibodies to monitor the effects on disease progression. Genetic models also are useful. Animals, such as mice, can be generated which mimic a disease or condition by the overexpression, underexpression or knock-out of one or more genes. Such animals can be generated by transgenic animal production techniques well-known in the art or using naturally-occurring or induced mutant strains. One of skill in the art is familiar with various animal models associated with particular targets.
Such animal model systems include, but are not limited to, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g. baboons, chimpanzees and monkey. Any animal system well-known in the art can be used. Several aspects of the procedure can vary; said aspects include, but are not limited to, the temporal regime of administering the antibodies (e.g., prophylactic and/or therapeutic agents), whether such antibodies are administered separately or as an admixture, and the frequency of administration of the antibodies.
Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals. Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g. baboons, chimpanzees and monkeys. Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al., (1985) Proc. Natl. Acad. Sci. USA 82:6148-615); gene targeting in embryonic stem cells (Thompson et al., (1989) Cell 56:313-321); electroporation of embryos (Lo, (1983) Mol. Cel. Biol. 3:1803-1814); sperm-mediated gene transfer (Lavitrano et al., (1989) Cell 57:717-73). For review, see, for example, U.S. Pat. No. 4,736,866.
Animal models can be used to assess the efficacy of an antibody, a composition, or a combination therapy provided herein. Examples of animal models for lung cancer include, but are not limited to, lung cancer animal models (see e.g. Zhang et al., (1994) In Vivo 8(5):755-69) and a transgenic mouse model with disrupted p53 function (see, e.g., Morris et al., (1998) J La State Med Soc 150(4):179-85). An example of an animal model for breast cancer includes, but is not limited to, a transgenic mouse that overexpresses cyclin D1 (see, e.g., Hosokawa et al., (2001) Transgenic Res 10(5):471-8). An example of an animal model for colon cancer includes, but is not limited to, a TCR b and p53 double knockout mouse (see, e.g., Kado et al. (2001), Cancer Res 61(6):2395-8). Examples of animal models for pancreatic cancer include, but are not limited to, a metastatic model of Panc02 murine pancreatic adenocarcinoma (see, e.g., Wang et al. (2001) Int J Pancreatol 29(1):37-46) and nu-nu mice generated in subcutaneous pancreatic tumors (see, e.g., Ghaneh et al., (2001) Gene Ther 8(3):199-208). Examples of animal models for non-Hodgkin's lymphoma include, but are not limited to, a severe combined immunodeficiency (“SCID”) mouse (see, e.g., Bryant et al. (2000) Lab Invest 80(4):553-73) and an IgHmu-HOX11 transgenic mouse (see, e.g., Hough et al. (1998) Proc Natl Acad Sci USA 95(23):13853-8). An example of an animal model for esophageal cancer includes, but is not limited to, a mouse transgenic for the human papillomavirus type 16 E7 oncogene (see, e.g., Herber et al., (1996) J Virol 70(3):1873-81). Examples of animal models for colorectal carcinomas include, but are not limited to, Apc mouse models (see, e.g., Fodde & Smits, (2001) Trends Mol Med 7(8):369-73 and Kuraguchi et al., (2000) Oncogene 19(50):5755-63).
Animal models for arthritis include, but are not limited to, rheumatoid arthritis rats (see e.g. Pearson, (1956) Proc. Soc. Exp. Biol. Med., 91:95-101) and collagen induced arthritis in mice and rats (see e.g. Current Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc., 1994). An example of an animal model for asthma, includes but is not limited to, a mouse model of pulmonary hypersensitivity (see e.g. Riese et al. (1998) J. Clin. Invest. 101:2351-2363 and Shi, et al. (1999) Immunity 10:197-206). Animal models for allogenic rejection include, but are not limited to, rat allogeneic heart transplant models (see e.g. Tanabe et al. (1994) Transplantation 58:23-27 and Tinubu et al. (1994) J. Immunol. 153:4330-4338) and rat heterocardiac allograft rejection (Jae-Hyuck Sim et al. (2002) Proc Natl Acad Sci U.S.A. 99(16):10617-10622). Steel mice are used as a model of human aplastic anemia (see e.g. Jones, (1983) Exp. Hematol., 11:571-580). An example of an animal model for anemia, includes but is not limited to, hemolytic anemia guinea pigs (see e.g. Schreiber, et al. (1972) J. Clin. Invest. 51:575). An example of an animal model for neutropenia, includes but is not limited to, neutropenia neutropenic CD rats (see, e.g. Nohynek et al. (1997) Cancer Chemother. Pharmacol. 39:259-266).
8. Articles of Manufacture/Kits
Pharmaceutical compounds of selected antibodies or nucleic acids encoding selected antibodies, or a derivative or a biologically active portion thereof can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating the disease or disorder, and a label that indicates that selected antibody or nucleic acid molecule is to be used for treating the disease or disorder.
The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, for example, U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any EPO-mediated disease or disorder or therapeutic polypeptide-mediated disease or disorder.
Antibodies and nucleic acid molecules encoding the antibodies thereof also can be provided as kits. Kits can include a pharmaceutical composition described herein and an item for administration. For example, a selected antibody can be supplied with a device for administration, such as a syringe, an inhaler, a dosage cup, a dropper, or an applicator. The kit can, optionally, include instructions for application including dosages, dosing regimens and instructions for modes of administration. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of the antibody in a subject.
9. Formulations/Administration and Uses of Antibodies and Polypeptides
The antibodies provided herein can be provided as a formulation for administration. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations contain at least one active ingredient, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations can conveniently be presented in unit dosage form and can be prepared by methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, N.Y.; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets Dekker, N.Y.; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, N.Y.
The route of antibody administration is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, topical or by sustained release systems as noted below. The antibody is preferably administered continuously by infusion or by bolus injection. One can administer the antibodies in a local or systemic manner.
The antibodies provided herein can be prepared in a mixture with a pharmaceutically acceptable carrier. Techniques for formulation and administration of the compounds of the instant application can be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. This therapeutic composition can be administered intravenously or through the nose or lung, preferably as a liquid or powder aerosol (lyophilized). The composition also can be administered parenterally or subcutaneously as desired. When administered systematically, the therapeutic composition should be sterile, pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art.
Therapeutic formulations can be administered in many conventional dosage formulations. Briefly, dosage formulations of the antibodies provided herein are prepared for storage or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are non-toxic to the recipients at the dosages and concentrations employed, and can include buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS or polyethyleneglycol.
When used for in vivo administration, the antibody formulation must be sterile and can be formulated according to conventional pharmaceutical practice. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The antibody ordinarily will be stored in lyophilized form or in solution. Other vehicles such as naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like can be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
Pharmaceutical compositions suitable for use include compositions wherein one or more rationally designed antibodies are contained in an amount effective to achieve their intended purpose. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Therapeutically effective dosages can be determined by using in vitro and in vivo methods.
An effective amount of antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. In addition, the attending physician takes into consideration various factors known to modify the action of drugs including severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
For any antibody containing a peptide, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC50 as determined in cell culture (e.g., the concentration of the test molecule which promotes or inhibits cellular proliferation or differentiation). Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the antibody molecules described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Molecules which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such molecules lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1, p. 1).
Dosage amount and interval can be adjusted individually to provide plasma levels of the antibody which are sufficient to promote or inhibit cellular proliferation or differentiation or minimal effective concentration (MEC). The MEC will vary for each antibody, but can be estimated from in vitro data using described assays. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Antibody molecules should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, typically between 30-90% and most typically between 50-90%.
In cases of local administration or selective uptake, the effective local concentration of the antibody can not be related to plasma concentration.
A typical daily dosage might range from about 1 /kg to up to 1000 mg/kg or more, depending on the factors mentioned above. Typically, the clinician will administer the molecule until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
Depending on the type and severity of the disease, from about 0.001 mg/kg to abut 1000 mg/kg, more typically about 0.01 mg to 100 mg/kg, more typically about 0.010 to 20 mg/kg of the antibody, for example an antagonist or agonist antibody, is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs or the desired improvement in the patient's condition is achieved. However, other dosage regimes also are contemplated.
H. EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1
Materials and Methods
1. Plasmids A and C
Plasmid A (SEQ ID NO:1), Plasmid C (SEQ ID NO:3), Plasmid D (SEQ ID NO:2) and Plasmid E (SEQ ID NO:4), set forth in FIGS. 3-6, are used to produce recombinant Fab antibodies. They contain high-copy, ColE1 replication origins that are compatible with E. coli. In addition, the ColE1 replication origins of plasmids A and D are compatible with the replication origins of plasmids C and E. Moreover, they replicate at similar copy numbers when co-cultured in the same E. coli cell. The plasmids contain a STII leader sequence that allows for the Fab to be translocated to the periplasm, to allow for better folding, and an Ara promoter for arabinose inducible gene expression. Plasmid A contains a heavy chain constant region for production of a Fab heavy chain and includes Flag and His tags for protein purification. Plasmid D contains a heavy chain constant region for production of a Fab heavy chain, and includes Flag, LPETG, and His tags for both protein purification and site specific modification using the protein ligase, sortase. Plasmid C contains a kappa light chain constant region for production of a kappa Fab light chain while Plasmid E contains a lambda light chain constant region for production of a lambda Fab light chain. Thus Plasmids A and D encode Fab heavy chain and Plasmids C and E encode Fab light chain. A Fab is produced upon co-transformation of E. coli with a plasmid encoding a heavy chain and a plasmid encoding a light chain, and induction of the promoter to express the heavy or light chain genes.
2. Cell Lines
E. coli strains BL21 (EMD Biosciences) and LMG194 (ATCC) were used for expression of the Fabs. Ramos B-Lymphocyte cells (ATCC) and Jurkat T-Lymphocyte cells (ATCC) were used for the Apo-ONE homogenous Caspase-3/7 Assay. The BaF3 mouse peripheral blood cell line (Harvey Lodish, Massachusetts Institute of Technology) was used for the EPO Fab library cell based assays.
3. DNA Sequence Compilation Software
The DNA Sequence Compilation Software is provided herein. It implements an in silico method for recombining heavy chain V, D, and J segment sequences and recombining light chain V and J segments while maintaining functional germline antibody sequences. The software systematically combines DNA encoding the VH, DH and JH and VL and JL germline segment sequences, or alternatively a subset of VH, DH and JH and VL and JL germline segment sequences, to produce sequences of recombined heavy chain and light chain variable regions from which nucleic acid molecules are produced for production of a Fab library. The germline segment sequences are inputted from a database. Example 2 provides a description of the software. Example 3 describes how to use the software, and Example 4 describes implementation of the software to compile recombined germline sequences and output the sequences for DNA synthesis.
4. PiccoloTM Automated System
PiccoloTM system is a fully automated system for the rapid optimization of recombinant protein production, sold by The Automation Partnership (TAP) (see e.g., Wollerton et al. (2006) JALA, 11:291-303, which describes the system). The system combines cell culture with automated harvesting, lysis, and purification units. Example 9 provides details about the specifics of the PiccoloTM system operation.
Example 2
DNA Sequence Compilation Software Design
This example describes the DNA Sequence Compilation Software.
A. General Description:
Sequences for VH, DH and JH and VL and JL germline segment sequences are inputted from a database file, i.e. SequenceDatabase.txt file described in detail below. The software 1) generates every possible recombination of VH, DH and JH and VL and JL germline segments to generate sequences encoding a variable heavy (VH) chain and sequences encoding a variable light (VL) chain; 2) translates and frame-checks the sequences; 3) stores the sequences in a SequenceHistory.txt file to keep track of sequences in nucleotide format; and 4) outputs sequences as files that represent 96-well plates for ordering for DNA synthesis. Output is generated in the form of a 96-well plate file that lists the distinct heavy chain or light chain sequences as an array designating each locus in the array corresponding to a well in a 96-well plate. DNA molecules can be synthesized in a 96-well plate based on this output. In addition, the software can rank the sequences and generate diversity scores. The software automatically generates all permutions of recombined nucleic acid sequences encoding VH chains and VL chains (kappa and lambda). If desired, the output file can be manually restricted by the user by selecting germline segments to be recombined by the software, or by choosing sequences that are sufficiently diverse from one another using the ranking function.
B. Software Description:
The DNA Sequence Compilation Software was written in the C# programming language using the Visual Studio 2005 Professional Edition development tool. The DNA Sequence compilation Software require the Microsoft .NET framework 2.0 to run under the Windows XP and Vista 32-bit Edition Operating System. The software is composed of ten modules (see FIG. 7.) These modules include (a) Blast to Diversity Converter; (b) DNA Sequence Compilation GUI; (c) DNA Sequence Compilation Controls; (d) DNA Sequence Compilation Rules; and (e) DNA Sequence Compilation Rules Test, which are provided herein. The remaining five modules are available from public domain software. They include (f) NUnit; (g) formatdb; (h) BLAST; (i) BLASTClust; and (j) XP Explorer Bar.
The modules contain classes. A class is the formal definition of an object. The class acts as the template from which an instance of an object is created at run time. The class defines the properties of the object and the methods used to control the object's behavior. A singleton class is a class that is globally available to be accessed by all other classes. In this case the singleton class is the sequence compiler module, which has every rule for sequence compilation. A static class contains data and functions that can be accessed without creating an instance of the class. Static class members can be used to separate data and behavior that is independent of any object identity; the data and functions do not change regardless of what happens to the object.
Additionally, the software is grouped into 4 layers: 1) the graphical user interface (GUI); 2) GUI controls; 3) Compilation rules; and 4) NCBI tools. The 4 layers are:

    
    
        GUI—The GUI is the presentation of information to and interaction with the computer operator. The GUI is made up of the Blast to Diversity Converter, the DNA Sequence Compilation GUI and NUnit.
        GUI controls—Custom visible Windows controls that are re-used by the GUI. GUI controls is made up of the XP Explorer Bar and the DNA Sequence Compilation Controls.
        Compilation Rules—Implementation of the rules for DNA sequence compilation. The compilation rules are made up of DNA Sequence Compilation Rules and DNA Sequence Compilation Rules Test.
        NCBI tools—A set of tools available from NCBI that allows the user to run searches on a local computer.
    
    


1. Modules
a. DNA Sequence Compilation GUI
DNA Sequence Compilation GUI is the main application. It interacts with the user and enables all the functionality to be available to the user. As a whole, the DNA Sequence Compilation GUI permits the user to selectively restrict germline segments to compile a sequence; automatically compile all sequences; automatically rank all the sequences in terms of sequence diversity, clusters or similarity; and select compiled DNA sequences to be placed in a 96-well plate.
The DNA Sequence Compilation GUI contains the following classes:

    
    
        Program—a static class automatically generated that contains the execution entry point to the application;
        SplashScreen—a user control that displays a splash screen when the application starts and exits;
        MainForm—a form that encapsulates all the functionality that is available to the user. The MainForm also displays the user controls that are implemented in the DNA Sequence Compilation Controls module and initializes the SequenceCompiler singleton defined in the DNA Sequence Compilation Rules module;
        Resources—a resource file containing the image for the splash screen;
        Settings—an automatically generated class that contains the user settings for the 5′ restriction site and 3′ restriction site to be placed with the sequence for the IDT order; and
        AboutBox—a form that displays the application information to the user. It interacts with the user and enables all the functionality to be available to the user.
    
    


b. DNA Sequence Compilation Controls
The DNA Sequence Compilation Controls module contains custom window controls to be used by the DNA Sequence Compilation GUI. It contains the following classes:

    
    
        FabrusDataGridView—a derived class of .NET framework DataGridView that displays the row number in the row header. A .NET framework is part of windows operating systems that contains a large library of pre-coded solutions to common programming problems and manages the execution of programs written specifically for the framework. The DataGridView control provides a customizable table for displaying data.
        WellPlateControl—a custom control that displays 96-well plate information;
        AutoHeavySequenceControl—a custom control that displays all of the automatically compiled DNA sequences in a data grid view for user selection into the 96-well plate, while also displaying rank, diversity score, cluster, V(D)J sequence identifier and DNA sequence protein sequence;
        AutoLightSequenceControl—a custom control that displays all the automatically compiled DNA sequences in a data grid view for user selection into the 96-well plate, while also displaying rank, diversity score, cluster, V(D)J sequence identifier and DNA sequence protein sequence;
        ManualHeavySequenceControl—a custom control that allows the user to manually select a VH, DH and JH sequence with which to compile into a DNA heavy chain sequence;
        ManualLightSequenceControl—a custom control that allows the user to manually select a VL and JL sequence with which to compile into a DNA light chain sequence;
        LightSequenceBlastForm—a custom control that displays in grid format the BLAST results of a light sequence against all other light chains; and
        HeavySequenceBlastForm—a custom control that displays in grid format the BLAST results of a heavy chain sequence against all the other heavy chains.
    
    


c. DNA Sequence Compilation Rules Module
The DNA Sequence Compilation Rules module contains all of the business logic for the compilation of the DNA sequences. This module includes all the rules for compiling functional recombined VDJ and VJ antibody sequences, including compilation, joint generation, stop codon removal, and sequence ranking.
There are six main classes within the DNA Sequence Compilation Module:

    
    
        SequenceCompiler is a singleton class that provides all the DNA sequence compilation functionality. This class provides the ability to automatically or manually generate combined DNA sequences from the V(D)J sequences and all its associated information;
        BlastTable provides all the information regarding diversity scores, cluster number and performs the BLAST search for a specified sequence;
        SequenceHistorian is a class that keeps track of the DNA sequences in nucleotide format that have been ordered from the DNA synthesis company (e.g., IDT, Genscript, Invitrogen, and the like). Persistence is achieved by using a text file named SequenceHistory.txt, which keeps track of all of the sequences in nucleotide format ordered from the DNA Synthesis Company and
        SequenceContainer is a class that holds all the V(D)J sequence information and restriction sites as specified in the text file SequencesDatabase.txt.
        DnaSequence is the parent class that models the heavy and light chain sequences through the use of HeavyChainDnaSequence and LightChainDnaSequence classes, respectively.
        WellPlate—8×12 is a class that models a 96-well plate. It holds references to the DNA sequence instances and allows them to be saved into an order file.
    
    


Additionally, there are three helper classes within the DNA Sequence Compilation Rules:

    
    
        CodonUsageTable is a class that encapsulates the codon usage table for E. coli K12;
        Translator is a static class that performs the translation of the sixty four codons to their respective amino acid equivalents based on the genetic code; and
        ProtoParam is a static class that computes the GRAVY value.
    
    


The following is a discussion of each of the six main classes (i-vi) and the three helper classes (vii-ix) of the DNA Sequence Compilation Rules Module.
i. SequenceCompiler
SequenceCompiler is a singleton class that provides all the DNA sequence compilation functionality. This class provides the ability to automatically or manually generate combined DNA sequences from the V(D)J sequences and all its associated information. Sequence Compiler includes the following functionalities:
1) Algorithm for Automatic DNA Sequence Compilation
SequenceCompiler uses the algorithm shown in FIG. 8 to generate compiled sequences by computing all the possible combinations of the available V(D)J sequences for the heavy and light chains. The algorithm equates to simply computing all the possible combinations of the available V(D)J sequences for the heavy and light sequences, respectively.
For example, if there are three VH sequences, three DH sequences and three JH sequences, the algorithm computes 3*3*3 combinations, thereby producing all 27 possible combined sequences. Similarly, if there are four Vκ sequences and four Jκ sequences, the algorithm computes 4*4 combinations, thereby producing 16 possible combined sequences. In addition, the algorithm computes lambda light chains and kappa light chains separately.
2) Sequence Compilation
Actual sequence compilation starts with the identification of a VH, DH, and JH sequence and a VL and JL sequence for a heavy and light chain respectively from the SequenceDatabase.txt file. Individual segments are identified as described in Example 4. Once the individual segments are identified, V(D)J joints are created, and finally all nucleotide pieces (the segments and the joints) are joined together thus compiling the sequence (see FIG. 9). The rules for joint generation, removal of STOP codons and restriction sites are set forth below under Section Headings “V(D)J Joint Generation,” “Stop Codon Removal,” and “Codon Usage Table,” respectively.
3) V(D)J Joint Generation
The joints in the compiled sequences between the different V(D)J sequences are selected so that the resulting sequences are in frame. SequenceCompiler uses the following three rules for determining reading frames.

    
    
        1. The V Sequence is always reading frame 1.
        2. The D Sequence has a frame that yields the most negative GRAVY (Grand Average of Hydropathy) value.
        3. The J Sequence has a reading frame as specified in the SequenceDatabase.txt file (see Example 4.1 entitled “Creating the V(D)J Sequence Database File,” which describes that the J sequences are set forth in three letter codons to designate the reading frame; also see Table 13).
    
    


Based on the rules listed above, in the joints between sequences, a codon will not automatically be generated by simply recombining the bases from either of the sequences. SequenceCompiler uses the rules listed in Table 19 to generate a new codon. For example, when creating a light chain V-J joint where the V sequence is in reading frame 1 and the J sequence is in reading frame 1, a “G” nucleotide will be inserted between the V-J joint, thereby maintaining the reading frame of the entire segment.







TABLE 19





V(D)J joint generation.


















V-D





Joints
(Heavy Chains)


V
D
Frame
Rule





0
0
0
NO MANIPULATION


0
1
1
Delete 1 nucleotide from 5′ end of D


0
2
2
Add a “G” between V-D


1
0
1
Delete 1 nucleotide from 3′ end of V


1
1
2
Add a “G” between V-D


1
2
3
NO MANIPULATION


2
0
2
Add a “G” between V-D


2
1
3
NO MANIPULATION


2
2
4
Delete 1 nucleotide from 3′ end of V





D-J



Joints
(Heavy Chains)


D
J
Frame
Rule





0
0
0
NO MANIPULATION


0
1
1
Delete 1 nucleotide from 5′ end of J


0
2
2
Add a “G” between D-J


1
0
1
Delete 1 nucleotide from 3′ end of D


1
1
2
Add a “G” between D-J


1
2
3
NO MANIPULATION


2
0
2
Add a “G” between D-J


2
1
3
NO MANIPULATION


2
2
4
Delete 1 nucleotide from 3′ end of D





V-J



Joints
(Light Chains)


V
J
Frame
Rule





0
0
0
NO MANIPULATION


0
1
1
Delete 1 nucleotide from 5′ end of J


0
2
2
Add a “G” between V-J


1
0
1
Delete 1 nucleotide from 3′ end of V


1
1
2
Add a “G” between V-J


1
2
3
NO MANIPULATION


2
0
2
Add a “G” between V-J


2
1
3
NO MANIPULATION


2
2
4
Delete 1 nucleotide from 3′ end of V









4) Stop Codon Removal
There are situations where V(D)J joint generation will generate a STOP codon (TAA, TAG or TGA). SequenceCompiler removes detrimental STOP codons according to the following rules, whereby nucleotides encoding a Stop Codon are replaced with the designated new codon:














STOP Codon
New Codon









TAA
TAT



TAG
TAT



TGA
TCA










Once SequenceCompiler has completed the successive steps of V(D)J joint generation and STOP codon removal, the protein sequence is complete and functional.
5) Algorithm for Ranking Sequences
Once sequences are compiled, each sequence is ranked and presented to the user in order of highest diversity to lowest diversity (See Table 20 and FIGS. 17 and 19). This presentation of ranked sequences allows the user several options when selecting sequences to be placed in the 96-well plate order file. The user can select the most diverse sequences, or sequences that are most similar to each other.
Compiled sequences are ranked by the software by using a combination of the diversity score and cluster number information for the sequences. Diversity scores are generated by the blast to diversity converter module, as described in Section B.1.e, and cluster information is generated by BLASTClust, as described in Section B.1.i. The sequence ranking algorithm flowchart is shown in FIG. 10. The algorithm picks from each cluster the sequence with the lowest diversity score, orders those sequences in terms of diversity from the lowest diversity number to the highest diversity number (a low diversity score indicates lack of similarity, and therefore highest diversity), ranks the sequences in that order, eliminates those sequences from the respective clusters, and then iteratively applies the previous procedure until all the sequences are ranked.
An example of the ranking of Sequences using this algorithm is set forth in Table 20. The sequences in the table are ordered from highest diversity to lowest diversity. The sequence with the lowest diversity score indicates the highest diversity and receives the rank of 1. The second sequence belongs to the same cluster as the first sequences, and receives the rank of 64. This data also indicates to the user that there are a total of 63 clusters defined, since sequence 001 and sequence 002 are both in cluster 39 and they have consecutive diversity scores.







TABLE 20







Ranking of Sequences



















Diversity
V
J
Amino Acid
SEQ ID



Selected
Rank
Cluster
Score
Sequence
Sequence
Sequence
NO





001

  1
39
73.202
V3-4
IGLJ6*01
QTWTQEPSFSVSP . . .
1905


 


002

 64
39
73.305
V3-4
IGLJ7*01
QTWTQEPSFSVSP . . .
1906


 


003

  2
21
73.560
V4-2
IGLJ6*01
QAVLTQPSSLSASP . . .
1907


 


004

 65
32
73.603
V4-2
IGLJ7*01
QAVLTQPSSLSASP . . .
1908


 


005

  3
32
73.876
V1-22
IGLJ6*01
NFMLTQPHSVSES . . .
1909


 


006

 66
24
74.017
V1-22
IGLJ7*01
NFMLTQPHSVSES . . .
1910


 


007

  4
24
74.040
V5-1
IGLJ7*01
LPVLTQPPSASALL . . .
1911


 


008

 67
39
74.056
V5-1
IGLJ6*01
LPVLTQPPSASALL . . .
1912


 


009

127
39
74.081
V3-4
IGLJ1*01
QTWTQEPSFSVSP . . .
1881


 


010

190
21
74.399
V3-4
IGLJ4*01
QTWTQEPSFSVSP . . .
1913


 


011

128
39
74.458
V4-2
IGLJ1*01
QAVLTQPSSLSASP . . .
1914


 


012

253
21
74.571
V3-4
IGLJ5*01
QTWTQEPSFSVSP . . .
1915


 


013

191
21
74.577
V4-2
IGLJ4*01
QAVLTQPSSLSASP . . .
1883









ii. BlastTable
BlastTable provides all the information regarding diversity scores, cluster number and performs the BLAST search for a specified sequence;
iii. SequenceHistorian
SequenceHistorian is a class that keeps track of the DNA sequences in nucleotide format that have been ordered for DNA synthesis. The persistence is achieved by using a text file named SequenceHistory.txt.
iv. SequenceContainer
SequenceContainer is a class that reads V(D)J sequences and restriction sites from a text file and stores them in memory for later retrieval for either manual (i.e. restricted selection) or automatic compilation.
v. DnaSequence
DnaSequence is the parent class that models the heavy and light chain sequences through the use of HeavyChainDnaSequence and LightChainDnaSequence classes, respectively. The only difference between the HeavyChainDnaSequence and LightChainDnaSequence classes is in the way the sequence is compiled which is implemented in the constructor of each class.
Each class contains Fields, Properties and Methods. In addition, the parent DnaSequence class contains events and Nested Types. Thus, DnaSequence implements all the functionality that is common to heavy chain and light chain sequences. This includes the fields such as AminoAcids, Cluster, DiversityScore, _SEQ_ID, Name, Notes, Nucleotides, Rank, and RestrictionSites and methods such as removing restriction sites, removing stop codons and silencing restriction sites. HeavyChainDnaSequence class contains fields for the VH, JH and DH sequences, the VH-JH and JH-DH joints and methods to generate the VH-JH and JH-DH joints. LightChainDnaSequence class contains fields for the VL and JL sequences, the VL-JL joint and methods to generate the VL-JL joint.
vi. WellPlate—8×12
WellPlate—8×12 is a class that models a 96-well plate. It holds references to the DNA sequence instances and allows them to be saved into an order file.
vii CodonUsageTable
CodonUsageTable is a class that encapsulates the codon usage table for E. coli K12 (see, Table 14). The CodonUsageTable is used by the software to modify reading frame problems as well as restriction site silencing. The software is programmed to recognize restriction sites that will be used to insert the DNA into the desired plasmid. When these restriction sites are observed in the compiled DNA sequence, the software modifies the nucleotide sequence to modify the undesired restriction site while maintaining the appropriate amino acid sequence. The above mentioned codon redundancy is used to modify codons. A codon change is used to provide a beneficial increase in the frequency of usage of the possible newly used codon. In addition, modifying the last base of a codon (the degenerate position) is preferred over modifying the first or second base of the codon.
viii. Translator
Translator is a static class that performs the translation of the sixty four codons to their respective amino acid equivalents based on the genetic code.







TABLE 20







DNA Codon table.









Amino Acid
SLC
DNA codons





Isoleucine
I
ATT, ATC, ATA


 


Leucine
L
CTT, CTC, CTA, CTG, TTA, TTG


 


Valine
V
GTT, GTC, GTA, GTG


 


Phenylalanine
F
TTT, TTC


 


Methionine
M
ATG


 


Cysteine
C
TGT, TGC


 


Alanine
A
GCT, GCC, GCA, GCG


 


Glycine
G
GGT, GGC, GGA, GGG


 


Proline
P
CCT, CCC, CCA, CCG


 


Threonine
T
ACT, ACC, ACA, ACG


 


Serine
S
TCT, TCC, TCA, TCG, AGT, AGC


 


Tyrosine
Y
TAT, TAC


 


Tryptophan
W
TGG


 


Glutamine
Q
CAA, CAG


 


Asparagine
N
AAT, AAC


 


Histidine
H
CAT, CAC


 


Glutamic acid
E
GAA, GAG


 


Aspartic acid
D
GAT, GAC


 


Lysine
K
AAA, AAG


 


Arginine
R
CGT, CGC, CGA, CGG, AGA, AGG


 


Stop codons
Stop
TAA, TAG, TGA





In this table, the twenty amino acids found in proteins are listed, along with the single-letter code used to represent these amino acids in protein data bases. The DNA codons representing each amino acid are also listed. All 64 possible 3-letter combinations of the DNA coding units T, C, A and G are used either to encode one of these amino acids or as one


of the three stop codons that signals the end of a sequence. While DNA can be decoded unambiguously, it is not possible to predict a DNA sequence from its protein sequence. Because most amino acids have multiple codons, a number of possible DNA sequences might represent the same protein sequence.






ix. ProtoParam
ProtoParam is a static class that computes the GRAVY value. The GRAVY (Grand Average of Hydropathy) value for a peptide or protein is calculated as the sum of hydropathy values of all the amino acids divided by the number of residues in the sequence (Table 21). In essence, the GRAVY score is the relative value for the hydrophobic residues of the protein. This information is used by the V(D)J joint generation software when computing the D sequence reading frame. This reading frame is determined by the most negative GRAVY value.







TABLE 21







Hydropathy Values










Amino Acid
Hydropathy Value














Ala
1.800



Arg
−4.500



Asn
−3.500



Asp
−3.500



Cys
2.500



Gln
−3.500



Glu
−3.500



Gly
−0.400



His
−3.200



Ile
4.500



Leu
3.800



Lys
−3.900



Met
1.900



Phe
2.800



Pro
−1.600



Ser
−0.800



Thr
−0.700



Trp
−0.900



Tyr
−1.300



Val
4.200










d. DNA Sequence Compilation Rules Test Module
The DNA Sequence Compilation Rules Test module is a collection of NUnit automated tests that perform unit tests for each class. These tests help to ensure the individual modules and classes listed are running properly. These nine classes include:
BlastTableTest;
CodonUsageTableTest;
HeavyChainDnaSequenceTester;
LightChainDnaSequenceTester;
ProtoParamTest;
SequenceCompilerTest,
SequenceContainerTest;
TranslatorTest; and
TranslatorWrapper.
e. Blast to Diversity Converter Module
The Blast to Diversity Converter is an executable that takes a BLAST output file and outputs a diversity score. It contains five classes:

    
    
        Program—a static class that is automatically generated and contains the execution entry point to the application;
        Main Form—a button for the operator to click on to select a file to be converted;
        Resources—an empty resource file that is created by default;
        Settings—an automatically generated class that contains the user settings; and
        BlastConverter—a class that contains the implementation of the conversion of the BLAST output file to a diversity score file.
    
    


i. BLAST Converter Algorithm
The BLAST converter algorithm takes a BLAST output file and applies the following equation to compute a sequence's diversity score.




  
    DiversityScore
    ⁡
    
      (
      
        seq
        i
      
      )
    
  
  =
  
    
      
        1
        N
      
      ⁢
      
        
          ∑
          
            
              k
              =
              1
            
            ,
            
              k
              ≠
              i
            
          
          N
        
        ⁢
        
          
            BLAST
            ⁡
            
              (
              
                i
                ,
                k
              
              )
            
          
          2
        
      
    
  




The algorithm computes a standard deviation for the BLAST scores by taking all the individual BLAST numbers, squaring them, adding up all the squares, normalizing by the number of samples (N), and then taking the square root.
For example, a sequence's diversity score was computed by applying the algorithm to the following BLAST output file (-m 9 BLAST switch, i.e.Alignment view options, tablular with comment lines; see Section B.1.h.):

    
    
        #BLASTP 2.2.17
        #Query: gnl|Fabrus|V4-2_IGLJ2*01
        #Database: AllLightChainSequences.txt
        #Fields: Query id, subject id, % identity, alignment length, mismatches, gap openings, q. start, q. end, s. start, s. end, e-value, bit score
        gnl|Fabrus|V4-2_IGLJ2*01 gnl|Fabrus|V4-2_IGLJ2*01 100.00 116 0 0 1 116 1 116 1e-050 186
        gnl|Fabrus|V4-2_IGLJ2*01 gnl|Fabrus|V4-2_IGLJ7*01 98.28 116 2 0 1 116 1 116 2e-049 182
        gnl|Fabrus|V4-2_IGLJ2*01 gnl|Fabrus|A5_IGKJ7*01 38.82 85 46 3 31 115 34 112 2e-007 43.1
        #BLASTP 2.2.17
        #Query: gnl|Fabrus|V4-2_IGLJ1*01
        #Database: AllLightChainSequences.txt
        #Fields: Query id, subject id, % identity, alignment length, mismatches, gap openings, q. start, q. end, s. start, s. and, e-value, bit score
        gnl|Fabrus|V4-2_IGLJ1*01 gnl|Fabrus|V4-2_IGLJ1*01 100.00 116 0 0 1 116 1 116 3e-050 185
        gnl|Fabrus|V4-2_IGLJ1*01 gnl|Fabrus|V4-2_IGLJ6*01 98.28 116 2 0 1 116 1 116 4e-049 181
    
    


The following output file is generated, which provide diversity scores for the respective variable heavy chain sequences. The lowest diversity score indicates the least similarity between the sequence and the next most related sequence, and therefore has the highest diversity.

