# Patent

## Title
Anti-ron antibodies

## Abstract

Monoclonal antibodies that bind and inhibit activation of human RON (Recepteur d'Origine Nantais) are disclosed. The antibodies can be used to treat certain forms of cancer that are associated with activation of RON.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 13/177,071, filed Jul. 6, 2011, which claims the benefit of and priority to U.S. Provisional Application No. 61/466,679, filed Mar. 23, 2011, and U.S. Provisional Application Ser. No. 61/361,808, filed Jul. 6, 2010; the contents of each application are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
The field of the invention is molecular biology, immunology and oncology. More particularly, the field is therapeutic antibodies.
BACKGROUND
Recepteur d'Origine Nantais (RON), also known as Macrophage Stimulating Protein Receptor (MSP R, or MST1-R), is a member of the MET family of receptor tyrosine kinases that binds the ligand known as Macrophage Stimulating Protein (MSP). RON is composed of a 40 kDa extracellular a chain and a 150 kDa transmembrane β chain. The β chain is responsible for the intrinsic kinase activity, and the extracellular portions of the two chains function together as the ligand binding domain (Wagh et al., 2008, ADV. CANCER RES. 100:1-33).
MSP binding to RON activates multiple downstream signaling pathways and mediates multiple cellular activities. RON pathway dysregulation is involved in inflammatory response, wound healing and liver regeneration. RON signaling can sustain tumor growth, survival, motility, invasion and angiogenesis in certain malignancies. The RON protein exists in several splice variants, some of which are tumorigenic in animal models of cancer. One such splice variant is delta 160 RON, which lacks exons 5 and 6 (Lu et al., 2007, CANCER LETT. 257:157-164).
When activated by ligand binding, RON activates the PI3K/AKT pathway and the MAPK pathway. RON also affects cells through interactions with other receptors, e.g., c-Met, integrins and EGFR. To date, no activating mutations in RON exons have been reported. Alternative splicing and overexpression appear to be the main mechanisms for constitutive activation of the receptor. Several small molecule inhibitors have been reported that inhibit multiple receptor tyrosine kinases, including RON, examples of which include EXCEL-2880, (Qian et al., 2009, CANCER RES. 69:8009-8016) and BMS-77607 (Schroeder et al., 2009 J. MED CHEM. 52:1251-1254). A dual c-met/RON inhibitor has also been reported, Amgen compound I (Zhang et al., 2008, CANCER RES. 68:6680-6687). A recent publication describes a selective RON small molecule inhibitor (Raeppel et al., 2010 BIOORG MED CHEM LETT 20:2745-9). Several antibodies that inhibit human RON activity have been reported (Huet et al., US 2009/0226442; Pereira et al., US 2009/0136510; Zhu et al., WO 2006/020258; Pereira et al., WO 2005/120557; and commercial antibody MAB691, R&D Systems, Minneapolis, Minn.).
Naturally occurring antibodies are multimeric proteins that contain four polypeptide chains (FIG. 1). Two of the polypeptide chains are called heavy chains (H chains), and two of the polypeptide chains are called light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (VL in FIG. 1) and one constant region (CL in FIG. 1). The heavy chain consists of one variable region (VH in FIG. 1) and at least three constant regions (CH1, CH2 and CH3 in FIG. 1). The variable regions determine the specificity of the antibody. Each variable region comprises three hypervariable regions also known as complementarity determining regions (CDRs) flanked by four relatively conserved framework regions (FRs). The three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies.
Although antibodies that bind RON are known in the art, there is still a need for improved RON antibodies that can be used as therapeutic agents.
SUMMARY
The invention is based, in part, upon the discovery of a family of antibodies that specifically bind human RON. The antibodies contain RON binding sites based on the CDRs of the antibodies. The antibodies can be used as therapeutic agents. When used as therapeutic agents, the antibodies are engineered, e.g., humanized, to reduce or eliminate an immune response when administered to a human patient.
The antibodies prevent or inhibit the activation of (i.e., neutralize) human RON. In some embodiments, the antibodies prevent RON from binding to its ligand, MSP, thereby neutralizing RON activity. In certain embodiments, the antibodies prevent RON activation without inhibiting RON binding to MSP. The antibodies can be used to inhibit the downstream signaling of the breast tumor cell line T47D. Furthermore, when administered to a mammal, the antibodies can inhibit or reduce tumor growth in the mammal.
These and other aspects and advantages of the invention will become apparent upon consideration of the following figures, detailed description, and claims. As used herein, “including” means without limitation, and examples cited are non-limiting.



DESCRIPTION OF THE DRAWINGS
The invention can be more completely understood with reference to the following drawings.
FIG. 1 (prior art) is a schematic representation of a typical naturally-occurring antibody.
FIG. 2 is a sequence alignment showing the amino acid sequence of the complete immunoglobulin heavy chain variable region of antibodies 07F01, 12B11, 17F06, 18H09 and 29B06. The amino acid sequences for each antibody are aligned against one another, and CDR1, CDR2, and CDR3, are identified in boxes. The unboxed sequences represent framework (FR). Alignment positioning (gaps) are based on Kabat numbering, rather than an alignment algorithm such as Clustal sequences.
FIG. 3 is a sequence alignment showing the CDR1, CDR2, and CDR3 sequences for each of the immunoglobulin heavy chain variable region sequences in FIG. 2.
FIG. 4 is a sequence alignment showing the amino acid sequence of the complete immunoglobulin light chain variable region of antibodies 07F01, 12B11, 17F06, 18H09 and 29B06. The amino acid sequences for each antibody are aligned against one another, and CDR1, CDR2, and CDR3, are identified in boxes. The unboxed sequences represent framework (FR) sequences. Alignment positioning (gaps) are based on Kabat numbering, rather than an alignment algorithm such as Clustal sequences.
FIG. 5 is a sequence alignment showing the CDR1, CDR2, and CDR3 sequences for each of the immunoglobulin light chain variable region sequences in FIG. 4.
FIG. 6 is a graph showing dose-response curves for inhibition of the MSP-RON binding interaction by antibodies 17F06 (▴), 07F01 (●), 12B11 (♦), 18H09 (▪), and 29B06 (x), as measured by electrochemiluminescence assay.
FIG. 7 is a graph showing dose-response curves for inhibition of MSP-dependent phosphorylation of ERK by antibodies 17F06 (▴), 07F01 (●), 12B11 (♦), 18H09 (▪), and 29B06 (x) by ELISA assay.
FIG. 8 is a histogram summarizing results from an experiment measuring inhibition of MSP induced HPAF-II cell migration by antibodies 07F01, 18H09, 29B06, 12B11, 17F06 and an IgG negative control (murine IgG) by transwell assay.
FIG. 9 is a graph summarizing data on inhibition of growth of a wild-type (wt) RON-dependent in vivo tumor model by antibodies 07F01 (●), 12B11 (♦), 18H09 (▪), 29B06 (*), and a murine IgG control (∘). The antibodies and IgG control were dosed at 20 mg/kg twice per week intraperitoneally.
FIG. 10 is a graph summarizing data on inhibition of growth of a delta 160 RON-dependent in vivo tumor model by antibodies 17F06 (▴), 07F01 (●), 12B11 (♦), 18H09 (▪), 29B06 (*), and a murine IgG control (∘). The antibodies and IgG control were dosed at 20 mg/kg twice per week intraperitoneally.
FIG. 11 is a graph summarizing data on inhibition of growth of an NCI-H358 xenograft tumor model by antibody 29B06 (*) and a murine IgG control (∘). The antibody and IgG control were dosed at 40 mg/kg (abbreviated as “mpk”) three per week intraperitoneally.
FIG. 12A is a schematic diagram showing the amino acid sequences of the complete immunoglobulin heavy chain variable region of 07F01 (SEQ ID NO: 2) and the complete heavy chain variable regions denoted as Chimeric 07F01 C102S (SEQ ID NO: 133), Sh07F01 Hv3-48 (SEQ ID NO: 135), and Sh07F01 Hv3-48 D28T T60A L63V E65G (SEQ ID NO: 137). The amino acid sequences for each heavy chain variable region are aligned against one another, and Complementary Determining Sequences (CDR) (Kabat definition), CDR1, CDR2, and CDR3, are identified in boxes. The unboxed sequences represent framework (FR) sequences.
FIG. 12B is a schematic diagram showing the amino acid sequences of the complete immunoglobulin heavy chain variable region of 29B06 (SEQ ID NO: 42) and the complete heavy chain variable regions denoted as Sh29B06_Hv4-59 (SEQ ID NO: 143), Hu29B06 Hv4-59 (SEQ ID NO: 145), and Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F (SEQ ID NO: 147). The amino acid sequences for each heavy chain variable region are aligned against one another, and CDR1, CDR2, and CDR3 sequences (Kabat definition) are identified in boxes. The unboxed sequences represent framework (FR) sequences.
FIG. 13A is a schematic diagram showing the CDR1, CDR2, and CDR3 sequences (Kabat definition) for each of the variable region sequences shown in FIG. 12A.
FIG. 13B is a schematic diagram showing the CDR1, CDR2, and CDR3 sequences (Kabat definition) for each of the variable region sequences shown in FIG. 12B.
FIG. 14A is a schematic diagram showing the amino acid sequences of the complete light chain variable region of 07F01 (SEQ ID NO: 4) and the complete light chain variable regions denoted as HE L 07F01 Kv1-9 (SEQ ID NO: 139) and Sh07F01 Kv1-9 F1 (SEQ ID NO: 141). The amino acid sequences for each light chain variable region are aligned against one another, and CDR1, CDR2, and CDR3 sequences (Kabat definition) are identified in boxes. The unboxed sequences represent framework (FR) sequences.
FIG. 14B is a schematic diagram showing the amino acid sequences of the complete light chain variable region of 29B06 (SEQ ID NO: 44) and the complete light chain variable region denoted as Sh29B06 Kv2-28 (SEQ ID NO: 149). The amino acid sequences for each light chain variable region are aligned against one another, and CDR1, CDR2, and CDR3 sequences (Kabat definition) are identified in boxes. The unboxed sequences represent framework (FR) sequences.
FIG. 15A is a sequence alignment showing the CDR1, CDR2, and CDR3 sequences (Kabat definition) for each of the variable region sequences shown in FIG. 14A.
FIG. 15B is a sequence alignment showing the CDR1, CDR2, and CDR3 sequences (Kabat definition) for each of the variable region sequences shown in FIG. 14B.
FIG. 16 is a histogram summarizing results from an experiment measuring inhibition of MSP induced HPAF-II cell migration by anti-RON antibodies Sh29B06-78 and Sh07F01-62, an IgG negative control (human IgG), and a no MSP control by transwell assay.
FIG. 17 is a histogram summarizing results from an experiment measuring inhibition of MSP induced HPAF-II cell invasion by anti-RON antibodies Sh29B06-78 and Sh07F01-62 and an IgG negative control (human IgG) at 0 and 1 nM MSP.
FIG. 18 is a graph summarizing data on inhibition of growth of an NCI-H358 xenograft tumor model by anti-RON antibodies mu07F01 (∘), Sh07F01-62 (▴), mu29B06 (♦), RON8 (▪), and Sh29B06-78 (●), and a human IgG control (+).
FIG. 19 depicts Western blots summarizing results from an experiment measuring RON receptor degradation by anti-RON antibodies mu07F01, Sh07F01-62, mu29B06, RON8, and Sh29B06-78.