    
    
        gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ2*01 119.675348536301
        gnl|Fabrus|VH3-15_IGHD5-24*01_IGHJ2*01 136.102291820977
        gnl|Fabrus|VH2-26_IGHD1-7*01_IGHJ4*01 111.073223319952
        gnl|Fabrus|VH3-13_IGHD1-14*01_IGHJ1*01 136.760646289454
        gnl|Fabrus|VH3-30_IGHD6-13*01_IGHJ6*01 139.423052600581
    
    


f. NUnit
NUnit is a unit-testing framework for all .Net languages. NUnit testing tools provide a means to test an object model, and is useful in ensuring that the application and its logic work correctly. Initially ported from JUnit, the current production release, version 2.4.2, is the fifth major release of this xUnit based unit testing tool for Microsoft .NET. It is written entirely in C# and has been completely redesigned to take advantage of many .NET language features, for example custom attributes and other reflection related capabilities. NUnit brings xUnit to all .NET languages. NUnit is available free to the public from the organization NUnit.
g. Formatdb
Formatdb must be used in order to format protein or nucleotide source databases before these databases can be searched by blastall, blastpgp or MegaBLAST. The source database can be in either FASTA or ASN.1 format. Once a source database has been formatted by formatdb it is not needed by BLAST.
The following Command Line Options are used when generating output files with formatdb:

    
    
        -i Input File for formatting
        -p Type of File (T—protein; F—nucleotide; Default =T)
        -o Parse options
        
            T—True: Parse SeqlD and create indexes
            F—False: Do not parse SeqID. Do not create indexes.
        
        
    
    


The version win32-ia32 (2.2.17) is currently used. Formatdb can be downloaded from the National Center for Biotechnology Information.
h. The Basic Local Alignment Search Tool (BLAST)
The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.
The following Command Line Options are used when generating output files with blastall:

    
    
        -p Program Name (“blastp”, “blastn”, “blastx”, “tblastn”, or “tblastx”)
        -d Database
        -l Query File
        -o BLAST report Output File
        -m Alignment View Options (0=default; 9=tabular with comment lines)
        -b Number of Database Sequence to show alignments for (B) [Integer]
    
    


The version win32-ia32 (2.2.17) is currently used. BLAST can be downloaded from the National Center for Biotechnology Information.
i. BLASTClust
BLASTClust is a program within the stand alone BLAST package used to cluster either protein or nucleotide sequences. The program begins with pairwise matches and places a sequence in a cluster if the sequence matches at least one sequence already in the cluster. In the case of proteins, the blastp algorithm is used to compute the pairwise matches; in the case of nucleotide sequences, the Megablast algorithm is used.
In the simplest case, BLASTClust takes as input a file containing concatenated FASTA format sequences, each with a unique identifier at the start of the definition line. BLASTClust formats the input sequence to produce a temporary BLAST database, performs the clustering, and removes the database at completion. Hence, there is no need to run formatdb in advance to use BLASTClust. The output of BLASTClust is a file, with one cluster to a line, of sequence identifiers separated by spaces. The clusters are sorted from the largest cluster to the smallest.
The following Command Line Options are used when generating output files with blastclust:

    
    
        -d Sequence Database Name
        -o Output File to save cluster list
        -S Similarity Threshold
        
            if <3 then the threshold is set as a BLAST score density (0.0 to 0.3; default=1.75)
            if >3 then the threshold is set as a percent of identical residues (3 to 100)
        
        
    
    


The version win32-ia32 (2.2.17) is currently used. BLASTClust can be downloaded from the National Center for Biotechnology Information
j. XPExplorerBar
The XPExplorerBar is a fully customizable Windows XP style Explorer Bar that supports Windows XP themes and animated expand/collapse with transparency. The XP Explorer Bar is available as free public domain software.
Example 3
Sequence Database
The DNA Sequence compilation software requires a file containing sequence information used by the program to perform sequence compilation in accordance with sequence compilation rules. This is called the SequenceDatabase.txt file. All recombined sequences are generated from the sequence data in the file. Hence, the file contains all sequences that are used in performing the sequence compilation by the software. For example, the file contains nucleotide sequences for all desired germline segments and also contains nucleotide recognition sequences for restriction enzyme. The sequences in the database include any known sequence, and can be updated manually at any time. V, D, and J antibody germline sequences of human origin were obtained from the NCBI IgBLAST database (available from the National Center for Biotechnology Information) and the International ImMunoGeneTics Information system (IMGT) and were entered into a sequence database file. The database file served as an input file for the Sequence Compilation Software.
The format of the SequenceDatabast.txt file is illustrated in FIG. 11. The SequencesDatabase.txt file is updated by simply manually adding new sequences according to the following rules:

    
    
        All sequences must be specified using the FASTA format.
        Three letter codons are specified for J sequences.
        All sequences must have a “blank line” between them.
        // is used a the beginning of any line containing extraneous comments.
        Section titles are indicated by the following designations:
        
            [VH]—List of VH sequence to follow.
            [DH]—List of DH sequence to follow.
            [JH]—List of JH sequence to follow.
            [VK]—List of Vκ sequence to follow.
            [JK]—List of Jκ sequence to follow.
            [VL]—List of VL sequence to follow.
            [JL]—List of JL sequence to follow.
            [Restriction Sites]—List of restriction sites to follow.
        
        
    
    


Example 4
Generation of Heavy and Light Chain DNA Files and Ranking Analysis
This example describes methods for generating recombined heavy and light chain sequence files that will be used by the DNA Sequence Compilation Software. The files were created immediately upon initiating the software. The files are created from the SequenceDatabase.txt file and are analyzed using public domain software, such as NCBI's formatdb, BLAST and BLASTclust, to compute diversity and clustering information for the sequences included in the database file. The ranking function of the software is performed automatically, but can optionally be used by the user to rank the sequences for selection and ordering for DNA synthesis.
To generate sequence files, a database file that contains DNA sequences for all desired V, (D), and J variable regions sequences was generated as described in Example 3. The DNA Sequence Compilation Software mechanically recombined all heavy chain variable germline segments and light chain variable germline segment sequences in the database into nucleic acid molecules encoding functional amino acid sequences. The software translated the nucleic acid sequence into an amino acid sequence to create sequence files. The recombined amino acid sequences were compared to determine the similarity of each compiled sequence to every other compiled sequence, which was subsequently used to generate a diversity score. Finally, the compiled sequences were clustered by sequence similarity. These various steps of the software were performed immediately upon initiating the software and are described below.
1. Compiling Heavy Chains and Light Chains
Once the database file was updated and contains all the desired V(D)J sequences, the database file was used as an input file by DNA Sequence Compilation software to mechanically compile full length heavy chain or light chain sequences in accordance with the DNA sequence compilation rules as described in Example 2, Section B.1.c. The software created files containing the full-length amino acid sequences encoded by compiled variable heavy and light chain germline segments. These are contained in the following two files:

    
    
        TempLightChainSequences.txt—This file contains all the lambda and kappa light chain sequence combinations in the amino-acid format.
        TempHeavyChainSequences.txt—This file contains all the heavy chain sequence combinations in the amino-acid format.
    
    


The compiled light and heavy chain amino acid sequences are each identified with a unique sequence ID (gnl|Fabrus|V_(D)_J), examples of which are illustrated below.








>gnl|Fabrus|V4-2_1GLJ2*01



(SEQ ID NO: 2027)



QAVLTQPSSLSASPGASASLTCTLRSGINVGTYRIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVPSRFSGSKDASANAGILLISGLQSEDE



 


ADYYCMIWHSSASVVFGGGTKLTVL


 


>gnl|Fabrus|VH1-2_IGHD2-21*01_IGHJ1*01


(SEQ ID NO: 2028)



QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRS



 


DDTAVYYCAREAYCGGDCYSAEYFQHWGQGTLVTVSS






2. Ranking Recombined Variable Heavy and Light Chains
Recombined variable heavy and light chain sequences were compared by the software for sequence similarity between and among all recombined sequences. This is performed using the NCBI utility BLAST. The Blast bit score is used to generate a diversity score. Recombined variable heavy and light chain sequences also were analyzed for sequence similarity using cluster analysis (e.g. BLASTclust).
As described in Example 2, under the heading “Alogrithm for ranking Sequences,” compiled sequences were ranked by the software by using a combination of the diversity score and cluster number information for the sequences. The ranking occurs automatically upon intiation of the program, and can be viewed by the user using the auto compilation function as described in Example 5 below. For example, FIGS. 17 and 19 exemplify screen shots following auto compilation, and depict columns indiating the cluster, diversity score and rank for exemplary heavy and light chain sequences.
The following sections describe the various steps for determining the diversity score and the cluster in order to rank the sequences using the Alogrithm described in Example 2.
a. Calculating Compiled Sequence Similarity and Diversity Scores
The compiled sequences were compared in order to determine the similarity of each compiled sequence to every other compiled sequence. This information was used to generate a diversity score. To start, the TempLightChainSequences.txt and TempHeavyChainSequences.txt files were copied to a folder (as created by the user) and a DOS command prompt window was opened. The NCBI DNA utility formatdb was used to prepare the files for use by BLAST by first using the following command prompts for the respective light chain and heavy chain files:
Run formatdb -i TempLightChainSequences.txt -p T -o F
Run formatdb -i TempHeavyChainSequences.txt -p T -o F
Formatdb must be installed in the folder for which the DOS command prompt window is opened or the utility will not be found. Alternatively, a path can be added to direct the DOS command to utilize formatdb from the folder it was previously installed in. For example, if all the NCBI utilities are downloaded into a folder entitled “NCBIUtilities” then the command prompts are be as follows:
Run NCBIUtilities\bin\formatdb -i TempLightChainSequences.txt -p T -o F
Run NCBIUtilities\bin\formatdb -i TempHeavyChainSequences.txt -p T -o F
BLAST command prompts were then used to generate output files LightChainBlastResults.txt and HeavyChainBlastResults.txt. The command prompts are as follows:

    
    
        Run blastall -p blastp -d TempLightChainSequences.txt -l TempLightChainSequences.txt -o LightChainBlastResults.txt -m 9 -b 500

(The -b value must be equal to or greater than the number of sequences to BLAST.)

        Run blastall -p blastp -d TempHeavyChainSequences.txt -l TempHeavyChainSequences.txt -o HeavyChainBlastResults.txt -m 9 -b 8000

(The -b value must be equal to or greater than the number of sequences to BLAST.)

    
    


Once the BLAST output files were generated, the BLAST bit score is used to calculate a diversity score for each sequence using the algorithm shown in Example 2, under the sub-section heading “Blast Converter Algorithm” using the following commands:

    
    
        (a) Run BlastToDiversityConverter.exe (This file runs automatically each time the software is started.)
        (b) Open the file LightChainBlastResults.txt
        (c) Save this file as AllLightChainSequencesDiversityScores.txt
        (d) Open the file HeavyChainBlastResults.txt
        (e) Save this file as AllHeavyChainSequencesDiversityScores.txt
    
    


b. Clustering Compiled Sequences by Sequence Similarity
Finally, the sequences are clustered based on sequence similarity by generating pairwise matches using the NCBI utility BLASTClust. BLASTClust is used to generate two output files, AllLightChainsBLastClust.txt and AllHeavyChainsBlastClust.txt. The current example dictates that two sequences must be either 95% or 96% identical to register a pairwise match. The command prompts are as follows:

    
    
        Run blastclust -d TempLightChainSequences.txt -o AllLightChainsBlastClust.txt -S 95
        Run blastclust -d TempHeavyChainSequences.txt -o AllHeavyChainsBlastClust.txt -S 96
    
    


The BlastClust setting (-S) can be modified to in order to better suit any individual requirements that are necessary.
Once all the files are generated (as indicated above), the following files are manually copied into the installation folder of the DNA Sequence Compilation Tool.

    
    
        (a) AllHeavyChainsBlastClust.txt
        (b) AllLightChainsBlastClust.txt
        (c) AllLightChainSequencesDiversityScores.txt
        (d) AllHeavyChainSequencesDiversityScores.txt
    
    


Example 5
Installation and Graphical User Interface of the DNA Sequence Compilation Tool Software
The DNA Sequence Compilation Software is used to recombine heavy and light chain antibody sequences and subsequently select desired sequences to populate a 96-well plate output file, which serves as an order file for purchasing DNA sequences in nucleotide format from a DNA Synthesis Company. This example illustrates the software requirements as well as the controls the user encounters when using the DNA Sequence Compilation Software, including Main Menu Options and Explorer Bar Options.
A. Software Overview
The software is designed to run on a computer that uses Windows XP or Windows Vista as the operating system. The software is installed by running the executable file setup.exe and installing the program in a folder of the users choice. Once installed, the user encounters Main Menu Options as well as Explorer Bar Options. The details are described below.
1. System Requirements
The DNA Sequence Compilation Software has been designed to run on a local machine as a stand alone application.

    
    
        Operating System: Windows XP/Vista 32-bit Edition
        Internet Connection: Not Required
        Database Server: None
    
    


2. Installation of the DNA Sequence Compilation Tool Program
To install the program, run the executable file setup.exe and install the program in a folder of your choice.
3. Main Menu Options (see FIG. 12)
There are three main menu options.

    
    
        File—The file menu is used to close the application.
        Well-Plate—The well-plate menu is used to generate an order, clear the entire plate or a selected well, or configure the restriction sites for the well-plate order generation.
        Help—The help menu is used to find out what version of the software you are running.
    
    


4. Explorer Bar Options
There are three explorer bar options (see FIG. 12; left column):

    
    
        Manual Compilation—Manual compilation contains items used to display the light chain or heavy chain manual compilation form, which allows users selection of germline segments.
        Auto Compilation—Auto compilation contains items used to display the light chain or heavy chain automatic compilation form.
        Well Plate—Well plate contains items used to display the well plate form.

C. Starting the Application

    
    


The software is launched either by clicking on the Fabrus DNA Compilation Tool on the All Programs Windows menu or by double-clicking the Fabrus DNA Compilation Tool shortcut on the desktop. A Splash Screen (FIG. 13) appears upon application startup and then fades into the background.
D. Generating an Output Plate Using the DNA Sequence Compilation Software
The DNA Sequence Compilation Software is used to recombine heavy and light chain antibody sequences and subsequently select desired sequences to populate a 96-well plate output file which serves as an order file for purchasing DNA sequences in nucleotide format from a DNA Synthesis Company. In addition to the plate file, a SequenceHistory.txt file is generated to keep track of all sequences ordered. Users can select either manual compilation or auto compilation in order to generate sequences and subsequently select individual sequences for inclusion into the 96-well plate grid, as described below in sections C.2-4. The software program also computes a diversity score for all sequences. The following sections describe user options for selecting and compiling germline sequences encoding variable heavy and light chains for DNA synthesis ordering.
1. The 96-Well Plate Screen
When the application launches, the 96 Well-Plate screen is displayed. As is shown in FIG. 12, the 96-well plate is empty at application startup. The Main Menu is observed horizontally along the top of the screen, underneath the title DNA Sequence Compilation. The Explorer Bar is observed vertically along the left side of the screen. The user must populate the empty 96-well plate with DNA sequences that will be selected for manufacture as described herein below using either the manual compilation function or the automatic compilation function. The operating functions within the 96-well plate screen that permit population of the screen include the following:
a. Selecting Well Plate Restriction Sites
Restriction enzyme sequences are selected by the user from the Sequencedatabase.txt file to add to the compiled DNA sequence at the 5′ and 3′ ends for cloning purposes. The following commands are used to specify the 5′ and 3′ restriction sites that are added to the plate order according to antibody chain type:
Heavy Chains:

    
    
        Well Plate>Configuration>Heavy Chains>5′ End→choose; and Well Plate>Configuration>Heavy Chains>3′ End→choose.
    
    


Kappa Light Chains:

    
    
        Well Plate>Configuration>Kappa Light Chains>5′ End→choose; and Well Plate>Configuration>Kappa Light Chains>3′ End→choose.
    
    


Lambda Light Chains:

    
    
        Well Plate>Configuration>Lambda Light Chains>5′ End→choose; and WellPlate>Configuration>Lambda Light Chains>3′ End→choose.
    
    


This defines the restriction site that is prefixed and suffixed to each sequence chain type in the order file and can be viewed in the IDT Order Well Plate Start/Stop Restriction Sites box (See FIG. 12 and FIG. 14). The compilation software includes a function to internally search compiled sequences for the chosen restriction site and modify the nucleotide sequence in accord with codon usage to remove any internal restriction sites that can be incompatible for cloning purposes. This is completed prior to placing the sequences in the well plate for ordering such that only those sequences “with restriction sites silenced” are ordered. This is described in Example 2 in the section entitled “Codon usage Tables,” and in Section 2 below.
b. Placing Sequences in the Well Plate
The selection of sequences is performed from either manual compilation or auto compilation screens as described below in Sections C.2 and C.3. As described below, in the manual compilation or auto compilation screens, following compilation, sequences are selected by checking the “Selected” column (for auto compilation) or clicking the “Add to Well Plate” button (for manual compilation).
c. Displaying Well Sequence Information
The sequence of a compiled sequence placed in a well is displayed by clicking on the well. The well for the selected sequence is highlighted. For example, FIG. 14 shows a model screen for a single 96-well plate containing compiled sequences which the user has selected for ordering. In row A of the Figure, columns 1-3 (shaded in medium gray) indicate a cell with a compiled heavy chain sequence; columns 3-6 (shaded in light gray) indicate a cell with a compiled light chain sequence. The highlighted cell (row A, column 5, indicated in dark shading) indicates the cell for which the individual data is observed in the Sequence Information box at the bottom of the display.
The Sequence Information text box is then updated with the sequence information for that well. The Sequence Information box lists all the pertinent information for a particular sequence, including the name, the nucleotide sequence, the amino acid sequence, the diversity score and individual component titles for the V(D)J recombination. For example, in FIG. 14, the highlighted sequence is a variable light chain compiled from the VL germline segment V3-4 and the JL germline segment IGLJ7*01.
d. Generating an Order
An order is generated for the 96-well plate regardless of how many samples are placed in the plate, i.e. from one (1) to 96 sequences can be ordered from a 96-well plate. To generate an order, the option Well Plate>Generate Order is chosen and a filename is inputted when requested. The order file is saved in a comma separated variable (.csv) format. Example 4 describes compilation, output and ordering of exemplary sequences compiled using the Software compilation software.
e. Clearing a Selected Well of a Well Plate
In order to clear a specific well in the plate, click on the selected well, then choose WellPlate>Clear>Selected Well or, alternatively, right click on the desired well and select “Clear Selected Well”.
f. Clearing all Sequences from an Entire Well Plate
The Well Plate is not automatically cleared when an order is generated. In order to clear the entire Well-Plate, choose Well Plate>Clear>Entire Plate.
2. Manual Compilation of Sequences
Compiled sequences can be generated manually by user selection using the Manual Compilation option.
a. Manual Compilation of Light Chains
To manually compile a light chain, choose the option Manual Compilation>Light Chain from the explorer bar. The manual compilation screen is displayed in FIG. 15. The manual compilation light chain screen allows the user to compile either a Lambda or Kappa sequence. This option is available under the heading Type and is chosen by selecting the circle in front of either Lambda or Kappa at the top of the screen. The software does not allow the combination of a Vl and a Jk sequence or vice-versa.
i. Compiling Lambda Light Chains
The following steps show how to compile a Lambda Light Chain.

    
    
        Select Type>Lambda.
        Select the desired Vλ Sequence from the VL Sequence Combo box. All of the available Vλ nucleotide sequences that are entered in the SequenceDatabase.txt file appear in the drop down menu box underneath.
        Select the desired Jλ Sequence from the JL Sequence Combo Box. All of the available Jλ nucleotide sequences that are entered in the SequenceDatabase.txt file appear in the drop down menu box underneath.
        Click “Add to Well Plate” button to add the compiled sequence to the well plate.
    
    


Two boxes are observed under the Dna Sequence heading. The “No Silencing of restricted Sites” box displays the individual Vλ and Jλ nucleotide sequences plus any V-J joints generated in accord with the sequence compilation rules. The corresponding encoded amino acid sequence also is displayed. For example, in FIG. 15, the first sequence displayed in the “no silencing of restricted sites” box corresponds to the selected Vλ sequence (in this case V1-11). Following this is a depiction of three nucleotides “cct” encoding proline (P), which correspond to the joint region created by performance of the compilation method in order to generate a functional nucleic acid molecule. The last sequence displayed corresponds to the selected Jλ sequence (in this case IGLJ1*01). The “With Restriction Sites Silenced” box displays the compiled sequence containing any necessary nucleotide modifications to the sequence to silence restriction sites, while maintaining codon usage. Any restriction sites silenced by the software are displayed in the “Notes” box. The sequence with the restriction sites silenced is the sequence that is placed in the order file.
(ii) Compiling Kappa Light Chains
The following steps show how to compile a Kappa Light Chain.

    
    
        Select Type>Kappa.
        Select the desired VK Sequence from the VK Sequence Combo box. All of the available Vκ nucleotide sequences that are entered in the SequenceDatabase.txt file appear in the box underneath.
        Select the desired JK Sequence from the JK Sequence Combo Box. All of the available Jκ nucleotide sequences that are entered in the SequenceDatabase.txt file appear in the box underneath.
        Click “Add to Well Plate” button to add the compiled sequence to the well plate. All compiled sequences are added to the well plate for ordering for DNA synthesis.
    
    


As above for compiling lambda light chain sequences, two boxes are observed under the Dna Sequence heading. The “No Silencing of restricted Sites” box displays the individual V and J sequences plus any V-J joints generated. The “With Restriction Sites Silenced” box displays the compiled sequence with any sequence modifications to silence selected restriction sites. Any restriction sites silenced by the software are displayed in the “Notes” box. The sequence with the restriction sites silenced is the sequence that is placed in the order file.
b. Manual Compilation of Heavy Chains
To manually compile a heavy chain, choose the option Manual Compilation>Heavy Chain on the explorer bar. The manual compilation screen is displayed in FIG. 16. The manual compilation heavy chain screen allows the user to concatenate VH, DH and JH sequences. The following steps show how to compile a heavy chain.

    
    
        Select the desired VH Sequence from the VH Sequence Combo box. All of the available VH nucleotide sequences that are entered in the SequenceDatabase.txt file appear in the drop down menu box underneath.
        Select the desired DH Sequence from the DH Sequence Combo Box. All of the available DH nucleotide sequences that are entered in the SequencDatabase.txt file appear in the drop down menu box underneath.
        Select the desired DH reading frame from the DH Reading Frame Box. The GRAVY value is updated according to the reading frame selection. The user should select the reading frame that corresponds to the lowest GRAVY value.
        Select the desired JH Sequence from the JH Sequence Combo Box. All of the available JH nucleotide sequences that are entered in the SequenceDatabase.txt file appear in the box underneath.
        Click “Add to Well Plate” button to add the compiled sequence to the well plate. All compiled sequences are added to the well plate for ordering for DNA synthesis.
    
    


Two boxes are observed in under the Dna Sequence heading. The “No Silencing of restricted Sites” box displays the individual VH, DH and JH sequences plus any V-D-J joints generated in accord with the sequence compilation rules. The corresponding encoded amino acid sequence also is displayed. For example, in FIG. 16, the first sequence displayed in the “no silencing of restricted sites” box corresponds to the selected VH sequence (in this case VH1-18). Following this is a depiction of three nucleotides “gag” encoding glutamic acid (E), which correspond to the V-D joint region created by performance of the compilation method in order to generate a functional nucleic acid molecule. The next sequence displayed corresponds to the selected DH sequence (in this case IGHD1-1*01). That sequence is followed by depiction of three nucleotides “acg” encoding threonine (T), which correspond to the D-J joint region created by performance of the compilation method in order to generate a functional nucleic acid molecule. The last sequence displayed corresponds to the JH selected sequence (in this case IGHJ1*01). The “With Restriction Sites Silenced” box displays the compiled sequence with any restriction sites that are present silenced. Any restriction sites silenced are displayed in the “Notes” box. The sequence with the restriction sites silenced is the sequence that is placed in the order file.
3. Auto Compilation of Sequences
Compiled chains are generated automatically by using the Auto Compilation option. As discussed in Example 4, compilation of all sequences is performed mechanically by the computer upon initiation and stored in files. The auto compilation function permits the user to view these sequences.
a. Auto Compilation of Light Chains
To automatically compile a light chain, choose the option Auto Compilation>Light Chain on the explorer bar. The auto compilation screen is displayed in FIG. 17. The auto compilation light chain screen displays all the possible combinations of Vκ and Jκ, and V and J sequences.
The auto compilation screen shows a grid with the following columns:

    
    
        Selected—This column contains a box that is either checked or unchecked to add or remove a sequence to or from the well plate.
        Rank—This column indicates the rank order in terms of diversity. A value of 1 indicates the most diverse sequence.
        Cluster—This column lists the cluster that the sequence is grouped into. The cluster number is simply an identifier and its value is not associated to any sort of ranking.
        Diversity Score—This column lists a score based on the root mean square of the BLAST Bit score results of this sequence against all other sequences in this list. A low value indicates a more diverse sequence.
        V Sequence—This column lists the V sequence identifier that is used for this sequence.
        J Sequence—This column lists the J sequence identifier that is used for this sequence.
        Amino Acid Sequence—This column lists the compiled sequence in protein format.
    
    


b. Auto Compilation of Heavy Chains
To automatically compile heavy chains, choose the option Auto Compilation>Heavy Chain on the explorer bar. The auto compilation screen is displayed in FIG. 19. The auto compilation heavy chain screen displays all the possible combinations of VH, DH and JH sequences.
The auto compilation screen shows a grid with the following columns:

    
    
        Selected—This column contains a box that is either checked or unchecked to add or remove a sequence to or from the well plate.
        Rank—This column indicates the rank order in terms of diversity. A value of 1 indicates the most diverse sequence.
        Cluster—This column lists the cluster that the sequence is grouped into. The cluster number is simply an identifier and its value is not associated to any sort of ranking.
        Diversity Score—This column lists a score based on the root mean square of the BLAST Bit score results of this sequence against all other sequences in this list. A low value indicates a more diverse sequence.
        V Sequence—This column lists the V sequence identifier that is used for this sequence.
        D Sequence—This column lists the D sequence identifier that is used for this sequence.
        J Sequence—This column lists the J sequence identifier that is used for this sequence.
        Amino Acid Sequence—This column lists the compiled sequence in protein format.
    
    


4. Other Functionalities Associated with Compilation of Light Chain and Heavy Chain Sequences
a. Changing the Sorting Option of the Auto Compilation Screen
The view of the sequences listed in the auto compilation screen can be reorganized by clicking on the desired column header. For example, to view sequences based on diversity score, the column header “Diversity Score” is clicked and the sequences are reordered in terms of lowest to highest diversity score.
b. Status Indicators
The auto compilation screen uses color conventions to identify the status of compiled sequences. For example, grey indicates the sequence has already been ordered, blue indicates the sequence has neither been ordered nor placed in the 96-Well Plate, and bold blue indicates the sequence has been placed in the 96-Well Plate. For example, the bolded items in rows 022, 023 and 024 in FIG. 17 indicate that the compiled sequences have been placed in the 96-well plate for ordering, which also is indicated by the check mark in the “selected” column. For example, the bolded items in rows 0103-0107 in FIG. 19 indicate that the compiled sequences have been placed in the 96-well plate for ordering, which also is indicated by the check mark in the “selected” column.
c. Placing Multiple Sequences in the 96-Well Plate
Multiple sequences can be placed into the 96-well plate by clicking on the box for multiple selected sequences. This can be down by selecting a first sequence with the mouse, and then, holding down the Shift key and clicking on the box for the last selected sequence. Alternatively, the Ctrl key can be used to select individual sequences by holding down the Ctrl key and clicking on the individual boxes for all selected sequences. Once VL sequences are selected to add to the 96-well plate, right Click on the row header and select “Copy to Well Plate”. All selected sequences are added to the well plate for ordering for DNA synthesis.
d. Placing a Single Sequence in the 96-Well Plate
To place a single sequences into the 96-well plate, click the box in the “Selected” Column; or alternatively, right Click on the Row Header for the sequence and click “Copy to Well Plate”. Only the selected sequence is added to the well place for ordering for DNA synthesis.
e. BLAST of a Selected Sequence Against all the Other Sequences in the Compilation.
Once the sequences are compiled by auto compilation, the software allows the user the option of performing a BLAST search of a single sequence against all the other sequences in the list. This function provides an output indicator, the BLAST bit score, which is a value calculated from the number of gaps and substitutions associated with each aligned sequence. The higher the score, the more significant the alignment. This data is useful if the user is trying to select sequences that have either the most diversity or alternatively, the least diversity to every other selected sequence. An example of generated data for selected VL sequences is illustrated in FIG. 18 and an example of generated data for selected VH sequences is illustrated in FIG. 20.
The compiled sequences are then compared in order to determine the similarity of each compiled sequence to every other compiled sequence and this information is used to generate a diversity score. For example, the NCBI utility BLAST can be used to find regions of local similarity between sequences, by comparing nucleotide or protein sequences and calculating the statistical significance of matches.
To perform BLAST, the sequence to be compared is selected by clicking on the sequence. The user can select the BLAST option by right clicking the sequence. For example, for VL sequences, FIG. 18 illustrates a BLAST grid form generated by selecting on the V4-2_IGLJ2*01 sequence on a manual compilation or auto compilation screen, followed by right clicking the sequence and selecting Blast. A new grid form was generated, illustrated in FIG. 18, which provides the Blast bit score of other compiled sequences aligned with the selected sequence. For example, for VH sequences, FIG. 20 illustrates a BLAST grid form generated by selecting on the VH4-39_IGHD5-24*01_IGHJ6*01 sequence on a manual compilation or auto compilation screen, followed by right clicking the sequence and selecting Blast. A new grid was generated, illustrated in FIG. 20, which provides the Blast bit score of other compiled sequences aligned with the selected sequence. Sequences can be selected from this new grid form for insertion into the 96-well plate file for ordering for DNA synthesis as indicated by checking the box in the “selected” column.
The BLAST form is a grid with the columns:

    
    
        Selected—This column contains a box that is either checked or unchecked to add or remove a sequence to or from the well plate.
        Rank—This column indicates the rank order in terms of diversity. A value of 1 indicates the most diverse sequence.
        BLAST Bit Score—This column indicates the BLAST Bit score for this particular sequence against the selected sequence.
        V Sequence—This column lists the V sequence identifier that is used for this sequence.
        J Sequence—This column lists the J sequence identifier that is used for this sequence.
        Amino Acid Sequence—This column lists the compiled sequence in protein format.
    
    


Example 6
Generation of Heavy and Light Chain Germline Recombined DNA Sequences
This example describes methods for generating heavy and light chain DNA sequences by the DNA Sequence Compilation Software. The first step in compilation is to select either manual compilation or automatic compilation of sequences as discussed in Example 5 above. Manual compilation is useful as it gives the user complete control over the selected sequences. This allows the user to cater a library to any desired circumstance, such as creating a library where every sequence contains a particular segment that is the same, or by controlling the diversity by selecting sequences that are different (e.g. from different gene families).
Using manual compilation germline segments were selected by selecting a germline segment from each gene family of a VH, DH, JH, V, J, V or J germline segment. Compilation of the variable heavy chain was performed separate from the light chain. Also compilation of the variable kappa light chain was performed separate from the variable lambda chain. Hence, separate 96-well plate files were created for the variable heavy chain, variable kappa light chain and variable lambda light chain.
For example, for a variable heavy chain, one VH germline segment from the IGHV1-18 gene family was selected, i.e. IGHV1-18*01; one VH germline segment from the IGHV1-2 gene family was selected, i.e. IGHV1-2*01; and so on. Similar selections were made for DH germline segments and JH germline segments. The DH germline segments were selected in a reading frame to give the lowest gravy score where possible. A combination of VHDHJH germline segments were made one at at time in the Manual compilation screen. In addition, the restriction enzyme sites NcoI and Nhe I were selected from the “well plate” menu for each VHDHJH combination for inclusion at the 5′ and 3′ ends of the recombined segments to allow for subcloning into appropriate vectors (see Example 8). This resulted in the software automatically viewing in the DNA Sequence Window the sequence of the selected sequence with “no silencing of restricted sites” and “with restriction sites silenced,” (i.e. in this case any NcoI or NheI restriction sites present internally in the sequence were silenced by modification using the codon usage table). Generated V-D and D-J joints also were created by the software in accordance with the sequence compilation rules and indicated in the DNA sequence window (an exemplary selection is set forth in FIG. 16). Once germline segment components for a rearranged sequence were selected, it was “added to the well plate.” This was repeated for all variable heavy chain germline segment combinations.
Similar selections also were made for the V and J light chain germline by selecting a combination of V and J germline segments (only one gene family member from each germline segment was ever selected) one at a time in the manual compilation screen. In addition, the restriction enzyme sites Nco I and BsiW I were selected from the “well plate” menu for each V and J combination for inclusion at the 5′ and 3′ ends of the recombined segments to allow for subcloning into appropriate vectors (see Example 8). This resulted in the software automatically displaying in the DNA Sequence Window the sequence of the selected sequence with “no silencing of restricted sites” and “with restriction sites silenced,” (i.e. in this case any Nco I or BsiW I restriction sites present internally in the sequence were silenced by nucleotide modification using the codon usage table.) Generated V-J joints also were created by the software in accordance with the sequence compilation rules and indicated in the DNA sequence window. Once germline segment components for a rearranged sequence were selected, it was “added to the well plate.” This was repeated for all variable kappa light chain germline segment combinations.
Similar selections also were made for the V or J light chain germline segments by selecting a combination of V or J germline segments (only one gene family member from each germline segment was ever selected) one at at time in the manual compilation screen. In addition, the restriction enzyme sites Nco I and Avr II were selected from the “well plate” menu for each V and J combination for inclusion at the 5′ and 3′ ends of the recombined segments to allow for subcloning into appropriate vectors (see Example 8) This resulted in the software automatically displaying in the DNA Sequence Window the sequence of the selected sequence with “no silencing of restricted sites” and “with restriction sites silenced,” (i.e. in this case any Nco I or Avr II restriction sites present internally in the sequence were silenced by nucleotide modification using the codon usage table.) Generated V-J joints also were created by the software in accordance with the sequence compilation rules and indicated in the DNA sequence window (an exemplary selection is set forth in FIG. 15). Once germline segment components for a rearranged sequence were selected, it was “added to the well plate.” This was repeated for all variable lambda light chain germline segment combinations.
The choice of Nhe I, Bsi WI and Avr II sites allow for complete amino acid conservation for their respective heavy and light chains thereby maintaining the natural human V and C regions.
Once all sequences were selected, the selected sequences were viewed under the 96-well plate view (see e.g., FIG. 12 and FIG. 14). A DNA synthesis order was generated using the Generate Order function. The DNA Compilation Software outputted the selected sequences in arrays that mapped to the 96-well format. The sequences in this format were sent to a DNA synthesis vendor (Genscript Corp.) to generate synthetic antibody variable heavy and light chain sequences. The synthesized nucleic acid molecules were returned and identified by the 96-well format. Table 22 lists sequences generated by the Sequence Compilation Software, ordered and synthesized.