DETAILED DESCRIPTION
The anti-RON antibodies disclosed herein are based on the antigen binding sites of certain monoclonal antibodies that have been selected on the basis of binding and neutralizing the activity of human RON. The antibodies contain immunoglobulin variable region CDR sequences that define a binding site for human RON.
In view of the neutralizing activity of these antibodies, they are useful for modulating the growth and/or proliferation of certain types of cancer cells. When used as a therapeutic agent, the antibodies can be engineered to minimize or eliminate an immune response when administered to a human patient. In some embodiments, the antibodies are fused or conjugated to other moieties, such as effector molecules (e.g., other proteins or small molecule therapeutics), a detectable label or a toxin moiety. Various features and aspects of the invention are discussed in more detail below.
As used herein, unless otherwise indicated, the term “antibody” means an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody or antigen-binding fragment that has been modified, engineered or chemically conjugated, or that is a human antibody. Examples of antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab′, F(ab′)2, Fv, single chain antibodies (e.g., scFv), minibodies and diabodies. An antibody conjugated to a toxin moiety is an example of a chemically conjugated antibody.
I. Antibodies that Bind RON
The antibodies disclosed herein comprise: (a) an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3 and (b) an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human RON protein.
In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3 and (b) an immunoglobulin light chain variable region, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human RON. A CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (07F01), SEQ ID NO: 51 (07F01), SEQ ID NO: 124 (Sh07F01 Hv3-48 D28T T60A L63V E65G), SEQ ID NO: 15 (12B11), SEQ ID NO: 53 (12B11), SEQ ID NO: 25 (17F06), SEQ ID NO: 55 (17F06), SEQ ID NO: 35 (18H09), SEQ ID NO: 57 (18H09), SEQ ID NO: 45 (29B06), SEQ ID NO: 59 (29B06), and SEQ ID NO: 126 (Sh29B06 Hv4-59, Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F); a CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (07F01), SEQ ID NO: 16 (12B11), SEQ ID NO: 26 (17F06), SEQ ID NO: 36 (18H09), SEQ ID NO: 46 (29B06), and SEQ ID NO: 122 (Sh07F01 Hv3-48 D28T T60A L63V E65G); and a CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7 (07F01), SEQ ID NO: 17 (12B11), SEQ ID NO: 27 (17F06), SEQ ID NO: 37 (18H09), SEQ ID NO: 47 (29B06), and SEQ ID NO: 123 (Chimeric 07F01 C102S, Sh07F01 Hv3-48, Sh07F01 Hv3-48 D28T T60A L63V E65G). Throughout the specification a particular SEQ ID NO. is followed in parentheses by the antibody that was the origin of that sequence. For example, “SEQ ID NO: 5 (07F01)” means that SEQ ID NO: 5 comes from antibody 07F01.
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 5 (07F01), SEQ ID NO: 51 (07F01), or SEQ ID NO: 124 (Sh07F01 Hv3-48 D28T T60A L63V E65G); a CDRH2 comprising the amino acid sequence of SEQ ID NO: 6 (07F01) or SEQ ID NO: 122 (Sh07F01 Hv3-48 D28T T60A L63V E65G), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 7 (07F01) or SEQ ID NO: 123 (Chimeric 07F01 C102S, Sh07F01 Hv3-48, Sh07F01 Hv3-48 D28T T60A L63V E65G).
In some embodiments, the heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 5 (07F01), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 122 (Sh07F01 Hv3-48 D28T T60A L63V E65G), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 123 (Chimeric 07F01 C102S, Sh07F01 Hv3-48, Sh07F01 Hv3-48 D28T T60A L63V E65G).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 15 (12B11) or SEQ ID NO: 53 (12B11), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 16 (12B11), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 17 (12B11).
In some embodiments, the heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 25 (17F06) or SEQ ID NO: 55 (17F06), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26 (17F06), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27 (17F06).
In some embodiments, the heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 35 (18H09) or SEQ ID NO: 57 (18H09), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 36 (18H09), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 37 (18H09).
In some embodiments, the heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 45 (29B06), SEQ ID NO: 59 (29B06), or SEQ ID NO: 126 (Sh29B06 Hv4-59, Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 46 (29B06), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 47 (29B06).
In some embodiments, the heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 45 (29B06) or SEQ ID NO: 126 (Sh29B06 Hv4-59, Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 46 (29B06), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 47 (29B06).
Preferably, the CDRH1, CDRH2, and CDRH3 sequences are interposed between human or humanized immunoglobulin FRs. The antibody can be an intact antibody or an antigen-binding antibody fragment.
In some embodiments, the antibody comprises (a) an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, and (b) an immunoglobulin heavy chain variable region, wherein the IgG light chain variable region and the IgG heavy chain variable region together define a single binding site for binding human RON. A CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (07F01), SEQ ID NO: 18 (12B11), SEQ ID NO: 28 (17F06), SEQ ID NO: 38 (18H09), SEQ ID NO: 48 (29B06), and SEQ ID NO: 130 (HE L 07F01 Kv1-9, Sh07F01 Kv1-9 F1); a CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (07F01), SEQ ID NO: 19 (12B11), SEQ ID NO: 29 (17F06), SEQ ID NO: 39 (18H09), SEQ ID NO: 49 (29B06), and SEQ ID NO: 131 (HE L 07F01 Kv1-9, Sh07F01 Kv1-9 F1); and a CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 (07F01), SEQ ID NO: 20 (12B11), SEQ ID NO:30 (17F06), SEQ ID NO: 40 (18H09), and SEQ ID NO: 50 (29B06).
In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 8 (07F01) or SEQ ID NO: 130 (HE L 07F01 Kv1-9, Sh07F01 Kv1-9 F1), a CDRL2 comprising the amino acid sequence of SEQ ID NO: 9 (07F01) or SEQ ID NO: 131 (HE L 07F01 Kv1-9, Sh07F01 Kv1-9 F1), and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 10 (07F01).
In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 130 (HE L 07F01 Kv1-9, Sh07F01 Kv1-9 F1); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 131 (HE L 07F01 Kv1-9, Sh07F01 Kv1-9 F1); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 10 (07F01).
In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 18 (12B11); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 19 (12B11); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 20 (12B11).
In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 28 (17F06); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 29 (17F06); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 30 (17F06).
In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 38 (18H09); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 39 (18H09); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 40 (18H09).
In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 48 (29B06); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 49 (29B06); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 50 (29B06).
Preferably, the CDRL1, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FRs. The antibody can be an intact antibody or an antigen-binding antibody fragment.
In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3 and (b) an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human RON. The CDRH1 is an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (07F01), SEQ ID NO: 51 (07F01), SEQ ID NO: 124 (Sh07F01 Hv3-48 D28T T60A L63V E65G), SEQ ID NO: 15 (12B11), SEQ ID NO: 53 (12B11), SEQ ID NO: 25 (17F06), SEQ ID NO: 55 (17F06), SEQ ID NO: 35 (18H09), SEQ ID NO: 57 (18H09), SEQ ID NO: 45 (29B06), SEQ ID NO: 59 (29B06), and SEQ ID NO: 126 (Sh29B06 Hv4-59, Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F); the CDRH2 is an amino acid sequence selected from the group consisting SEQ ID NO: 6 (07F01), SEQ ID NO: 16 (12B11), SEQ ID NO: 26 (17F06), SEQ ID NO: 36 (18H09), SEQ ID NO: 46 (29B06), and SEQ ID NO: 122 (Sh07F01 Hv3-48 D28T T60A L63V E65G); and the CDRH3 is an amino acid sequence selected from the group consisting of SEQ ID NO:7 (07F01), SEQ ID NO: 17 (12B11), SEQ ID NO: 27 (17F06), SEQ ID NO: 37 (18H09), SEQ ID NO: 47 (29B06), and SEQ ID NO: 123 (Chimeric 07F01 C102S, Sh07F01 Hv3-48, Sh07F01 Hv3-48 D28T T60A L63V E65G). The CDRL1 is an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (07F01), SEQ ID NO: 18 (12B11), SEQ ID NO: 28 (17F06), SEQ ID NO: 38 (18H09), SEQ ID NO: 48 (29B06), and SEQ ID NO: 130 (HE L 07F01 Kv1-9, Sh07F01 Kv1-9 F1); the CDRL2 is an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (07F01), SEQ ID NO: 19 (12B11), SEQ ID NO: 29 (17F06), SEQ ID NO: 39 (18H09), SEQ ID NO: 49 (29B06), and SEQ ID NO: 131 (HE L 07F01 Kv1-9, Sh07F01 Kv1-9 F1); and the CDRL3 is an amino acid sequence selected from the group consisting of SEQ ID NO: 10 (07F01), SEQ ID NO: 20 (12B11), SEQ ID NO: 30 (17F06), SEQ ID NO: 40 (18H09), and SEQ ID NO: 50 (29B06).
The antibodies disclosed herein comprise an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 2 (07F01), SEQ ID NO: 12 (12B11), SEQ ID NO: 22 (17F06), SEQ ID NO: 32 (18H09), SEQ ID NO: 42 (29B06), SEQ ID NO: 133 (Chimeric 07F01 C102S), SEQ ID NO: 135 (Sh07F01 Hv3-48), SEQ ID NO: 137 (Sh07F01 Hv3-48 D28T T60A L63V E65G), SEQ ID NO: 143 (Sh29B06 Hv4-59), SEQ ID NO: 145 (Hu29B06 Hv4-59), and SEQ ID NO: 147 (Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F), and an immunoglobulin light chain variable region.
In other embodiments, the antibody comprises an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO: 4 (07F01), SEQ ID NO: 14 (12B11), SEQ ID NO: 24 (17F06), SEQ ID NO: 34 (18H09), SEQ ID NO: 44 (29B06), SEQ ID NO: 139 (HE L 07F01 Kv1-9), SEQ ID NO: 141 (Sh07F01 Kv1-9 F1), and SEQ ID NO: 149 (Sh29B06 Kv2-28), and an immunoglobulin heavy chain variable region.
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 2 (07F01), SEQ ID NO: 12 (12B11), SEQ ID NO: 22 (17F06), SEQ ID NO: 32 (18H09), SEQ ID NO: 42 (29B06), SEQ ID NO: 133 (Chimeric 07F01 C102S), SEQ ID NO: 135 (Sh07F01 Hv3-48), SEQ ID NO: 137 (Sh07F01 Hv3-48 D28T T60A L63V E65G), SEQ ID NO: 143 (Sh29B06 Hv4-59), SEQ ID NO: 145 (Hu29B06 Hv4-59), and SEQ ID NO: 147 (Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F), and an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO: 4 (07F01), SEQ ID NO: 14 (12B11), SEQ ID NO: 24 (17F06), SEQ ID NO: 34 (18H09), SEQ ID NO: 44 (29B06), SEQ ID NO: 139 (HE L 07F01 Kv1-9), SEQ ID NO: 141 (Sh07F01 Kv1-9 F1), and SEQ ID NO: 149 (Sh29B06 Kv2-28).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (07F01), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (07F01).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (12B11), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (12B11).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 (17F06), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 24 (17F06).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32 (18H09), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 34 (18H09).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 (29B06), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 44 (29B06).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 137 (Sh07F01 Hv3-48 D28T T60A L63V E65G), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 139 (HE L 07F01 Kv1-9).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 147 (Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 149 (Sh29B06 Kv2-28).
In certain embodiments, the antibodies disclosed herein comprise an immunoglobulin heavy chain and an immunoglobulin light chain. In some embodiments, the antibody comprises an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO: 93 (07F01), SEQ ID NO: 97 (12B11), SEQ ID NO: 101 (17F06), SEQ ID NO: 105 (18H09), SEQ ID NO: 109 (29B06), SEQ ID NO: 156 (Chimeric 07F01 C102S IgG1), SEQ ID NO: 160 (Chimeric 29B06 IgG1), SEQ ID NO: 164 (Sh07F01 Hv3-48 IgG1), SEQ ID NO: 166 (Sh07F01 Hv3-48 D28T T60A L63V E65G IgG1), SEQ ID NO: 172 (Sh29B06 Hv4-59 IgG1), SEQ ID NO: 174 (Hu29B06 Hv4-59 IgG1), and SEQ ID NO: 176 (Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F IgG1), and an immunoglobulin light chain.
In other embodiments, the antibody comprises an immunoglobulin light chain selected from the group consisting of SEQ ID NO: 95 (07F01), SEQ ID NO: 99 (12B11), SEQ ID NO: 103 (17F06), SEQ ID NO: 107 (18H09), SEQ ID NO: 111 (29B06), SEQ ID NO: 158 (Chimeric 07F01 Kappa), SEQ ID NO: 162 (Chimeric 29B06 Kappa), SEQ ID NO: 168 (HE L 07F01 Kv1-9 Kappa), SEQ ID NO: 170 (Sh07F01 Kv1-9 F1 Kappa), and SEQ ID NO: 178 (Sh29B06 Kv2-28 Kappa), and an immunoglobulin heavy chain.
In some embodiments, the antibody comprises (i) an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO: 93 (07F01), SEQ ID NO: 97 (12B11), SEQ ID NO: 101 (17F06), SEQ ID NO: 105 (18H09), SEQ ID NO: 109 (29B06), SEQ ID NO: 156 (Chimeric 07F01 C102S IgG1), SEQ ID NO: 160 (Chimeric 29B06 IgG1), SEQ ID NO: 164 (Sh07F01 Hv3-48 IgG1), SEQ ID NO: 166 (Sh07F01 Hv3-48 D28T T60A L63V E65G IgG1), SEQ ID NO: 172 (Sh29B06 Hv4-59 IgG1), SEQ ID NO: 174 (Hu29B06 Hv4-59 IgG1), and SEQ ID NO: 176 (Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F IgG1), and (ii) an immunoglobulin light chain selected from the group consisting of SEQ ID NO: 95 (07F01), SEQ ID NO: 99 (12B11), SEQ ID NO: 103 (17F06), SEQ ID NO: 107 (18H09), SEQ ID NO: 111 (29B06), SEQ ID NO: 158 (Chimeric 07F01 Kappa), SEQ ID NO: 162 (Chimeric 29B06 Kappa), SEQ ID NO: 168 (HE L 07F01 Kv1-9 Kappa), SEQ ID NO: 170 (Sh07F01 Kv1-9 F1 Kappa), and SEQ ID NO: 178 (Sh29B06 Kv2-28 Kappa).
In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 93 (07F01), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 95 (07F01).
In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 97 (12B11), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 99 (12B11).
In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 101 (17F06), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 103 (17F06).
In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 105 (18H09), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 107 (18H09).
In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 109 (29B06), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 111 (29B06).
In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 166 (Sh07F01 Hv3-48 D28T T60A L63V E65G IgG1), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 168 (HE L 07F01 Kv1-9 Kappa).
In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 176 (Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F IgG1), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 178 (Sh29B06 Kv2-28 Kappa).
In certain embodiments, an isolated antibody that binds human RON comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire variable region or the framework region sequence of SEQ ID NO: 2 (07F01), SEQ ID NO: 12 (12B11), SEQ ID NO: 22 (17F06), SEQ ID NO: 32 (18H09), SEQ ID NO: 42 (29B06), SEQ ID NO: 133 (Chimeric 07F01 C102S), SEQ ID NO: 135 (Sh07F01 Hv3-48), SEQ ID NO: 137 (Sh07F01 Hv3-48 D28T T60A L63V E65G), SEQ ID NO: 143 (Sh29B06 Hv4-59), SEQ ID NO: 145 (Hu29B06 Hv4-59), or SEQ ID NO: 147 (Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F).
In certain embodiments, an isolated antibody that binds human RON comprises an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire variable region or the framework region sequence of SEQ ID NO: 4 (07F01), SEQ ID NO: 14 (12B11), SEQ ID NO: 24 (17F06), SEQ ID NO: 34 (18H09), SEQ ID NO: 44 (29B06), SEQ ID NO: 139 (HE L 07F01 Kv1-9), SEQ ID NO: 141 (Sh07F01 Kv1-9 F1), or SEQ ID NO: 149 (Sh29B06 Kv2-28).
Homology or identity may be determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87, 2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25, 3389-3402, incorporated by reference) are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases see Altschul et al., (1994) NATURE GENETICS 6, 119-129 which is fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89, 10915-10919, fully incorporated by reference). Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings may be Q=9; R=2; wink=1; and gapw=32. Searches may also be conducted using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameter (e.g.: -G, Cost to open gap [Integer]: default=5 for nucleotides/11 for proteins; -E, Cost to extend gap [Integer]: default=2 for nucleotides/1 for proteins; -q, Penalty for nucleotide mismatch [Integer]: default=−3; -r, reward for nucleotide match [Integer]: default=1; -e, expect value [Real]: default=10; -W, wordsize [Integer]: default=11 for nucleotides/28 for megablast/3 for proteins; -y, Dropoff (X) for blast extensions in bits: default=20 for blastn/7 for others; -X, X dropoff value for gapped alignment (in bits): default=15 for all programs, not applicable to blastn; and -Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty=10 and Gap Extension Penalty=0.1). A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.
In each of the foregoing embodiments, it is contemplated herein that immunoglobulin heavy chain variable region sequences and/or light chain variable region sequences that together bind human RON may contain amino acid alterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the heavy and/or light chain variable regions.
In certain embodiments, the antibody binds human RON with a KD of 1 nM, 900 pM, 750 pM, 650 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM or lower. Unless otherwise specified, KD values are determined by surface plasmon resonance methods under the conditions described in Examples 5 and 14.
Antibody Sh29B06-78 binds human RON with a KD of 500 pM, 250 pM, 200 pM, 150 pM, 100 pM or lower as measured by surface plasmon resonance methods under the conditions described in Examples 5 and 14. In an exemplary embodiment, antibody Sh29B06-78 binds human RON with a KD of 150 pM or lower as measured by surface plasmon resonance methods at 37° C. under the conditions described in Examples 5 and 14.
Antibody SH07F01-62 binds human RON with a KD of 500 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM or lower as measured by surface plasmon resonance methods under the conditions described in Examples 5 and 14. In an exemplary embodiment, antibody SH07F01-62 binds human RON with a KD of 250 pM to 350 pM or lower as measured by surface plasmon resonance methods at 37° C. under the conditions described in Examples 5 and 14.
In certain embodiments, the antibodies inhibit human MSP binding to human RON. For example, the antibodies can have an IC50 (concentration at 50% of maximum inhibition) of about 5 nM, 2 nM, 1 nM or lower, when assayed using the protocol described in Examples 8 and 15.
Although the embodiments illustrated in the Examples comprise pairs of variable regions, pairs of full length antibody chains, or pairs of CDR1, CDR2 and CDR3 regions, one from a heavy chain and one from a light chain, a skilled artisan will recognize that alternative embodiments may comprise single heavy chain variable regions or single light chain variable regions, single full length antibody chains, or CDR1, CDR2 and CDR3 regions from one antibody chain, either heavy or light. The single variable region, full length antibody chain or CDR1, CDR2 and CDR3 region of one chain can be used to screen for corresponding domains in another chain, the two chains capable of forming an antibody that binds antigen. The screening may be accomplished by phage display screening methods using, e.g., a hierarchical dual combinatorial approach disclosed in PCT Publ. No. WO92/01047. In this approach, an individual colony containing either a heavy or light chain clone is used to infect a complete library of clones encoding the other chain (light or heavy), and the resulting two-chain specific antigen-binding domain is selected in accordance with phage display techniques as described.
II. Production of Antibodies
Methods for producing antibodies, such as those disclosed herein, are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art. Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein, or prior art sequence information regarding genes encoding the heavy and light chains of murine antibodies in hybridoma cells.
Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions.
Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile or inclusion bodies, and can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art.
If the engineered gene is to be expressed in eukayotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, IgG enhancers, and various introns. This expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be introduced into eukaryotic host cells using conventional techniques. The host cells express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a moiety having another function (e.g., cytotoxicity). In some embodiments, a host cell is transfected with a single vector expressing a polypeptide expressing an entire, or part of, a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In other embodiments, a host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In still other embodiments, a host cell is co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide comprising an entire, or part of, a heavy chain or heavy chain variable region, and another expression vector expressing a polypeptide comprising an entire, or part of, a light chain or light chain variable region).
A polypeptide comprising an immunoglobulin heavy chain variable region or light chain variable region can be produced by growing a host cell transfected with an expression vector encoding such variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified using techniques well known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) and histidine tags.
A monoclonal antibody that binds human RON, or an antigen-binding fragment of the antibody, can be produced by growing a host cell transfected with: (a) an expression vector that encodes a complete or partial immunoglobulin heavy chain, and a separate expression vector that encodes a complete or partial immunoglobulin light chain; or (b) a single expression vector that encodes both chains (e.g., complete or partial heavy and light chains), under conditions that permit expression of both chains. The intact antibody (or antigen-binding fragment) can be harvested and purified using techniques well known in the art, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags. It is within ordinary skill in the art to express the heavy chain and the light chain from a single expression vector or from two separate expression vectors.
III. Antibody Modifications
Methods for reducing or eliminating the antigenicity of antibodies and antibody fragments are known in the art. When the antibodies are to be administered to a human, the antibodies preferably are “humanized” to reduce or eliminate antigenicity in humans. Preferably, the humanized antibodies have the same or substantially the same affinity for the antigen as the non-humanized mouse antibody from which it was derived.
In one humanization approach, chimeric proteins are created in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, e.g., Morrison et al., 1984, PROC. NAT. ACAD. SCI. 81:6851-6855, Neuberger et al., 1984, NATURE 312:604-608; U.S. Pat. No. 6,893,625 (Robinson); U.S. Pat. No. 5,500,362 (Robinson); and U.S. Pat. No. 4,816,567 (Cabilly).
In an approach known as CDR grafting, the CDRs of the light and heavy chain variable regions are grafted into frameworks from another species. For example, murine CDRs can be grafted into human FRs. In some embodiments, the CDRs of the light and heavy chain variable regions of an anti-RON antibody are grafted into human FRs or consensus human FRs. To create consensus human FRs, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR grafting is described in U.S. Pat. No. 7,022,500 (Queen); U.S. Pat. No. 6,982,321 (Winter); U.S. Pat. No. 6,180,370 (Queen); U.S. Pat. No. 6,054,297 (Carter); U.S. Pat. No. 5,693,762 (Queen); U.S. Pat. No. 5,859,205 (Adair); U.S. Pat. No. 5,693,761 (Queen); U.S. Pat. No. 5,565,332 (Hoogenboom); U.S. Pat. No. 5,585,089 (Queen); U.S. Pat. No. 5,530,101 (Queen); Jones et al. (1986) NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332: 323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter (1998) FEBS LETT 430: 92-94.
In an approach called “SUPERHUMANIZATIONTM,” human CDR sequences are chosen from human germline genes, based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. See, e.g., U.S. Pat. No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL. 169:1119-1125.
Other methods to reduce immunogenicity include “reshaping,” “hyperchimerization,” and “veneering/resurfacing.” See, e.g., Vaswami et al., 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81:105; Roguska et al., 1996, PROT. ENGINEER 9:895-904; and U.S. Pat. No. 6,072,035 (Hardman). In the veneering/resurfacing approach, the surface accessible amino acid residues in the murine antibody are replaced by amino acid residues more frequently found at the same positions in a human antibody. This type of antibody resurfacing is described, e.g., in U.S. Pat. No. 5,639,641 (Pedersen).
Another approach for converting a mouse antibody into a form suitable for medical use in humans is known as ACTIVMABTM technology (Vaccinex, Inc., Rochester, N.Y.), which involves a vaccinia virus-based vector to express antibodies in mammalian cells. High levels of combinatorial diversity of IgG heavy and light chains are said to be produced. See, e.g., U.S. Pat. No. 6,706,477 (Zauderer); U.S. Pat. No. 6,800,442 (Zauderer); and U.S. Pat. No. 6,872,518 (Zauderer).
Another approach for converting a mouse antibody into a form suitable for use in humans is technology practiced commercially by KaloBios Pharmaceuticals, Inc. (Palo Alto, Calif.). This technology involves the use of a proprietary human “acceptor” library to produce an “epitope focused” library for antibody selection.
Another approach for modifying a mouse antibody into a form suitable for medical use in humans is HUMAN ENGINEERINGTM technology, which is practiced commercially by XOMA (US) LLC. See, e.g., PCT Publication No. WO 93/11794 and U.S. Pat. No. 5,766,886 (Studnicka); U.S. Pat. No. 5,770,196 (Studnicka); U.S. Pat. No. 5,821,123 (Studnicka); and U.S. Pat. No. 5,869,619 (Studnicka).
Any suitable approach, including any of the above approaches, can be used to reduce or eliminate human immunogenicity of an antibody.
In addition, it is possible to create fully human antibodies in mice. Fully human mAbs lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice by techniques referenced in, e.g., Lonberg et al., NATURE 368:856-859, 1994; Fishwild et al., NATURE BIOTECHNOLOGY 14:845-851, 1996; and Mendez et al., NATURE GENETICS 15:146-156, 1997. Human mAbs can also be prepared and optimized from phage display libraries by techniques referenced in, e.g., Knappik et al., J. MOL. BIOL. 296:57-86, 2000; and Krebs et al., J. Immunol. Meth. 254:67-84 2001).
If the antibody is for use as a therapeutic, it can be conjugated to an effector agent such as a small molecule toxin or a radionuclide using standard in vitro conjugation chemistries. If the effector agent is a polypeptide, the antibody can be chemically conjugated to the effector or joined to the effector as a fusion protein. Construction of fusion proteins is within ordinary skill in the art.
IV. Use of Antibodies
The antibodies disclosed herein can be used to treat various forms of cancer, e.g., non-small cell lung cancer, breast, ovarian, prostate, cervical, colorectal, lung, pancreatic, gastric, and head and neck cancers. The cancer cells are exposed to a therapeutically effective amount of the antibody so as to inhibit or reduce proliferation of the cancer cell. In some embodiments, the antibodies inhibit cancer cell proliferation by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%.