TABLE 22







Exemplary Sequences Generated by DNA Compilation Software











SEQ ID


Number
Name
NO.










Heavy Chain









3
gnl|Fabrus|VH1-18_IGHD1-26*01_IGHJ2*01
454


4
gnl|Fabrus|VH1-18_IGHD2-21*01_IGHJ2*01
455


5
gnl|Fabrus|VH1-18_IGHD3-16*01_IGHJ6*01
456


6
gnl|Fabrus|VH1-18_IGHD3-22*01_IGHJ4*01
457


7
gnl|Fabrus|VH1-18_IGHD4-23*01_IGHJ1*01
458


8
gnl|Fabrus|VH1-18_IGHD5-12*01_IGHJ4*01
459


9
gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01
460


10
gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01
461


11
gnl|Fabrus|VH1-24_IGHD1-7*01_IGHJ4*01
462


12
gnl|Fabrus|VH1-24_IGHD2-15*01_IGHJ2*01
463


13
gnl|Fabrus|VH1-24_IGHD3-10*01_IGHJ4*01
464


14
gnl|Fabrus|VH1-24_IGHD3-16*01_IGHJ4*01
465


15
gnl|Fabrus|VH1-24_IGHD4-23*01_IGHJ2*01
466


16
gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01
467


17
gnl|Fabrus|VH1-24_IGHD5-18*01_IGHJ6*01
468


18
gnl|Fabrus|VH1-24_IGHD6-19*01_IGHJ4*01
469


19
gnl|Fabrus|VH1-3_IGHD2-15*01_IGHJ2*01
470


20
gnl|Fabrus|VH1-3_IGHD2-2*01_IGHJ5*01
471


21
gnl|Fabrus|VH1-3_IGHD3-9*01_IGHJ6*01
472


22
gnl|Fabrus|VH1-3_IGHD4-23*01_IGHJ4*01
473


23
gnl|Fabrus|VH1-3_IGHD5-18*01_IGHJ4*01
474


24
gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01
475


25
gnl|Fabrus|VH1-3_IGHD7-27*01_IGHJ4*01
476


26
gnl|Fabrus|VH1-45_IGHD1-26*01_IGHJ4*01
477


27
gnl|Fabrus|VH1-45_IGHD2-15*01_IGHJ6*01
478


28
gnl|Fabrus|VH1-45_IGHD2-8*01_IGHJ3*01
479


29
gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01
480


30
gnl|Fabrus|VH1-45_IGHD3-16*01_IGHJ2*01
481


31
gnl|Fabrus|VH1-45_IGHD4-23*01_IGHJ4*01
482


32
gnl|Fabrus|VH1-45_IGHD5-24*01_IGHJ4*01
483


33
gnl|Fabrus|VH1-45_IGHD6-19*01_IGHJ4*01
484


34
gnl|Fabrus|VH1-45_IGHD7-27*01_IGHJ6*01
485


35
gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01
486


36
gnl|Fabrus|VH1-46_IGHD2-15*01_IGHJ2*01
487


37
gnl|Fabrus|VH1-46_IGHD3-10*01_IGHJ4*01
488


38
gnl|Fabrus|VH1-46_IGHD4-17*01_IGHJ4*01
489


39
gnl|Fabrus|VH1-46_IGHD5-18*01_IGHJ4*01
490


40
gnl|Fabrus|VH1-46_IGHD6-13*01_IGHJ4*01
491


41
gnl|Fabrus|VH1-46_IGHD6-6*01_IGHJ1*01
492


42
gnl|Fabrus|VH1-46_IGHD7-27*01_IGHJ2*01
493


43
gnl|Fabrus|VH1-58_IGHD1-26*01_IGHJ4*01
494


44
gnl|Fabrus|VH1-58_IGHD2-15*01_IGHJ2*01
495


45
gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01
496


46
gnl|Fabrus|VH1-58_IGHD4-17*01_IGHJ4*01
497


47
gnl|Fabrus|VH1-58_IGHD5-18*01_IGHJ4*01
498


48
gnl|Fabrus|VH1-58_IGHD6-6*01_IGHJ1*01
499


49
gnl|Fabrus|VH1-58_IGHD7-27*01_IGHJ5*01
500


50
gnl|Fabrus|VH1-69_IGHD1-1*01_IGHJ6*01
501


51
gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01
502


52
gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01
503


53
gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01
504


54
gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01
505


55
gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01
506


56
gnl|Fabrus|VH1-69_IGHD3-9*01_IGHJ6*01
507


57
gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01
508


58
gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01
509


59
gnl|Fabrus|VH1-69_IGHD5-24*01_IGHJ6*01
510


60
gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01
511


61
gnl|Fabrus|VH1-69_IGHD6-6*01_IGHJ1*01
512


62
gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01
513


63
gnl|Fabrus|VH1-8_IGHD1-26*01_IGHJ4*01
514


64
gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01
515


65
gnl|Fabrus|VH1-8_IGHD2-2*01_IGHJ6*01
516


66
gnl|Fabrus|VH1-8_IGHD3-10*01_IGHJ4*01
517


67
gnl|Fabrus|VH1-8_IGHD4-17*01_IGHJ4*01
518


68
gnl|Fabrus|VH1-8_IGHD5-5*01_IGHJ4*01
519


69
gnl|Fabrus|VH1-8_IGHD7-27*01_IGHJ4*01
520


70
gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01
521


71
gnl|Fabrus|VH2-26_IGHD2-15*01_IGHJ2*01
522


72
gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01
523


73
gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01
524


74
gnl|Fabrus|VH2-26_IGHD3-9*01_IGHJ6*01
525


75
gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01
526


76
gnl|Fabrus|VH2-26_IGHD5-12*01_IGHJ4*01
527


77
gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01
528


78
gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01
529


79
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
530


80
gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01
531


81
gnl|Fabrus|VH2-5_IGHD1-1*01_IGHJ5*01
532


82
gnl|Fabrus|VH2-5_IGHD2-15*01_IGHJ6*01
533


83
gnl|Fabrus|VH2-5_IGHD3-16*01_IGHJ4*01
534


84
gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01
535


85
gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01
536


86
gnl|Fabrus|VH2-5_IGHD6-13*01_IGHJ4*01
537


87
gnl|Fabrus|VH2-5_IGHD7-27*01_IGHJ2*01
538


88
gnl|Fabrus|VH2-70_IGHD1-1*01_IGHJ2*01
539


89
gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01
540


90
gnl|Fabrus|VH2-70_IGHD3-22*01_IGHJ4*01
541


91
gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01
542


92
gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01
543


93
gnl|Fabrus|VH2-70_IGHD7-27*01_IGHJ2*01
544


94
gnl|Fabrus|VH3-11_IGHD1-26*01_IGHJ4*01
545


95
gnl|Fabrus|VH3-11_IGHD2-2*01_IGHJ6*01
546


96
gnl|Fabrus|VH3-11_IGHD3-16*01_IGHJ4*01
547


97
gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01
548


98
gnl|Fabrus|VH3-11_IGHD4-23*01_IGHJ5*01
549


99
gnl|Fabrus|VH3-11_IGHD5-18*01_IGHJ4*01
550


100
gnl|Fabrus|VH3-11_IGHD6-19*01_IGHJ6*01
551


101
gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01
552


102
gnl|Fabrus|VH3-11_IGHD7-27*01_IGHJ4*01
553


103
gnl|Fabrus|VH3-13_IGHD1-26*01_IGHJ4*01
554


104
gnl|Fabrus|VH3-13_IGHD2-8*01_IGHJ5*01
555


105
gnl|Fabrus|VH3-13_IGHD3-3*01_IGHJ1*01
556


106
gnl|Fabrus|VH3-13_IGHD3-9*01_IGHJ6*01
557


107
gnl|Fabrus|VH3-13_IGHD4-23*01_IGHJ5*01
558


108
gnl|Fabrus|VH3-13_IGHD5-5*01_IGHJ4*01
559


109
gnl|Fabrus|VH3-13_IGHD6-6*01_IGHJ1*01
560


110
gnl|Fabrus|VH3-13_IGHD7-27*01_IGHJ5*01
561


111
gnl|Fabrus|VH3-15_IGHD1-26*01_IGHJ4*01
562


112
gnl|Fabrus|VH3-15_IGHD2-15*01_IGHJ2*01
563


113
gnl|Fabrus|VH3-15_IGHD2-15*01_IGHJ6*01
564


114
gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01
565


115
gnl|Fabrus|VH3-15_IGHD3-9*01_IGHJ2*01
566


116
gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01
567


117
gnl|Fabrus|VH3-15_IGHD6-6*01_IGHJ1*01
568


118
gnl|Fabrus|VH3-16_IGHD1-1*01_IGHJ1*01
569


119
gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01
570


120
gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01
571


121
gnl|Fabrus|VH3-16_IGHD2-2*01_IGHJ2*01
572


122
gnl|Fabrus|VH3-16_IGHD3-10*01_IGHJ4*01
573


123
gnl|Fabrus|VH3-16_IGHD4-4*01_IGHJ2*01
574


124
gnl|Fabrus|VH3-16_IGHD5-24*01_IGHJ4*01
575


125
gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01
576


126
gnl|Fabrus|VH3-16_IGHD7-27*01_IGHJ2*01
577


127
gnl|Fabrus|VH3-20_IGHD1-14*01_IGHJ4*01
578


128
gnl|Fabrus|VH3-20_IGHD2-15*01_IGHJ2*01
579


129
gnl|Fabrus|VH3-20_IGHD2-8*01_IGHJ4*01
580


130
gnl|Fabrus|VH3-20_IGHD3-10*01_IGHJ4*01
581


131
gnl|Fabrus|VH3-20_IGHD3-9*01_IGHJ6*01
582


132
gnl|Fabrus|VH3-20_IGHD4-23*01_IGHJ4*01
583


133
gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01
584


134
gnl|Fabrus|VH3-20_IGHD6-13*01_IGHJ4*01
585


135
gnl|Fabrus|VH3-20_IGHD7-27*01_IGHJ2*01
586


136
gnl|Fabrus|VH3-21_IGHD1-26*01_IGHJ4*01
587


137
gnl|Fabrus|VH3-21_IGHD2-2*01_IGHJ5*01
588


138
gnl|Fabrus|VH3-21_IGHD3-22*01_IGHJ4*01
589


139
gnl|Fabrus|VH3-21_IGHD4-23*01_IGHJ5*01
590


140
gnl|Fabrus|VH3-21_IGHD5-24*01_IGHJ5*01
591


141
gnl|Fabrus|VH3-21_IGHD6-19*01_1GHJ1*01
592


142
gnl|Fabrus|VH3-21_IGHD7-27*01_IGHJ4*01
593


143
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ1*01
594


144
gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01
595


145
gnl|Fabrus|VH3-23_IGHD1-20*01_IGHJ3*01
596


146
gnl|Fabrus|VH3-23_IGHD1-26*01_IGHJ4*01
597


147
gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01
598


148
gnl|Fabrus|VH3-23_IGHD2-21*01_IGHJ1*01
599


149
gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01
600


150
gnl|Fabrus|VH3-23_IGHD3-16*01_IGHJ4*01
601


151
gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01
602


152
gnl|Fabrus|VH3-23_IGHD3-3*01_IGHJ5*01
603


153
gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01
604


154
gnl|Fabrus|VH3-23_IGHD4-23*01_IGHJ2*01
605


155
gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01
606


156
gnl|Fabrus|VH3-23_IGHD5-24*01_IGHJ1*01
607


157
gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01
608


158
gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01
609


159
gnl|Fabrus|VH3-23_IGHD6-25*01_IGHJ2*01
610


160
gnl|Fabrus|VH3-23_IGHD6-6*01_IGHJ1*01
611


161
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01
612


162
gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01
613


163
gnl|Fabrus|VH3-30_IGHD1-1*01_IGHJ6*01
614


164
gnl|Fabrus|VH3-30_IGHD1-26*01_IGHJ1*01
615


165
gnl|Fabrus|VH3-30_IGHD1-26*01_IGHJ4*01
616


166
gnl|Fabrus|VH3-30_IGHD2-15*01_IGHJ2*01
617


167
gnl|Fabrus|VH3-30_IGHD2-2*01_IGHJ6*01
618


168
gnl|Fabrus|VH3-30_IGHD3-10*01_IGHJ1*01
619


169
gnl|Fabrus|VH3-30_IGHD3-16*01_IGHJ6*01
620


170
gnl|Fabrus|VH3-30_IGHD4-17*01_IGHJ4*01
621


171
gnl|Fabrus|VH3-30_IGHD5-12*01_IGHJ4*01
622


172
gnl|Fabrus|VH3-30_IGHD5-18*01_IGHJ1*01
623


173
gnl|Fabrus|VH3-30_IGHD6-13*01_IGHJ4*01
624


174
gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01
625


175
gnl|Fabrus|VH3-35_IGHD1-1*01_IGHJ2*01
626


176
gnl|Fabrus|VH3-35_IGHD1-20*01_IGHJ6*01
627


177
gnl|Fabrus|VH3-35_IGHD2-15*01_IGHJ2*01
628


178
gnl|Fabrus|VH3-35_IGHD2-21*01_IGHJ6*01
629


179
gnl|Fabrus|VH3-35_IGHD3-10*01_IGHJ4*01
630


180
gnl|Fabrus|VH3-35_IGHD3-9*01_IGHJ6*01
631


181
gnl|Fabrus|VH3-35_IGHD5-12*01_IGHJ4*01
632


182
gnl|Fabrus|VH3-35_IGHD6-13*01_IGHJ4*01
633


183
gnl|Fabrus|VH3-35_IGHD7-27*01_IGHJ1*01
634


184
gnl|Fabrus|VH3-38_IGHD1-14*01_IGHJ5*01
635


185
gnl|Fabrus|VH3-38_IGHD1-20*01_IGHJ6*01
636


186
gnl|Fabrus|VH3-38_IGHD2-15*01_IGHJ6*01
637


187
gnl|Fabrus|VH3-38_IGHD2-2*01_IGHJ1*01
638


188
gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01
639


189
gnl|Fabrus|VH3-38_IGHD3-16*01_IGHJ1*01
640


190
gnl|Fabrus|VH3-38_IGHD4-17*01_IGHJ2*01
641


191
gnl|Fabrus|VH3-38_IGHD5-24*01_IGHJ3*01
642


192
gnl|Fabrus|VH3-38_IGHD6-6*01_IGHJ1*01
643


193
gnl|Fabrus|VH3-38_IGHD7-27*01_IGHJ6*01
644


194
gnl|Fabrus|VH3-43_IGHD1-26*01_IGHJ5*01
645


195
gnl|Fabrus|VH3-43_IGHD1-7*01_IGHJ6*01
646


196
gnl|Fabrus|VH3-43_IGHD2-2*01_IGHJ3*01
647


197
gnl|Fabrus|VH3-43_IGHD2-21*01_IGHJ6*01
648


198
gnl|Fabrus|VH3-43_IGHD3-16*01_IGHJ6*01
649


199
gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01
650


200
gnl|Fabrus|VH3-43_IGHD4-23*01_IGHJ3*01
651


201
gnl|Fabrus|VH3-43_IGHD5-18*01_IGHJ5*01
652


202
gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01
653


203
gnl|Fabrus|VH3-43_IGHD7-27*01_IGHJ1*01
654


204
gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01
655


205
gnl|Fabrus|VH3-49_IGHD1-26*01_IGHJ4*01
656


206
gnl|Fabrus|VH3-49_IGHD1-7*01_IGHJ6*01
657


207
gnl|Fabrus|VH3-49_IGHD2-2*01_IGHJ6*01
658


208
gnl|Fabrus|VH3-49_IGHD2-8*01_IGHJ4*01
659


209
gnl|Fabrus|VH3-49_IGHD3-22*01_IGHJ4*01
660


210
gnl|Fabrus|VH3-49_IGHD3-9*01_IGHJ6*01
661


211
gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01
662


212
gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01
663


213
gnl|Fabrus|VH3-49_IGHD7-27*01_IGHJ1*01
664


214
gnl|Fabrus|VH3-53_IGHD1-14*01_IGHJ6*01
665


215
gnl|Fabrus|VH3-53_IGHD1-7*01_IGHJ1*01
666


216
gnl|Fabrus|VH3-53_IGHD2-2*01_IGHJ2*01
667


217
gnl|Fabrus|VH3-53_IGHD3-22*01_IGHJ3*01
668


218
gnl|Fabrus|VH3-53_IGHD4-23*01_IGHJ1*01
669


219
gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01
670


220
gnl|Fabrus|VH3-53_IGHD6-13*01_IGHJ3*01
671


221
gnl|Fabrus|VH3-53_IGHD7-27*01_IGHJ4*01
672


222
gnl|Fabrus|VH3-64_IGHD1-26*01_IGHJ4*01
673


223
gnl|Fabrus|VH3-64_IGHD1-7*01_IGHJ6*01
674


224
gnl|Fabrus|VH3-64_IGHD2-2*01_IGHJ5*01
675


225
gnl|Fabrus|VH3-64_IGHD3-3*01_IGHJ4*01
676


226
gnl|Fabrus|VH3-64_IGHD4-17*01_IGHJ4*01
677


227
gnl|Fabrus|VH3-64_IGHD5-12*01_IGHJ4*01
678


228
gnl|Fabrus|VH3-64_IGHD6-19*01_IGHJ1*01
679


229
gnl|Fabrus|VH3-64_IGHD7-27*01_IGHJ4*01
680


230
gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01
681


231
gnl|Fabrus|VH3-7_IGHD1-20*01_IGHJ3*01
682


232
gnl|Fabrus|VH3-7_IGHD1-7*01_IGHJ6*01
683


233
gnl|Fabrus|VH3-7_IGHD2-21*01_IGHJ5*01
684


234
gnl|Fabrus|VH3-7_IGHD2-8*01_IGHJ6*01
685


235
gnl|Fabrus|VH3-7_IGHD3-22*01_IGHJ3*01
686


236
gnl|Fabrus|VH3-7_IGHD3-9*01_IGHJ6*01
687


237
gnl|Fabrus|VH3-7_IGHD4-17*01_IGHJ4*01
688


238
gnl|Fabrus|VH3-7_IGHD5-12*01_IGHJ4*01
689


239
gnl|Fabrus|VH3-7_IGHD5-24*01_IGHJ4*01
690


240
gnl|Fabrus|VH3-7_IGHD6-19*01_IGHJ6*01
691


241
gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01
692


242
gnl|Fabrus|VH3-7_IGHD7-27*01_IGHJ2*01
693


243
gnl|Fabrus|VH3-72_IGHD1-1*01_IGHJ4*01
694


244
gnl|Fabrus|VH3-72_IGHD2-15*01_IGHJ1*01
695


245
gnl|Fabrus|VH3-72_IGHD3-22*01_IGHJ4*01
696


246
gnl|Fabrus|VH3-72_IGHD3-9*01_IGHJ6*01
697


247
gnl|Fabrus|VH3-72_IGHD4-23*01_IGHJ2*01
698


248
gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01
699


249
gnl|Fabrus|VH3-72_IGHD5-24*01_IGHJ6*01
700


250
gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01
701


251
gnl|Fabrus|VH3-72_IGHD7-27*01_IGHJ2*01
702


252
gnl|Fabrus|VH3-73_IGHD1-1*01_IGHJ5*01
703


253
gnl|Fabrus|VH3-73_IGHD2-8*01_IGHJ2*01
704


254
gnl|Fabrus|VH3-73_IGHD3-22*01_IGHJ4*01
705


255
gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01
706


256
gnl|Fabrus|VH3-73_IGHD4-11*01_IGHJ6*01
707


257
gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01
708


258
gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01
709


259
gnl|Fabrus|VH3-73_IGHD6-19*01_IGHJ1*01
710


260
gnl|Fabrus|VH3-73_IGHD7-27*01_IGHJ5*01
711


261
gnl|Fabrus|VH3-74_IGHD1-1*01_IGHJ6*01
712


262
gnl|Fabrus|VH3-74_IGHD1-26*01_IGHJ4*01
713


263
gnl|Fabrus|VH3-74_IGHD2-2*01_IGHJ5*01
714


264
gnl|Fabrus|VH3-74_IGHD3-22*01_IGHJ5*01
715


265
gnl|Fabrus|VH3-74_IGHD4-17*01_IGHJ1*01
716


266
gnl|Fabrus|VH3-74_IGHD5-12*01_IGHJ4*01
717


267
gnl|Fabrus|VH3-74_IGHD6-6*01_IGHJ1*01
718


268
gnl|Fabrus|VH3-74_IGHD7-27*01_IGHJ4*01
719


269
gnl|Fabrus|VH3-9_IGHD1-1*01_IGHJ6*01
720


270
gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01
721


271
gnl|Fabrus|VH3-9_IGHD2-2*01_IGHJ4*01
722


272
gnl|Fabrus|VH3-9_IGHD3-16*01_IGHJ6*01
723


273
gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01
724


274
gnl|Fabrus|VH3-9_IGHD4-11*01_IGHJ4*01
725


275
gnl|Fabrus|VH3-9_IGHD5-24*01_IGHJ1*01
726


276
gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01
727


277
gnl|Fabrus|VH3-9_IGHD6-25*01_IGHJ6*01
728


278
gnl|Fabrus|VH3-9_IGHD7-27*01_IGHJ2*01
729


279
gnl|Fabrus|VH4-28_IGHD1-20*01_IGHJ1*01
730


280
gnl|Fabrus|VH4-28_IGHD1-7*01_IGHJ6*01
731


281
gnl|Fabrus|VH4-28_IGHD2-15*01_IGHJ6*01
732


282
gnl|Fabrus|VH4-28_IGHD3-16*01_IGHJ2*01
733


283
gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01
734


284
gnl|Fabrus|VH4-28_IGHD4-4*01_IGHJ4*01
735


285
gnl|Fabrus|VH4-28_IGHD5-5*01_IGHJ1*01
736


286
gnl|Fabrus|VH4-28_IGHD6-13*01_IGHJ4*01
737


287
gnl|Fabrus|VH4-28_IGHD7-27*01_IGHJ1*01
738


288
gnl|Fabrus|VH4-31_IGHD1-26*01_IGHJ2*01
739


289
gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01
740


290
gnl|Fabrus|VH4-31_IGHD2-2*01_IGHJ6*01
741


291
gnl|Fabrus|VH4-31_IGHD3-10*01_IGHJ4*01
742


292
gnl|Fabrus|VH4-31_IGHD3-9*01_IGHJ6*01
743


293
gnl|Fabrus|VH4-31_IGHD4-17*01_IGHJ5*01
744


294
gnl|Fabrus|VH4-31_IGHD5-12*01_IGHJ4*01
745


295
gnl|Fabrus|VH4-31_IGHD6-13*01_IGHJ4*01
746


296
gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01
747


297
gnl|Fabrus|VH4-31_IGHD7-27*01_IGHJ5*01
748


298
gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01
749


299
gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01
750


300
gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01
751


301
gnl|Fabrus|VH4-34_IGHD3-22*01_IGHJ6*01
752


302
gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01
753


303
gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01
754


304
gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01
755


305
gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01
756


306
gnl|Fabrus|VH4-34_IGHD6-6*01_IGHJ6*01
757


307
gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01
758


308
gnl|Fabrus|VH4-39_IGHD1-14*01_IGHJ1*01
759


309
gnl|Fabrus|VH4-39_IGHD1-20*01_IGHJ6*01
760


310
gnl|Fabrus|VH4-39_IGHD2-21*01_IGHJ3*01
761


311
gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01
762


312
gnl|Fabrus|VH4-39_IGHD3-16*01_IGHJ2*01
763


313
gnl|Fabrus|VH4-39_IGHD3-9*01_IGHJ6*01
764


314
gnl|Fabrus|VH4-39_IGHD4-23*01_IGHJ2*01
765


315
gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01
766


316
gnl|Fabrus|VH4-39_IGHD6-6*01_IGHJ1*01
767


317
gnl|Fabrus|VH4-4_IGHD1-20*01_IGHJ3*01
768


318
gnl|Fabrus|VH4-4_IGHD2-8*01_IGHJ4*01
769


319
gnl|Fabrus|VH4-4_IGHD3-22*01_IGHJ2*01
770


320
gnl|Fabrus|VH4-4_IGHD4-23*01_IGHJ4*01
771


321
gnl|Fabrus|VH4-4_IGHD5-12*01_IGHJ5*01
772


322
gnl|Fabrus|VH4-4_IGHD6-6*01_IGHJ4*01
773


323
gnl|Fabrus|VH4-4_IGHD7-27*01_IGHJ6*01
774


324
gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01
775


325
gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01
776


326
gnl|Fabrus|VH5-51_IGHD1-26*01_IGHJ6*01
777


327
gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01
778


328
gnl|Fabrus|VH5-51_IGHD3-10*01_IGHJ6*01
779


329
gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01
780


330
gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01
781


331
gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01
782


332
gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01
783


333
gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01
784


334
gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01
785


335
gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01
786


336
gnl|Fabrus|VH6-1_IGHD1-20*01_IGHJ6*01
787


337
gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01
788


338
gnl|Fabrus|VH6-1_IGHD2-21*01_IGHJ6*01
789


339
gnl|Fabrus|VH6-1_IGHD3-16*01_IGHJ5*01
790


340
gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01
791


341
gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01
792


342
gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01
793


343
gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01
794


344
gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01
795


345
gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01
796


346
gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01
797


347
gnl|Fabrus|VH7-81_IGHD1-14*01_IGHJ4*01
798


348
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01
799


349
gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01
800


350
gnl|Fabrus|VH7-81_IGHD3-16*01_IGHJ6*01
801


351
gnl|Fabrus|VH7-81_IGHD4-23*01_IGHJ1*01
802


352
gnl|Fabrus|VH7-81_IGHD5-12*01_IGHJ6*01
803


353
gnl|Fabrus|VH7-81_IGHD6-25*01_IGHJ4*01
804


354
gnl|Fabrus|VH7-81_IGHD7-27*01_IGHJ4*01
805







Light Chain









1
gnl|Fabrus|A14_IGKJ1*01
806


2
gnl|Fabrus|A17_IGKJ1*01
807


3
gnl|Fabrus|A2_IGKJ1*01
808


4
gnl|Fabrus|A20_IGKJ1*01
809


5
gnl|Fabrus|A23_IGKJ1*01
810


6
gnl|Fabrus|A26_IGKJ1*01
811


7
gnl|Fabrus|A27_IGKJ1*01
812


8
gnl|Fabrus|A27_IGKJ3*01
813


9
gnl|Fabrus|A30_IGKJ1*01
814


10
gnl|Fabrus|B2_IGKJ1*01
815


11
gnl|Fabrus|B2_IGKJ3*01
816


12
gnl|Fabrus|B3_IGKJ1*01
817


14
gnl|Fabrus|L11_IGKJ1*01
819


15
gnl|Fabrus|L12_IGKJ1*01
820


16
gnl|Fabrus|L14_IGKJ1*01
821


17
gnl|Fabrus|L2_IGKJ1*01
822


18
gnl|Fabrus|L22_IGKJ3*01
823


19
gnl|Fabrus|L23_IGKJ1*01
824


20
gnl|Fabrus|L25_IGKJ1*01
825


21
gnl|Fabrus|L25_IGKJ3*01
826


22
gnl|Fabrus|L4/18a_IGKJ1*01
827


23
gnl|Fabrus|L5_IGKJ1*01
828


24
gnl|Fabrus|L6_IGKJ1*01
829


25
gnl|Fabrus|L8_IGKJ1*01
830


26
gnl|Fabrus|L9_IGKJ2*01
831


27
gnl|Fabrus|O1_IGKJ1*01
832


28
gnl|Fabrus|O12_IGKJ1*01
833


29
gnl|Fabrus|O18_IGKJ1*01
834


31
gnl|Fabrus|V1-11_IGLJ2*01
836


32
gnl|Fabrus|V1-13_IGLJ5*01
837


33
gnl|Fabrus|V1-16_IGLJ6*01
838


34
gnl|Fabrus|V1-18_IGLJ2*01
839


35
gnl|Fabrus|V1-2_IGLJ7*01
840


36
gnl|Fabrus|V1-20_IGLJ6*01
841


37
gnl|Fabrus|V1-3_IGLJ1*01
842


38
gnl|Fabrus|V1-4_IGLJ4*01
843


39
gnl|Fabrus|V1-5_IGLJ2*01
844


40
gnl|Fabrus|V1-7_IGLJ1*01
845


41
gnl|Fabrus|V1-9_IGLJ6*01
846


42
gnl|Fabrus|V2-1_IGLJ6*01
847


43
gnl|Fabrus|V2-11_IGLJ7*01
848


44
gnl|Fabrus|V2-13_IGLJ2*01
849


45
gnl|Fabrus|V2-14_IGLJ4*01
850


46
gnl|Fabrus|V2-15_IGLJ7*01
851


47
gnl|Fabrus|V2-17_IGLJ2*01
852


48
gnl|Fabrus|V2-19_IGLJ4*01
853


49
gnl|Fabrus|V2-6_IGLJ4*01
854


50
gnl|Fabrus|V2-7_IGLJ2*01
855


51
gnl|Fabrus|V2-7_IGLJ7*01
856


52
gnl|Fabrus|V2-8_IGLJ6*01
857


53
gnl|Fabrus|V3-2_IGLJ4*01
858


54
gnl|Fabrus|V3-3_IGLJ7*01
859


55
gnl|Fabrus|V3-4_IGLJ1*01
860


56
gnl|Fabrus|V4-1_IGLJ4*01
861


57
gnl|Fabrus|V4-2_IGLJ4*01
862


58
gnl|Fabrus|V4-3_IGLJ4*01
863


59
gnl|Fabrus|V4-4_IGLJ5*01
864


60
gnl|Fabrus|V4-6_IGLJ4*01
865


61
gnl|Fabrus|V5-4_IGLJ2*01
866


62
gnl|Fabrus|V5-6_IGLJ1*01
867









Example 7
Comparison of Fab Secretion by Different Leader Sequences
In this Example, the effect of different leader sequences on expression of encoded Fabs was assessed. In order to assure properly folded Fab proteins, disulfide bonds must form in an oxidizing environment and therefore it is necessary to translocate the Fab proteins into the periplasm by using a pathway such as Sec, SRP and TAT. The SRP pathway does not require unfolding of any proteins. SRP leader sequences are shown in Table 23.







TABLE 23







SRP Leader Sequences











Name
SEQ ID NO.
Sequence







DsbA
5
MKKIWLALAGLVLAFSASA



 



SfmC
6
MMTKIKLLMLIIFYLIISASAHA



 



ToIB
7
MKQALRVAFGFLILWASVLHA



 



TorT
8
MRVLLFLLLSLRMLPAFS










Plasmid A (SEQ ID NO:1) and plasmid C (SEQ ID NO:3) were modified to contain either a DsbA leader sequence (SEQ ID NO:5) or a mutant DsbA leader sequence (SEQ ID NO:965) and a ribosomal binding site (RBS) variation. Overlap PCR was performed using three forward primers containing RBS variations and the sequence corresponding to the N-terminus of DsbA (set forth in SEQ ID NOS:966-968) and two reverse primers corresponding to the C-terminus of either DsbA or mutant DsbA (set forth in SEQ ID NOS:969-970). The PCR resulted in six different leader sequences (Table 24). These leader sequences were then inserted into Plasmid A and Plasmid C between EcoRI and NcoI sites upstream of the start codon, ATG. Plasmids A and C containing the DsbA leader sequence with the RBS variations are set forth in SEQ ID NO:1015-1017 and 1021-1023, respectively. Plasmids A and C containing the mutant DsbA leader sequence with the RBS variations are set forth in SEQ ID NO:1018-1020 and 1024-1026, respectively.







TABLE 24







DsbA and mutant DsbA leader sequences with RBS variations









Name
Leader Sequence
SEQ ID NO.