In some embodiments, the antibody (e.g., 07F01, 29B06, 17F06, 18H09, 12B11, sh29B06, sh07F01) inhibits or reduces proliferation of a tumor cell by inhibiting binding of human RON to its ligand, MSP. In some embodiments, the antibody (e.g., 07F01, 29B06, 17F06, 18H09, 12B11, sh29B06, sh07F01) inhibits or reduces proliferation of a tumor cell without inhibiting RON binding to MSP. The antibody (e.g., 07F01, 29B06, 17F06, 18H09, 12B11, sh29B06, sh07F01) can also be used in therapy. The antibody (e.g., 07F01, 29B06, 17F06, 18H09, 12B11, sh29B06, sh07F01) can be used to inhibit tumor growth in a mammal (e.g., a human patient). In some embodiments, use of the antibody to inhibit tumor growth in a mammal comprises administering to the mammal a therapeutically effective amount of the antibody.
In certain embodiments, antibody Sh29B06-78 is used in therapy. For example, antibody Sh29B06-78 can be used for inhibiting or reducing proliferation of a tumor cell. Antibody Sh29B06-78 can also be used for inhibiting or reducing tumor growth in a mammal.
In other embodiments, antibody Sh07F01-62 is used in therapy. For example, antibody Sh07F01-62 can be used for inhibiting or reducing proliferation of a tumor cell. Antibody Sh07F01-62 can also be used for inhibiting or reducing tumor growth in a mammal.
Cancers associated with overexpression or inappropriate activation of RON include non-small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and some forms of brain cancer, melanomas, and gastrointestinal cancers.
As used herein, “treat,” “treating” and “treatment” mean the treatment of a disease in a mammal, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state.
Generally, a therapeutically effective amount of active component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level. Alternatively, the initial dosage can be smaller than the optimum, and the dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. In some embodiments, dosing is once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. Formulation of monoclonal antibody-based drugs is within ordinary skill in the art. In some embodiments, the antibody is lyophilized and reconstituted in buffered saline at the time of administration.
For therapeutic use, an antibody preferably is combined with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be “acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
Pharmaceutical compositions containing antibodies, such as those disclosed herein, can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration. A preferred route of administration for monoclonal antibodies is IV infusion. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
EXAMPLES
The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.
Example 1
Production of Human RON Extracellular Domain (ECD)
This Example describes the production of the antigen, hRON ECD protein. The use of the full length ECD as the immunogen, allowed for the selection of two classes of hybridomas: (a) those producing antibodies that interact with the ligand binding domain, thereby inhibiting contact of the ligand to the receptor; and (b) those producing antibodies that bind outside the ligand binding domain, thereby inhibiting the receptor functions through mechanisms other than inhibition of ligand binding.
DNA encoding the extracellular domain of human RON (hRON ECD) (ref seq. NM—002447) was amplified by PCR and subcloned using the XmaI/EcoRI restriction sites in-frame into the pEE14.4 vector (Lonza, Basel, Switzerland) containing THXmFC (Thrombin/His tag/Factor Xa-AJ mouse IgG-Fc), to produce a fusion protein. The resulting clone was linearized using the PvuI enzyme (NEBiolabs, Cat. No. R0150), then electroporated into CHO K1SVcells (Lonza). The electroporated cells were diluted in 200 ml CD CHO media (Gibco Cat. No. 10743-011). The next day, CD CHO media containing methionine sulfoximine (MSX) for a final concentration of 50 μM was added to the cells. After four weeks, positive clones were selected by sandwich ELISA in which the immobilized antibody was commercial monoclonal anti-hRON antibody MAB691 (R&D Systems), and the detection antibody was commercial polyclonal anti-hRON antibody AF691 (R&D Systems). Positive clones were re-transfected using LIPOFECTAMINETM 2000 in a standard protocol. Cells were aliquoted into four separate shaker flasks and selected using 50 uM, 100 uM, 200 uM, and 400 uM MSX. After two weeks of selection, the individual flasks were checked for hRON-ECD protein expression by ELISA. The highest selection pressure, 400 μM MSX, yielded good protein expression and was chosen for scale-up and purification. Cells were grown for 2 weeks at 37° C. in BELLOCELL® Bottles (Bellco Glass, Vineland, N.J.) at a concentration of 2-2.5×106 cells/ml in CD CHO media, with a final concentration of 80 μM MSX for protein production. The resulting cells were spun down in 500 ml conical tubes for 15 minutes. The supernatant was filtered using vacuum filtration using a 0.45 micron filter and then a 0.22 micron filter. The protein was then batch bound to PROSEP®-A beads (Millipore) at 4° C. overnight with rotation after adjusting the pH to 7.5. The beads were washed with 1×PBS and loaded onto disposable protein A affinity columns (Bio-Rad ECONO-PAC® columns; Bio-Rad cat. No. 732-1010). The beads were washed with 10 column volumes (CV) of glycine binding buffer (3M glycine ph 9.0, 1M NaCl). The protein was then eluted off the column using 5-10 CV of 200 mM glycine pH 2.5 acid elution buffer. The samples were then neutralized using 1.3 mL of 1.0 M Tris pH 8.0 neutralization buffer concentrated using VIVASPIN® concentrators (Sartorius Stedim Biotech).
Example 2
Anti-RON Antibodies
This Example describes the production of anti-hRON monoclonal antibodies. Immunizations, fusions, and primary screens were conducted at Maine Biotechnology Services Inc. (Portland, Me.), following the Repetitive Immunization Multiple Sites (RIMMS) protocol. Five AJ mice and five Balb/c mice were immunized with recombinant human RON extracellular domain (hRON-ECD). Two Balb/c mice with sera displaying the highest anti-RON activity by Enzyme Linked Immunosorbent Assay (ELISA) were chosen for subsequent fusion. Spleens and lymph nodes from the appropriate mice were harvested. B-cells were harvested and fused with a myeloma line. Fusion products were serially diluted onto forty 96-well plates to near clonality.
Approximately 4,000 supernatants from the cell fusions were screened by ELISA for binding to recombinant hRON-ECD. A total of 158 supernatants containing antibodies against RON were further characterized by in vitro biochemical and cell-based assays, as described below. A panel of hybridomas was selected, subcloned and expanded. Hybridoma cell lines were transferred to BioXCell (West Lebanon, N.H.) for antibody expression and purification by affinity chromatography on Protein G resin, under standard conditions.
Example 3
Screening Assays
A biochemical assay was carried out to identify antibodies that inhibit ligand binding. A cell-based assay was carried out to identify antibodies that inhibit MSP induced phosphoERK downstream signaling of the receptor. Antibodies that inhibited RON mediated cellular signaling were selected for further characterization regardless of whether they blocked ligand binding in the neutralization assay.
The biochemical neutralization assay measures inhibition of MSP binding to hRON by antibodies in hybridoma supernatants, using electrochemiluminescence (ECL). MA2400 96-well high binding plates (Meso Scale Discovery) were coated with 25 μl of 0.42 μg/mL hRON SEMA+PSI (an N-terminal portion of the ECD of hRON; R&D Systems) in PBS for one hour at room temperature with agitation. The plates were washed four times with PBS+0.1% TWEEN-20TM (PBST), and blocked with 150 μl of charcoal-stripped fetal bovine serum (FBS) (Gibco). The hybridoma supernatant were added and incubated for 45 minutes at room temperature. After incubation, 5 μl of MSP (3 μg/mL) in charcoal stripped FBS was added to each well, and incubated for 45 minutes. The plate was washed four times with PBST, and 25 μl of 1 μg/mL biotinylated anti-MSP antibody (R&D Systems) was added to the plates for one hour at room temperature with agitation. The plates were washed four times with PBST, and incubated with 25 μl of 1 μg/mL ST-streptavidin (Meso Scale Discovery) for one hour at room temperature with agitation. The plates were washed four times with PBST, and 150 μl read buffer (Meso Scale Discovery) was added to each well before the plates were analyzed on a SECTOR® IMAGER 2400 (Meso Scale Discovery) instrument. Antibodies 07F01, 18H09 and 29B06 each blocked MSP binding to hRON SEMA+PSI in this neutralization assay.
In the cell-based assay, antibodies in the hybridoma supernatant were tested for inhibition of MSP-induced phosphorylation of ERK, which is a RON downstream signaling molecule. T47D cells were cultured in 96-well plates in RPMI 1640+10% FBS+insulin. Medium was removed, and cells were incubated in serum-free medium for 24 hours. Hybridoma supernatants containing RON antibodies were added to the cells at a dilution of 1:4 in-serum-free medium, and incubated for one hour at 37° C. MSP (5 nM) was added to the wells and incubated for 15 minutes. Medium was removed, and cells were fixed in 4% paraformaldehyde (PFA) in PBS. Total ERK and phospho-ERK were measured according to the vendor's instructions (R&D Systems, DY1018). Antibodies 07F01, 12B11, 17F06, 18H09 and 29B06 each inhibited MSP induced ERK phosphorylation in T47D cells.
As discussed herein (see Examples 8 and 9), antibodies 07F01, 12B11, 17F06, 18H09 and 29B06 each inhibited MSP induced ERK phosphorylation in T47D cells, while only antibodies 07F01, 18H09 and 29B06 each blocked MSP binding to hRON SEMA+PSI in the neutralization assay. This suggests that antibodies 12B11 and 17F06 do not neutralize binding of MSP to the hRON SEMA+PSI domain, neutralize binding of MSP to RON in the context of the full RON extracellular domain, or function by a mechanism other than blocking MSP binding to RON.
Example 4
Antibody Sequence Analysis
The light chain isotype and heavy chain isotype of each monoclonal antibody in Example 2 was determined using the ISOSTRIPTM Mouse Monoclonal Antibody Isotyping Kit according the kit vendor's instructions (Roche Applied Science, Indianapolis, Ind.). All antibodies were found to be kappa or lambda light chain and IgG1 or IgG2a heavy chain.
The heavy and light chain variable regions of the mouse monoclonal antibodies were sequenced using 5′ RACE (Rapid Amplification of cDNA Ends). Total RNA was extracted from each monoclonal hybridoma cell line using the RNEASY® Miniprep kit according to the kit vendor's instructions (Qiagen, Valencia, Calif.). Full-length first strand cDNA containing 5′ ends was generated using either the GENERACERTM Kit (Invitrogen, Carlsbad, Calif.) or SMARTERTM RACE cDNA Amplification Kit (Clontech, Mountain View, Calif.) according to the kit vendor's instructions using random primers for 5′ RACE.
The variable regions of the light (kappa or lambda) and heavy (IgG1 or IgG2b) chains were amplified by PCR, using KOD Hot Start Polymerase (EMD Chemicals, Gibbstown, N.J.), Expand High Fidelity PCR System (Roche Applied Science), or Advantage 2 Polymerase Mix (Clontech) according to the kit vendor's instructions. For amplification of 5′ cDNA ends in conjunction with the GENERACERTM Kit, the GENERACERTM 5′ Primer, 5′ cgactggagcacgaggacactga 3′ (SEQ ID NO: 112) (Invitrogen) was used as a 5′ primer. For amplification of 5′ cDNA ends in conjunction with the SMARTERTM RACE cDNA Amplification Kit, the Universal Primer Mix A primer (Clontech), a mix of: 5′ CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3′ (SEQ ID NO: 113) and 5′ CTAATACGACTCACTATAGGGC 3′ (SEQ ID NO: 114), was used as a 5′ primer. Heavy chain variable regions were amplified using the above 5′ primers and a 3′ IgG1 constant region specific primer, 5′ TATGCAAGGCTTACAACCACA 3′ (SEQ ID NO: 115), or a 3′ IgG2a constant region specific primer, 5′ AGGACAGGGCTTGATTGTGGG 3′ (SEQ ID NO: 116). Kappa chain variable regions were amplified with the above 5′ primers and a 3′ kappa constant region specific primer, 5′ CTCATTCCTGTTGAAGCTCTTGACAAT 3′ (SEQ ID NO: 117). Lambda chain variable regions were amplified with the above 5′ primers and a mix of 3′ lambda constant region specific primers, 5′ GCACGGGACAAACTCTTCTC 3′ (SEQ ID NO: 118) and 5′ CACAGTGTCCCCTTCATGTG 3′ (SEQ ID NO: 119).
Individual PCR products were isolated by agarose gel electrophoresis and purified using the QIAQUICK® Gel Purification kit according to the kit vendor's instructions (Qiagen). The PCR products were subsequently cloned into the PCR® 4Blunt TOPO® plasmid or PCR2.1® TOPO plasmid using the ZERO BLUNT® TOPO® PCR Cloning Kit or the TOPO® TA Cloning Kit, respectively, according to the kit vendor's instructions (Invitrogen) and transformed into DH5-α bacteria (Invitrogen) through standard molecular biology techniques. Plasmid DNA isolated from transformed bacterial clones was sequenced using M13 Forward (5′ GTAAAACGACGGCCAGT 3′) (SEQ ID NO: 120) and M13 Reverse primers (5′ CAGGAAACAGCTATGACC 3′) (SEQ ID NO: 121) by Beckman Genomics (Danvers, Mass.), using standard dideoxy DNA sequencing methods to identify the sequence of the variable region sequences. The sequences were analyzed using VECTOR NTI® software (Invitrogen) and the IMGT/V-Quest web server (imgt.cines.fr) to identify and confirm variable region sequences.
The nucleic acid sequences encoding and the protein sequences defining variable regions of the murine monoclonal antibodies are shown below (amino terminal signal peptide sequences are not shown). CDR sequences (Kabat definition) are indicated by bold font and underlining in the amino acid sequences.