DS1
GAATTCTAAGGAGGTTTCACCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGC
971



GTTTAGC GCC ATG G



 


DS2
GAATTCTAAGGAGTATTCACCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGC
972



GTTTAGC GCC ATG G



 


DS3
GAATTCTTAGGATTATTCACCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCG
973



TTTAGC GCC ATG G



 


DM1
GAATTCTAAGGAGGTTTCACCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGC
974



GTTTAGCGCATCG GCG GCC ATG GCA



 


DM2
GAATTCTAAGGAGTATTCACCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGC
975



GTTTAGCGCATCG GCG GCC ATG GCA



 


DM3
GAATTCTTAGGATTATTCACCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCG
976



TTTAGCGCATCG GCG GCC ATG GCA









To assess the effect of the different leader sequences on the expression of a Fab antibody, expression of Rituxan was assessed. Rituxan heavy chain (SEQ ID NO:453) was cloned in-frame to the CH sequence present on the respective Plasmid A vector and Rituxan light chain (SEQ ID NO:835) was cloned in-frame to the C sequence present in the respective Plasmid C vector. Briefly, the VH and V chains were cloned into the Plasmid A or C vectors (containing an STII leader sequence, a DsbA leader sequence or a mutant DsbA leader sequence) by digestion of the plasmids with NheI and NcoI (for the heavy chain DNA) or with NcoI and BsiWI (for the light chain DNA) followed by ligation. Plasmids encoding a heavy and light chain were co-transformed into LMG194 cells as described in Example 8 below and grown for 36 hours at 20° C. in terrific broth (TB) medium in the presence of 0.4% glucose and 0.008% arabinose. Expressed Fabs were extracted from the whole cells using Bugbuster® (Novagen), and purified using a Ni2+ affinity column (EMD), and analyzed by SDS-PAGE followed by Western Blot. The results show that in the absence of an inducer little to no protein was expressed. Upon induction, expression of the Fabs under the control of the DsbA leader sequence (for each of the three RBS variations) was equal to expression under the control of the STII leader sequence. Mutated DsbA resulted in lower expression. There was no significant difference in expression among the various RBS variations of the DsbA leader sequence.
Example 8
Cloning and Co-Transformation of Synthesized Variable Heavy and Light Chains
In this Example, a Fab library was created by cloning heavy or light chain DNA into their respective Plasmids followed by co-transformation and protein growth/purification. Following synthesis of the DNA molecules generated from the Sequence Compilation Software as described in Example 6, the DNA molecules were cloned into plasmids containing constant heavy or light chains as appropriate for co-transformation and expression of combinatorial Fabs. Plasmid A (SEQ ID NO:1) and plasmid D (SEQ ID NO:2) contain heavy chain constant regions sequences. Plasmid C (SEQ ID NO:3) contains a kappa light chain constant region sequence and Plasmid E (SEQ ID NO:4) contains a lambda light chain constant region sequence.
Synthetic recombined nucleic acid encoding a variable heavy chain were digested with Nhe I and Nco I and ligated into Plasmid A with a StII leader sequence using standard molecular techniques. Synthetic recombined nucleic acid encoding a variable kappa light chain were digested with NcoI and BsiWI and synthetic recombined nucleic acid encoding a variable lambda chain were digested with NcoI and AvrII, and were ligated into Plasmid C or Plasmid E, respectively, with a StII leader sequence, using standard molecular biology techniques.
Plasmid A and one of either Plasmid C or Plasmid E, each containing various combinations of variable heavy and light chains, were co-transformed into E. coli. The process was repeated for all combinations of heavy and light chains. Briefly, plasmid A (encoding a Fab heavy chain) and plasmid C or Plasmid E (encoding a Fab light chain) were resuspended separately in TE buffer to a final concentration of 1 ng/μl. One (1) μL of heavy chain plasmid and 1 μL of light chain plasmid were combined in a PCR tube or a PCR plate and were mixed with 20 μL ice cold LMG194 competent cells. The transformation reaction was incubated on ice for 10 minutes followed by heat shock in a preheated PCR block at 42° C. for 45 seconds. The tube was then placed on ice for an additional 2 minutes followed by addition of 200 μL SOC medium. The cells were allowed to recover for 1.5 hours at 37° C. A 100 μL aliquot of the transformation culture was used to inoculate 0.9 mL LB (Luria-Bertani Broth) containing 0.4% (w/v) glucose, 17 μg/mL kanamycin (Sigma Aldrich) and 34 μg/mL chloramphenicol (Sigma Aldrich). The culture was grown at 30° C. with vigorous shaking for 20 hours. The transformation culture was grown and purified using the PiccoloTM system as described in Example 9.
Example 9
High Throughput Growth and Purification of Fab Libraries
In this Example, Fab libraries are generated and purified using high throughput techniques. High throughput transformation of pairs of heavy chains (plasmid A) and light chains (plasmid C or E) was performed as described in Example 8, except that a 96-well PCR plate was used instead of individual PCR tubes. After transformation, the cells were grown overnight in 2 ml deep well 96-well plates (VWR) block covered with breathable tape. The overnight culture was used directly for inoculation in PiccoloTM (Wollerton et al. (2006) JALA, 11:291-303.)
High throughput, parallel expression and purification of antibody Fab fragments was performed using PiccoloTM (The Automation Partnership (TAP)), which automates protein expression and purification. The expression and purification parameters for PiccoloTM were prepared using Run Composer software (TAP). A ‘Strain File’ was generated mapping the location of each clone in the seed culture plate. This was submitted to the Run Composer software and the basic machine settings were set as follows: Pre-induction Incubator set at 30° C.; Expression Incubator 1 set at 16° C.; Centrifuge set at 6° C. and 5000×g; Media Pump 1 primed with TB (Terrific Broth; per liter contains 12 g tryptone, 24 g yeast extract, 9.4 g potassium phosphate, dibasic, and 2.2 g potassium phosphate, monobasic) (EMD Biosciences; catalog No. 71754), 50 μg/mL kanamycin (Sigma Aldrich), 35 μg/mL chloramphenicol (Sigma Aldrich), 0.4% (w/v) glucose (Sigma Aldrich) and 0.015% (v/v) Antifoam 204 (Sigma Aldrich); Inducer Pump 1 primed with 0.2% (w/v) arabinose (EMD Biosciences); Incubator Gassing Rate set at 2 sec with 51% oxygen, 0.1 mL inoculation volume; Induction Statistic Mean set w/o Outliers (i.e. block mean OD600 determined after excluding the 3 highest and 3 lowest values); culture vessel blocks (CVB) pre-induction delay set at 1 hr 20 min and Expression Incubator Acclimatization set at 30 min.
The seed cultures were prepared and loaded into PiccoloTM along with the necessary labware: 24-well culture vessel blocks (CVBs; The Automation Partnership), 24-well Filter Plates (The Automation Partnership), 24-well Output Plates (Seahorse Bioscience) and Pipette Tip Boxes (MBP) as specified by the manufacturer. The TB media supplemented as described above, arabinose inducer and associated pumps were prepared under sterile conditions and attached to the machine. The centrifuge counterbalance weight was set and placed inside the centrifuge. Lastly, purification reagents were prepared and attached to the system pumps (lysis buffer, resin, wash buffer and elution buffer as described below). Once this was complete, the machine was started and processing began.
Before inoculation, the inocula were mapped to specific wells of 24-well CVB, and expression and induction conditions were set as described in Table 25 below. Each well of the CVBs were filled with 10 mL of TB media supplemented as described above prior to inoculation from the seed plate. Each well of each CVB was inoculated with 0.1 mL seed culture and then returned to the storage carousel to await scheduled admission to pre-induction incubation. Once a CVB was queued to begin pre-induction incubation it was removed from the storage carousel and coupled to an aeration assembly (which provides agitation, well sealing and a means for controlled administration of oxygen/air) and then placed in the pre-induction incubator. OD600 readings were taken upon commencement of incubation and approximately every 30 minutes thereafter. Piccolo operation control software monitors the OD600 measurements to predict when each CVB will reach the 1.0 OD600 set point. Approximately 30 minutes prior to the CVB reaching the OD600 set point the assembly was moved to the expression incubator to equilibrate to the desired expression temperature, and then the cultures in the CVB were induced by addition of the predetermined volume of arabinose as set forth in Table 25. Cell growth was monitored by measuring the OD600 every 30 minutes after induction and plotting the data showing total incubation time versus optical density reading. The 6 CVB expressions/purifications schemes from Table 25 were analyzed. CVB01 through CVB04 exhibited the same growth patterns with the OD600 maxing out around 20 after approximately 1700 minutes. In these plates, growth of the Fab cultures in each of the individual wells of the plates were similar, with a somewhat greater variability in growth rates per well observed in the CVB03 and CVB04 conditions. CVB05 and CVB06 showed slower growth, with the OD600 still increasing at the last time point tested, i.e. total incubation time of 2300 minutes after induction. The growth of the Fab cultures varied between wells of each plate; the maximum OD600 between wells varied between OD600=10 to OD600=16-18.







TABLE 25







Growth Curves of CVB plates Under Various Conditions













Culture

Post-






Vessel
Inocula Wells of 96-
establishment chill
Pre-Induction

Expression
Induction


Block
well plate
Incubation
Incubation
Induction Time
Conditions
Conditions





CVB01
A1, 3, 5, 7, 9, 11
41 min 47 s
7 hr 56 min @ 30° C.
509 min
30 hours @ 16° C.
0.008%(w/v)



B1, 3, 5, 7, 9, 11




arabinose at OD = 2



C1, 3, 5, 7, 9, 11



D1, 3, 5, 7, 9, 11


CVB02
A1, 3, 5, 7, 9, 11
4 hr 26 min 25 s
7 hr 40 min @ 30° C.
492 min
36 hours @ 16° C.
0.008%(w/v)



B1, 3, 5, 7, 9, 11




arabinose at OD = 2



C1, 3, 5, 7, 9, 11



D1, 3, 5, 7, 9, 11


CVB03
E1, 3, 5, 7, 9, 11
1 hr 45 min 40 s
8 hr 11 min @ 30° C.
525 min
30 hours @ 16° C.
0.008%(w/v)



F1, 3, 5, 7, 9, 11




arabinose at OD = 2



G1, 3, 5, 7, 9, 11



H1, 3, 5, 7, 9, 11


CVB04
E1, 3, 5, 7, 9, 11
5 hr 31 min 34 s
8 hr 12 min @ 30° C.
525 min
36 hours @ 16° C.
0.008%(w/v)



F1, 3, 5, 7, 9, 11




arabinose at OD = 2



G1, 3, 5, 7, 9, 11



H1, 3, 5, 7, 9, 11


CVB05
E2, 4, 6, 8, 10, 12
3 hr 11 min 23 s
8 hr 18 min @ 30° C.
532 min
30 hours @ 16° C.
0.008%(w/v)



F2, 4, 6, 8, 10, 12




arabinose at OD = 2



G2, 4, 6, 8, 10, 12



H2, 4, 6, 8, 10, 12


CVB06
E2, 4, 6, 8, 10, 12
6 hr 46 min 46 s
8 hr 35 min @ 30° C.
549 min
36 hours @ 16° C.
0.008%(w/v)



F2, 4, 6, 8, 10, 12




arabinose at OD = 2



G2, 4, 6, 8, 10, 12



H2, 4, 6, 8, 10, 12









Following culture inoculation and growth induction of cultures, the cells were harvested and lysed for purification of Fabs. PiccoloTM was used for purification of the expressed Fab proteins using an automated expression and purification ‘Lifecycle’ of a whole culture purification. After controlled expression, CVBs were chilled for 30 minutes at 6° C. in the storage carousel prior to lysis. The CVB was moved to the liquid handling bed and lysis buffer (2.5 mL of Popculture with 1:1000 Lysonase (EMD Biosciences)) was added to each well with thorough mixing. The lysis proceeded for 10 minutes and then the CVB was centrifuged for 10 minutes at 5000×g to pellet cell debris. During centrifugation, a Filter Plate was placed in the filter bed and resin (2 mL of a 50% slurry of Ni-charged His-Bind resin (EMD Biosciences)) was added to each well. Soluble lysate was added to the corresponding wells of the filter plate containing resin and allowed to bind for 10 minutes prior to draining to waste. Wash buffer (12 mL of wash buffer (50 mM Sodium Phosphate, 300 mM NaCl, 30 mM Imidazole, pH 8.0)) was added in two steps to each well and allowed to drain to waste. Finally, an Output Plate was placed under the Filter Plate in the filter bed and IMAC elution buffer ((50 mM Sodium Phosphate, 300 mM NaCl, 500 mM Imidazole)) was added in two steps draining into the output plate. The output plate was returned to the storage carousel as was all other labware. Once this process was complete for each CVB in the designed run, the machine was unloaded.
Using similar PiccoloTM runs with the same clones allowed for the optimization of incubation temperatures for expression, concentration of arabinose inducer and time of expression. Global optimal results were obtained by use of a two-step temperature incubation where the pre-induction samples are incubated at 30° C. Following induction, protein expression was carried out at 20° C. Optimal expression yields were obtained using 0.032% arabinose inducer followed by 45 hours of expression.
Example 10
Orthogonal Secondary Purification of Fab Antibodies
To rapidly further purify partially pure Fabs generated after the PiccoloTM process, an orthogonal method of purification was developed. Fabs were expressed and purified as described above in Example 9 using the PiccoloTM machine. Approximately 1.8 mL of the IMAC elution per Fab sample obtained from PiccoloTM purification was further purified on a 1 mL Hi-Trap Protein G column (GE Healthcare) at 4° C. using the Akta purifier (GE Healthcare) and A-905 autosampler (GE Healthcare) according to the manufacturer's protocol. The protein samples were transferred to a deep well 96-well block (VWR), which was covered by aluminum foil tape to prevent evaporation. The autosampler was set for multiple injections (typically four injections of 450 μL per sample) onto the Hi-Trap Protein G column. The column was then washed with 2 column volumes of 50 mM sodium phosphate pH7.2, 150 mM NaCl. The Fab was eluted with six column volumes of 100 mM glycine pH2.8. The elution peak fractions (approximately 0.8 mL) were collected in a deep well 96-well plate block. The eluted protein was immediately neutralized with 100 μL saturated dibasic sodium phosphate pH9.0. Protein concentration was determined by measuring absorbance at A280 on a Molecular Dynamic plate reader and calculated from the extinction coefficient of the corresponding Fab. Extinction coefficients are calculated based on the total numbers of Tyrosine+Tryptophane+Phenylalanine in the Fab heavy and light chains. Following purification using the PiccoloTM system, expressed protein was generally less than 20% pure. After orthogonal purification with protein G, Fab purity was greater than 95% pure as indicated by SDS-PAGE.
Table 17 sets forth Fab antibodies generated by Piccolo and further purified in a secondary purification. The amino acid sequences of the Fab antibodies correspond to sequences containing variable heavy chain sequences set forth in any of SEQ ID NOS: 1475-1826 and variable light chain sequences set forth in SEQ ID NO: 1827-1838, 1840-1855 and 1857-188. The sequences of the heavy and light chain Fab library members also further contains a sequence for a constant region included in Plasmid A, C or E.
Example 11
Lymphoma Apoptosis Assay
To identify unique Fabs from an antibody library, assays can be performed to assess any desired function, property or activity. As an example of an assay for protein function, cross-linked anti-CD20 Fab (heavy chain set forth in SEQ ID NO:453 and light chain set forth in SEQ ID NO:835) was tested in a cell based assay for the ability to induce apoptosis in lymphoma cells. Anti-CD20 Fabs were cross-linked by adding equal molar concentration of polyclonal anti human kappa light chain antibodies that recognize different parts of the anti-CD20 kappa light chain.
Apoptosis was determined using the Apo-ONE homogenous Caspase-3/7 Assay (Promega). Ramos B-Lymphocyte cells were grown in RPMI1640 media containing 10% FBS (fetal bovine serum), P-S (penicillin-streptomycin) and arrayed into a clear 96-well plate (Costar 3595, Corning, 2.5×104 cells per well). Jurkat T-Lymphocyte cells were grown in RPMI1640 with 10% FBS and P-S, and arrayed into a clear 96-well plate (Costar 3595, Corning, 2.5×104 cells per well). Crosslinked anti-CD20 Fab was added to each well at concentrations of 0, 25, 50, 100 and 200 nM together with equal molar concentrations of polyclonal antibody against human kappa light chain (Sigma). Anti-Fas monoclonal antibody (20-100 ng/ml) or staurosporine (1.0 μM) were added for positive controls and trastuzumab Fab was used as a negative control. An equal volume of Apo-ONE Caspase-3/7 Reagent (rhodamine 110, bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide, Z-DEVD-R110) was added and the plate was incubated for one hour at room temperature. The presence of rhodamine 110 was detected by measuring fluorescence in a fluorescent plate reader at 485 nm exitation/521 nm emission. The increase in the percentage of apoptosis was calculated according to the equation: 100×[(Fluorescence for Fab)−(Fluorescence for background)]/(Fluorescence for background).
Example 12
Erythropoietin Binding Fab Library
As an alternative to “naïve” libraries, directed Fab libraries are constructed to a known target. In this example, a directed library was constructed in which a 16 amino acid erythropoietin peptide (EPO) was inserted into various CDRs of an antibody to identify Fabs that induce activation of recombinant EPO (EpoR). Because there are variations in the number of amino acid residues that occur in a CDR (see, e.g., Table 26), CDRs with larger inserts were selected for insertion of the EPO peptide. These CDRs include CDR-H2, CDR-H3, CDR-L1, and CDR-L3.







TABLE 26







Amino Acid Length of CDR










Heavy Chain
Light Chain















CDR1
10-12
10-17



CDR2
16-19
7



CDR3
 3-25
 7-11










To randomize the orientation of the peptide insert to expose the active surface of the peptide, two extra amino acid residues, either proline or glycine, were added to the N-terminus and C-terminus of the EPO peptide, yielding sixteen different peptides (SEQ ID NO:874-889). A Fab, containing heavy chain VH3-23 (SEQ ID NO:869) and light chain A17 (SEQ ID NO:871), served as a parent antibody for the EPO peptide library. BsaI restriction sites were introduced into the nucleic acid sequences of the EPO peptides and the nucleic acid sequences of the heavy and light chain variable regions in order to allow the cloning of the DNA encoding the EPO peptides into the respective CDRs. Heavy chain VH3-23 DNA was modified to create VH3-23B (SEQ ID NO:896) containing BsaI sites at CDR2 and VH3-23R (SEQ ID NO:913) containing BsaI sites at CDR3. Light chain A17 DNA was modified to create A17P (SEQ ID NO:872) containing BsaI sites at CDR1 and A17Q (SEQ ID NO:873) containing BsaI sites at CDR3. Sixteen different genes encoding the EPO peptides were cloned into the respective heavy (CDR2 and CDR3) or light (CDR1 and CDR3) chain sequences and the resulting Fabs were expressed and purified as described in Example 9. Table 27 lists the resulting 64 EPO containing Fabs.







TABLE 27







EPO Peptide containing Fab Library








HEAVY CHAIN
LIGHT CHAIN










NAME
SEQ ID NO
NAME
SEQ ID NO





H2-EP14 (VH3-23B)
897
A17
871


H2-EP18 (VH3-23B)
898
A17
871


H2-EP19 (VH3-23B)
899
A17
871


H2-EP20 (VH3-23B)
900
A17
871


H2-EP21 (VH3-23B)
901
A17
871


H2-EP22 (VH3-23B)
902
A17
871


H2-EP23 (VH3-23B)
903
A17
871


H2-EP24 (VH3-23B)
904
A17
871


H2-EP25 (VH3-23B)
905
A17
871


H2-EP26 (VH3-23B)
906
A17
871


H2-EP27 (VH3-23B)
907
A17
871


H2-EP28 (VH3-23B)
908
A17
871


H2-EP29 (VH3-23B)
909
A17
871


H2-EP30 (VH3-23B)
910
A17
871


H2-EP31 (VH3-23B)
911
A17
871


H2-EP32 (VH3-23B)
912
A17
871


H3-EP14 (VH3-23R)
914
A17
871


H3-EP18 (VH3-23R)
915
A17
871


H3-EP19 (VH3-23R)
916
A17
871


H3-EP20 (VH3-23R)
917
A17
871


H3-EP21 (VH3-23R)
918
A17
871


H3-EP22 (VH3-23R)
919
A17
871


H3-EP23 (VH3-23R)
920
A17
871


H3-EP24 (VH3-23R)
921
A17
871


H3-EP25 (VH3-23R)
922
A17
871


H3-EP26 (VH3-23R)
923
A17
871


H3-EP27 (VH3-23R)
924
A17
871


H3-EP28 (VH3-23R)
925
A17
871


H3-EP29 (VH3-23R)
926
A17
871


H3-EP30 (VH3-23R)
927
A17
871


H3-EP31 (VH3-23R)
928
A17
871


H3-EP32 (VH3-23R)
929
A17
871


VH3-23
869
L1-EP14 (A17P)
930


VH3-23
869
L1-EP18 (A17P)
931


VH3-23
869
L1-EP19 (A17P)
932


VH3-23
869
L1-EP20 (A17P)
933


VH3-23
869
L1-EP21 (A17P)
934


VH3-23
869
L1-EP22 (A17P)
935


VH3-23
869
L1-EP23 (A17P)
936


VH3-23
869
L1-EP24 (A17P)
937


VH3-23
869
L1-EP25 (A17P)
938


VH3-23
869
L1-EP26 (A17P)
939


VH3-23
869
L1-EP27 (A17P)
940


VH3-23
869
L1-EP28 (A17P)
941


VH3-23
869
L1-EP29 (A17P)
942


VH3-23
869
L1-EP30 (A17P)
943


VH3-23
869
L1-EP31 (A17P)
944


VH3-23
869
L1-EP32 (A17P)
945


VH3-23
869
L3-EP14 (A17Q)
946


VH3-23
869
L3-EP18 (A17Q)
947


VH3-23
869
L3-EP19 (A17Q)
948


VH3-23
869
L3-EP20 (A17Q)
949


VH3-23
869
L3-EP21 (A17Q)
950


VH3-23
869
L3-EP22 (A17Q)
951


VH3-23
869
L3-EP23 (A17Q)
952


VH3-23
869
L3-EP24 (A17Q)
953


VH3-23
869
L3-EP25 (A17Q)
954


VH3-23
869
L3-EP26 (A17Q)
955


VH3-23
869
L3-EP27 (A17Q)
956


VH3-23
869
L3-EP28 (A17Q)
957


VH3-23
869
L3-EP29 (A17Q)
958


VH3-23
869
L3-EP30 (A17Q)
959


VH3-23
869
L3-EP31 (A17Q)
960


VH3-23
869
L3-EP32 (A17Q)
961









Screening to identify Fabs that modulated activation of the Epo Receptor (EpoR, SEQ ID NO:962) was performed in BaF3 cells stably transfected with EpoR-encoding cDNA.
a. MTT Cell Proliferation Assay
The tetrazolium ring of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) is cleaved by mitochondrial dehydrogenase of viable cells to produce purple MTT formazan crystals that are insoluble in aqueous solution but can be solubilized in acidified isopropanol. BaF3/EpoR cells (5×103-5×105) were incubated for one or two days in the presence and in the absence of the Epo Fabs. MTT solution (10% of the culture volume) was added to cells and incubated 3-4 hrs at 37° C. in a CO2 incubator. An equal volume of 0.1 N HCl in isopropanol was added. Absorbance was measured at 570 nm and the number of cells was calculated based on the standard curve.
b. Luciferase Reporter Assay
Activation of EpoR leads to activation of STAT5 transcription factor, which in turn induces transcription of genes including c-myc, bcl-2, pim-1 and cyclin-D. Transcription can be detected by linking the promotor of any one of the above genes to a reporter gene, thus creating a reporter plasmid. Two reporter plasmids were created to assess activation of the EpoR. The first reporter plasmid was constructed by placing the mouse cyclin-D promoter (SEQ ID NO:963) at the 5′ end of the luciferase gene in the pGL4.70 vector (Promega; SEQ ID NO:1997). Briefly, pGL4.70 was digested with restriction enzymes NheI and HindIII and the cyclin D promoter was inserted between nucleotides 28-66 of the plasmid using standard molecular biology protocols.
An EPO Fab that is capable of activating EpoR will cause activation of STAT5, thereby activating the cyclin-D promoter causing induction and expression of the luciferase gene. Hence, a second reporter plasmid was created for STAT5 by using the pGL4.23 vector (Promega; SEQ ID NO: 1998), a vector that contains a minimal promoter at the 5′ end of the translation start site for luciferase that allows for activation of a promoterless-response element to drive expression. Six repeats of a DNA element for STAT5 binding (SEQ ID NO:964) were cloned directly upstream of the minimal promoter. Briefly, pGL4.23 was digested with restriction enzymes NheI and HindIII and the DNA element repeats of STAT5 was inserted between nucleotides 28-66 of the plasmid using standard molecular biology protocols.
An EPO Fab capable of activating EpoR will cause the activation of STAT5 and the direct expression of the luciferase gene. BaF3 cells, previously transfected with EpoR-encoding cDNA, were transiently transfected with one of the reporter plasmids above. The resulting cells are incubated in the presence and absence of EPO Fabs. An equal volume of lysis buffer containing luciferase substrate is added to the cell culture. Relative luminescence was measured for 10 seconds using a luminometer after 5 min incubation with the substrate.
Example 13
Electrochemiluminescence Binding Assay
In this example, an electrochemiluminescence (ECL) binding assay was used to screen a 960 member Fab library for antibodies capable of binding to one of nine different antigens, including the human epidermal growth factor 2 receptor (ErbB2), epidermal growth factor receptor (EGF R), hepatocyte growth factor receptor (HGF R/c-Met), Notch-1, CD44, insulin-like growth factor-1 soluble receptor (IGF-1 sR), P-cadherin, erythropoietin receptor (Epo R) and delta-like protein 4 (DLL4). In an ECL assay, an antigen-antibody interaction is detected by addition of a detection antibody labeled with ruthenium tri-bispyridine-(4-methysulfone) (Ru(bpy)22+). Upon application of an electric current, the Ru(bpy)22+-label undergoes an oxidation-reduction cycle in the presence of a co-reactant and light is emitted. A signal is only generated when the Ru(bpy)22+-label is in close proximity to the electrode, eliminating the need for washing. Detected light intensity is proportional to the amount of captured protein.
Recombinant human proteins were obtained from R&D Systems and included: rHuman ErbB2/Fc Chimera, CF (Cat#1129-ER); rHuman EGF R/Fc Chimera, CF (Cat#344-ER); rHuman HGF R/c-MET/Fc Chimera, CF (Cat#358-MT/CF); rHuman Notch-1/Fc Chimera, CF (Cat#3647-TK); rHuman CD44/Fc Chimera, CF (Cat#3660-CD); rHuman IGF-1 sR,(IGF-1 sR), CF (Cat#391-GR); rHuman P-Cadherin/Fc Chimera, CF (Cat#861-PC); rHuman Erythropoietin R/Fc Chimera, CF (Cat#963-ER); and Recombinant Human DLL4 (Cat#1506-D4/CF). The proteins were immobilized onto each well of 10 plates by spotting 50 nanoliters (nl) of each protein (of a 60 μg/mL antigen) on the surface of a 96-well Multi-Spot 10 Highbind plate (Meso Scale Discovery; Gaithersburg Md.). Spot 10 was left blank as a control.
An 150 μl aliquot of 1% Bovine Serum Albumin (BSA) in Tris-buffered Saline Tween (TBST) was added to each well and allowed to incubate for 30 min at 20° C. followed by washing and tap drying to completely remove any residual solution. Subsequently, a 12.5 μl aliquot of 1% BSA TBST was added to each well followed by the addition of a 12.5 μl aliquot of a purified Fab. The plate was sealed and incubated for 1 hour at 20° C. with shaking.
Detection antibodies were prepared by individually conjugating both goat anti-human Kappa light chain polyclonal antibody (K3502-1MG, Sigma-Aldrich) and goat anti-human Lambda light chain polyclonal antibody (L1645-1ML, Sigma-Aldrich) with Ruthenium (II) tris-bipyridine-(4-methylsulfone)-N-hydroxysuccinimide (SULFO-TAG NHS-ester, Meso Scale Discovery) according to the manufacturer's instructions. TAG-detection antibody at 25 μl was added to each well and allowed to incubate for 1 hour at 20° C. with shaking. Finally, 15 μl of Read Buffer P with Surfactant (Cat #R92PC-1, Meso Scale Discovery) was added to each well. The electrochemiluminescence was measured using a Sector Imager 2400 (Meso Scale Discovery). Data was analyzed by comparing the ECL signals for an antigen to the blank of each well. A signal to blank ratio of 4 or more was considered a “Hit” Fab.
Ten plates, each containing 96 different Fabs, were screened using the ECL assay. The results of the initial screen are shown in Tables 28-28B, below. Table 28, below, lists the 6 Fabs (including the heavy chain and light chain) that were identified as “hits” in the initial ECL screen. A “hit” was a Fab antibody with signal to blank ratio of greater than 4. Three Fabs were identified that bind to recombinant human delta-like protein 4 (DLL4). One Fab was identified that binds to recombinant human epidermal growth factor 2 (ErbB2) and one Fab was identified that binds to recombinant human erythropoietin receptor (Epo R). An additional Fab was identified that binds to both ErbB2 and EpoR. The results of the initial MSD assay screen at a single Fab concentration are listed in Table 28B below. Table 28B lists the 6 Fabs (the Fab No. corresponds each of the Fabs identified in Table 28), the Fab concentration, the 9 recombinant human target/protein antigens, and the ECL signals from the initial MSD assay screen at the given Fab concentration.
To confirm a “Hit” from the intial ECL screening, a Fab concentration dependent titration was carried out to determine the Fab-antigen binding affinity. The assay procedure was the same as described above, except that the concentration of Fab antibody was varied between wells from 0.1 nM to 2.4 μM. The data are set forth in Tables 29-34 below. The data were graphed using Microsoft Excel and the binding affinity was estimated from the 50% binding signal. As noted, binding affinity can be assay dependent (see Example 16).
The results show that Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 specifically binds Human DLL4 with high affinity in the lower nanomolar range at or about 10 nM or lower, whereas Fabs VH5-51_IGHD5-18*01>3-IGHJ4*01 & V3-4_IGLJ1*01, and VH6-1-IGHD3-3*01-IGHJ4*01 & V4-3-IGLJ4*01 bind Human DLL4 in the micromolar range. Since the three Fabs contain different heavy and light chains, the results suggest that the binding epiotopes on DL44 recognized by the antibodies can be different.
The results further show that Fab VH4-31IGHD1-26*01_IGHJ2*01 & A27_IGKJ1*01 binds Human ErbB2/Fc chimera at a concentration of approximately 100 nM and Fab VH1-46_IGHD3-10*01_IGHJ4*01 & B3_IGKJ1*01 binds Human Erythropoietin R/Fc chimera at a concentration of approximately 100 nM. One Fab, VH1-46_IGHD6-13*01_IGH41*01 & B3_IGKJ1*01, shows affinity for both Human ErbB2/Fc and Human Erythropoietin R/Fc chimeras. It is unlikely that this Fab is binding to the Fc region of the chimera proteins, since no binding was observed to five other antigens that were Fc fusion proteins.







TABLE 28







Identified Fab “Hits”












Fab No.
Target
Heavy Chain
SEQ ID NO
Light Chain
SEQ ID NO





1
rHuman DLL4
VH1-46_IGHD6-
1513
L6_IGKJ1*01
1850




6*01_IGHJ1*01


2
rHuman DLL4
VH5-51_IGHD5-
1803
V3-4_IGLJ1*01
1881




18*01>3_IGHJ4*01


3
rHuman DLL4
VH6-1_IGHD3-
1812
V4-3_IGLJ4*01
1884




3*01_IGHJ4*01


4
rHuman ErbB2/
VH4-31_IGHD1-
1760
A27_IGKJ1*01
1833



Fc chimera
26*01_IGHJ2*01


5
rHuman Epo R/
VH1-46_IGHD3-
1509
B3_IGKJ1*01
1838



Fc chimera
10*01_IGHJ4*01


6
rHuman ErbB2/Fc
VH1-46_IGHD6-
1512
B3_IGKJ1*01
1838



chimera and rHuman
13*01_IGHJ4*01



Epo R/Fc chimera
















TABLE 28B







ECL Signals for Identified Fab “Hits” from 960 Fab Library Screen









Fab
[Fab]
rHuman Target


















No.
uM
ErbB2
EGFR
HGFR
Notch-1
CD44
IGF-1
P-cad
EPOR
DLL4
Blank





















1
0.14
870
823
848
614
515
663
423
693
19293
237


2
2.38
594
681
931
636
666
691
1224
834
12172
526


3
0.21
9229
815
744
949
837
763
890
1115
1069
529


4
0.23
1251
942
954
722
675
982
508
777
2808
363


5
0.74
562
652
633
980
682
593
1155
4108
1000
692


6
0.43
16499
1233
1138
1793
1497
1139
5245
12985
2538
879
















TABLE 29







Binding affinity of Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01















Fab[nM]
2383
595.8
148.9
37.2
9.3
2.3
0.6
0.1


















ErbB2/Fc
454
321
247
384
354
291
215
306


EGF R/Fc
621
403
290
228
424
289
309
311


HGF R/Fc
762
353
205
207
324
253
256
286


Notch-1/Fc
690
306
375
402
492
333
337
378


CD44/FC
559
372
348
356
396
317
238
323


IGF-1 sR
527
335
322
295
315
231
313
241


P-Cadherin/Fc
728
617
687
649
452
401
321
235


EPO R/Fc
658
378
373
315
306
429
337
373


DLL4
11794
17203
16253
16717
13210
3055
508
317


Blank
344
285
218
199
287
234
226
201
















TABLE 30







Binding affinity of Fab VH5-51_IGHD5-


18*01>3_IGHJ4*01 & V3-4_IGLJ1*01













Fab[nM]
154
51
17
6

















ErbB2/Fc
1593
1248
1033
873



EGF R/Fc
1398
816
805
742



HGF R/Fc
1520
1044
914
831



Notch-1/Fc
929
685
558
464



CD44/Fc
960
651
518
547



IGF-1 sR
1396
1051
872
854



P-Cadherin/Fc
1733
854
542
358



EPO R/Fc
1195
750
620
548



DLL4
40392
17025
7158
1946



Blank
447
335
143
191

















TABLE 31







Binding affinity of Fab VH6-1_IGHD3-


3*01_IGHJ4*01 & V4-3_IGLJ4*01















Fab[nM]
480
240
120
60
30
15
7.5
3.8


















ErbB2/Fc
965
833
822
777
726
713
695
714


EGF R/Fc
877
690
658
679
585
584
582
511


HGF R/Fc
951
834
785
623
640
694
558
519


Notch-1/Fc
545
368
472
415
425
508
392
383


CD44/FC
541
470
442
434
484
454
444
419


IGF-1 sR
741
625
813
654
697
705
642
463


P-Cadherin/Fc
596
383
450
372
440
351
352
281


EPO R/Fc
621
478
431
423
325
397
443
407


DLL4
1532
1273
938
875
736
690
598
462


Blank
362
316
363
237
213
261
217
198
















TABLE 32







Binding affinity of Fab VH4-31_IGHD1-


26*01_IGHJ2*01 & A27_IGKJ1*01















Fab[nM]
410
205
102.5
51.3
25.6
12.8
6.4
3.2


















ErbB2/Fc
5422
5260
4355
3588
2992
2255
1796
868


EGF R/Fc
734
595
455
379
373
320
249
254


HGF R/Fc
753
735
425
456
382
258
234
294


Notch-1/Fc
804
722
607
408
270
249
279
275


CD44/FC
767
613
461
409
332
273
240
295


IGF-1 sR
600
565
443
316
311
323
209
313


P-Cadherin/Fc
814
769
714
424
323
245
197
206


EPO R/Fc
797
595
587
498
409
338
264
233


DLL4
859
599
550
474
384
268
256
242


Blank
637
430
437
337
345
227
133
172
















TABLE 33







Binding affinity of Fab VH1-46_IGHD3-


10*01_IGHJ4*01 & B3_IGKJ1*01















Fab[nM]
1410
705
352.5
176.3
88.1
44.1
22
11


















ErbB2/Fc
932
671
514
448
200
347
363
216


EGF R/Fc
1071
692
769
428
376
428
312
201


HGF R/Fc
903
839
606
418
392
336
203
268


Notch-1/Fc
1034
958
715
664
440
331
389
404


CD44/FC
885
693
556
376
340
302
317
296


IGF-1 sR
426
630
528
393
273
309
347
289


P-Cadherin/Fc
1059
827
649
532
278
343
215
270


EPO R/Fc
4314
4894
4105
3519
3368
2387
2241
1824


DLL4
1265
981
660
460
434
388
342
254


Blank
709
483
494
346
301
200
289
212
















TABLE 34







Binding affinity of Fab VH1-46_IGHD6-13*01_IGHJ4*01 & B3_IGKJ1*01















Fab[nM]
1000
500
250
125
62.5
31.3
15.6
7.8


















ErbB2/Fc
8731
10241
11026
12956
13124
13911
14791
13220


EGF R/Fc
2236
1468
1138
860
602
447
346
379


HGF R/Fc
2109
1371
1221
778
578
299
293
282


Notch-1/Fc
2267
1975
1241
802
536
563
418
486


CD44/FC
1966
1685
1175
764
591
439
473
409


IGF-1 sR
1667
1334
993
654
491
385
349
353


P-Cadherin/Fc
4495
3447
2784
1481
1173
1105
971
695


EPO R/Fc
8594
10305
8535
9237
7749
7878
8357
6765


DLL4
2785
2319
1560
912
715
528
525
407


Blank
1133
680
590
403
268
250
294
316









Example 14
VH3-23 Heavy Chain Library
In this example, a library of 690 different VH3-23 containing heavy chains was generated using standard molecular biology protocols. Library diversity was generated by 1) use of 6 different JH segments; 2) including the DIRECT sequence and INVERTED sequence (or reverse complement) of each DH segment; and 3) translating each DIRECT and INVERTED DH segment in all 3 reading frames. This resulted in the initial generation of six pF-VH3-23-IGHJ plasmids and115 different DH segments. Simultaneous cloning of each individual DH segment into each pF-VH3-23-IGHJ plasmid resulted in the 690 member heavy chain library. The library was then transformed with various previously generated light chains (see Example 8) to generate a 3012 member Fab library.
A. Generation of pF-VH3-23-IGHJ1 to pF-VH3-23-IGHJ6 Plasmids
In this example, 6 plasmids, each encoding the VH3-23 VH segment and one of six JH segments, were generated. The plasmids were modified such that BsaI sites were incorporated to 1) allow the cloning of individual JH segments (see Table 35); and 2) subsequently allow the cloning of any individual DH segment (see Table 37). To this end, Plasmid A (SEQ ID NO:1) and VH segment VH3-23R (SEQ ID NO:2050) were first modified to remove internal BsaI sites (SEQ ID NO:3719). Subsequently, VH3-23R was further modified to add two BsaI sites, including 1) at the 3′ end of the VH3-23 VH segment; and 2) at the 5′ end of the nucleotides encoding framework region 4 (amino acids WGQGTLVTVSSAS of SEQ ID NOS:3456-3461, see Table 35 below) of the JH segment. VH3-23R (SEQ ID NO:2050) was then synthesized using standard DNA synthesis protocols. VH3-23R was digested with NcoI (SEQ ID NO:977) and NheI (SEQ ID NO:978) and ligated into the modified Plasmid A creating plasmid pF-VH3-23R (SEQ ID NO:2051).
A series of forward and reverse oligos (see Table 36, below) encoding segments IGHJ1-IGHJ6 (see Table 35, and SEQ ID NOS:3450-3455 and SEQ ID NOS:3456-3461) were generated by standard DNA synthesis protocols. Pairs of oligos (encoding a particular IGHJ segment) were digested with BsaI and ligated into similarly digested plasmid pF-VH3-23R (SEQ ID NO:2051), thereby generating 6 new plasmids: pF-VH3-23-IGHJ1 (SEQ ID NO:2064), pF-VH3-23-IGHJ2 (SEQ ID NO:2065), pF-VH3-23-IGHJ3 (SEQ ID NO:2066), pF-VH3-23-IGHJ4 (SEQ ID NO:2067), pF-VH3-23-IGHJ5 (SEQ ID NO:2068), and pF-VH3-23-IGHJ6 (SEQ ID NO:2069). For each of the new plasmids, this resulted in the removal of the BsaI site at the 5′ end of the nucleotides encoding framework region 4 and the generation of a new BsaI site at the 5′ end of the JH segment to allow subsequent cloning of a single DH segment into all six vectors simultaneously thereby creating six different genes per each DH segment.