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the



07F01 Antibody


(SEQ ID NO: 1)










  1
gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc cgggtggatc cctgaaactc



 


 61
tcctgtgcag cctcaggatt cgattttagt agacactgga tgagttgggt ccggctggct


 


121
ccagggaaag ggctagaatg gatcgcagaa attaatccag atagcagaac gataaactat


 


181
acgccatctc taaaggagaa attcatcatc tccagagaca acgccaaaaa ttcgctgttt


 


241
ctgcaaatga acagagtgag atctgaggac acagcccttt attactgtgc aagacgggta


 


301
agaattcatt actacggcgc tatggactgc tggggtcaag gaacctcagt caccgtctcc


 


361
tca


 








Protein Sequence Defining the Heavy Chain Variable Region of the



07F01 Antibody 


(SEQ ID NO: 2)










  1
evkllesggg lvqpggslkl scaasgfdfs rhwmswvrla pgkglewiae inpdsrtiny



 


 61
tpslkekfii srdnaknslf lqmnrvrsed talyycarrv rihyygamdc wgqgtsvtvs


 


121
s


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the



07F01 Antibody 


(SEQ ID NO: 3)










  1
gacattgtgt tgacccagtc tcaaaaaatc gtgtccacat cagtaggagc cagggtcagc



 


 61
gtcacctgca aggccagtca gaatgtgggt tctagtttag tctggtatca acagaaacca


 


121
ggtcaatctc ctaaaacact gatttactcg gcatccttcc ggtacagtgg agtccctgat


 


181
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct


 


241
gaagacttgg cagattattt ctgtcaacaa tataataact atccgctcac gttcggtgct


 


301
gggaccaagc tggagctgaa a


 








Protein Sequence Defining the Kappa Chain Variable Region of the



07F01 Antibody 


(SEQ ID NO: 4)










  1
divltqsqki vstsvgarvs vtckasqnvg sslvwyqqkp gqspktliys asfrysgvpd



 


 61
rftgsgsgtd ftltisnvqs edladyfcqq ynnypltfga gtklelk


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the



12B11 Antibody 


(SEQ ID NO: 11)










  1
gaggtgcagt tagtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc



 


 61
tcctgtgcag cctctggatt cactttcagt acctatgcca tgtcttggat tcgccagact


 


121
ccggagaaga ggctggagtg ggtcgcagga atcactaatg gtggtagttt cacctactat


 


181
ccagacactg tgaagggacg attcaccatc tccagagaca atgccaggaa catcctatac


 


241
ctgcaaatga gcggtctgag gtctgaggac acggccatgt attattgtgc aagacagggt


 


301
tactatggtg ttaactttga ctactggggc caaggcacca ctctcacagt ctcctca


 








Protein Sequence Defining the Heavy Chain Variable Region of the



 


12B11 Antibody 


(SEQ ID NO: 12)










  1
evqlvesggg lvkpggslkl scaasgftfs tyamswirqt pekrlewvag itnggsftyy



 


 61
pdtvkgrfti srdnarnily lqmsglrsed tamyycarqg yygvnfdywg qgttltvss


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the



12B11 Antibody 


(SEQ ID NO: 13)










  1
gatgctgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc



 


 61
atctcttgca ggtctagtca gagccttgaa aacagtaacg gaaacactta tttgaactgg


 


121
tacctccaga aaccaggcca gtctccacag ctcctgatct acagggtttc caaccgattt


 


181
tctggggtcc cagacaggtt cagtggtagt ggatcaggga cagatttcac actgaaaatc


 


241
atcagagtgg aggctgagga tttgggactt tatttctgcc tccaagttac acatgtcccg


 


301
cacacgttcg gaggggggac caaactggaa ttaaaa


 








Protein Sequence Defining the Kappa Chain Variable Region of the



12B11 Antibody 


(SEQ ID NO: 14)










  1
davmtqtpls lpvslgdqas iscrssqsle nsngntylnw ylqkpgqspq lliyrvsnrf



 


 61
sgvpdrfsgs gsgtdftlki irveaedlgl yfclqvthvp htfgggtkle lk


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the



17F06 Antibody 


(SEQ ID NO: 21)










  1
gaagtgaagc tggtggagtc ggggggaggc ttagtgaagc ctggagcgtc tctgaaactc



 


 61
tcctgtgcag cctctggatt cattttcagt tcctatggca tgtcttgggt tcgccagact


 


121
tcagacaaga ggctggagtg ggtcgcttcc attagtagtg gtggtggtac cacctactat


 


181
ctagacactg taaagggccg attcaccatc tccagagaga atgccaagga caccctgtac


 


241
ctgcaaatga gtggtctgaa gtctgaagac acggccttgt attactgtac aagaggccaa


 


301
tggttactaa agtttgctta ctggggccaa gggactctgg tcactgtctc tgca


 








Protein Sequence Defining the Heavy Chain Variable Region of the



17F06 Antibody 


(SEQ ID NO: 22)










  1
evklvesggg lvkpgaslkl scaasgfifs sygmswvrqt sdkrlewvas issgggttyy



 


 61
ldtvkgrfti srenakdtly lqmsglksed talyyctrgq wllkfaywgq gtlvtvsa


 








Nucleic Acid Sequence Encoding the Lambda Chain Variable Region of the



17F06 Antibody 


(SEQ ID NO: 23)










  1
caacttgtgc tcactcagtc atcttcagcc tctttctccc tgggagcctc agcaaaactc



 


 61
acgtgcacct tgagtagtca gcacactacg tacaccattg aatggtatca gcaactgcca


 


121
ctcaagcctc ctaagtatgt gatggagctt aagaaagatg gaagccacag cacaggtgtt


 


181
gggattcctg atcgcttctc tggatccagc tctggtgctg atcgctacct taccatttcc


 


241
aacatccagc ctgaagatga agcaatatac atctgtggtg tgggtgagac aattgaggac


 


301
caatttgtgt atgttttcgg cggtggcacc aaggtcactg tccta


 








Protein Sequence Defining the Lambda Chain Variable Region of the



17F06 Antibody 


(SEQ ID NO: 24)










  1
qlvltqsssa sfslgasakl tctlssqhtt ytiewyqqlp lkppkyvmel kkdgshstgv



 


 61
gipdrfsgss sgadryltis niqpedeaiy icgvgetied qfvyvfgggt kvtvl


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the



18H09 Antibody 


(SEQ ID NO: 31)










  1
gaggtgcagc ttcaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc



 


 61
acctgttatg tcactggcga ctccatcacc agtgattact ggaattggat ccggaaattc


 


121
ccaggaaata aacttgagta catgggatat atcagctaca gtggtagcac ttactacaat


 


181
ccatctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca gttctacctt


 


241
cggttgaatt ctgtgactac tgaggacaca gccacatatt actgtgcaag aacccatata


 


301
cttacgattg cttactgggg ccaagggact ctggtcactg tctctgca


 








Protein Sequence Defining the Heavy Chain Variable Region of the



18H09 Antibody 


(SEQ ID NO: 32)










  1
evqlqesgps lvkpsqtlsl tcyvtgdsit sdywnwirkf pgnkleymgy isysgstyyn



 


 61
pslksrisit rdtsknqfyl rlnsvttedt atyycarthi ltiaywgqgt lvtvsa


 








Nucleic Acid Sequence Encoding the Lambda Chain Variable Region of the



18H09 Antibody 


(SEQ ID NO: 33)










  1
caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc



 


 61
acttgtcgct caagtgccgg ggctgttaca actagtaact ttgccaactg ggtccaagaa


 


121
aaaccagatc atttattcac tggtctaata ggtgatacca acatccgagc tccaggtgtt


 


181
cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca


 


241
cagactgagg atgaggcaat atatttctgt gctctttggt acagcaacca ttactgggtg


 


301
ttcggtggag gaaccaaact gactgtccta


 








Protein Sequence Defining the Lambda Chain Variable Region of the



18H09 Antibody 


(SEQ ID NO: 34)










  1
qavvtqesal ttspgetvtl tcrssagavt tsnfanwvqe kpdhlftgli gdtnirapgv



 


 61
parfsgslig dkaaltitga qtedeaiyfc alwysnhywv fgggtkltvl


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the



29B06 Antibody 


(SEQ ID NO: 41)










  1
gaggtgcagc ttcaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc



 


 61
acctgttctg tcactggcga ctccatcacc agtggttact ggaactggat ccggaaattc


 


121
ccagggaata aacttgagta catggggtac ataagctaca gtggtaaaac ttactacaat


 


181
ccatctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca ttactacctg


 


241
cagttgattt ctgtgactgc tgaggacaca gccacatatt actgtgcaag gtctaagtac


 


301
gactatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a


 








Protein Sequence Defining the Heavy Chain Variable Region of the



29B06 Antibody 


(SEQ ID NO: 42)










  1
evqlqesgps lvkpsqtlsl tcsvtgdsit sgywnwirkf pgnkleymgy isysgktyyn



 


 61
pslksrisit rdtsknhyyl qlisvtaedt atyycarsky dyamdywgqg tsvtvss


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the



29B06 Antibody 


(SEQ ID NO: 43)










  1
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctaggaca gagggccacc



 


 61
atctcctgca gagccagcga aattgttgat aattttggca ttagttttat gaactggttc


 


121
caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc


 


181
ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat


 


241
cctgtggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttcctccg


 


301
acgttcggtg gaggcaccaa gctggaaatc aaa


 








Protein Sequence Defining the Kappa Chain Variable Region of the



29B06 Antibody 


(SEQ ID NO: 44)










  1
divltqspas lavslgqrat iscraseivd nfgisfmnwf qqkpgqppkl liyaasnqgs



 


 61
gvparfsgsg sgtdfslnih pveeddtamy fcqqskevpp tfgggtklei k






The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 2 are aligned in FIG. 2. Amino terminal signal peptide sequences (for expression/secretion) are not shown. CDR1, CDR2, and CDR3 (Kabat definition) are identified by boxes. FIG. 3 shows an alignment of the separate CDR1, CDR2, and CDR3 sequences for each antibody.
The amino acid sequences defining the immunoglobulin light chain variable regions of the antibodies in Example 2 are aligned in FIG. 4. Amino terminal signal peptide sequences (for expression/secretion) are not shown. CDR1, CDR2 and CDR3 are identified by boxes. FIG. 5 shows an alignment of the separate CDR1, CDR2, and CDR3 sequences for each antibody.
Table 1 shows the SEQ ID NO. of each sequence discussed in this Example.








TABLE 1





SEQ. ID NO.
Nucleic Acid or Protein
















1
07F01 Heavy Chain Variable Region-nucleic acid


2
07F01 Heavy Chain Variable Region-protein


3
07F01 Light (kappa) Chain Variable Region-nucleic



acid


4
07F01 Light (kappa) Chain Variable Region-protein


5
07F01 Heavy Chain CDR1


6
07F01 Heavy Chain CDR2


7
07F01 Heavy Chain CDR3


8
07F01 Light (kappa) Chain CDR1


9
07F01 Light (kappa) Chain CDR2


10
07F01 Light (kappa) Chain CDR3


11
12B11 Heavy Chain Variable Region-nucleic acid


12
12B11 Heavy Chain Variable Region-protein


13
12B11 Light (kappa) Chain Variable Region-nucleic



acid


14
12B11 Light (kappa) Chain Variable Region-protein


15
12B11 Heavy Chain CDR1


16
12B11 Heavy Chain CDR2


17
12B11 Heavy Chain CDR3


18
12B11 Light (kappa) Chain CDR1


19
12B11 Light (kappa) Chain CDR2


20
12B11 Light (kappa) Chain CDR3


21
17F06 Heavy Chain Variable Region-nucleic acid


22
17F06 Heavy Chain Variable Region-protein


23
17F06 Light (lambda) Chain Variable Region-nucleic



acid


24
17F06 Light (lambda) Chain Variable Region-protein


25
17F06 Heavy Chain CDR1


26
17F06 Heavy Chain CDR2


27
17F06 Heavy Chain CDR3


28
17F06 Light (lambda) Chain CDR1


29
17F06 Light (lambda) Chain CDR2


30
17F06 Light (lambda) Chain CDR3


31
18H09 Heavy Chain Variable Region-nucleic acid


32
18H09 Heavy Chain Variable Region-protein


33
18H09 Light (lambda) Chain Variable Region-nucleic



acid


34
18H09 Light (lambda) Chain Variable Region-protein


35
18H09 Heavy Chain CDR1


36
18H09 Heavy Chain CDR2


37
18H09 Heavy Chain CDR3


38
18H09 Light (lambda) Chain CDR1


39
18H09 Light (lambda) Chain CDR2


40
18H09 Light (lambda) Chain CDR3


41
29B06 Heavy Chain Variable Region-nucleic acid


42
29B06 Heavy Chain Variable Region-protein


43
29B06 Light (kappa) Chain Variable Region-nucleic



acid


44
29B06 Light (kappa) Chain Variable Region-protein


45
29B06 Heavy Chain CDR1


46
29B06 Heavy Chain CDR2


47
29B06 Heavy Chain CDR3


48
29B06 Light (kappa) Chain CDR1


49
29B06 Light (kappa) Chain CDR2


50
29B06 Light (kappa) Chain CDR3









Mouse monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 2.