TABLE 35







IGHJ1 to IGHJ6 Segments








Segment
Nucleotide Sequence





IGHJ1 
GCT GAA TAC TTC CAG CAC TGG GGC CAA GGA ACC CTG GTC ACC GTC TCC


(SEQ ID NO: 3450)
TCA G


 


IGHJ2 
C TAC TGG TAC TTC GAT TCC TGG GGC CAA GGA ACC CTG GTC ACC GTC


(SEQ ID NO: 3451)
TCC TCA G


 


IGHJ3 
T GCT TTT GAT GTC TGG GGC CAA GGA ACC CTG GTC ACC GTC TCC TCA G


(SEQ ID NO: 3452)



 


IGHJ4 
AC TAC TTT GAC TAC TGG GGC CAA GGA ACC CTG GTC ACC GTC TCC TCA G


(SEQ ID NO: 3453)



 


IGHJ5 
AC AAC TGG TTC GAC TAC TGG GGC CAA GGA ACC CTG GTC ACC GTC TCC


(SEQ ID NO: 3454)
TCA G


 


IGHJ6 
AT TAC TAC TAC TAC TAC GGT ATG GAC GTC TGG GGC CAA GGA ACC CTG


(SEQ ID NO: 3455)
GTC ACC GTC TCC TCA G


 


IGHJ1 
AEYFQHWGQGTLVTVSSAS


(SEQ ID NO: 3456)



 


IGHJ2 
YWYFDSWGQGTLVTVSSAS


(SEQ ID NO: 3457)



 


IGHJ3 
AFDVWGQGTLVTVSSAS


(SEQ ID NO: 3458)



 


IGHJ4 
YFDYWGQGTLVTVSSAS


(SEQ ID NO: 3459)



 


IGHJ5 
NWFDYWGQGTLVTVSSAS


(SEQ ID NO: 3460)



 


IGHJ6 
YYYYYGMDVWGQGTLVTVSSAS


(SEQ ID NO: 3461)
















TABLE 36







Oligos used to generate J1 to J6 plasmids








Oligo
Nucleotide Sequence





J1_F
CGAAAGAGACCTACTATGGTTCGGGTCTCTCCTACGCTGAATACTTCCAGCACT


(SEQ ID NO: 2052)



 


J1_R
CCCCAGTGCTGGAAGTATTCAGCGTAGGAGAGACCCGAACCATAGTAGGTCTCT


(SEQ ID NO: 2053)



 


J2_F
CGAAAGAGACCTACTATGGTTCGGGTCTCTCCTACTACTGGTACTTCGATTCCT


(SEQ ID NO: 2054)



 


J2_R
CCCCAGGAATCGAAGTACCAGTAGTAGGAGAGACCCGAACCATAGTAGGTCTCT


(SEQ ID NO: 2055)



 


J3_F
CGAAAGAGACCTACTATGGTTCGGGTCTCTCCTATGCTTTTGATGTCT


(SEQ ID NO: 2056)



 


J3_R
CCCCAGACATCAAAAGCATAGGAGAGACCCGAACCATAGTAGGTCTCT


(SEQ ID NO: 2057)



 


J4_F
CGAAAGAGACCTACTATGGTTCGGGTCTCTCCTACTACTTTGACTACT


(SEQ ID NO: 2058)



 


J4_R
CCCCAGTAGTCAAAGTAGTAGGAGAGACCCGAACCATAGTAGGTCTCT


(SEQ ID NO: 2059)



 


J5_F
CGAAAGAGACCTACTATGGTTCGGGTCTCTCCTACAACTGGTTCGACTACT


(SEQ ID NO: 2060)



 


J5_R
CCCCAGGAGTCGAACCAGTTGTAGGAGAGACCCGAACCATAGTAGGTCTCT


(SEQ ID NO: 2061)



 


J6_F
CGAAAGAGACCTACTATGGTTCGGGTCTCTCCTATTACTACTACTACTACGGTATGGACGTCT


(SEQ ID NO: 2062)



 


J6_R
CCCCAGACGTCCATACCGTAGTAGTAGTAGTAATAGGAGAGACCCGAACCATAGTAGGTCTCT


(SEQ ID NO: 2063)









B. Generation and Cloning of Modified DH Oligos into pF-VH3-23-IGHJ Plasmids
In this example, pairs of DH oligos were generated and cloned into the pF-VH3-23-IGHJ plasmids thereby generating 690 new VH3-23 heavy chains as follows. Twenty seven (27) DH segments (see Table 37 below) were selected for cloning into each modified pF-VH3-23-IGHJ plasmid. Library diversity in the CDR3 region was generated by 1) including the DIRECT sequence and INVERTED sequence (or reverse complement) of each DH segment; and 2) translating each DIRECT and INVERTED DH segment in all 3 reading frames. If the translation of a reading frame for any given DH segment resulted in a stop codon, that particular sequence and reading frame were excluded from the library. Table 37 below indicates the open reading frames for each particular DH segment that were included in the library, thereby resulting in 115 different DH segments.







TABLE 37







DH segments included in heavy chain library














DIRECT

INVERTED





(SEQ
Reading
(SEQ
Reading


Number
D segment
ID NO)
Frames
ID NO)
Frames















1
IGHD1-1*01
239
1, 2, 3
3462
1, 2, 3


2
IGHD1-7*01
243
1, 3
3463
1, 3


3
IGHD1-14*01
240
1, 3
3464
1, 2, 3


4
IGHD1-20*01
241
1, 3
3465
1, 2, 3


5
IGHD1-26*01
242
1, 3
3466
1, 3


6
IGHD2-2*01
245
2, 3
3467
1, 3


7
IGHD2-8*01
250
2, 3
3468
1


8
IGHD2-15*01
244
2, 3
3469
1, 3


9
IGHD2-21*01
248
2, 3
3470
1, 3


10
IGHD3-3*01
258
1, 2, 3
3471
1, 3


11
IGHD3-9*01
259
2
3472
1, 3


12
IGHD3-10*01
252
2, 3
3473
1, 3


13
IGHD3-16*01
254
2, 3
3474
1, 3


14
IGHD3-22*01
256
2, 3
3475
1, 3


15
IGHD4-4*01
263
2, 3
3476
1, 3


16
IGHD4-11*01
260
2, 3
3477
1, 3


17
IGHD4-17*01
261
2, 3
3478
1, 3


18
IGHD4-23*01
262
2, 3
3479
1, 3


19
IGHD5-5*01
267
1, 2, 3
3480
1, 3


20
IGHD5-12*01
264
1, 3
3481
1, 3


21
IGHD5-18*01
265
1, 2, 3
3482
1, 3


22
IGHD5-24*01
266
1, 3
3483
1, 3


23
IGHD6-6*01
271
1, 2
3484
1, 2, 3


24
IGHD6-13*01
268
1, 2
3485
1, 2, 3


25
IGHD6-19*01
269
1, 2
3486
1, 2, 3


26
IGHD6-25*01
270
1, 2
3487
1, 3


27
IGHD7-27*01
272
1, 3
3488
1, 2









The following rules (see Table 38) were used to design pairs of D oligos for cloning into the six different vectors to ensure that the resulting heavy chain sequences are in frame. The rules were applied to both the direct and inverted DH segments. In order to facilite cloning into the BsaI sites of the pF-VH3-23-IGHJ plasmids, the following nucleotides were added to the DH segment oligonucleotides:










DIRECT 5′ End:
CGAAA;



 



DIRECT 3′ End:
T;



 



INDIRECT 5′ End:
CGAAA;



 



INDIRECT 3′ End:
T.






The resulting oligos for each particular DH segment are listed in Tables 39-40 below. The nucleotides added to facilite cloning are in lower case letters. The nucleotides encoding the DH segment are in upper case letters. The oligos were synthesized by standard DNA synthesis technology. Pairs of oligos (encoding a particular DH segment) were digested with BsaI and ligated into similarly digested plasmids pF-VH3-23-IGHJ1 to pF-VH3-23-IGHJ6 thereby generating a VH3-23 library of 690 members, listed in Table 41 below. The VH3-23 heavy chain library was co-transformed with various light chains (as described in Example 8) creating a Fab library of 3012 members.







TABLE 38







Rules for generation of DH segment oligos










No. of



Frame
nucleotides
Manipulation





1
3N
NONE


1
3N + 1
Remove nucleotide at 3′ end


1
3N + 2
Add G to 3′ end


2
3N
Remove nucleotide at 5′ end, add G to 3′ end


2
3N + 1
Remove nucleotide at 5′ end


2
3N + 2
Remove nucleotide at 5′ end, add GG to 3′ end


3
3N
Add G to 5′ end, remove nucleotide at 3′ end


3
3N + 1
Add G to 5′ end, remove 2 nucleotides at 3′ end


3
3N + 2
Add G to 5′ end
















TABLE 39







Direct DH segment oligos













SEQ

SEQ




ID

ID


D segment
Forward
NO
Reverse
NO





1_IGHD1-1*01
cgaaaGGTACAACTGGAACGACGt
3489
taggaCGTCGTTCCAGTTGTACCt
3490


 


2_IGHD1-1*01
cgaaaGTACAACTGGAACGACGGt
3491
taggaCCGTCGTTCCAGTTGTACt
3492


 


3_IGHD1-1*01
cgaaaGGGTACAACTGGAACGACt
3493
taggaGTCGTTCCAGTTGTACCCt
3494


 


1_IGHD1-7*01
cgaaaGGTATAACTGGAACTACGt
3495
taggaCGTAGTTCCAGTTATACCt
3496


 


3_IGHD1-7*01
cgaaaGGGTATAACTGGAACTACt
3497
taggaGTAGTTCCAGTTATACCCt
3498


 


1_IGHD1-14*01
cgaaaGGTATAACCGGAACCACGt
3499
taggaCGTGGTTCCGGTTATACCt
3500


 


3_IGHD1-14*01
cgaaaGGGTATAACCGGAACCACt
3501
taggaGTGGTTCCGGTTATACCCt
3502


 


1_IGHD1-20*01
cgaaaGGTATAACTGGAACGACGt
3503
taggaCGTCGTTCCAGTTATACCt
3504


 


3_IGHD1-20*01
cgaaaGGGTATAACTGGAACGACt
3505
taggaGTCGTTCCAGTTATACCCt
3506


 


1_IGHD1-26*01
cgaaaGGTATAGTGGGAGCTACT
3507
taggaCGTAGTAGCTCCCACTATAC
3508



ACGt

Ct



 


3_IGHD1-26*01
cgaaaGGGTATAGTGGGAGCTAC
3509
taggaGTAGTAGCTCCCACTATACC
3510



TACt

Ct



 


2_IGHD2-2*01
cgaaaGGATATTGTAGTAGTACCA
3511
taggaGGCATAGCAGCTGGTACTA
3512



GCTGCTATGCCt

CTACAATATCCt



 


3_IGHD2-2*01
cgaaaGAGGATATTGTAGTAGTAC
3513
taggaCATAGCAGCTGGTACTACTA
3514



CAGCTGCTATGt

CAATATCCTCt



 


2_IGHD2-8*01
cgaaaGGATATTGTACTAATGGTG
3515
taggaGGTATAGCATACACCATTAG
3516



TATGCTATACCt

TACAATATCCt



 


3_IGHD2-8*01
cgaaaGAGGATATTGTACTAATGG
3517
taggaTATAGCATACACCATTAGTA
3518



TGTATGCTATAt

CAATATCCTCt



 


2_IGHD2-15*01
cgaaaGGATATTGTAGTGGTGGT
3519
taggaGGAGTAGCAGCTACCACCA
3520



AGCTGCTACTCCt

CTACAATATCCt



 


3_IGHD2-15*01
cgaaaGAGGATATTGTAGTGGTG
3521
taggaAGTAGCAGCTACCACCACTA
3522



GTAGCTGCTACTt

CAATATCCTCt



 


2_IGHD2-21*01
cgaaaGCATATTGTGGTGGTGATT
3523
taggaGGAATAGCAATCACCACCAC
3524



GCTATTCCt

AATATGCt



 


3_IGHD2-21*01
cgaaaGAGCATATTGTGGTGGTG
3525
taggaAATAGCAATCACCACCACAA
3526



ATTGCTATTt

TATGCTCt



 


1_IGHD3-3*01
cgaaaGTATTACGATTTTTGGAGT
3527
taggaGTATAATAACCACTCCAAAA
3528



GGTTATTATACt

ATCGTAATACt



 


2_IGHD3-3*01
cgaaaTATTACGATTTTTGGAGTG
3529
taggaGGTATAATAACCACTCCAAA
3530



GTTATTATACCt

AATCGTAATAt



 


3_IGHD3-3*01
cgaaaGGTATTACGATTTTTGGAG
3531
taggaTATAATAACCACTCCAAAAAT
3532



TGGTTATTATAt

CGTAATACCt



 


2_IGHD3-9*01
cgaaaTATTACGATATTTTGACTG
3533
taggaGTTATAATAACCAGTCAAAA
3534



GTTATTATAACt

TATCGTAATAt



 


2_IGHD3-10*01
cgaaaTATTACTATGGTTCGGGGA
3535
taggaGTTATAATAACTCCCCGAAC
3536



GTTATTATAACt

CATAGTAATAt



 


3_IGHD3-10*01
cgaaaGGTATTACTATGGTTCGG
3537
taggaTATAATAACTCCCCGAACCA
3538



GGAGTTATTATAt

TAGTAATACCt



 


2_IGHD3-16*01
cgaaaTATTATGATTACGTTTGGG
3539
taggaGGTATAAGCATAACTCCCCC
3540



GGAGTTATGCTTATACCt

AAACGTAATCATAATAt



 


3_IGHD3-16*01
cgaaaGGTATTATGATTACGTTTG
3541
taggaTATAAGCATAACTCCCCCAA
3542



GGGGAGTTATGCTTATAt

ACGTAATCATAATACCt



 


2_IGHD3-22*01
cgaaaTATTACTATGATAGTAGTG
3543
taggaGTAGTAATAACCACTACTAT
3544



GTTATTACTACt

CATAGTAATAt



 


3_IGHD3-22*01
cgaaaGGTATTACTATGATAGTAG
3545
taggaAGTAATAACCACTACTATCA
3546



TGGTTATTACTt

TAGTAATACCt



 


2_IGHD4-4*01 (1)
cgaaaGACTACAGTAACTACt
3547
taggaGTAGTTACTGTAGTCt
3548


 


3_IGHD4-4*01 (1)
cgaaaGTGACTACAGTAACTt
3549
taggaAGTTACTGTAGTCACt
3550


 


2_IGHD4-11*01 (1)
cgaaaGACTACAGTAACTACt
3551
taggaGTAGTTACTGTAGTCt
3552


 


3_IGHD4-11*01 (1)
cgaaaGTGACTACAGTAACTt
3553
taggaAGTTACTGTAGTCACt
3554


 


2_IGHD4-17*01
cgaaaGACTACGGTGACTACt
3555
taggaGTAGTCACCGTAGTCt
3556


 


3_IGHD4-17*01
cgaaaGTGACTACGGTGACTt
3557
taggaAGTCACCGTAGTCACt
3558


 


2_IGHD4-23*01
cgaaaGACTACGGTGGTAACTCCt
3559
taggaGGAGTTACCACCGTAGTCt
3560


 


3_IGHD4-23*01
cgaaaGTGACTACGGTGGTAACTt
3561
taggaAGTTACCACCGTAGTCACt
3562


 


1_IGHD5-5*01 (2)
cgaaaGTGGATACAGCTATGGTTA
3563
taggaCGTAACCATAGCTGTATCCA
3564



CGt

Ct



 


2_IGHD5-5*01 (2)
cgaaaTGGATACAGCTATGGTTAC
3565
taggaCCGTAACCATAGCTGTATCC
3566



GGt

At



 


3_IGHD5-5*01 (2)
cgaaaGGTGGATACAGCTATGGT
3567
taggaGTAACCATAGCTGTATCCAC
3568



TACt

Ct



 


1_IGHD5-12*01
cgaaaGTGGATATAGTGGCTACG
3569
taggaCGTAATCGTAGCCACTATAT
3570



ATTACGt

CCACt



 


3_IGHD5-12*01
cgaaaGGTGGATATAGTGGCTAC
3571
taggaGTAATCGTAGCCACTATATC
3572



GATTACt

CACCt



 


1_IGHD5-18*01 (2)
cgaaaGTGGATACAGCTATGGTTA
3573
taggaCGTAACCATAGCTGTATCCA
3574



CGt

Ct



 


2_IGHD5-18*01 (2)
cgaaaTGGATACAGCTATGGTTAC
3575 
taggaCCGTAACCATAGCTGTATCC
3576



GGt

At



 


3_IGHD5-18*01 (2)
cgaaaGGTGGATACAGCTATGGT
3577
taggaGTAACCATAGCTGTATCCAC
3578



TACt

Ct



 


1_IGHD5-24*01
cgaaaGTAGAGATGGCTACAATTA
3579
taggaCGTAATTGTAGCCATCTCTA
3580



CGt

Ct



 


3_IGHD5-24*01
cgaaaGGTAGAGATGGCTACAAT
3581
taggaGTAATTGTAGCCATCTCTAC
3582



TACt

Ct



 


1_IGHD6-6*01
cgaaaGAGTATAGCAGCTCGTCCt
3583
taggaGGACGAGCTGCTATACTCt
3584


 


2_IGHD6-6*01
cgaaaAGTATAGCAGCTCGTCCGt
3585
taggaCGGACGAGCTGCTATACTt
3586


 


1_IGHD6-13*01
cgaaaGGGTATAGCAGCAGCTGG
3587
taggaGTACCAGCTGCTGCTATACC
3588



TACt

Ct



 


2_IGHD6-13*01
cgaaaGGTATAGCAGCAGCTGGT
3589
taggaCGTACCAGCTGCTGCTATAC
3590



ACGt

Ct



 


1_IGHD6-19*01
cgaaaGGGTATAGCAGTGGCTGG
3591
taggaGTACCAGCCACTGCTATACC
3592



TACt

Ct



 


2_IGHD6-19*01
cgaaaGGTATAGCAGTGGCTGGT
3593
taggaCGTACCAGCCACTGCTATAC
3594



ACGt

Ct



 


1_IGHD6-25*01
cgaaaGGGTATAGCAGCGGCTACt
3595
taggaGTAGCCGCTGCTATACCCt
3596


 


2_IGHD6-25*01
cgaaaGGTATAGCAGCGGCTACGt
3597
taggaCGTAGCCGCTGCTATACCt
3598


 


1_IGHD7-27*01
cgaaaCTAACTGGGGAGt
3599
taggaCTCCCCAGTTAGt
3600


 


3_IGHD7-27*01
cgaaaGCTAACTGGGGAt
3601
taggaTCCCCAGTTAGCt
3602
















TABLE 40







Inverted DH segment oligos













SEQ

SEQ




ID

ID


D segment
Forward
NO
Reverse
NO





1′_IGHD1-1*01
cgaaaGTCGTTCCAGTTGTACCGt
3603
taggaCGGTACAACTGGAACGACt
3604


 


2′_IGHD1-1*01
cgaaaTCGTTCCAGTTGTACCGGt
3605
taggaCCGGTACAACTGGAACGAt
3606


 


3′_IGHD1-1*01
cgaaaGGTCGTTCCAGTTGTACCt
3607
taggaGGTACAACTGGAACGACCt
3608


 


1′_IGHD1-7*01
cgaaaGTAGTTCCAGTTATACCGt
3609
taggaCGGTATAACTGGAACTACt
3610


 


3′_IGHD1-7*01
cgaaaGGTAGTTCCAGTTATACCt
3611
taggaGGTATAACTGGAACTACCt
3612


 


1′_IGHD1-14*01
cgaaaGTGGTTCCGGTTATACCGt
3613
taggaCGGTATAACCGGAACCACt
3614


 


2′_IGHD1-14*01
cgaaaTGGTTCCGGTTATACCGGt
3615
taggaCCGGTATAACCGGAACCAt
3616


 


3′_IGHD1-14*01
cgaaaGGTGGTTCCGGTTATACCt
3617
taggaGGTATAACCGGAACCACCt
3618


 


1′_IGHD1-20*01
cgaaaGTCGTTCCAGTTATACCGt
3619
taggaCGGTATAACTGGAACGACt
3620


 


2′_IGHD1-20*01
cgaaaTCGTTCCAGTTATACCGGt
3621
taggaCCGGTATAACTGGAACGAt
3622


 


3′_IGHD1-20*01
cgaaaGGTCGTTCCAGTTATACCt
3623
taggaGGTATAACTGGAACGACCt
3624


 


1′_IGHD1-26*01
cgaaaGTAGTAGCTCCCACTATAC
3625
taggaCGGTATAGTGGGAGCTACTA
3626



CGt

Ct



 


3′_IGHD1-26*01
cgaaaGGTAGTAGCTCCCACTATA
3627
taggaGGTATAGTGGGAGCTACTAC
3628



CCt

Ct



 


1′_IGHD2-2*01
cgaaaGGCATAGCAGCTGGTACT
3629
taggaGGATATTGTAGTAGTACCAG
3630



ACTACAATATCCt

CTGCTATGCCt



 


3′_IGHD2-2*01
cgaaaGGGCATAGCAGCTGGTAC
3631
taggaGATATTGTAGTAGTACCAGC
3632



TACTACAATATCt

TGCTATGCCCt



 


1′_IGHD2-8*01
cgaaaGGTATAGCATACACCATTA
3633
taggaGGATATTGTACTAATGGTGT
3634



GTACAATATCCt

ATGCTATACCt



 


1′_IGHD2-15*01
cgaaaGGAGTAGCAGCTACCACC
3635
taggaGGATATTGTAGTGGTGGTAG
3636



ACTACAATATCCt

CTGCTACTCCt



 


3′_IGHD2-15*01
cgaaaGGGAGTAGCAGCTACCAC
3637
taggaGATATTGTAGTGGTGGTAGC
3638



CACTACAATATCt

TGCTACTCCCt



 


1′_IGHD2-21*01
cgaaaGGAATAGCAATCACCACC
3639
taggaGCATATTGTGGTGGTGATTG
3640



ACAATATGCt

CTATTCCt



 


3′_IGHD2-21*01
cgaaaGGGAATAGCAATCACCAC
3641
taggaCATATTGTGGTGGTGATTGC
3642



CACAATATGt

TATTCCCt



 


1′_IGHD3-3*01
cgaaaGGTATAATAACCACTCCAA
3643
taggaTATTACGATTTTTGGAGTGG
3644



AAATCGTAATAt

TTATTATACCt



 


3′_IGHD3-3*01
cgaaaGGGTATAATAACCACTCCA
3645
taggaATTACGATTTTTGGAGTGGT
3646



AAAATCGTAATt

TATTATACCCt



 


1′_IGHD3-9*01
cgaaaGTTATAATAACCAGTCAAA
3647
taggaTATTACGATATTTTGACTGGT
3648



ATATCGTAATAt

TATTATAACt



 


3′_IGHD3-9*01
cgaaaGGTTATAATAACCAGTCAA
3649
taggaATTACGATATTTTGACTGGTT
3650



AATATCGTAATt

ATTATAACCt



 


1′_IGHD3-10*01
cgaaaGTTATAATAACTCCCCGAA
3651
taggaTATTACTATGGTTCGGGGAG
3652



CCATAGTAATAt

TTATTATAACt



 


3′_IGHD3-10*01
cgaaaGGTTATAATAACTCCCCGA
3653
taggaATTACTATGGTTCGGGGAGT
3654



ACCATAGTAATt

TATTATAACCt



 


1′_IGHD3-16*01
cgaaaGGTATAAGCATAACTCCCC
3655
taggaTATTATGATTACGTTTGGGG
3656



CAAACGTAATCATAATAt

GAGTTATGCTTATACCt



 


3′_IGHD3-16*01
cgaaaGGGTATAAGCATAACTCC
3657
taggaATTATGATTACGTTTGGGGG
3658



CCCAAACGTAATCATAATt

AGTTATGCTTATACCCt



 


1′_IGHD3-22*01
cgaaaGTAGTAATAACCACTACTA
3659
taggaTATTACTATGATAGTAGTGG
3660



TCATAGTAATAt

TTATTACTACt



 


1′_IGHD4-4*01 (1)
cgaaaGTAGTTACTGTAGTCt
3661
taggaGACTACAGTAACTACt
3662


 


3′_IGHD4-4*01 (1)
cgaaaGGTAGTTACTGTAGTt
3663
taggaACTACAGTAACTACCt
3664


 


1′_IGHD4-11*01 (1)
cgaaaGTAGTTACTGTAGTCt
3665
taggaGACTACAGTAACTACt
3666


 


3′_IGHD4-11*01 (1)
cgaaaGGTAGTTACTGTAGTt
3667
taggaACTACAGTAACTACCt
3668


 


1′_IGHD4-17*01
cgaaaGTAGTCACCGTAGTCt
3669
taggaGACTACGGTGACTACt
3670


 


3′_IGHD4-17*01
cgaaaGGTAGTCACCGTAGTt
3671
taggaACTACGGTGACTACCt
3672


 


1′_IGHD4-23*01
cgaaaGGAGTTACCACCGTAGTCt
3673
taggaGACTACGGTGGTAACTCCt
3674


 


3′_IGHD4-23*01
cgaaaGGGAGTTACCACCGTAGTt
3675
taggaACTACGGTGGTAACTCCCt
3676


 


1′_IGHD5-5*01 (2)
cgaaaGTAACCATAGCTGTATCCA
3677
taggaCGTGGATACAGCTATGGTTA
3678



CGt

Ct



 


3′_IGHD5-5*01 (2)
cgaaaGGTAACCATAGCTGTATCC
3679
taggaGTGGATACAGCTATGGTTAC
3680



ACt

Ct



 


1′_IGHD5-12*01
cgaaaGTAATCGTAGCCACTATAT
3681
taggaCGTGGATATAGTGGCTACGA
3682



CCACGt

TTACt



 


3′_IGHD5-12*01
cgaaaGGTAATCGTAGCCACTATA
3683
taggaGTGGATATAGTGGCTACGAT
3684



TCCACt

TACCt



 


1′_IGHD5-18*01 (2)
cgaaaGTAACCATAGCTGTATCCA
3685
taggaCGTGGATACAGCTATGGTTA
3686



CGt

Ct



 


3′_IGHD5-18*01 (2)
cgaaaGGTAACCATAGCTGTATCC
3687
taggaGTGGATACAGCTATGGTTAC
3688



ACt

Ct



 


1′_IGHD5-24*01
cgaaaGTAATTGTAGCCATCTCTA
3689
taggaCGTAGAGATGGCTACAATTA
3690



CGt

Ct



 


3′_IGHD5-24*01
cgaaaGGTAATTGTAGCCATCTCT
3691
taggaGTAGAGATGGCTACAATTAC
3692



ACt

Ct



 


1′_IGHD6-6*01
cgaaaGGACGAGCTGCTATACTCt
3693
taggaGAGTATAGCAGCTCGTCCt
3694


 


2′_IGHD6-6*01
cgaaaGACGAGCTGCTATACTCGt
3695
taggaCGAGTATAGCAGCTCGTCt
3696


 


3′_IGHD6-6*01
cgaaaGGGACGAGCTGCTATACTt
3697
taggaAGTATAGCAGCTCGTCCCt
3698


 


1′_IGHD6-13*01
cgaaaGTACCAGCTGCTGCTATA
3699
taggaGGGTATAGCAGCAGCTGGT
3700



CCCt

ACt



 


2′_IGHD6-13*01
cgaaaTACCAGCTGCTGCTATACC
3701
taggaCGGGTATAGCAGCAGCTGG
3702



CGt

TAt



 


3′_IGHD6-13*01
cgaaaGGTACCAGCTGCTGCTAT
3703
taggaGGTATAGCAGCAGCTGGTA
3704



ACCt

CCt



 


1′_IGHD6-19*01
cgaaaGTACCAGCCACTGCTATA
3705
taggaGGGTATAGCAGTGGCTGGT
3706



CCCt

ACt



 


2′_IGHD6-19*01
cgaaaTACCAGCCACTGCTATACC
3707
taggaCGGGTATAGCAGTGGCTGG
3708



CGt

TAt



 


3′_IGHD6-19*01
cgaaaGGTACCAGCCACTGCTAT
3709
taggaGGTATAGCAGTGGCTGGTA
3710



ACCt

CCt



 


1′_IGHD6-25*01
cgaaaGTAGCCGCTGCTATACCCt
3711
taggaGGGTATAGCAGCGGCTACt
3712


 


3′_IGHD6-25*01
cgaaaGGTAGCCGCTGCTATACCt
3713
taggaGGTATAGCAGCGGCTACCt
3714


 


1′_IGHD7-27*01
cgaaaTCCCCAGTTAGGt
3715
taggaCCTAACTGGGGAt
3716


 