TABLE 2






CDR1
CDR2
CDR3















Kabat










07F01
RHWMS
EINPDSRTINYTPSLKE
RVRIHYYGAMDC



(SEQ ID
(SEQ ID NO: 6)
(SEQ ID NO: 7)



NO: 5)




 


12B11
TYAMS
GITNGGSFTYYPDTVKG
QGYYGVNFDY



(SEQ ID
(SEQ ID NO: 16)
(SEQ ID NO: 17)



NO: 15)




 


17F06
SYGMS
SISSGGGTTYYLDTVKG
GQWLLKFAY



(SEQ ID
(SEQ ID NO: 26)
(SEQ ID NO: 27)



NO: 25)




 


18H09
SDYWN
YISYSGSTYYNPSLK
THILTIAY



(SEQ ID
(SEQ ID NO: 36)
(SEQ ID NO: 37)



NO: 35)




 


29B06
SGYWN
YISYSGKTYYNPSLKS
SKYDYAMDY



(SEQ ID
(SEQ ID NO: 46)
(SEQ ID NO: 47)



NO: 45)




 







Chothia










07F01
GFDFSRH
NPDSRT
RVRIHYYGAMDC



(SEQ ID
(SEQ ID NO: 52)
(SEQ ID NO: 7)



NO: 51)




 


12B11
GFTFSTY
TNGGSF
QGYYGVNFDY



(SEQ ID
(SEQ ID NO: 54)
(SEQ ID NO: 17)



NO: 53)




 


17F06
GFIFSSY
SSGGGT
GQWLLKFAY



(SEQ ID
(SEQ ID NO: 56)
(SEQ ID NO: 27)



NO: 55)




 


18H09
GDSITSD
SYSGS
THILTIAY



(SEQ ID
(SEQ ID NO: 58)
(SEQ ID NO: 37)



NO: 57)




 


29B06
GDSITSG
SYSGK
SKYDYAMDY



(SEQ ID
(SEQ ID NO: 60)
(SEQ ID NO: 47)



NO: 59)




 







IMGT










07F01
GFDFSRHW
INPDSRTI
ARRVRIHYYGAMDC



(SEQ ID
(SEQ ID NO: 62)
(SEQ ID NO: 63)



NO: 61)




 


12B11
GFTFSTYA
ITNGGSFT
ARQGYYGVNFDY



(SEQ ID
(SEQ ID NO: 65)
(SEQ ID NO: 66)



NO: 64)




 


17F06
GFIFSSYG
ISSGGGTT
TRGQWLLKFAY



(SEQ ID
(SEQ ID NO: 68)
(SEQ ID NO: 69)



NO: 67)




 


18H09
GDSITSDY
ISYSGST
ARTHILTIAY



(SEQ ID
(SEQ ID NO: 71)
(SEQ ID NO: 72)



NO: 70)




 


29B06
GDSITSGY
ISYSGKT
ARSKYDYAMDY



(SEQ ID
(SEQ ID NO: 74)
(SEQ ID NO: 75)



NO: 73)









Mouse monoclonal antibody Kappa light chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 3.










TABLE 3






CDR1
CDR2
CDR3















Kabat/Chothia










07F01
KASQNVGSSLV
SASFRYS
QQYNNYPLT



(SEQ ID NO: 8)
(SEQ ID
(SEQ ID NO: 10)




NO: 9)



 


12B11
RSSQSLENSNGNTYLN
RVSNRFS
LQVTHVPHT



(SEQ ID NO: 18)
(SEQ ID
(SEQ ID NO: 20)




NO: 19)



 


17F06
TLSSQHTTYTIE
LKKDGSHSTGV
GVGETIEDQFVYV



(SEQ ID NO: 28)
(SEQ ID
(SEQ ID NO: 30)




NO: 29)



 


18H09
RSSAGAVTTSNFAN
DTNIRAP
ALWYSNHYWV



(SEQ ID NO: 38)
(SEQ ID
(SEQ ID NO: 40)




NO: 39)



 


29B06
RASEIVDNFGISFMN
AASNQGS
QQSKEVPPT



(SEQ ID NO: 48)
(SEQ ID
(SEQ ID NO: 50)




NO: 49)



 







IMGT










07F01
QNVGSS
SAS
QQYNNYPLT



(SEQ ID NO: 76)

(SEQ ID NO: 10)


 


12B11
QSLENSNGNTY
RVS
LQVTHVPHT



(SEQ ID NO: 77)

(SEQ ID NO: 20)


 


17F06
SQHTTYT
LKKDGSH
GVGETIEDQFVYV



(SEQ ID NO: 78)
(SEQ ID
(SEQ ID NO: 30)




NO: 79)



 


18H09
AGAVTTSNF
DTN
ALWYSNHYWV



(SEQ ID NO: 80)

(SEQ ID NO: 40)


 


29B06
EIVDNFGISF
AAS
QQSKEVPPT



(SEQ ID NO: 81)

(SEQ ID NO: 50)









To create the complete heavy or kappa chain antibody sequences, each variable sequence above is combined with its respective constant region. For example, a complete heavy chain comprises a heavy variable sequence followed by the murine IgG1 or IgG2a heavy chain constant sequence, a complete kappa chain comprises a kappa variable sequence followed by the murine kappa light chain constant sequence, and a complete lambda chain comprises a lambda variable sequence followed by the murine lambda IGLC1 or IGLC2 light chain constant sequence.








Nucleic Acid Sequence Encoding the Murine IgG1 Heavy Chain Constant Region



(SEQ ID NO: 82)










  1
gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac



 


 61
tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc


 


121
tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac


 


181
ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc


 


241
acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg


 


301
gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc


 


361
cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg


 


421
gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag


 


481
gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc


 


541
agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc


 


601
aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg


 


661
aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc


 


721
agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg


 


781
aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct


 


841
tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc


 


901
acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac


 


961
tctcctggta aa


 








Protein Sequence Defining the Murine IgG1 Heavy Chain Constant Region



(SEQ ID NO: 83)










  1
akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd



 


 61
lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif


 


121
ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv


 


181
selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv


 


241
sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf


 


301
tcsvlheglh nhhtekslsh spgk


 








Nucleic Acid Sequence Encoding the Murine IgG2a Heavy Chain Constant Region



(SEQ ID NO: 84)










  1
gccaaaacaa cagccccatc ggtctatcca ctggcccctg tgtgtggaga tacaactggc



 


 61
tcctcggtga ctctaggatg cctggtcaag ggttatttcc ctgagccagt gaccttgacc


 


121
tggaactctg gatccctgtc cagtggtgtg cacaccttcc cagctgtcct gcagtctgac


 


181
ctctacaccc tcagcagctc agtgactgta acctcgagca cctggcccag ccagtccatc


 


241
acctgcaatg tggcccaccc ggcaagcagc accaaggtgg acaagaaaat tgagcccaga


 


301
gggcccacaa tcaagccctg tcctccatgc aaatgcccag cacctaacct cttgggtgga


 


361
ccatccgtct tcatcttccc tccaaagatc aaggatgtac tcatgatctc cctgagcccc


 


421
atagtcacat gtgtggtggt ggatgtgagc gaggatgacc cagatgtcca gatcagctgg


 


481
tttgtgaaca acgtggaagt acacacagct cagacacaaa cccatagaga ggattacaac


 


541
agtactctcc gggtggtcag tgccctcccc atccagcacc aggactggat gagtggcaag


 


601
gagttcaaat gcaaggtcaa caacaaagac ctcccagcgc ccatcgagag aaccatctca


 


661
aaacccaaag ggtcagtaag agctccacag gtatatgtct tgcctccacc agaagaagag


 


721
atgactaaga aacaggtcac tctgacctgc atggtcacag acttcatgcc tgaagacatt


 


781
tacgtggagt ggaccaacaa cgggaaaaca gagctaaact acaagaacac tgaaccagtc


 


841
ctggactctg atggttctta cttcatgtac agcaagctga gagtggaaaa gaagaactgg


 


901
gtggaaagaa atagctactc ctgttcagtg gtccacgagg gtctgcacaa tcaccacacg


 


961
actaagagct tctcccggac tccgggtaaa


 








Protein Sequence Defining the Murine IgG2a Heavy Chain Constant Region



(SEQ ID NO: 85)










  1
akttapsvyp lapvcgdttg ssvtlgclvk gyfpepvtlt wnsgslssgv htfpavlqsd



 


 61
lytlsssvtv tsstwpsqsi tcnvahpass tkvdkkiepr gptikpcppc kcpapnllgg


 


121
psvfifppki kdvlmislsp ivtcvvvdvs eddpdvqisw fvnnvevhta qtqthredyn


 


181
stlrvvsalp iqhqdwmsgk efkckvnnkd lpapiertis kpkgsvrapq vyvlpppeee


 


241
mtkkqvtltc mvtdfmpedi yvewtnngkt elnykntepv ldsdgsyfmy sklrvekknw


 


301
vernsyscsv vheglhnhht tksfsrtpgk


 








Nucleic Acid Sequence Encoding the Murine Kappa Light Chain Constant Region



(SEQ ID NO: 86)










  1
cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct



 


 61
ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag


 


121
tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac


 


181
agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa


 


241
cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag


 


301
agcttcaaca ggaatgagtg t


 








Protein Sequence Defining the Murine Kappa Light Chain Constant Region



(SEQ ID NO: 87)










  1
radaaptvsi fppsseqlts ggasvvcfln nfypkdinvk wkidgserqn gvlnswtdqd



 


 61
skdstysmss tltltkdeye rhnsytceat hktstspivk sfnrnec


 








Nucleic Acid Sequence Encoding the Murine Lambda (IGLC1) Light Chain Constant



Region


(SEQ ID NO: 88)










  1
ggccagccca agtcttcgcc atcagtcacc ctgtttccac cttcctctga agagctcgag



 


 61
actaacaagg ccacactggt gtgtacgatc actgatttct acccaggtgt ggtgacagtg


 