2′_IGHD7-27*01
cgaaaCCCCAGTTAGGGt
3717
taggaCCCTAACTGGGGt
3718
















TABLE 41







VH3-23 Library












Nuc. SEQ
Prot. SEQ


No.
Heavy Chain
ID NO
ID NO













1
>gi|Fabrus|VH3-23_IGHD1-1*01>1_IGHJ1*01
2070
2760


2
>gi|Fabrus|VH3-23_IGHD1-1*01>2_IGHJ1*01
2071
2761


3
>gi|Fabrus|VH3-23_IGHD1-1*01>3_IGHJ1*01
2072
2762


4
>gi|Fabrus|VH3-23_IGHD1-7*01>1_IGHJ1*01
2073
2763


5
>gi|Fabrus|VH3-23_IGHD1-7*01>3_IGHJ1*01
2074
2764


6
>gi|Fabrus|VH3-23_IGHD1-14*01>1_IGHJ1*01
2075
2765


7
>gi|Fabrus|VH3-23_IGHD1-14*01>3_IGHJ1*01
2076
2766


8
>gi|Fabrus|VH3-23_IGHD1-20*01>1_IGHJ1*01
2077
2767


9
>gi|Fabrus|VH3-23_IGHD1-20*01>3_IGHJ1*01
2078
2768


10
>gi|Fabrus|VH3-23_IGHD1-26*01>1_IGHJ1*01
2079
2769


11
>gi|Fabrus|VH3-23_IGHD1-26*01>3_IGHJ1*01
2080
2770


12
>gi|Fabrus|VH3-23_IGHD2-2*01>2_IGHJ1*01
2081
2771


13
>gi|Fabrus|VH3-23_IGHD2-2*01>3_IGHJ1*01
2082
2772


14
>gi|Fabrus|VH3-23_IGHD2-8*01>2_IGHJ1*01
2083
2773


15
>gi|Fabrus|VH3-23_IGHD2-8*01>3_IGHJ1*01
2084
2774


16
>gi|Fabrus|VH3-23_IGHD2-15*01>2_IGHJ1*01
2085
2775


17
>gi|Fabrus|VH3-23_IGHD2-15*01>3_IGHJ1*01
2086
2776


18
>gi|Fabrus|VH3-23_IGHD2-21*01>2_IGHJ1*01
2087
2777


19
>gi|Fabrus|VH3-23_IGHD2-21*01>3_IGHJ1*01
2088
2778


20
>gi|Fabrus|VH3-23_IGHD3-3*01>1_IGHJ1*01
2089
2779


21
>gi|Fabrus|VH3-23_IGHD3-3*01>2_IGHJ1*01
2090
2780


22
>gi|Fabrus|VH3-23_IGHD3-3*01>3_IGHJ1*01
2091
2781


23
>gi|Fabrus|VH3-23_IGHD3-9*01>2_IGHJ1*01
2092
2782


24
>gi|Fabrus|VH3-23_IGHD3-10*01>2_IGHJ1*01
2093
2783


25
>gi|Fabrus|VH3-23_IGHD3-10*01>3_IGHJ1*01
2094
2784


26
>gi|Fabrus|VH3-23_IGHD3-16*01>2_IGHJ1*01
2095
2785


27
>gi|Fabrus|VH3-23_IGHD3-16*01>3_IGHJ1*01
2096
2786


28
>gi|Fabrus|VH3-23_IGHD3-22*01>2_IGHJ1*01
2097
2787


29
>gi|Fabrus|VH3-23_IGHD3-22*01>3_IGHJ1*01
2098
2788


30
>gi|Fabrus|VH3-23_IGHD4-4*01(1)>2_IGHJ1*01
2099
2789


31
>gi|Fabrus|VH3-23_IGHD4-4*01 (1)>3_IGHJ1*01
2100
2790


32
>gi|Fabrus|VH3-23_IGHD4-11*01(1)>2_IGHJ1*01
2101
2791


33
>gi|Fabrus|VH3-23_IGHD4-11*01(1)>3_IGHJ1*01
2102
2792


34
>gi|Fabrus|VH3-23_IGHD4-17*01>2_IGHJ1*01
2103
2793


35
>gi|Fabrus|VH3-23_IGHD4-17*01>3_IGHJ1*01
2104
2794


36
>gi|Fabrus|VH3-23_IGHD4-23*01>2_IGHJ1*01
2105
2795


37
>gi|Fabrus|VH3-23_IGHD4-23*01>3_IGHJ1*01
2106
2796


38
>gi|Fabrus|VH3-23_IGHD5-5*01(2)>1_IGHJ1*01
2107
2797


39
>gi|Fabrus|VH3-23_IGHD5-5*01(2)>2_IGHJ1*01
2108
2798


40
>gi|Fabrus|VH3-23_IGHD5-5*01(2)>3_IGHJ1*01
2109
2799


41
>gi|Fabrus|VH3-23_IGHD5-12*01>1_IGHJ1*01
2110
2800


42
>gi|Fabrus|VH3-23_IGHD5-12*01>3_IGHJ1*01
2111
2801


43
>gi|Fabrus|VH3-23_IGHD5-18*01(2)>1_IGHJ1*01
2112
2802


44
>gi|Fabrus|VH3-23_IGHD5-18*01(2)>2_IGHJ1*01
2113
2803


45
>gi|Fabrus|VH3-23_IGHD5-18*01(2)>3_IGHJ1*01
2114
2804


46
>gi|Fabrus|VH3-23_IGHD5-24*01>1_IGHJ1*01
2115
2805


47
>gi|Fabrus|VH3-23_IGHD5-24*01>3_IGHJ1*01
2116
2806


48
>gi|Fabrus|VH3-23_IGHD6-6*01>1_IGHJ1*01
2117
2807


49
>gi|Fabrus|VH3-23_IGHD6-6*01>2_IGHJ1*01
2118
2808


50
>gi|Fabrus|VH3-23_IGHD6-13*01>1_IGHJ1*01
2119
2809


51
>gi|Fabrus|VH3-23_IGHD6-13*01>2_IGHJ1*01
2120
2810


52
>gi|Fabrus|VH3-23_IGHD6-19*01>1_IGHJ1*01
2121
2811


53
>gi|Fabrus|VH3-23_IGHD6-19*01>2_IGHJ1*01
2122
2812


54
>gi|Fabrus|VH3-23_IGHD6-25*01>1_IGHJ1*01
2123
2813


55
>gi|Fabrus|VH3-23_IGHD6-25*01>2_IGHJ1*01
2124
2814


56
>gi|Fabrus|VH3-23_IGHD7-27*01>1_IGHJ1*01
2125
2815


57
>gi|Fabrus|VH3-23_IGHD7-27*01>3_IGHJ1*01
2126
2816


58
>gi|Fabrus|VH3-23_IGHD1-1*01>1′_IGHJ1*01
2127
2817


59
>gi|Fabrus|VH3-23_IGHD1-1*01>2′_IGHJ1*01
2128
2818


60
>gi|Fabrus|VH3-23_IGHD1-1*01>3′_IGHJ1*01
2129
2819


61
>gi|Fabrus|VH3-23_IGHD1-7*01>1′_IGHJ1*01
2130
2820


62
>gi|Fabrus|VH3-23_IGHD1-7*01>3′_IGHJ1*01
2131
2821


63
>gi|Fabrus|VH3-23_IGHD1-14*01>1′_IGHJ1*01
2132
2822


64
>gi|Fabrus|VH3-23_IGHD1-14*01>2′_IGHJ1*01
2133
2823


65
>gi|Fabrus|VH3-23_IGHD1-14*01>3′_IGHJ1*01
2134
2824


66
>gi|Fabrus|VH3-23_IGHD1-20*01>1′_IGHJ1*01
2135
2825


67
>gi|Fabrus|VH3-23_IGHD1-20*01>2′_IGHJ1*01
2136
2826


68
>gi|Fabrus|VH3-23_IGHD1-20*01>3′_IGHJ1*01
2137
2827


69
>gi|Fabrus|VH3-23_IGHD1-26*01>1′_IGHJ1*01
2138
2828


70
>gi|Fabrus|VH3-23_IGHD1-26*01>3′_IGHJ1*01
2139
2829


71
>gi|Fabrus|VH3-23_IGHD2-2*01>1′_IGHJ1*01
2140
2830


72
>gi|Fabrus|VH3-23_IGHD2-2*01>3′_IGHJ1*01
2141
2831


73
>gi|Fabrus|VH3-23_IGHD2-8*01>1′_IGHJ1*01
2142
2832


74
>gi|Fabrus|VH3-23_IGHD2-15*01>1′_IGHJ1*01
2143
2833


75
>gi|Fabrus|VH3-23_IGHD2-15*01>3′_IGHJ1*01
2144
2834


76
>gi|Fabrus|VH3-23_IGHD2-21*01>1′_IGHJ1*01
2145
2835


77
>gi|Fabrus|VH3-23_IGHD2-21*01>3′_IGHJ1*01
2146
2836


78
>gi|Fabrus|VH3-23_IGHD3-3*01>1′_IGHJ1*01
2147
2837


79
>gi|Fabrus|VH3-23_IGHD3-3*01>3′_IGHJ1*01
2148
2838


80
>gi|Fabrus|VH3-23_IGHD3-9*01>1′_IGHJ1*01
2149
2839


81
>gi|Fabrus|VH3-23_IGHD3-9*01>3′_IGHJ1*01
2150
2840


82
>gi|Fabrus|VH3-23_IGHD3-10*01>1′_IGHJ1*01
2151
2841


83
>gi|Fabrus|VH3-23_IGHD3-10*01>3′_IGHJ1*01
2152
2842


84
>gi|Fabrus|VH3-23_IGHD3-16*01>1′_IGHJ1*01
2153
2843


85
>gi|Fabrus|VH3-23_IGHD3-16*01>3′_IGHJ1*01
2154
2844


86
>gi|Fabrus|VH3-23_IGHD3-22*01>1′_IGHJ1*01
2155
2845


87
>gi|Fabrus|VH3-23_IGHD4-4*01(1)>1′_IGHJ1*01
2156
2846


88
>gi|Fabrus|VH3-23_IGHD4-4*01(1)>3′_IGHJ1*01
2157
2847


89
>gi|Fabrus|VH3-23_IGHD4-11*01(1)>1′_IGHJ1*01
2158
2848


90
>gi|Fabrus|VH3-23_IGHD4-11*01(1)>3′_IGHJ1*01
2159
2849


91
>gi|Fabrus|VH3-23_IGHD4-17*01>1′_IGHJ1*01
2160
2850


92
>gi|Fabrus|VH3-23_IGHD4-17*01>3′_IGHJ1*01
2161
2851


93
>gi|Fabrus|VH3-23_IGHD4-23*01>1′_IGHJ1*01
2162
2852


94
>gi|Fabrus|VH3-23_IGHD4-23*01>3′_IGHJ1*01
2163
2853


95
>gi|Fabrus|VH3-23_IGHD5-5*01(2)>1′_IGHJ1*01
2164
2854


96
>gi|Fabrus|VH3-23_IGHD5-5*01(2)>3′_IGHJ1*01
2165
2855


97
>gi|Fabrus|VH3-23_IGHD5-12*01>1′_IGHJ1*01
2166
2856


98
>gi|Fabrus|VH3-23_IGHD5-12*01>3′_IGHJ1*01
2167
2857


99
>gi|Fabrus|VH3-23_IGHD5-18*01(2)>1′_IGHJ1*01
2168
2858


100
>gi|Fabrus|VH3-23_IGHD5-18*01(2)>3′_IGHJ1*01
2169
2859


101
>gi|Fabrus|VH3-23_IGHD5-24*01>1′_IGHJ1*01
2170
2860


102
>gi|Fabrus|VH3-23_IGHD5-24*01>3′_IGHJ1*01
2171
2861


103
>gi|Fabrus|VH3-23_IGHD6-6*01>1′_IGHJ1*01
2172
2862


104
>gi|Fabrus|VH3-23_IGHD6-6*01>2′_IGHJ1*01
2173
2863


105
>gi|Fabrus|VH3-23_IGHD6-6*01>3′_IGHJ1*01
2174
2864


106
>gi|Fabrus|VH3-23_IGHD6-13*01>1′_IGHJ1*01
2175
2865


107
>gi|Fabrus|VH3-23_IGHD6-13*01>2′_IGHJ1*01
2176
2866


108
>gi|Fabrus|VH3-23_IGHD6-13*01>3′_IGHJ1*01
2177
2867


109
>gi|Fabrus|VH3-23_IGHD6-19*01>1′_IGHJ1*01
2178
2868


110
>gi|Fabrus|VH3-23_IGHD6-19*01>2′_IGHJ1*01
2179
2869


111
>gi|Fabrus|VH3-23_IGHD6-19*01>3′_IGHJ1*01
2180
2870


112
>gi|Fabrus|VH3-23_IGHD6-25*01>1′_IGHJ1*01
2181
2871


113
>gi|Fabrus|VH3-23_IGHD6-25*01>3′_IGHJ1*01
2182
2872


114
>gi|Fabrus|VH3-23_IGHD7-27*01>1′_IGHJ1*01
2183
2873


115
>gi|Fabrus|VH3-23_IGHD7-27*01>2′_IGHJ1*01
2184
2874


116
>gi|Fabrus|VH3-23_IGHD1-1*01>1_IGHJ2*01
2185
2875


117
>gi|Fabrus|VH3-23_IGHD1-1*01>2_IGHJ2*01
2186
2876


118
>gi|Fabrus|VH3-23_IGHD1-1*01>3_IGHJ2*01
2187
2877


119
>gi|Fabrus|VH3-23_IGHD1-7*01>1_IGHJ2*01
2188
2878


120
>gi|Fabrus|VH3-23_IGHD1-7*01>3_IGHJ2*01
2189
2879


121
>gi|Fabrus|VH3-23_IGHD1-14*01>1_IGHJ2*01
2190
2880


122
>gi|Fabrus|VH3-23_IGHD1-14*01>3_IGHJ2*01
2191
2881


123
>gi|Fabrus|VH3-23_IGHD1-20*01>1_IGHJ2*01
2192
2882


124
>gi|Fabrus|VH3-23_IGHD1-20*01>3_IGHJ2*01
2193
2883


125
>gi|Fabrus|VH3-23_IGHD1-26*01>1_IGHJ2*01
2194
2884


126
>gi|Fabrus|VH3-23_IGHD1-26*01>3_IGHJ2*01
2195
2885


127
>gi|Fabrus|VH3-23_IGHD2-2*01>2_IGHJ2*01
2196
2886


128
>gi|Fabrus|VH3-23_IGHD2-2*01>3_IGHJ2*01
2197
2887


129
>gi|Fabrus|VH3-23_IGHD2-8*01>2_IGHJ2*01
2198
2888


130
>gi|Fabrus|VH3-23_IGHD2-8*01>3_IGHJ2*01
2199
2889


131
>gi|Fabrus|VH3-23_IGHD2-15*01>2_IGHJ2*01
2200
2890


132
>gi|Fabrus|VH3-23_IGHD2-15*01>3_IGHJ2*01
2201
2891


133
>gi|Fabrus|VH3-23_IGHD2-21*01>2_IGHJ2*01
2202
2892


134
>gi|Fabrus|VH3-23_IGHD2-21*01>3_IGHJ2*01
2203
2893


135
>gi|Fabrus|VH3-23_IGHD3-3*01>1_IGHJ2*01
2204
2894


136
>gi|Fabrus|VH3-23_IGHD3-3*01>2_IGHJ2*01
2205
2895


137
>gi|Fabrus|VH3-23_IGHD3-3*01>3_IGHJ2*01
2206
2896


138
>gi|Fabrus|VH3-23_IGHD3-9*01>2_IGHJ2*01
2207
2897


139
>gi|Fabrus|VH3-23_IGHD3-10*01>2_IGHJ2*01
2208
2898


140
>gi|Fabrus|VH3-23_IGHD3-10*01>3_IGHJ2*01
2209
2899


141
>gi|Fabrus|VH3-23_IGHD3-16*01>2_IGHJ2*01
2210
2900


142
>gi|Fabrus|VH3-23_IGHD3-16*01>3_IGHJ2*01
2211
2901


143
>gi|Fabrus|VH3-23_IGHD3-22*01>2_IGHJ2*01
2212
2902


144
>gi|Fabrus|VH3-23_IGHD3-22*01>3_IGHJ2*01
2213
2903


145
>gi|Fabrus|VH3-23_IGHD4-4*01(1)>2_IGHJ2*01
2214
2904


146
>gi|Fabrus|VH3-23_IGHD4-4*01(1)>3_IGHJ2*01
2215
2905


147
>gi|Fabrus|VH3-23_IGHD4-11*01(1)>2_IGHJ2*01
2216
2906


148
>gi|Fabrus|VH3-23_IGHD4-11*01(1)>3_IGHJ2*01
2217
2907


149
>gi|Fabrus|VH3-23_IGHD4-17*01>2_IGHJ2*01
2218
2908


150
>gi|Fabrus|VH3-23_IGHD4-17*01>3_IGHJ2*01
2219
2909


151
>gi|Fabrus|VH3-23_IGHD4-23*01>2_IGHJ2*01
2220
2910


152
>gi|Fabrus|VH3-23_IGHD4-23*01>3_IGHJ2*01
2221
2911


153
>gi|Fabrus|VH3-23_IGHD5-5*01(2)>1_IGHJ2*01
2222
2912


154
>gi|Fabrus|VH3-23_IGHD5-5*01(2)>2_IGHJ2*01
2223
2913


155
>gi|Fabrus|VH3-23_IGHD5-5*01(2)>3_IGHJ2*01
2224
2914


156
>gi|Fabrus|VH3-23_IGHD5-12*01>1_IGHJ2*01
2225
2915


157
>gi|Fabrus|VH3-23_IGHD5-12*01>3_IGHJ2*01
2226
2916


158
>gi|Fabrus|VH3-23_IGHD5-18*01(2)>1_IGHJ2*01
2227
2917


159
>gi|Fabrus|VH3-23_IGHD5-18*01(2)>2_IGHJ2*01
2228
2918


160
>gi|Fabrus|VH3-23_IGHD5-18*01(2)>3_IGHJ2*01
2229
2919


161
>gi|Fabrus|VH3-23_IGHD5-24*01>1_IGHJ2*01
2230
2920


162
>gi|Fabrus|VH3-23_IGHD5-24*01>3_IGHJ2*01
2231
2921


163
>gi|Fabrus|VH3-23_IGHD6-6*01>1_IGHJ2*01
2232
2922


164
>gi|Fabrus|VH3-23_IGHD6-6*01>2_IGHJ2*01
2233
2923


165
>gi|Fabrus|VH3-23_IGHD6-13*01>1_IGHJ2*01
2234
2924


166
>gi|Fabrus|VH3-23_IGHD6-13*01>2_IGHJ2*01
2235
2925


167
>gi|Fabrus|VH3-23_IGHD6-19*01>1_IGHJ2*01
2236
2926


168
>gi|Fabrus|VH3-23_IGHD6-19*01>2_IGHJ2*01
2237
2927


169
>gi|Fabrus|E06_VH3-23_IGHD6-25*01>1_IGHJ2*01
2238
2928


170
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>2_IGHJ2*01
2239
2929


171
>gi|Fabrus|E08_VH3-23_IGHD7-27*01>1_IGHJ2*01
2240
2930


172
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>3_IGHJ2*01
2241
2931


173
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1′_IGHJ2*01
2242
2932


174
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2′_IGHJ2*01
2243
2933


175
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3′_IGHJ2*01
2244
2934


176
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1′_IGHJ2*01
2245
2935


177
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3′_IGHJ2*01
2246
2936


178
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1′_IGHJ2*01
2247
2937


179
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>2′_IGHJ2*01
2248
2938


180
>gi|Fabrus|A08_VH3-23_IGHD1-14*01>3′_IGHJ2*01
2249
2939


181
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>1′_IGHJ2*01
2250
2940


182
>gi|Fabrus|A10_VH3-23_IGHD1-20*01>2′_IGHJ2*01
2251
2941


183
>gi|Fabrus|A11_VH3-23_IGHD1-20*01>3′_IGHJ2*01
2252
2942


184
>gi|Fabrus|A12_VH3-23_IGHD1-26*01>1′_IGHJ2*01
2253
2943


185
>gi|Fabrus|B01_VH3-23_IGHD1-26*01>3′_IGHJ2*01
2254
2944


186
>gi|Fabrus|B02_VH3-23_IGHD2-2*01>1′_IGHJ2*01
2255
2945


187
>gi|Fabrus|B03_VH3-23_IGHD2-2*01>3′_IGHJ2*01
2256
2946


188
>gi|Fabrus|B04_VH3-23_IGHD2-8*01>1′_IGHJ2*01
2257
2947


189
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>1′_IGHJ2*01
2258
2948


190
>gi|Fabrus|B06_VH3-23_IGHD2-15*01>3′_IGHJ2*01
2259
2949


191
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>1′_IGHJ2*01
2260
2950


192
>gi|Fabrus|B08_VH3-23_IGHD2-21*01>3′_IGHJ2*01
2261
2951


193
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>1′_IGHJ2*01
2262
2952


194
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3′_IGHJ2*01
2263
2953


195
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>1′_IGHJ2*01
2264
2954


196
>gi|Fabrus|B12_VH3-23_IGHD3-9*01>3′_IGHJ2*01
2265
2955


197
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>1′_IGHJ2*01
2266
2956


198
>gi|Fabrus|C02_VH3-23_IGHD3-10*01>3′_IGHJ2*01
2267
2957


199
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>1′_IGHJ2*01
2268
2958


200
>gi|Fabrus|C04_VH3-23_IGHD3-16*01>3′_IGHJ2*01
2269
2959


201
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>1′_IGHJ2*01
2270
2960


202
>gi|Fabrus|C06_VH3-23_IGHD4-4*01(1)>1′_IGHJ2*01
2271
2961


203
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3′_IGHJ2*01
2272
2962


204
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>1′_IGHJ2*01
2273
2963


205
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3′_IGHJ2*01
2274
2964


206
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>1′_IGHJ2*01
2275
2965


207
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3′_IGHJ2*01
2276
2966


208
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>1′_IGHJ2*01
2277
2967


209
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3′_IGHJ2*01
2278
2968


210
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1′_IGHJ2*01
2279
2969


211
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>3′_IGHJ2*01
2280
2970


212
>gi|Fabrus|D04_VH3-23_IGHD5-12*01>1′_IGHJ2*01
2281
2971


213
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>3′_IGHJ2*01
2282
2972


214
>gi|Fabrus|D06_VH3-23_IGHD5-18*01(2)>1′_IGHJ2*01
2283
2973


215
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>3′_IGHJ2*01
2284
2974


216
>gi|Fabrus|D08_VH3-23_IGHD5-24*01>1′_IGHJ2*01
2285
2975


217
>gi|Fabrus|D09_VH3-23_IGHD5-24*01>3′_IGHJ2*01
2286
2976


218
>gi|Fabrus|D10_VH3-23_IGHD6-6*01>1′_IGHJ2*01
2287
2977


219
>gi|Fabrus|D11_VH3-23_IGHD6-6*01>2′_IGHJ2*01
2288
2978


220
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>3′_IGHJ2*01
2289
2979


221
>gi|Fabrus|E01_VH3-23_IGHD6-13*01>1′_IGHJ2*01
2290
2980


222
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>2′_IGHJ2*01
2291
2981


223
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>3′_IGHJ2*01
2292
2982


224
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1′_IGHJ2*01
2293
2983


225
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2′_IGHJ2*01
2294
2984


226
>gi|Fabrus|E06_VH3-23_IGHD6-19*01>3′_IGHJ2*01
2295
2985


227
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>1′_IGHJ2*01
2296
2986


228
>gi|Fabrus|E08_VH3-23_IGHD6-25*01>3′_IGHJ2*01
2297
2987


229
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>1′_IGHJ2*01
2298
2988


230
>gi|Fabrus|E10_VH3-23_IGHD7-27*01>2′_IGHJ2*01
2299
2989


231
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1_IGHJ3*01
2300
2990


232
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2_IGHJ3*01
2301
2991


233
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3_IGHJ3*01
2302
2992


234
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1_IGHJ3*01
2303
2993


235
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3_IGHJ3*01
2304
2994


236
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1_IGHJ3*01
2305
2995


237
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>3_IGHJ3*01
2306
2996


238
>gi|Fabrus|A08_VH3-23_IGHD1-20*01>1_IGHJ3*01
2307
2997


239
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>3_IGHJ3*01
2308
2998


240
>gi|Fabrus|A10_VH3-23_IGHD1-26*01>1_IGHJ3*01
2309
2999


241
>gi|Fabrus|A11_VH3-23_IGHD1-26*01>3_IGHJ3*01
2310
3000


242
>gi|Fabrus|A12_VH3-23_IGHD2-2*01>2_IGHJ3*01
2311
3001


243
>gi|Fabrus|B01_VH3-23_IGHD2-2*01>3_IGHJ3*01
2312
3002


244
>gi|Fabrus|B02_VH3-23_IGHD2-8*01>2_IGHJ3*01
2313
3003


245
>gi|Fabrus|B03_VH3-23_IGHD2-8*01>3_IGHJ3*01
2314
3004


246
>gi|Fabrus|B04_VH3-23_IGHD2-15*01>2_IGHJ3*01
2315
3005


247
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>3_IGHJ3*01
2316
3006


248
>gi|Fabrus|B06_VH3-23_IGHD2-21*01>2_IGHJ3*01
2317
3007


249
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>3_IGHJ3*01
2318
3008


250
>gi|Fabrus|B08_VH3-23_IGHD3-3*01>1_IGHJ3*01
2319
3009


251
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>2_IGHJ3*01
2320
3010


252
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3_IGHJ3*01
2321
3011


253
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>2_IGHJ3*01
2322
3012


254
>gi|Fabrus|B12_VH3-23_IGHD3-10*01>2_IGHJ3*01
2323
3013


255
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>3_IGHJ3*01
2324
3014


256
>gi|Fabrus|C02_VH3-23_IGHD3-16*01>2_IGHJ3*01
2325
3015


257
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>3_IGHJ3*01
2326
3016


258
>gi|Fabrus|C04_VH3-23_IGHD3-22*01>2_IGHJ3*01
2327
3017


259
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>3_IGHJ3*01
2328
3018


260
>gi|Fabrus|C06_VH3-23_IGHD4-4*01(1)>2_IGHJ3*01
2329
3019


261
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3_IGHJ3*01
2330
3020


262
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>2_IGHJ3*01
2331
3021


263
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3_IGHJ3*01
2332
3022


264
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>2_IGHJ3*01
2333
3023


265
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3_IGHJ3*01
2334
3024


266
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>2_IGHJ3*01
2335
3025


267
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3_IGHJ3*01
2336
3026


268
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1_IGHJ3*01
2337
3027


269
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>2_IGHJ3*01
2338
3028


270
>gi|Fabrus|D04_VH3-23_IGHD5-5*01(2)>3_IGHJ3*01
2339
3029


271
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>1_IGHJ3*01
2340
3030


272
>gi|Fabrus|D06_VH3-23_IGHD5-12*01>3_IGHJ3*01
2341
3031


273
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>1_IGHJ3*01
2342
3032


274
>gi|Fabrus|D08_VH3-23_IGHD5-18*01(2)>2_IGHJ3*01
2343
3033


275
>gi|Fabrus|D09_VH3-23_IGHD5-18*01(2)>3_IGHJ3*01
2344
3034


276
>gi|Fabrus|D10_VH3-23_IGHD5-24*01>1_IGHJ3*01
2345
3035


277
>gi|Fabrus|D11_VH3-23_IGHD5-24*01>3_IGHJ3*01
2346
3036


278
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>1_IGHJ3*01
2347
3037


279
>gi|Fabrus|E01_VH3-23_IGHD6-6*01>2_IGHJ3*01
2348
3038


280
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>1_IGHJ3*01
2349
3039


281
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>2_IGHJ3*01
2350
3040


282
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1_IGHJ3*01
2351
3041


283
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2_IGHJ3*01
2352
3042


284
>gi|Fabrus|E06_VH3-23_IGHD6-25*01>1_IGHJ3*01
2353
3043


285
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>2_IGHJ3*01
2354
3044


286
>gi|Fabrus|E08_VH3-23_IGHD7-27*01>1_IGHJ3*01
2355
3045


287
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>3_IGHJ3*01
2356
3046


288
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1′_IGHJ3*01
2357
3047


289
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2′_IGHJ3*01
2358
3048


290
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3′_IGHJ3*01
2359
3049


291
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1′_IGHJ3*01
2360
3050


292
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3′_IGHJ3*01
2361
3051


293
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1′_IGHJ3*01
2362
3052


294
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>2′_IGHJ3*01
2363
3053


295
>gi|Fabrus|A08_VH3-23_IGHD1-14*01>3′_IGHJ3*01
2364
3054


296
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>1′_IGHJ3*01
2365
3055


297
>gi|Fabrus|A10_VH3-23_IGHD1-20*01>2′_IGHJ3*01
2366
3056


298
>gi|Fabrus|A11_VH3-23_IGHD1-20*01>3′_IGHJ3*01
2367
3057


299
>gi|Fabrus|A12_VH3-23_IGHD1-26*01>1′_IGHJ3*01
2368
3058


300
>gi|Fabrus|B01_VH3-23_IGHD1-26*01>3′_IGHJ3*01
2369
3059


301
>gi|Fabrus|B02_VH3-23_IGHD2-2*01>1′_IGHJ3*01
2370
3060


302
>gi|Fabrus|B03_VH3-23_IGHD2-2*01>3′_IGHJ3*01
2371
3061


303
>gi|Fabrus|B04_VH3-23_IGHD2-8*01>1′_IGHJ3*01
2372
3062


304
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>1′_IGHJ3*01
2373
3063


305
>gi|Fabrus|B06_VH3-23_IGHD2-15*01>3′_IGHJ3*01
2374
3064


306
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>1′_IGHJ3*01
2375
3065


307
>gi|Fabrus|B08_VH3-23_IGHD2-21*01>3′_IGHJ3*01
2376
3066


308
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>1′_IGHJ3*01
2377
3067


309
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3′_IGHJ3*01
2378
3068


310
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>1′_IGHJ3*01
2379
3069


311
>gi|Fabrus|B12_VH3-23_IGHD3-9*01>3′_IGHJ3*01
2380
3070


312
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>1′_IGHJ3*01
2381
3071


313
>gi|Fabrus|C02_VH3-23_IGHD3-10*01>3′_IGHJ3*01
2382
3072


314
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>1′_IGHJ3*01
2383
3073


315
>gi|Fabrus|C04_VH3-23_IGHD3-16*01>3′_IGHJ3*01
2384
3074


316
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>1′_IGHJ3*01
2385
3075


317
>gi|Fabrus|C06_VH3-23_IGHD4-4*01(1)>1′_IGHJ3*01
2386
3076


318
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3′_IGHJ3*01
2387
3077


319
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>1′_IGHJ3*01
2388
3078


320
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3′_IGHJ3*01
2389
3079


321
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>1′_IGHJ3*01
2390
3080


322
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3′_IGHJ3*01
2391
3081


323
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>1′_IGHJ3*01
2392
3082


324
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3′_IGHJ3*01
2393
3083


325
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1′_IGHJ3*01
2394
3084


326
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>3′_IGHJ3*01
2395
3085


327
>gi|Fabrus|D04_VH3-23_IGHD5-12*01>1′_IGHJ3*01
2396
3086


328
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>3′_IGHJ3*01
2397
3087


329
>gi|Fabrus|D06_VH3-23_IGHD5-18*01(2)>1′_IGHJ3*01
2398
3088


330
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>3′_IGHJ3*01
2399
3089


331
>gi|Fabrus|D08_VH3-23_IGHD5-24*01>1′_IGHJ3*01
2400
3090


332
>gi|Fabrus|D09_VH3-23_IGHD5-24*01>3′_IGHJ3*01
2401
3091


333
>gi|Fabrus|D10_VH3-23_IGHD6-6*01>1′_IGHJ3*01
2402
3092


334
>gi|Fabrus|D11_VH3-23_IGHD6-6*01>2′_IGHJ3*01
2403
3093


335
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>3′_IGHJ3*01
2404
3094


336
>gi|Fabrus|E01_VH3-23_IGHD6-13*01>1′_IGHJ3*01
2405
3095


337
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>2′_IGHJ3*01
2406
3096


338
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>3′_IGHJ3*01
2407
3097


339
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1′_IGHJ3*01
2408
3098


340
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2′_IGHJ3*01
2409
3099


341
>gi|Fabrus|E06_VH3-23_IGHD6-19*01>3′_IGHJ3*01
2410
3100


342
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>1′_IGHJ3*01
2411
3101


343
>gi|Fabrus|E08_VH3-23_IGHD6-25*01>3′_IGHJ3*01
2412
3102


344
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>1′_IGHJ3*01
2413
3103


345
>gi|Fabrus|E10_VH3-23_IGHD7-27*01>2′_IGHJ3*01
2414
3104


346
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1_IGHJ4*01
2415
3105


347
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2_IGHJ4*01
2416
3106


348
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3_IGHJ4*01
2417
3107


349
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1_IGHJ4*01
2418
3108


350
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3_IGHJ4*01
2419
3109


351
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1_IGHJ4*01
2420
3110


352
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>3_IGHJ4*01
2421
3111


353
>gi|Fabrus|A08_VH3-23_IGHD1-20*01>1_IGHJ4*01
2422
3112


354
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>3_IGHJ4*01
2423
3113


355
>gi|Fabrus|A10_VH3-23_IGHD1-26*01>1_IGHJ4*01
2424
3114


356
>gi|Fabrus|A11_VH3-23_IGHD1-26*01>3_IGHJ4*01
2425
3115


357
>gi|Fabrus|A12_VH3-23_IGHD2-2*01>2_IGHJ4*01
2426
3116


358
>gi|Fabrus|B01_VH3-23_IGHD2-2*01>3_IGHJ4*01
2427
3117


359
>gi|Fabrus|B02_VH3-23_IGHD2-8*01>2_IGHJ4*01
2428
3118


360
>gi|Fabrus|B03_VH3-23_IGHD2-8*01>3_IGHJ4*01
2429
3119


361
>gi|Fabrus|B04_VH3-23_IGHD2-15*01>2_IGHJ4*01
2430
3120


362
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>3_IGHJ4*01
2431
3121


363
>gi|Fabrus|B06_VH3-23_IGHD2-21*01>2_IGHJ4*01
2432
3122


364
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>3_IGHJ4*01
2433
3123


365
>gi|Fabrus|B08_VH3-23_IGHD3-3*01>1_IGHJ4*01
2434
3124


366
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>2_IGHJ4*01
2435
3125


367
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3_IGHJ4*01
2436
3126


368
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>2_IGHJ4*01
2437
3127


369
>gi|Fabrus|B12_VH3-23_IGHD3-10*01>2_IGHJ4*01
2438
3128


370
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>3_IGHJ4*01
2439
3129


371
>gi|Fabrus|C02_VH3-23_IGHD3-16*01>2_IGHJ4*01
2440
3130


372
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>3_IGHJ4*01
2441
3131


373
>gi|Fabrus|C04_VH3-23_IGHD3-22*01>2_IGHJ4*01
2442
3132


374
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>3_IGHJ4*01
2443
3133


375
>gi|Fabrus|C06_VH3-23_IGHD4-4*01(1)>2_IGHJ4*01
2444
3134


376
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3_IGHJ4*01
2445
3135


377
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>2_IGHJ4*01
2446
3136


378
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3_IGHJ4*01
2447
3137


379
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>2_IGHJ4*01
2448
3138


380
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3_IGHJ4*01
2449
3139


381
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>2_IGHJ4*01
2450
3140


382
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3_IGHJ4*01
2451
3141


383
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1_IGHJ4*01
2452
3142


384
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>2_IGHJ4*01
2453
3143


385
>gi|Fabrus|D04_VH3-23_IGHD5-5*01(2)>3_IGHJ4*01
2454
3144


386
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>1_IGHJ4*01
2455
3145


387
>gi|Fabrus|D06_VH3-23_IGHD5-12*01>3_IGHJ4*01
2456
3146


388
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>1_IGHJ4*01
2457
3147


389
>gi|Fabrus|D08_VH3-23_IGHD5-18*01(2)>2_IGHJ4*01
2458
3148


390
>gi|Fabrus|D09_VH3-23_IGHD5-18*01(2)>3_IGHJ4*01
2459
3149


391
>gi|Fabrus|D10_VH3-23_IGHD5-24*01>1_IGHJ4*01
2460
3150


392
>gi|Fabrus|D11_VH3-23_IGHD5-24*01>3_IGHJ4*01
2461
3151


393
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>1_IGHJ4*01
2462
3152


394
>gi|Fabrus|E01_VH3-23_IGHD6-6*01>2_IGHJ4*01
2463
3153


395
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>1_IGHJ4*01
2464
3154


396
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>2_IGHJ4*01
2465
3155


397
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1_IGHJ4*01
2466
3156


398
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2_IGHJ4*01
2467
3157


399
>gi|Fabrus|E06_VH3-23_IGHD6-25*01>1_IGHJ4*01
2468
3158


400
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>2_IGHJ4*01
2469
3159


401
>gi|Fabrus|E08_VH3-23_IGHD7-27*01>1_IGHJ4*01
2470
3160


402
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>3_IGHJ4*01
2471
3161


403
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1′_IGHJ4*01
2472
3162


404
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2′_IGHJ4*01
2473
3163


405
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3′_IGHJ4*01
2474
3164


406
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1′_IGHJ4*01
2475
3165


407
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3′_IGHJ4*01
2476
3166


408
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1′_IGHJ4*01
2477
3167


409
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>2′_IGHJ4*01
2478
3168


410
>gi|Fabrus|A08_VH3-23_IGHD1-14*01>3′_IGHJ4*01
2479
3169


411
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>1′_IGHJ4*01
2480
3170


412
>gi|Fabrus|A10_VH3-23_IGHD1-20*01>2′_IGHJ4*01
2481
3171


413
>gi|Fabrus|A11_VH3-23_IGHD1-20*01>3′_IGHJ4*01
2482
3172


414
>gi|Fabrus|A12_VH3-23_IGHD1-26*01>1′_IGHJ4*01
2483
3173


415
>gi|Fabrus|B01_VH3-23_IGHD1-26*01>3′_IGHJ4*01
2484
3174


416
>gi|Fabrus|B02_VH3-23_IGHD2-2*01>1′_IGHJ4*01
2485
3175


417
>gi|Fabrus|B03_VH3-23_IGHD2-2*01>3′_IGHJ4*01
2486
3176


418
>gi|Fabrus|B04_VH3-23_IGHD2-8*01>1′_IGHJ4*01
2487
3177


419
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>1′_IGHJ4*01
2488
3178


420
>gi|Fabrus|B06_VH3-23_IGHD2-15*01>3′_IGHJ4*01
2489
3179


421
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>1′_IGHJ4*01
2490
3180


422
>gi|Fabrus|B08_VH3-23_IGHD2-21*01>3′_IGHJ4*01
2491
3181


423
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>1′_IGHJ4*01
2492
3182


424
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3′_IGHJ4*01
2493
3183


425
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>1′_IGHJ4*01
2494
3184


426
>gi|Fabrus|B12_VH3-23_IGHD3-9*01>3′_IGHJ4*01
2495
3185


427
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>1′_IGHJ4*01
2496
3186


428
>gi|Fabrus|C02_VH3-23_IGHD3-10*01>3′_IGHJ4*01
2497
3187


429
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>1′_IGHJ4*01
2498
3188


430
>gi|Fabrus|C04_VH3-23_IGHD3-16*01>3′_IGHJ4*01
2499
3189


431
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>1′_IGHJ4*01
2500
3190


432
>gi|Fabrus|C06_VH3-23_IGHD4-4*01(1)>1′_IGHJ4*01
2501
3191


433
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3′_IGHJ4*01
2502
3192


434
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>1′_IGHJ4*01
2503
3193


435
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3′_IGHJ4*01
2504
3194


436
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>1′_IGHJ4*01
2505
3195


437
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3′_IGHJ4*01
2506
3196


438
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>1′_IGHJ4*01
2507
3197


439
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3′_IGHJ4*01
2508
3198


440
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1′_IGHJ4*01
2509
3199


441
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>3′_IGHJ4*01
2510
3200


442
>gi|Fabrus|D04_VH3-23_IGHD5-12*01>1′_IGHJ4*01
2511
3201


443
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>3′_IGHJ4*01
2512
3202


444
>gi|Fabrus|D06_VH3-23_IGHD5-18*01(2)>1′_IGHJ4*01
2513
3203


445
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>3′_IGHJ4*01
2514
3204


446
>gi|Fabrus|D08_VH3-23_IGHD5-24*01>1′_IGHJ4*01
2515
3205


447
>gi|Fabrus|D09_VH3-23_IGHD5-24*01>3′_IGHJ4*01
2516
3206


448
>gi|Fabrus|D10_VH3-23_IGHD6-6*01>1′_IGHJ4*01
2517
3207


449
>gi|Fabrus|D11_VH3-23_IGHD6-6*01>2′_IGHJ4*01
2518
3208


450
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>3′_IGHJ4*01
2519
3209


451
>gi|Fabrus|E01_VH3-23_IGHD6-13*01>1′_IGHJ4*01
2520
3210


452
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>2′_IGHJ4*01
2521
3211


453
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>3′_IGHJ4*01
2522
3212


454
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1′_IGHJ4*01
2523
3213


455
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2′_IGHJ4*01
2524
3214


456
>gi|Fabrus|E06_VH3-23_IGHD6-19*01>3′_IGHJ4*01
2525
3215


457
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>1′_IGHJ4*01
2526
3216


458
>gi|Fabrus|E08_VH3-23_IGHD6-25*01>3′_IGHJ4*01
2527
3217


459
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>1′_IGHJ4*01
2528
3218


460
>gi|Fabrus|E10_VH3-23_IGHD7-27*01>2′_IGHJ4*01
2529
3219


461
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1_IGHJ5*01
2530
3220


462
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2_IGHJ5*01
2531
3221


463
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3_IGHJ5*01
2532
3222


464
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1_IGHJ5*01
2533
3223


465
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3_IGHJ5*01
2534
3224


466
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1_IGHJ5*01
2535
3225


467
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>3_IGHJ5*01
2536
3226


468
>gi|Fabrus|A08_VH3-23_IGHD1-20*01>1_IGHJ5*01
2537
3227


469
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>3_IGHJ5*01
2538
3228


470
>gi|Fabrus|A10_VH3-23_IGHD1-26*01>1_IGHJ5*01
2539
3229


471
>gi|Fabrus|A11_VH3-23_IGHD1-26*01>3_IGHJ5*01
2540
3230


472
>gi|Fabrus|A12_VH3-23_IGHD2-2*01>2_IGHJ5*01
2541
3231


473
>gi|Fabrus|B01_VH3-23_IGHD2-2*01>3_IGHJ5*01
2542
3232


474
>gi|Fabrus|B02_VH3-23_IGHD2-8*01>2_IGHJ5*01
2543
3233


475
>gi|Fabrus|B03_VH3-23_IGHD2-8*01>3_IGHJ5*01
2544
3234


476
>gi|Fabrus|B04_VH3-23_IGHD2-15*01>2_IGHJ5*01
2545
3235


477
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>3_IGHJ5*01
2546
3236


478
>gi|Fabrus|B06_VH3-23_IGHD2-21*01>2_IGHJ5*01
2547
3237


479
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>3_IGHJ5*01
2548
3238


480
>gi|Fabrus|B08_VH3-23_IGHD3-3*01>1_IGHJ5*01
2549
3239


481
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>2_IGHJ5*01
2550
3240


482
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3_IGHJ5*01
2551
3241


483
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>2_IGHJ5*01
2552
3242


484
>gi|Fabrus|B12_VH3-23_IGHD3-10*01>2_IGHJ5*01
2553
3243


485
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>3_IGHJ5*01
2554
3244


486
>gi|Fabrus|C02_VH3-23_IGHD3-16*01>2_IGHJ5*01
2555
3245


487
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>3_IGHJ5*01
2556
3246


488
>gi|Fabrus|C04_VH3-23_IGHD3-22*01>2_IGHJ5*01
2557
3247


489
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>3_IGHJ5*01
2558
3248


490
>gi|Fabrus|C06_VH3-23_IGHD4-4*01(1)>2_IGHJ5*01
2559
3249


491
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3_IGHJ5*01
2560
3250


492
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>2_IGHJ5*01
2561
3251


493
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3_IGHJ5*01
2562
3252


494
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>2_IGHJ5*01
2563
3253


495
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3_IGHJ5*01
2564
3254


496
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>2_IGHJ5*01
2565
3255


497
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3_IGHJ5*01
2566
3256


498
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1_IGHJ5*01
2567
3257


499
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>2_IGHJ5*01
2568
3258


500
>gi|Fabrus|D04_VH3-23_IGHD5-5*01(2)>3_IGHJ5*01
2569
3259


501
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>1_IGHJ5*01
2570
3260


502
>gi|Fabrus|D06_VH3-23_IGHD5-12*01>3_IGHJ5*01
2571
3261


503
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>1_IGHJ5*01
2572
3262


504
>gi|Fabrus|D08_VH3-23_IGHD5-18*01(2)>2_IGHJ5*01
2573
3263


505
>gi|Fabrus|D09_VH3-23_IGHD5-18*01(2)>3_IGHJ5*01
2574
3264


506
>gi|Fabrus|D10_VH3-23_IGHD5-24*01>1_IGHJ5*01
2575
3265


507
>gi|Fabrus|D11_VH3-23_IGHD5-24*01>3_IGHJ5*01
2576
3266


508
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>1_IGHJ5*01
2577
3267


509
>gi|Fabrus|E01_VH3-23_IGHD6-6*01>2_IGHJ5*01
2578
3268


510
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>1_IGHJ5*01
2579
3269


511
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>2_IGHJ5*01
2580
3270


512
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1_IGHJ5*01
2581
3271


513
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2_IGHJ5*01
2582
3272


514
>gi|Fabrus|E06_VH3-23_IGHD6-25*01>1_IGHJ5*01
2583
3273


515
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>2_IGHJ5*01
2584
3274


516
>gi|Fabrus|E08_VH3-23_IGHD7-27*01>1_IGHJ5*01
2585
3275


517
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>3_IGHJ5*01
2586
3276


518
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1′_IGHJ5*01
2587
3277


519
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2′_IGHJ5*01
2588
3278


520
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3′_IGHJ5*01
2589
3279


521
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1′_IGHJ5*01
2590
3280


522
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3′_IGHJ5*01
2591
3281


523
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1′_IGHJ5*01
2592
3282


524
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>2′_IGHJ5*01
2593
3283


525
>gi|Fabrus|A08_VH3-23_IGHD1-14*01>3′_IGHJ5*01
2594
3284


526
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>1′_IGHJ5*01
2595
3285


527
>gi|Fabrus|A10_VH3-23_IGHD1-20*01>2′_IGHJ5*01
2596
3286


528
>gi|Fabrus|A11_VH3-23_IGHD1-20*01>3′_IGHJ5*01
2597
3287


529
>gi|Fabrus|A12_VH3-23_IGHD1-26*01>1′_IGHJ5*01
2598
3288


530
>gi|Fabrus|B01_VH3-23_IGHD1-26*01>3′_IGHJ5*01
2599
3289


531
>gi|Fabrus|B02_VH3-23_IGHD2-2*01>1′_IGHJ5*01
2600
3290


532
>gi|Fabrus|B03_VH3-23_IGHD2-2*01>3′_IGHJ5*01
2601
3291


533
>gi|Fabrus|B04_VH3-23_IGHD2-8*01>1′_IGHJ5*01
2602
3292


534
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>1′_IGHJ5*01
2603
3293


535
>gi|Fabrus|B06_VH3-23_IGHD2-15*01>3′_IGHJ5*01
2604
3294


536
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>1′_IGHJ5*01
2605
3295


537
>gi|Fabrus|B08_VH3-23_IGHD2-21*01>3′_IGHJ5*01
2606
3296


538
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>1′_IGHJ5*01
2607
3297


539
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3′_IGHJ5*01
2608
3298


540
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>1′_IGHJ5*01
2609
3299


541
>gi|Fabrus|B12_VH3-23_IGHD3-9*01>3′_IGHJ5*01
2610
3300


542
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>1′_IGHJ5*01
2611
3301


543
>gi|Fabrus|C02_VH3-23_IGHD3-10*01>3′_IGHJ5*01
2612
3302


544
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>1′_IGHJ5*01
2613
3303


545
>gi|Fabrus|C04_VH3-23_IGHD3-16*01>3′_IGHJ5*01
2614
3304


546
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>1′_IGHJ5*01
2615
3305


547
>gi|Fabrus|C06_VH3-23_IGHD4-4*01(1)>1′_IGHJ5*01
2616
3306


548
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3′_IGHJ5*01
2617
3307


549
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>1′_IGHJ5*01
2618
3308


550
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3′_IGHJ5*01
2619
3309


551
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>1′_IGHJ5*01
2620
3310


552
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3′_IGHJ5*01
2621
3311


553
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>1′_IGHJ5*01
2622
3312


554
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3′_IGHJ5*01
2623
3313


555
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1′_IGHJ5*01
2624
3314


556
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>3′_IGHJ5*01
2625
3315


557
>gi|Fabrus|D04_VH3-23_IGHD5-12*01>1′_IGHJ5*01
2626
3316


558
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>3′_IGHJ5*01
2627
3317


559
>gi|Fabrus|D06_VH3-23_IGHD5-18*01(2)>1′_IGHJ5*01
2628
3318


560
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>3′_IGHJ5*01
2629
3319


561
>gi|Fabrus|D08_VH3-23_IGHD5-24*01>1′_IGHJ5*01
2630
3320


562
>gi|Fabrus|D09_VH3-23_IGHD5-24*01>3′_IGHJ5*01
2631
3321


563
>gi|Fabrus|D10_VH3-23_IGHD6-6*01>1′_IGHJ5*01
2632
3322


564
>gi|Fabrus|D11_VH3-23_IGHD6-6*01>2′_IGHJ5*01
2633
3323


565
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>3′_IGHJ5*01
2634
3324


566
>gi|Fabrus|E01_VH3-23_IGHD6-13*01>1′_IGHJ5*01
2635
3325


567
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>2′_IGHJ5*01
2636
3326


568
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>3′_IGHJ5*01
2637
3327


569
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1′_IGHJ5*01
2638
3328


570
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2′_IGHJ5*01
2639
3329


571
>gi|Fabrus|E06_VH3-23_IGHD6-19*01>3′_IGHJ5*01
2640
3330


572
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>1′_IGHJ5*01
2641
3331


573
>gi|Fabrus|E08_VH3-23_IGHD6-25*01>3′_IGHJ5*01
2642
3332


574
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>1′_IGHJ5*01
2643
3333


575
>gi|Fabrus|E10_VH3-23_IGHD7-27*01>2′_IGHJ5*01
2644
3334


576
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1_IGHJ6*01
2645
3335


577
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2_IGHJ6*01
2646
3336


578
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3_IGHJ6*01
2647
3337


579
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1_IGHJ6*01
2648
3338


580
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3_IGHJ6*01
2649
3339


581
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1_IGHJ6*01
2650
3340


582
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>3_IGHJ6*01
2651
3341


583
>gi|Fabrus|A08_VH3-23_IGHD1-20*01>1_IGHJ6*01
2652
3342


584
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>3_IGHJ6*01
2653
3343


585
>gi|Fabrus|A10_VH3-23_IGHD1-26*01>1_IGHJ6*01
2654
3344


586
>gi|Fabrus|A11_VH3-23_IGHD1-26*01>3_IGHJ6*01
2655
3345


587
>gi|Fabrus|A12_VH3-23_IGHD2-2*01>2_IGHJ6*01
2656
3346


588
>gi|Fabrus|B01_VH3-23_IGHD2-2*01>3_IGHJ6*01
2657
3347


589
>gi|Fabrus|B02_VH3-23_IGHD2-8*01>2_IGHJ6*01
2658
3348


590
>gi|Fabrus|B03_VH3-23_IGHD2-8*01>3_IGHJ6*01
2659
3349


591
>gi|Fabrus|B04_VH3-23_IGHD2-15*01>2_IGHJ6*01
2660
3350


592
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>3_IGHJ6*01
2661
3351


593
>gi|Fabrus|B06_VH3-23_IGHD2-21*01>2_IGHJ6*01
2662
3352


594
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>3_IGHJ6*01
2663
3353


595
>gi|Fabrus|B08_VH3-23_IGHD3-3*01>1_IGHJ6*01
2664
3354


596
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>2_IGHJ6*01
2665
3355


597
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3_IGHJ6*01
2666
3356


598
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>2_IGHJ6*01
2667
3357


599
>gi|Fabrus|B12_VH3-23_IGHD3-10*01>2_IGHJ6*01
2668
3358


600
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>3_IGHJ6*01
2669
3359


601
>gi|Fabrus|C02_VH3-23_IGHD3-16*01>2_IGHJ6*01
2670
3360


602
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>3_IGHJ6*01
2671
3361


603
>gi|Fabrus|C04_VH3-23_IGHD3-22*01>2_IGHJ6*01
2672
3362


604
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>3_IGHJ6*01
2673
3363


605
>gi|Fabrus|C06_VH3-23_IGHD4-4*01 (1)>2_IGHJ6*01
2674
3364


606
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3_IGHJ6*01
2675
3365


607
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>2_IGHJ6*01
2676
3366


608
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3_IGHJ6*01
2677
3367


609
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>2_IGHJ6*01
2678
3368


610
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3_IGHJ6*01
2679
3369


611
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>2_IGHJ6*01
2680
3370


612
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3_IGHJ6*01
2681
3371


613
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1_IGHJ6*01
2682
3372


614
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>2_IGHJ6*01
2683
3373


615
>gi|Fabrus|D04_VH3-23_IGHD5-5*01(2)>3_IGHJ6*01
2684
3374


616
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>1_IGHJ6*01
2685
3375


617
>gi|Fabrus|D06_VH3-23_IGHD5-12*01>3_IGHJ6*01
2686
3376


618
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>1_IGHJ6*01
2687
3377


619
>gi|Fabrus|D08_VH3-23_IGHD5-18*01(2)>2_IGHJ6*01
2688
3378


620
>gi|Fabrus|D09_VH3-23_IGHD5-18*01(2)>3_IGHJ6*01
2689
3379


621
>gi|Fabrus|D10_VH3-23_IGHD5-24*01>1_IGHJ6*01
2690
3380


622
>gi|Fabrus|D11_VH3-23_IGHD5-24*01>3_IGHJ6*01
2691
3381


623
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>1_IGHJ6*01
2692
3382


624
>gi|Fabrus|E01_VH3-23_IGHD6-6*01>2_IGHJ6*01
2693
3383


625
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>1_IGHJ6*01
2694
3384


626
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>2_IGHJ6*01
2695
3385


627
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1_IGHJ6*01
2696
3386


628
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2_IGHJ6*01
2697
3387


629
>gi|Fabrus|E06_VH3-23_IGHD6-25*01>1_IGHJ6*01
2698
3388


630
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>2_IGHJ6*01
2699
3389


631
>gi|Fabrus|E08_VH3-23_IGHD7-27*01>1_IGHJ6*01
2700
3390


632
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>3_IGHJ6*01
2701
3391


633
>gi|Fabrus|A01_VH3-23_IGHD1-1*01>1′_IGHJ6*01
2702
3392


634
>gi|Fabrus|A02_VH3-23_IGHD1-1*01>2′_IGHJ6*01
2703
3393


635
>gi|Fabrus|A03_VH3-23_IGHD1-1*01>3′_IGHJ6*01
2704
3394


636
>gi|Fabrus|A04_VH3-23_IGHD1-7*01>1′_IGHJ6*01
2705
3395


637
>gi|Fabrus|A05_VH3-23_IGHD1-7*01>3′_IGHJ6*01
2706
3396


638
>gi|Fabrus|A06_VH3-23_IGHD1-14*01>1′_IGHJ6*01
2707
3397


639
>gi|Fabrus|A07_VH3-23_IGHD1-14*01>2′_IGHJ6*01
2708
3398


640
>gi|Fabrus|A08_VH3-23_IGHD1-14*01>3′_IGHJ6*01
2709
3399


641
>gi|Fabrus|A09_VH3-23_IGHD1-20*01>1′_IGHJ6*01
2710
3400


642
>gi|Fabrus|A10_VH3-23_IGHD1-20*01>2′_IGHJ6*01
2711
3401


643
>gi|Fabrus|A11_VH3-23_IGHD1-20*01>3′_IGHJ6*01
2712
3402


644
>gi|Fabrus|A12_VH3-23_IGHD1-26*01>1′_IGHJ6*01
2713
3403


645
>gi|Fabrus|B01_VH3-23_IGHD1-26*01>3′_IGHJ6*01
2714
3404


646
>gi|Fabrus|B02_VH3-23_IGHD2-2*01>1′_IGHJ6*01
2715
3405


647
>gi|Fabrus|B03_VH3-23_IGHD2-2*01>3′_IGHJ6*01
2716
3406


648
>gi|Fabrus|B04_VH3-23_IGHD2-8*01>1′_IGHJ6*01
2717
3407


649
>gi|Fabrus|B05_VH3-23_IGHD2-15*01>1′_IGHJ6*01
2718
3408


650
>gi|Fabrus|B06_VH3-23_IGHD2-15*01>3′_IGHJ6*01
2719
3409


651
>gi|Fabrus|B07_VH3-23_IGHD2-21*01>1′_IGHJ6*01
2720
3410


652
>gi|Fabrus|B08_VH3-23_IGHD2-21*01>3′_IGHJ6*01
2721
3411


653
>gi|Fabrus|B09_VH3-23_IGHD3-3*01>1′_IGHJ6*01
2722
3412


654
>gi|Fabrus|B10_VH3-23_IGHD3-3*01>3′_IGHJ6*01
2723
3413


655
>gi|Fabrus|B11_VH3-23_IGHD3-9*01>1′_IGHJ6*01
2724
3414


656
>gi|Fabrus|B12_VH3-23_IGHD3-9*01>3′_IGHJ6*01
2725
3415


657
>gi|Fabrus|C01_VH3-23_IGHD3-10*01>1′_IGHJ6*01
2726
3416


658
>gi|Fabrus|C02_VH3-23_IGHD3-10*01>3′_IGHJ6*01
2727
3417


659
>gi|Fabrus|C03_VH3-23_IGHD3-16*01>1′_IGHJ6*01
2728
3418


660
>gi|Fabrus|C04_VH3-23_IGHD3-16*01>3′_IGHJ6*01
2729
3419


661
>gi|Fabrus|C05_VH3-23_IGHD3-22*01>1′_IGHJ6*01
2730
3420


662
>gi|Fabrus|C06_VH3-23_IGHD4-4*01(1)>1′_IGHJ6*01
2731
3421


663
>gi|Fabrus|C07_VH3-23_IGHD4-4*01(1)>3′_IGHJ6*01
2732
3422


664
>gi|Fabrus|C08_VH3-23_IGHD4-11*01(1)>1′_IGHJ6*01
2733
3423


665
>gi|Fabrus|C09_VH3-23_IGHD4-11*01(1)>3′_IGHJ6*01
2734
3424


666
>gi|Fabrus|C10_VH3-23_IGHD4-17*01>1′_IGHJ6*01
2735
3425


667
>gi|Fabrus|C11_VH3-23_IGHD4-17*01>3′_IGHJ6*01
2736
3426


668
>gi|Fabrus|C12_VH3-23_IGHD4-23*01>1′_IGHJ6*01
2737
3427


669
>gi|Fabrus|D01_VH3-23_IGHD4-23*01>3′_IGHJ6*01
2738
3428


670
>gi|Fabrus|D02_VH3-23_IGHD5-5*01(2)>1′_IGHJ6*01
2739
3429


671
>gi|Fabrus|D03_VH3-23_IGHD5-5*01(2)>3′_IGHJ6*01
2740
3430


672
>gi|Fabrus|D04_VH3-23_IGHD5-12*01>1′_IGHJ6*01
2741
3431


673
>gi|Fabrus|D05_VH3-23_IGHD5-12*01>3′_IGHJ6*01
2742
3432


674
>gi|Fabrus|D06_VH3-23_IGHD5-18*01(2)>1′_IGHJ6*01
2743
3433


675
>gi|Fabrus|D07_VH3-23_IGHD5-18*01(2)>3′_IGHJ6*01
2744
3434


676
>gi|Fabrus|D08_VH3-23_IGHD5-24*01>1′_IGHJ6*01
2745
3435


677
>gi|Fabrus|D09_VH3-23_IGHD5-24*01>3′_IGHJ6*01
2746
3436


678
>gi|Fabrus|D10_VH3-23_IGHD6-6*01>1′_IGHJ6*01
2747
3437


679
>gi|Fabrus|D11_VH3-23_IGHD6-6*01>2′_IGHJ6*01
2748
3438


680
>gi|Fabrus|D12_VH3-23_IGHD6-6*01>3′_IGHJ6*01
2749
3439


681
>gi|Fabrus|E01_VH3-23_IGHD6-13*01>1′_IGHJ6*01
2750
3440


682
>gi|Fabrus|E02_VH3-23_IGHD6-13*01>2′_IGHJ6*01
2751
3441


683
>gi|Fabrus|E03_VH3-23_IGHD6-13*01>3′_IGHJ6*01
2752
3442


684
>gi|Fabrus|E04_VH3-23_IGHD6-19*01>1′_IGHJ6*01
2753
3443


685
>gi|Fabrus|E05_VH3-23_IGHD6-19*01>2′_IGHJ6*01
2754
3444


686
>gi|Fabrus|E06_VH3-23_IGHD6-19*01>3′_IGHJ6*01
2755
3445


687
>gi|Fabrus|E07_VH3-23_IGHD6-25*01>1′_IGHJ6*01
2756
3446


688
>gi|Fabrus|E08_VH3-23_IGHD6-25*01>3′_IGHJ6*01
2757
3447


689
>gi|Fabrus|E09_VH3-23_IGHD7-27*01>1′_IGHJ6*01
2758
3448


690
>gi|Fabrus|E10_VH3-23_IGHD7-27*01>2′_IGHJ6*01
2759
3449









Example 15
Electrochemiluminescence Binding Assay
The electrochemiluminescence (ECL) binding assay described in Example 13 was further used to screen a 5,376 member Fab library and 15 modified Fab antibodies set forth in rows 22-36 in Table 77 below for antibodies capable of binding to one of the nine different antigens. As noted in Example 13 above, data were analyzed by comparing the ECL signals for an antigen to the blank of each well. A signal to blank ratio of 4 or more was considered a “Hit” Fab.
Fifty-six (56) plates, each containing 96 different Fabs, were screened using the ECL assay. Thirty-six (36) Fabs were identified with specific binding affinity to one or more of the protein antigens, as indicated in Tables 42-44. Table 42, below, summarizes the results of the ECL assay, including the recombinant human target/protein antigen, the number of antibody hits per target and the % of hits per target (number of hits/5376 antibodies screened). Eleven Fabs were identified that bind to recombinant human delta-like protein 4 (DLL4) whereas only 2 hits were identified that bind to to recombinant human epidermal growth factor 2 (ErbB2). Ten Fabs were identified that bind to recombinant human erythropoietin receptor (Epo R) and 6 Fabs were identified that bind to recombinant human P-cadherin. Additionally, 3 Fabs were identified that bind to recombinant human epidermal growth factor 2 (ErbB2) and 3 Fabs were identified that bind to recombinant human Notch-1. Table 43, below, lists the 21 Fabs (including the heavy chain and light chain) that were identified as “hits” in the initial ECL screen. A “hit” was a Fab antibody with signal to blank ratio of greater than 4. The results of the initial MSD assay screen at a single Fab concentration are listed in Table 44 below. Table 44 lists the 21 Fabs (the Fab No. corresponds each of the Fabs identified in Table 43), the Fab concentration, the 9 recombinant human target/protein antigens, and the ECL signals from the initial MSD assay screen at the given Fab concentration.







TABLE 42







Summary of 5376 Fab Library ECL Screen











rHuman Antigen
Number of Hits
% Per Target















ErbB2/Fc chimera
2
0.037



EGF R/Fc chimera
3
0.056



HGF R/Fc chimera
0
n/a



Notch-1/Fc chimera
3
0.056



CD44/Fc chimera
0
n/a



IGF-1 sR
0
n/a



P-cadherin/Fc chimera
6
0.112



Epo R/Fc chimera
10
0.186



DLL4
11
0.205

















TABLE 43







Fab Antibody “Hits” identified in ECL Screen









Fab No.
Heavy Chain
Light Chain












1
VH4-28_IGHD7-27*01_IGHJ1*01
L2_IGKJ1*01


2
VH4-31_IGHD7-27*01_IGHJ5*01
L2_IGKJ1*01


3
VH2-5_IGHD7-27*01_IGHJ2*01
L2_IGKJ1*01


4
VH1-46_IGHD7-27*01_IGHJ2*01
A27_IGKJ1*01


5
VH1-69_IGHD1-1*01_IGHJ6*01
A17_IGKJ1*01


6
VH1-46_IGHD2-15*01_IGHJ2*01
L2_IGKJ1*01


7
VH1-46_IGHD6-13*01_IGHJ4*01
L2_IGKJ1*01


8
VH4-34_IGHD7-27*01_IGHJ4*01
L5_IGKJ1*01


9
VH1-46_IGHD6-13*01_IGHJ4*01
A27_IGKJ1*01


10
VH1-46_IGHD7-27*01_IGHJ2*01
L6_IGKJ1*01


11
VH1-3_IGHD4-23*01_IGHJ4*01
L12_IGKJ1*01


12
VH1-46_IGHD2-15*01_IGHJ2*01
L12_IGKJ1*01


13
VH1-46_IGHD3-10*01_IGHJ4*01
L12_IGKJ1*01


14
VH1-8_IGHD2-2*01_IGHJ6*01
L12_IGKJ1*01


15
VH1-46_IGHD3-10*01_IGHJ4*01
O1_IGKJ1*01


16
VH1-46_IGHD6-13*01_IGHJ4*01
O1_IGKJ1*01


17
VH4-34_IGHD7-27*01_IGHJ4*01
V1-4_IGLJ4*01


18
VH4-31_IGHD2-15*01_IGHJ2*01
V1-4_IGLJ4*01


19
VH4-34_IGHD7-27*01_IGHJ4*01
V4-6_IGLJ4*01


20
VH3-23_IGHD3-10*01>3_IGHJ6*01
O12_IGKJ1*01


21
VH3-23_IGHD3-10*01>1′_IGHJ3*01
O12_IGKJ1*01
















TABLE 44







ECL Signals for Identified Fab “Hits” from 5376 Fab Library Screen









Fab
[Fab]
rHuman Target


















No.
μM
ErbB2
EGFR
HGFR
Notch-1
CD44
IGF-1
P-cad
EPOR
DLL4
Blank





















1
0.36
2028
3803
2034
3813
2482
1703
2741
19375
3911
1948


2
0.61
581
1222
539
1195
1048
596
782
3065
1006
570


3
0.03
2556
381
311
563
421
344
352
492
476
373


4
0.36
1474
1405
1225
1921
1542
1081
1795
2434
9082
928


5
0.65
2940
662
437
1336
797
656
695
922
849
441


6
0.25
15169
88015
5912
10346
10436
4929
16145
30118
14728
3693


7
1.19
18514
32030
17322
41739
27596
14089
34551
141492
39645
11543


8
0.51
1705
1807
1738
2876
1879
1742
3176
2563
5648
1393


9
0.48
6940
9043
6313
12759
9979
5468
15301
67194
13726
2877


10
1.56
2452
2497
3116
3953
2281
1919
5418
4129
3770
1129


11
0.06
1569
1755
1910
2232
1683
2325
2432
1973
13614
842


12
0.28
4314
8327
5182
5281
4561
4643
8057
8071
8151
1773


13
0.93
10795
15559
13790
23657
10634
13930
26366
16729
74294
4760


14
0.13
840
859
969
1324
932
1167
1641
1106
9848
786


15
1.57
904
1403
1039
1256
836
985
2029
6393
1496
797


16
0.93
1610
1855
2596
1918
1453
1787
2422
4241
9632
859


17
0.58
1262
1022
1706
739
761
1198
725
768
3128
403


18
0.21
1308
1297
1531
809
868
1557
915
885
3332
489


19
0.34
900
738
914
912
694
871
935
907
2561
356


20
0.12
4578
4060
5472
5501
4213
3137
17528
11210
6646
2225


21
0.42
702
759
935
919
748
844
3178
1504
922
450









To confirm a “Hit” identified in the intial ECL screening (see Tables 42-44 above), a Fab concentration dependent titration was carried out to determine the Fab-antigen binding affinity. The assay procedure was the same as described Example 13 above, except that the concentration of Fab antibody was varied between wells from 0.0628 nM to 1.57 μM. The results of the dose response assays are set forth in Tables 45-61 below. Tables 62-76 below list the results of the dose response assays for 15 modified anti-DLL4 antibodies (set forth in rows 22-36 of Table 77, below). The modified anti-DLL4 antibodies have at least one mutation in the heavy or light chain as compared to the previously identified germline antibodies.







TABLE 45







Binding affinity of Fab VH4-28_IGHD7-


27*01_IGHJ1*01 & L2_IGKJ1*01











Fab[nM]
360
36















ErbB2/Fc
647
600



EGF R/Fc
957
711



HGF R/Fc
581
613



Notch-1/Fc
1026
773



CD44/Fc
740
679



IGF-1 sR
535
486



P-Cadherin/Fc
636
693



EPO R/Fc
4715
2977



DLL4
866
799



Blank
462
413

















TABLE 46







Binding affinity of Fab VH1-46_IGHD2-


15*01_IGHJ2*01 & L2_IGKJ1*01













Fab[μM]
0.25
0.0625
0.01563
0.00391

















ErbB2/Fc
29608
9033
4495
1667



EGF R/Fc
116674
94778
70836
35936



HGF R/Fc
13427
4108
1998
913



Notch-1/Fc
21447
5848
2800
1282



CD44/Fc
23015
6746
3182
1295



IGF-1 sR
11050
3150
1742
822



P-Cadherin/Fc
25459
7739
4945
1962



EPO R/Fc
49177
21136
11342
5022



DLL4
27691
8051
4015
1551



Blank
6344
1738
906
576

















TABLE 47







Binding affinity of Fab VH1-46_IGHD6-


13*01_IGHJ4*01 & L2_IGKJ1*01













Fab[μM]
1.19
0.2975
0.07438
0.01859

















ErbB2/Fc
38410
15111
7551
5531



EGF R/Fc
62454
42213
16605
11750



HGF R/Fc
45494
17396
6611
4566



Notch-1/Fc
72018
37503
21990
17565



CD44/Fc
47145
28601
10922
7322



IGF-1 sR
35187
17389
5804
3779



P-Cadherin/Fc
69710
26043
14807
11672



EPO R/Fc
192967
167064
153692
188065



DLL4
74900
34726
20719
18888



Blank
24999
5019
2504
1776

















TABLE 48







Binding affinity of Fab VH4-34_IGHD7-


27*01_IGHJ4*01 & L5_IGKJ1*01













Fab[μM]
0.51
0.1275
0.03188
0.00797

















ErbB2/Fc
1532
857
584
493



EGF R/Fc
2363
1061
694
530



HGF R/Fc
1989
853
693
419



Notch-1/Fc
2773
1497
849
654



CD44/Fc
2012
926
653
490



IGF-1 sR
2236
1045
765
564



P-Cadherin/Fc
2389
957
775
502



EPO R/Fc
2624
1067
789
566



DLL4
5183
2382
1282
872



Blank
1096
530
536
364

















TABLE 49







Binding affinity of Fab VH1-46_IGHD6-


13*01_IGHJ4*01 & A27_IGKJ1*01












Fab[μM]
0.48
0.096
0.0192
















ErbB2/Fc
11287
3365
2313



EGF R/Fc
14638
4509
3115



HGF R/Fc
8002
2328
1582



Notch-1/Fc
15931
4802
3041



CD44/Fc
13445
4320
2915



IGF-1 sR
8927
2449
1826



P-Cadherin/Fc
15595
6654
5040



EPO R/Fc
70938
57356
62037



DLL4
16065
5586
3555



Blank
2945
917
751

















TABLE 50







Binding affinity of Fab VH1-46_IGHD7-


27*01_IGHJ2*01 & L6_IGKJ1*01












Fab[μM]
1.56
0.312
0.0624
















ErbB2/Fc
7577
3659
2146



EGF R/Fc
7832
4328
2415



HGF R/Fc
10267
4691
2453



Notch-1/Fc
9447
4462
2352



CD44/Fc
7595
4171
2110



IGF-1 sR
6913
3508
2034



P-Cadherin/Fc
15016
7098
4226



EPO R/Fc
9480
5020
2678



DLL4
10897
5484
2585



Blank
4357
1977
960

















TABLE 51







Binding affinity of Fab VH1-3_IGHD4-


23*01_IGHJ4*01 & L12_IGKJ1*01













Fab[nM]
60
15
3.75
0.9375

















ErbB2/Fc
2155
740
291
268



EGF R/Fc
2563
842
371
224



HGF R/Fc
2298
743
394
243



Notch-1/Fc
2886
1058
375
348



CD44/Fc
2355
748
307
251



IGF-1 sR
2666
859
314
204



P-Cadherin/Fc
2662
837
331
191



EPO R/Fc
3214
970
358
238



DLL4
17270
7728
1569
453



Blank
1433
536
191
153

















TABLE 52







Binding affinity of Fab VH1-46_IGHD2-


15*01_IGHJ2*01 & L12_IGKJ1*01













Fab[nM]
280
70
17.5
4.375

















ErbB2/Fc
3953
1358
541
384



EGF R/Fc
6667
2574
1305
542



HGF R/Fc
3564
1289
565
193



Notch-1/Fc
4382
1492
680
480



CD44/Fc
4069
1370
664
424



IGF-1 sR
3533
1319
626
369



P-Cadherin/Fc
5400
1817
949
469



EPO R/Fc
8496
2485
1262
594



DLL4
8111
2747
1219
558



Blank
1691
635
304
305

















TABLE 53







Binding affinity of Fab VH1-46_IGHD3-


10*01_IGHJ4*01 & L12_IGKJ1*01













Fab[nM]
920
230
57.5
14.375

















ErbB2/Fc
10924
4078
2447
1594



EGF R/Fc
13406
5723
3858
2672



HGF R/Fc
10708
3934
2297
1600



Notch-1/Fc
20086
9737
5886
4206



CD44/Fc
9698
3817
2313
1488



IGF-1 sR
10246
4764
2833
1746



P-Cadherin/Fc
16666
6484
4110
2318



EPO R/Fc
16429
6949
4038
2718



DLL4
73638
119436
144126
125422



Blank
4082
1656
954
738

















TABLE 54







Binding affinity of Fab VH1-8_IGHD2-


2*01_IGHJ6*01 & L12_IGKJ1*01













Fab[nM]
130
32.5
8.1
2.0

















ErbB2/Fc
1533
556
557
382



EGF R/Fc
1746
645
560
424



HGF R/Fc
1882
525
551
356



Notch-1/Fc
1759
706
612
539



CD44/Fc
1754
573
528
447



IGF-1 sR
1973
561
518
367



P-Cadherin/Fc
1845
556
573
250



EPO R/Fc
2151
673
660
433



DLL4
7738
2989
1548
605



Blank
1153
473
435
316

















TABLE 55







Binding affinity of Fab FabVH1-46_IGHD3-


10*01_IGHJ4*01 & O1_IGKJ1*01













Fab[nM]
1570
392.5
98.1
24.5

















ErbB2/Fc
1263
539
247
241



EGF R/Fc
2481
744
4386
317



HGF R/Fc
1638
581
335
211



Notch-1/Fc
1639
749
313
434



CD44/Fc
1381
498
265
267



IGF-1 sR
1428
466
309
239



P-Cadherin/Fc
1793
459
347
257



EPO R/Fc
6121
5863
5628
4531



DLL4
2701
735
402
339



Blank
866
338
210
149

















TABLE 56







Binding affinity of Fab VH1-46_IGHD6-


13*01_IGHJ4*01 & O1_IGKJ1*01













Fab[nM]
930
232.5
58.1
14.5

















ErbB2/Fc
2225
779
322
274



EGF R/Fc
3110
803
444
357



HGF R/Fc
2344
790
432
373



Notch-1/Fc
2206
778
388
317



CD44/Fc
1917
607
375
212



IGF-1 sR
1915
569
343
234



P-Cadherin/Fc
2438
655
478
277



EPO R/Fc
3009
1472
829
660



DLL4
8162
3586
1876
1149



Blank
1206
460
225
117

















TABLE 57







Binding affinity of Fab VH4-34_IGHD7-


27*01_IGHJ4*01 & V1-4_IGLJ4*01













Fab[nM]
580
145
36.3
9.1

















ErbB2/Fc
1712
1123
1029
987



EGF R/Fc
1631
856
831
800



HGF R/Fc
2341
1173
1065
894



Notch-1/Fc
1585
860
633
754



CD44/Fc
1228
692
629
607



IGF-1 sR
1364
794
799
788



P-Cadherin/Fc
2240
850
684
589



EPO R/Fc
1579
845
722
697



DLL4
4420
2140
1399
1030



Blank
679
357
314
276

















TABLE 58







Binding affinity of Fab VH4-31_IGHD2-


15*01_IGHJ2*01 & V1-4_IGLJ4*01













Fab[nM]
210
52.5
13.1
3.3

















ErbB2/Fc
1977
1511
930
1031



EGF R/Fc
1617
1109
824
847



HGF R/Fc
2060
1286
981
849



Notch-1/Fc
1972
1323
669
726



CD44/Fc
1395
897
708
621



IGF-1 sR
1431
911
814
743



P-Cadherin/Fc
4410
2161
1062
678



EPO R/Fc
2123
1319
776
695



DLL4
4108
1951
1107
922



Blank
833
467
376
359

















TABLE 59







Binding affinity of Fab VH4-34_IGHD7-


27*01_IGHJ4*01 & V4-6_IGLJ4*01













Fab[nM]
340
170
85.0
42.5

















ErbB2/Fc
1226
964
844
866



EGF R/Fc
1208
826
1001
528



HGF R/Fc
1238
757
998
607



Notch-1/Fc
1209
816
780
649



CD44/Fc
959
660
693
522



IGF-1 sR
1042
832
891
646



P-Cadherin/Fc
1160
744
709
421



EPO R/Fc
1255
790
817
494



DLL4
2332
1462
1311
877



Blank
554
262
292
162

















TABLE 60







Binding affinity of Fab VH3-23_IGHD3-


10*01>3_IGHJ6*01 & O12_IGKJ1*01













Fab[nM]
120
12
1.2
0.12

















ErbB2/Fc
17294
4358
677
287



EGF R/Fc
14925
1984
464
272



HGF R/Fc
15917
2703
412
287



Notch-1/Fc
14382
2582
660
218



CD44/Fc
13519
1321
341
291



IGF-1 sR
13265
1135
181
175



P-Cadherin/Fc
61714
28490
1684
318



EPO R/Fc
33268
10966
1014
260



DLL4
20627
2510
319
210



Blank
6749
573
227
264

















TABLE 61







Binding affinity of Fab VH3-23_IGHD3-


10*01>1′_IGHJ3*01 & O12_IGKJ1*01











Fab[nM]
421.12
42.112















ErbB2/Fc
868
524



EGF R/Fc
765
422



HGF R/Fc
1202
565



Notch-1/Fc
1061
437



CD44/Fc
903
360



IGF-1 sR
1065
364



P-Cadherin/Fc
2949
1546



EPO R/Fc
1299
759



DLL4
1090
404



Blank
639
323

















TABLE 62







Binding affinity of Fab VH5-51_IGHD5-


18*01>3_IGHJ4*01_G100K & V3-4_IGLJ1*01












Fab[nM]
1000
200
40
8
1.6















ErbB2/Fc
1251
1467
1394
1232
1320


EGF R/Fc
1029
1371
1149
1033
1180


HGF R/Fc
1199
1428
1306
1260
1438


Notch-1/Fc
1176
1247
875
831
816


CD44/Fc
1025
1162
857
853
938


IGF-1 sR
1134
1320
1271
1203
1437


P-Cadherin/Fc
1043
1126
744
788
811


EPO R/Fc
1122
1226
979
881
899


DLL4
29554
45421
35876
11408
2423


Blank
630
588
518
489
565
















TABLE 63







Binding affinity of Fab VH5-51_IGHD5-


18*01>3_IGHJ4*01_G100R & V3-4_IGLJ1*01













Fab[nM]
1000
200
40
8
1.6
0.32
















ErbB2/Fc
945
1448
1421
1326
1439
1485


EGF R/Fc
721
1017
1113
1078
1204
1221


HGF R/Fc
778
1164
1234
1240
1206
1287


Notch-1/Fc
761
1330
1014
916
1052
983


CD44/FC
559
895
824
786
997
877


IGF-1 sR
733
1110
1243
1107
1329
1253


P-Cadherin/Fc
762
815
1020
669
666
656


EPO R/Fc
666
1175
994
911
920
851


DLL4
23398
29772
19481
5472
1541
1212


Blank
427
529
502
479
449
513
















TABLE 64







Binding affinity of Fab VH1-46_IGHD6-


6*01>1_IGHJ1*01_S104F & L6_IGKJ1*01













Fab[nM]
1000
200
40
8
1.6
0.32
















ErbB2/Fc
7472
5434
3428
1775
1008
611


EGF R/Fc
3575
2921
2297
1473
987
709


HGF R/Fc
3314
2836
2110
1619
861
710


Notch-1/Fc
9798
6377
3990
2003
1114
684


CD44/FC
5105
4071
2711
1523
1016
679


IGF-1 sR
3074
2778
2141
1517
966
630


P-Cadherin/Fc
5955
6263
5103
2634
1369
750


EPO R/Fc
5162
4593
3233
2040
1132
673


DLL4
35253
58743
62035
44781
5920
916


Blank
2186
1787
1442
1117
713
518
















TABLE 65







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S104A & L6_IGKJ1*01













Fab[nM]
1000
200
40
8
1.6
0.32
















ErbB2/Fc
7419
4038
2231
1902
1168
766


EGF R/Fc
3481
2951
1843
1298
862
660


HGF R/Fc
3531
2443
1545
1305
830
574


Notch-1/Fc
8058
5705
3479
2196
1166
739


CD44/FC
4734
3189
2677
1487
1017
684


IGF-1 sR
3348
2603
1897
1287
815
611


P-Cadherin/Fc
5632
3993
2602
1916
1139
649


EPO R/Fc
8292
3997
3510
1982
1117
739


DLL4
14331
14838
13637
9329
2130
646


Blank
2142
1659
1229
1188
648
428
















TABLE 66







Binding affinity of Fab VH5-51_IGHD5-


18*01>3_IGHJ4*01_G104T & V3-4_IGLJ1*01











Fab[nM]
1000
10















ErbB2/Fc
3532
1517



EGF R/Fc
3937
1188



HGF R/Fc
3769
1392



Notch-1/Fc
3342
933



CD44/Fc
3378
841



IGF-1 sR
3992
1118



P-Cadherin/Fc
3426
705



EPO R/Fc
3376
910



DLL4
29776
5229



Blank
1742
427

















TABLE 67







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S103P & L6_IGKJ1*01











Fab[nM]
1000
10















ErbB2/Fc
1276
847



EGF R/Fc
1909
773



HGF R/Fc
1359
859



Notch-1/Fc
1210
792



CD44/Fc
1220
746



IGF-1 sR
1336
814



P-Cadherin/Fc
1376
846



EPO R/Fc
1511
802



DLL4
29752
55127



Blank
760
550

















TABLE 68







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S102A & L6_IGKJ1*01











Fab[nM]
1000
10















ErbB2/Fc
946
670



EGF R/Fc
1601
677



HGF R/Fc
1658
698



Notch-1/Fc
1253
647



CD44/Fc
1087
681



IGF-1 sR
1167
657



P-Cadherin/Fc
1502
691



EPO R/Fc
1320
761



DLL4
22225
38724



Blank
1121
418

















TABLE 69







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S102A_S103P_S104F & L6_IGKJ1*01











Fab[nM]
1000
10















ErbB2/Fc
1292
842



EGF R/Fc
2434
809



HGF R/Fc
1581
691



Notch-1/Fc
1620
1144



CD44/Fc
1314
887



IGF-1 sR
1175
720



P-Cadherin/Fc
2499
894



EPO R/Fc
1656
1109



DLL4
73307
345030



Blank
731
680

















TABLE 70







Binding affinity of Fab VH5-51_IGHD5-


18*01>3_IGHJ4*01_G100K_G104T & V34_IGLJ1*01













Fab[nM]
500
25
1.25
0.0625

















ErbB2/Fc
957
927
695
692



EGF R/Fc
1106
821
729
736



HGF R/Fc
1299
805
774
643



Notch-1/Fc
703
440
312
439



CD44/Fc
923
570
444
394



IGF-1 sR
1114
780
674
537



P-Cadherin/Fc
815
269
374
331



EPO R/Fc
828
597
448
332



DLL4
25941
77931
38837
1014



Blank
480
206
256
239

















TABLE 71







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S102A_S103P_S104F_H111F & L6_IGKJ1*01












Fab[nM]
500
50
5
















ErbB2/Fc
2621
2114
1377



EGF R/Fc
2514
2042
1158



HGF R/Fc
2043
1282
1016



Notch-1/Fc
4376
2726
1170



CD44/Fc
2912
1610
1132



IGF-1 sR
1872
1226
917



P-Cadherin/Fc
3665
2491
1663



EPO R/Fc
5518
2462
1835



DLL4
29919
94200
149972



Blank
1016
774
626

















TABLE 72







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S102A_S103P_S104F_H111Y & L6_IGKJ1*01













Fab[nM]
500
50
5
0.5

















ErbB2/Fc
713
589
315
331



EGF R/Fc
898
523
311
273



HGF R/Fc
523
325
323
319



Notch-1/Fc
644
475
383
263



CD44/Fc
664
433
413
279



IGF-1 sR
592
460
320
164



P-Cadherin/Fc
939
626
437
215



EPO R/Fc
1212
631
377
359



DLL4
93071
206919
84945
2971



Blank
410
433
367
365

















TABLE 73







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S102A_S103P_S104Y_H111Y & L6_IGKJ1*01













Fab[nM]
500
50
5
0.5

















ErbB2/Fc
596
470
2855
1721



EGF R/Fc
758
489
2980
1751



HGF R/Fc
841
312
3101
1688



Notch-1/Fc
689
389
3232
1756



CD44/Fc
604
345
2756
1563



IGF-1 sR
747
399
3033
1662



P-Cadherin/Fc
1195
470
4162
2628



EPO R/Fc
1273
533
3582
1840



DLL4
105743
201050
73428
5150



Blank
436
329
2104
1309

















TABLE 74







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S102A_S103P_S104F & L6_IGKJ1*01_S28P













Fab[nM]
50
10
2
0.4

















ErbB2/Fc
384
136
176
275



EGF R/Fc
328
207
352
246



HGF R/Fc
183
136
255
199



Notch-1/Fc
477
280
307
248



CD44/Fc
340
212
266
257



IGF-1 sR
355
152
205
172



P-Cadherin/Fc
431
216
223
191



EPO R/Fc
527
392
251
147



DLL4
37653
26851
6103
1140



Blank
274
146
130
138

















TABLE 75







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S102A_S103P_S104F & L6_IGKJ1*01_S30N













Fab[nM]
50
10
2
0.4

















ErbB2/Fc
395
274
138
203



EGF R/Fc
294
245
171
173



HGF R/Fc
135
239
207
120



Notch-1/Fc
279
253
274
197



CD44/Fc
271
286
209
176



IGF-1 sR
240
233
296
141



P-Cadherin/Fc
143
283
395
111



EPO R/Fc
500
420
294
224



DLL4
37208
33374
11375
1039



Blank
299
165
205
179

















TABLE 76







Binding affinity of Fab VH1-46_IGHD6-


6*01_IGHJ1*01_S102A_S103P_S104F & L6_IGKJ1*01_S31K













Fab[nM]
50
10
2
0.4

















ErbB2/Fc
397
303
337
159



EGF R/Fc
350
297
197
175



HGF R/Fc
257
271
183
119



Notch-1/Fc
355
388
202
197



CD44/Fc
262
352
146
190



IGF-1 sR
257
214
222
65



P-Cadherin/Fc
411
240
351
151



EPO R/Fc
574
392
326
217



DLL4
35274
29781
10534
1259



Blank
183
267
197
255










Summary
Table 77, below, summarizes the results of the MSD assay. Table 77 lists the recombinant human target/protein antigen(s) and the Fabs, as designated by their respective heavy and light chains (including SEQ ID NOS). As is indicated in Table 77, below, several Fabs were identified that bind to multiple targets. For example, Fab VH1-46_IGHD2-15*01_IGHJ2*01 & L12_IGKJ1*01 binds to EGF R, Epo R and DLL4 while Fab VH1-46_IGHD3-10*01_IGHJ4*01 & L12_IGKJ1*01 binds to Notch-1, P-cadherin and DLL4. Table 77 below also lists the 15 additional modified Fabs (set forth in rows 22-36) that bind to DLL4.







TABLE 77







Selected Fabs and their targets












Fab


SEQ ID

SEQ ID


No.
rHuman Target
Heavy Chain
NO
Light Chain
NO















1
Epo R/Fc chimera
VH4-28_IGHD7-27*01_IGHJ1*01
1759
L2_IGKJ1*01
1843


2
Epo R/Fc chimera
VH4-31_IGHD7-27*01_IGHJ5*01
1769
L2_IGKJ1*01
1843


3
ErbB2/Fc chimera
VH2-5_IGHD7-27*01_IGHJ2*01
1559
L2_IGKJ1*01
1843


4
Epo R/Fc chimera
VH1-46_IGHD7-27*01_IGHJ2*01
1514
A27_IGKJ1*01
1833


5
ErbB2/Fc chimera
VH1-69_IGHD1-1*01_IGHJ6*01
1522
A17_IGKJ1*01
1828


6
Epo R/Fc chimera and
VH1-46_IGHD2-15*01_IGHJ2*01
1508
L2_IGKJ1*01
1843



EGF R/Fc chimera


7
EGF R/Fc chimera, Notch-
VH1-46_IGHD6-13*01_IGHJ4*01
1512
L2_IGKJ1*01
1843



1/Fc chimera, P-cadherin/



Fc chimera, Epo



R/Fc chimera and DLL4


8
DLL4
VH4-34_IGHD7-27*01_IGHJ4*01
1779
L5_IGKJ1*01
1849


9
Notch-1/Fc chimera, P-
VH1-46_IGHD6-13*01_IGHJ4*01
1512
A27_IGKJ1*01
1833



cadherin/Fc chimera, Epo



R/Fc chimera and DLL4


10
P-cadherin/Fc chimera
VH1-46_IGHD7-27*01_IGHJ2*01
1514
L6_IGKJ1*01
1850


11
DLL4
VH1-3_IGHD4-23*01_IGHJ4*01
1494
L12_IGKJ1*01
1841


12
EGF R/Fc chimera, Epo
VH1-46_IGHD2-15*01_IGHJ2*01
1508
L12_IGKJ1*01
1841



R/Fc chimera and DLL4


13
Notch-1/Fc chimera, P-
VH1-46_IGHD3-10*01_IGHJ4*01
1509
L12_IGKJ1*01
1841



cadherin/Fc chimera and



DLL4


14
DLL4
VH1-8_IGHD2-2*01_IGHJ6*01
1537
L12_IGKJ1*01
1841


15
Epo R/Fc chimera
VH1-46_IGHD3-10*01_IGHJ4*01
1509
O1_IGKJ1*01
1853


16
Epo R/Fc chimera and
VH1-46_IGHD6-13*01_IGHJ4*01
1512
O1_IGKJ1*01
1853



DLL4


17
DLL4
VH4-34_IGHD7-27*01_IGHJ4*01
1779
V1-4_IGLJ4*01
1864



DLL4
VH4-31_IGHD2-15*01_IGHJ2*01
1761
V1-4_IGLJ4*01
1864


18


19
DLL4
VH4-34_IGHD7-27*01_IGHJ4*01
1779
V4-6_IGLJ4*01
1886


20
P-cadherin/Fc chimera and
VH3-23_IGHD3-10*01>3_IGHJ6*01
3359
O12_IGKJ1*01
1854



Epo R/Fc chimera


21
P-cadherin/Fc chimera
VH3-23_IGHD3-10*01>1′_IGHJ3*01
3071
O12_IGKJ1*01
1854


22
DLL4
VH5-51_IGHD5-18*01>3_IGHJ4*01_G100K
3720
V3-4_IGLJ1*01
1881


23
DLL4
VH5-51_IGHD5-18*01>3_IGHJ4*01_G100R
3721
V3-4_IGLJ1*01
1881


24
DLL4
VH1-46_IGHD6-6*01>1_IGHJ1*01_S104F
3722
L6_IGKJ1*01
1850


25
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S104A
3723
L6_IGKJ1*01
1850


26
DLL4
VH5-51_IGHD5-18*01>3_IGHJ4*01_G104T
3724
V3-4_IGLJ1*01
1881


27
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S103P
3725
L6_IGKJ1*01
1850


28
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S102A
3726
L6_IGKJ1*01
1850


29
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F
3727
L6_IGKJ1*01
1850


30
DLL4
VH5-51_IGHD5-18*01>3_IGHJ4*01_G100K_G104T
3728
V3-4_IGLJ1*01
1881


31
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F_H111F
3729
L6_IGKJ1*01
1850


32
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F_H111Y
3730
L6_IGKJ1*01
1850


33
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104Y_H111Y
3731
L6_IGKJ1*01
1850


34
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F
3727
L6_IGKJ1*01_S28P
3732


35
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F
3727
L6_IGKJ1*01_S30N
3733


36
DLL4
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F
3727
L6_IGKJ1*01_S31K
3734









Example 16
Surface Plasmon Resonance
In this example, the binding affinities of selected Fabs (see Tables 78-79) to recombinant human DLL4 (R&D Systems) were analyzed using Surface Plasmon Resonance (SPR) (Biosensor Tools, Salt Lake City, Utah). The Fabs (see Table 78) include germline antibodies identified in the initial ECL screen as binding to DLL4 (as shown in Example 13) and modified Fabs that contain one or more mutations in the heavy or light chain as compared to the initially identified anti-DLL4 Fabs.
The results are shown in Table 79 below. Table 79 lists Fab (by Fab No.), the ka (M−1s−1), the kd (s−1), and the KD (nM) and the standard deviation (in parentheses). The results indicate that the Fabs have binding affinity for DLL4 ranging from 48.5 nM to 38 uM. Germline Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 has an average KD of 4.8 uM while variant Fab VH5-51_IGHD5-18*01>3_IGHJ4*01_G100K_G104T & V3-4_IGLJ1*01 has an improved KD of 355 nM. Germline Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 binds DLL4 with an average KD of 730 nM while the variant Fabs (rows 5-6 and 8-10 of Tables 78 and 79 below) bind DLL4 with improved KDS ranging from 70.6 nM to 388 nM. Germline Fab VH6-1_IGHD3-3*01_IGHJ4*01 & V4-3_IGLJ4*01 has an average binding affinity of 38 uM while germline Fab VH1-46_IGHD3-10*01_IGHJ4*01 & L12_IGKJ1*01 has an average KD of 500 nM.







TABLE 78







DLL4 Fabs for Surface Plasmon Resonance









Fab No
Heavy Chain
Light Chain












1
VH5-51_IGHD5-18*01>3_IGHJ4*01
V3-4_IGLJ1*01


2
VH1-46_IGHD6-6*01_IGHJ1*01
L6_IGKJ1*01


3
VH6-1_IGHD3-3*01_IGHJ4*01
V4-3_IGLJ4*01


4
VH1-46_IGHD3-10*01_IGHJ4*01
L12_IGKJ1*01


5
VH1-46_IGHD6-6*01>1_IGHJ1*01_S104F
L6_IGKJ1*01


6
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F
L6_IGKJ1*01


7
VH5-51_IGHD5-18*01>3_IGHJ4*01_G100K_G104T
V3-4_IGLJ1*01


8
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F_H111F
L6_IGKJ1*01


9
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F_H111Y
L6_IGKJ1*01


10
VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F
L6_IGKJ1*01_S31K
















TABLE 79







Binding affinity of DLL4 Fabs












Fab No
ka (M−1s−1)
kd (s−1)
KD (nM)
















1
n/a
n/a
 4800(200)



2
1.63(3)e5
0.101(2)
 730(130)



3
n/a
n/a
 38000(4000)



4
  5(1)e5
0.29(2)
 500(100)



5
 5.0(8)e5
0.19(1)
 380(60)



6
4.05(5)e5
0.0492(4)
122(1)



7
0.645(0.92)e5   
0.023
355(7)



8
4.25(4)e5
0.0300(2)
70.6(7) 



9
3.40(3)e5
0.0317(2)
93.1(9) 



10
3.50(5)e5
0.0392(4)
112(2)










Example 17
Inhibition of DLL4-Notch Interaction
In this example, four Fabs previously identified as binding to DLL4 were functionally screened for their ability to block the binding of Notch-Fc to DLL4.
In this ELISA assay, recombinant human DLL4 bound to the plate followed by the addition of both the Fab and Notch-Fc. An anti-human FC-HRP conjugated antibody was used as a detection molecule therefore if Notch-Fc binds to DLL4, a strong signal will be observed at A450. Alternatively , if the Fab is capable of blocking the bind of Notch-Fc to DLL4, no signal should be observed. The Fabs that were assayed included Fab VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F & L6_IGKJ1*01, Fab VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F_H111F & L6_IGKJ1*01, Fab VH5-51_IGHD5-18*01>3_IGHJ4*01_G100K_G104T & V3-4_IGLJ1*01 and Fab VH1-46_IGHD3-10*01_IGHJ4*01 & L12_IGKJ1*01.
In short, Maxisorp Nunc 96-well plates were coated with 0.5 μg/ml recombinant human DLL4 extracellular domain (R&D Systems) for at least 2 hours. The wells were washed and then blocked with 4% BSA. Following blocking, Fabs VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F & L6_IGKJ1*01, VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F_H111F & L6_IGKJ1*01, VH5-51_IGHD5-18*01>3_IGHJ4*01_G100K_G104T & V3-4_IGLJ1*01 and VH1-46_IGHD3-10*01_IGHJ4*01 & L12_IGKJ1*01 at concentrations from 0.004 and 5 μM were added together with recombinant human Fc-Notch extracellular domain (R&D Systems) at a concentration of 10 nM. After incubation for one to two hours, the wells were washed and Notch binding was measure using a mouse anti-human FC-HRP conjugated antibody (Southern
Biotech) at an 1:1000 dilution. HRP activity was detected using TMB substrate (Pierce) followed by acid neutralization. The A450 was measured on a SpectraMax Plus 384.
Results show that the addition of Fabs VH1-46_IGHD6-6*01_IGHJ1*01 _S102A_S103P_S104F & L6_IGKJ1*01, VH1-46_IGHD6-6*01_IGHJ1*01 _S102A_S103P_S104F_H111F & L6_IGKJ1*01 or VH5-51_IGHD5-18*01>3_IGHJ4*01_G100K_G104T & V3-4_IGL1*01 resulted in a decreased signal therefore indicating their ability to block the binding of Notch-Fc to DLL4. The addition of Fab VH1-46_IGHD3-10*01_IGHJ4*01 & L12_IGKJ1*01did not result an any loss of activity, indicating that Fab VH1-46_IGHD3-10*01_IGHJ4*01 & L12_IGKJ1*01does not block the Notch-DLL4 interaction. This result also indicates that Fabs VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F & L6_IGKJ1*01, VH1-46_IGHD6-6*01_IGHJ1*01_S102A_S103P_S104F_H111F & L6_IGKJ1*01 or VHS-51_IGHD5-18*01>3_IGHJ4*01_G100K_G104T & V3-4_IGLJ1*01 bind different epitopes of DLL4 than Fab VH1-46_IGHD3-10*01IGHJ4*01 & L12_IGKJ1*01.
Example 18
EpoR Cell Based Assay for Receptor Stimulation
In this example, Fabs were analyzed for their ability to stimulate the erythropoietin receptor using a cell based assay. The cell lines used included Ba/F3 cells transfected with the human erythropoietin receptor (EpoR) and parental Ba/F3 cells cells lacking EpoR. The parental Ba/F3 cells do not respond to receptor agonist and both cell lines required IL-3 for growth.
In short, Ba/F3 cells (with and without EpoR) were propagated in RPMI 1640 media with 10% FBS, antibiotics, and 5 ng/L recombinant mouse IL-3, washed into equivalent media lacking IL-3 and plated into 96 well plates at 5000 cells/well in 50 μl. Following plating, cells were treated with 10 μl of Fabs, agonist control EMP16 (TYSCHFGPLTWVCKPQ, SEQ ID NO:3735), or vehicle, and grown for 4 days at 37° C. in humid 5% CO2 atmosphere. To measure cell viability and proliferation, a resazurin-based viability assay reagent was added to test wells for 24 hours. Reduction of the reagent by metabolically active cells produced the readily-quantifiable fluorescent molecule resorufin. Average fluorescence for each treatment was divided by the average fluorescence of vehicle controls to give fold proliferation.
The results show that 40 nM Fab VH1-46_IGHD2-15*01_IGHJ2*01 & L2_IGKJ1*01 showed receptor dependent proliferation, while 54 nM Fab VH1-46_IGHD6-13*01_IGHJ4*01 & O1_IGKJ1*01 showed little proliferation over vehicle in both receptor-expressing cells and parental cells. The known receptor agonist peptide EMP16 (TYSCHFGPLTWVCKPQ, SEQ ID NO:3735), added at a concentration of 2.5 μM, showed strong cell proliferation.
Example 19
Inhibition of DLL4/Jag1
In this example, a cellular assay is described in which activation of the Notch pathway prevents C2C12 myoblast cell differentiation (see e.g., Jarriault et al., 1998 Molecular and Cellular Biology, 18:7423-7431). In order to activate the Notch pathway, Notch ligands, such as DLL4 or Jag1, must be expressed as full-length proteins on the cell surface. To achieve this Notch activation, non-adherent cells naturally or ectopically expressing Notch ligands DLL4 or Jag1 are co-cultured with C2C12 cells and selected Fabs. Functional inhibition of DLL4 or Jag1 is assessed by the ability of the Fab to promote differentiation, indicating Notch pathway inactivation. The differentiation into tube-like structures is easily discernible morphologically and additionally can be detected with an antibody against troponin t (Sigma-Aldrich).
In short, C2C12 mouse myoblast cells are cultured in the presence and absence of Jag1-expressing IM9 cells (a human lymphoblast cell line) and Fabs. The cells are plated onto glass coverslips in 12 well dishes in DMEM containing 10% FBS (fetal bovine serum). The next day attached C2C12 cells are transferred into DMEM containing 1% FBS to induce differentiation. Following incubation, the cells are visualized to observe whether differentiation into myotubes occurred. Low serum conditions will induce the differentiation of myotubes while Jag1-expressing IM9 cells maintain C2C12 cells in an undifferentiated state in low serum conditions.
Example 20
Inhibition of p-Cadherin
In this example, a cellular assay is described in which the ability of an antibody to inhibit P-cadherin is observed by the failure of cells to “clump”. P-cadherin is involved in cell-to-cell adhesion and therefore inhibition of P-cadherin leads to cell scattering.
In short, A431 epidermoid carcinoma cells are plated at 10,000 cells/well (96-well in DMEM with 10% FBS into 96-well dishes. The next day, Fab is added to wells at 100 mg/ml. Function blocking p-cadherin antibody (Abcam) is used as a positive control and DMEM with 10% FBS alone is used as a negative control (see e.g. Shimoyama, Y. et al., 1989 Cancer Research, 49:2128-2133). After 3.5 hours cells are examined for “scattering” and photographed. Cells that are incubated with media only exhibit significant “clumping” while cells that are incubated with an anti-P-cadherin Fab or antibody are scattered.
Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

## Claims

1. A method of generating a combinatorial human antibody library comprising a plurality of antibody or antigen-binding antibody fragment species, comprising:
a) for each antibody or antigen-binding antibody fragment species in the library, combining a human VH and a JH or a human VH, a DH, and a JH germline segment or portion thereof in frame to generate a nucleic acid molecule encoding a VH chain or a portion thereof, wherein, optionally, the VH chain is modified by replacement or insertion of one amino acid, optionally the replacement or insertion of at least one amino acid into at least one complementarity determining region (CDR) selected from among a CDRH1, CDRH2, CDRH3, wherein the CDR optionally is CDRH3;
b) for each antibody or antigen-binding antibody fragment species in the library, combining a human Vκ and a Jκ germline segment or portion thereof, or a human Vλ and a Jλ germline segment or portion thereof in frame to generate a nucleic acid molecule encoding a VL chain or a portion thereof, wherein, optionally, the VL chain is modified by replacement or insertion of one amino acid, optionally the replacement or insertion of at least one amino acid into at least one complementarity determining region (CDR) selected from among a CDRL1, CDRL2, and CDRL3,
wherein each nucleic acid molecule of part (a) encoding the VH chain or portion thereof comprises a different combination of VH and JH or VH, DH and JH germline segments yielding a different nucleic acid sequence encoding the VH chain or portion thereof and/or each nucleic acid molecule of part (b) encoding the VL chain or portion thereof comprises a different combination of Vκ and Jκ germline segments or Vλ and Jλ germline segments yielding a different nucleic acid sequence encoding the VL chain; and
c) expressing nucleic acid molecules of parts (a) and (b) and generating the antibody or antigen-binding antibody fragment species that contain a VL chain and a VH chain or a sufficient portion thereof to form an antigen binding site, thereby generating the combinatorial human antibody library,
wherein the library comprises at least about 50 or 100 or more different antibody or antigen-binding antibody fragment species in an addressable format, wherein the antibody or antigen-binding antibody fragment species at each address is the same antibody or antigen-binding antibody fragment species and is different from the antibody or antigen-binding antibody fragment species at all other addresses, and wherein the amino acid sequence identity of each antibody or antigen-antibody fragment species is known based on the address of the antibody or antigen-binding antibody fragment species; and optionally further comprising,
d) purifying the antibody or antigen-binding antibody fragment species, optionally to about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity.

2. A method according to claim 1, wherein the antibody species are full-length antibody species and the antigen-binding antibody fragment species are Fab fragments.