121
gactggaagg tagatggtac ccctgtcact cagggtatgg agacaaccca gccttccaaa


 


181
cagagcaaca acaagtacat ggctagcagc tacctgaccc tgacagcaag agcatgggaa


 


241
aggcatagca gttacagctg ccaggtcact catgaaggtc acactgtgga gaagagtttg


 


301
tcccgtgctg actgttcc


 








Protein Sequence Defining the Murine Lambda (IGLC1) Light Chain Constant



Region


(SEQ ID NO: 89)










  1
gqpksspsvt lfppsseele tnkatlvcti tdfypgvvtv dwkvdgtpvt qgmettqpsk



 


 61
qsnnkymass yltltarawe rhssyscqvt heghtveksl sradcs


 








Nucleic Acid Sequence Encoding the Murine Lambda (IGLC2) Light Chain Constant



Region


(SEQ ID NO: 90)










  1
ggtcagccca agtccactcc cactctcacc gtgtttccac cttcctctga ggagctcaag



 


 61
gaaaacaaag ccacactggt gtgtctgatt tccaactttt ccccgagtgg tgtgacagtg


 


121
gcctggaagg caaatggtac acctatcacc cagggtgtgg acacttcaaa tcccaccaaa


 


181
gagggcaaca agttcatggc cagcagcttc ctacatttga catcggacca gtggagatct


 


241
cacaacagtt ttacctgtca agttacacat gaaggggaca ctgtggagaa gagtctgtct


 


301
cctgcagaat gtctc


 








Protein Sequence Defining the Murine Lambda (IGLC2) Light Chain Constant



Region


(SEQ ID NO: 91)










  1
gqpkstptlt vfppsseelk enkatlvcli snfspsgvtv awkangtpit qgvdtsnptk



 


 61
egnkfmassf lhltsdqwrs hnsftcqvth egdtveksls paecl






The following sequences represent the actual or contemplated full length heavy and light chain sequence (i.e., containing both the variable and constant regions sequences) for each antibody described in this Example. Signal sequences for proper secretion of the antibodies (e.g., signal sequences at the 5′ end of the DNA sequences or the amino terminal end of the protein sequences) are not shown in the full length heavy and light chain sequences disclosed herein and are not included in the final secreted protein. Also not shown are stop codons for termination of translation required at the 3′ end of the DNA sequences. It is within ordinary skill in the art to select a signal sequence and/or a stop codon for expression of the disclosed full length IgG heavy chain and light chain sequences. It is also contemplated that the variable region sequences can be ligated to other constant region sequences to produce active full length IgG heavy and light chains.








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy



Chain Variable Region and IgG1 Constant Region) of 07F01


(SEQ ID NO: 92)










   1
gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc cgggtggatc cctgaaactc



 


  61
tcctgtgcag cctcaggatt cgattttagt agacactgga tgagttgggt ccggctggct


 


 121
ccagggaaag ggctagaatg gatcgcagaa attaatccag atagcagaac gataaactat


 


 181
acgccatctc taaaggagaa attcatcatc tccagagaca acgccaaaaa ttcgctgttt


 


 241
ctgcaaatga acagagtgag atctgaggac acagcccttt attactgtgc aagacgggta


 


 301
agaattcatt actacggcgc tatggactgc tggggtcaag gaacctcagt caccgtctcc


 


 361
tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact


 


 421
aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg


 


 481
acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct


 


 541
gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc


 


 601
gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc


 


 661
agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc


 


 721
ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt


 


 781
gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg


 


 841
gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca


 


 901
gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg


 


 961
gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga


 


1021
ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa


 


1081
gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag


 


1141
tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc


 


1201
tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact


 


1261
ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc


 


1321
cactctcctg gtaaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain



Variable Region and IgG1 Constant Region) of 07F01


(SEQ ID NO: 93)










   1
evkllesggg lvqpggslkl scaasgfdfs rhwmswvrla pgkglewiae inpdsrtiny



 


  61
tpslkekfii srdnaknslf lqmnrvrsed talyycarrv rihyygamdc wgqgtsvtvs


 


 121
sakttppsvy plapgsaaqt nsmvtlgclv kgyfpepvtv twnsgslssg vhtfpavlqs


 


 181
dlytlsssvt vpsstwpset vtcnvahpas stkvdkkivp rdcgckpcic tvpevssvfi


 


 241
fppkpkdvlt itltpkvtcv vvdiskddpe vqfswfvddv evhtaqtqpr eeqfnstfrs


 


 301
vselpimhqd wlngkefkcr vnsaafpapi ektisktkgr pkapqvytip ppkeqmakdk


 


 361
vsltcmitdf fpeditvewq wngqpaenyk ntqpimdtdg syfvysklnv qksnweagnt


 


 421
ftcsvlhegl hnhhteksls hspgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Kappa



Chain Variable Region and Constant Region) of 07F01


(SEQ ID NO: 94)










   1
gacattgtgt tgacccagtc tcaaaaaatc gtgtccacat cagtaggagc cagggtcagc



 


  61
gtcacctgca aggccagtca gaatgtgggt tctagtttag tctggtatca acagaaacca


 


 121
ggtcaatctc ctaaaacact gatttactcg gcatccttcc ggtacagtgg agtccctgat


 


 181
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct


 


 241
gaagacttgg cagattattt ctgtcaacaa tataataact atccgctcac gttcggtgct


 


 301
gggaccaagc tggagctgaa acgggctgat gctgcaccaa ctgtatccat cttcccacca


 


 361
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac


 


 421
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg


 


 481
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg


 


 541
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca


 


 601
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gt


 








Protein Sequence Defining the Full Length Light Chain Sequence (Kappa Chain



Variable Region and Constant Region) of 07F01


(SEQ ID NO: 95)










   1
divltqsqki vstsvgarvs vtckasqnvg sslvwyqqkp gqspktliys asfrysgvpd



 


  61
rftgsgsgtd ftltisnvqs edladyfcqq ynnypltfga gtklelkrad aaptvsifpp


 


 121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt


 


 181
ltkdeyerhn sytceathkt stspivksfn rnec


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy



Chain Variable Region and IgG1 Constant Region) of 12B11


(SEQ ID NO: 96)










   1
gaggtgcagt tagtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc



 


  61
tcctgtgcag cctctggatt cactttcagt acctatgcca tgtcttggat tcgccagact


 


 121
ccggagaaga ggctggagtg ggtcgcagga atcactaatg gtggtagttt cacctactat


 


 181
ccagacactg tgaagggacg attcaccatc tccagagaca atgccaggaa catcctatac


 


 241
ctgcaaatga gcggtctgag gtctgaggac acggccatgt attattgtgc aagacagggt


 


 301
tactatggtg ttaactttga ctactggggc caaggcacca ctctcacagt ctcctcagcc


 


 361
aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc


 


 421
atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg


 


 481
aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc


 


 541
tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc


 


 601
tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat


 


 661
tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc


 


 721
ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta


 


 781
gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg


 


 841
cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt


 


 901
gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac


 


 961
agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag


 


1021
gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt


 


1081
ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat


 


1141
gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac


 


1201
ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc


 


1261
tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct


 


1321
cctggtaaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain



Variable Region and IgG1 Constant Region) of 12B11


(SEQ ID NO: 97)










   1
evqlvesggg lvkpggslkl scaasgftfs tyamswirqt pekrlewvag itnggsftyy



 


  61
pdtvkgrfti srdnarnily lqmsglrsed tamyycarqg yygvnfdywg qgttltvssa


 


 121
kttppsvypl apgsaaqtns mvtlgclvkg yfpepvtvtw nsgslssgvh tfpavlqsdl


 


 181
ytlsssvtvp sstwpsetvt cnvahpasst kvdkkivprd cgckpcictv pevssvfifp


 


 241
pkpkdvltit ltpkvtcvvv diskddpevq fswfvddvev htaqtqpree qfnstfrsvs


 


 301
elpimhqdwl ngkefkcrvn saafpapiek tisktkgrpk apqvytippp keqmakdkvs


 


 361
ltcmitdffp editvewqwn gqpaenyknt qpimdtdgsy fvysklnvqk snweagntft


 


 421
csvlheglhn hhtekslshs pgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Kappa



Chain Variable Region and Constant Region) of 12B11


(SEQ ID NO: 98)










   1
gatgctgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc



 


  61
atctcttgca ggtctagtca gagccttgaa aacagtaacg gaaacactta tttgaactgg


 


 121
tacctccaga aaccaggcca gtctccacag ctcctgatct acagggtttc caaccgattt


 


 181
tctggggtcc cagacaggtt cagtggtagt ggatcaggga cagatttcac actgaaaatc


 


 241
atcagagtgg aggctgagga tttgggactt tatttctgcc tccaagttac acatgtcccg


 


 301
cacacgttcg gaggggggac caaactggaa ttaaaacggg ctgatgctgc accaactgta


 


 361
tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc


 


 421
ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga


 


 481
caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg


 


 541
agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag


 


 601
gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacaggaa tgagtgt


 








Protein Sequence Defining the Full Length Light Chain Sequence (Kappa Chain



Variable Region and Constant Region) of 12B11


(SEQ ID NO: 99)










   1
davmtqtpls lpvslgdqas iscrssqsle nsngntylnw ylqkpgqspq lliyrvsnrf



 


  61
sgvpdrfsgs gsgtdftlki irveaedlgl yfclqvthvp htfgggtkle lkradaaptv


 


 121
sifppsseql tsggasvvcf lnnfypkdin vkwkidgser qngvinswtd qdskdstysm


 


 181
sstltltkde yerhnsytce athktstspi vksfnrnec


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy



Chain Variable Region and IgG2A Constant Region) of 17F06 


(SEQ ID NO: 100)










   1
gaagtgaagc tggtggagtc ggggggaggc ttagtgaagc ctggagcgtc tctgaaactc



 


  61
tcctgtgcag cctctggatt cattttcagt tcctatggca tgtcttgggt tcgccagact


 


 121
tcagacaaga ggctggagtg ggtcgcttcc attagtagtg gtggtggtac cacctactat


 


 181
ctagacactg taaagggccg attcaccatc tccagagaga atgccaagga caccctgtac


 


 241
ctgcaaatga gtggtctgaa gtctgaagac acggccttgt attactgtac aagaggccaa


 


 301
tggttactaa agtttgctta ctggggccaa gggactctgg tcactgtctc tgcagccaaa


 


 361
acaacagccc catcggtcta tccactggcc cctgtgtgtg gagatacaac tggctcctcg


 


 421
gtgactctag gatgcctggt caagggttat ttccctgagc cagtgacctt gacctggaac


 


 481
tctggatccc tgtccagtgg tgtgcacacc ttcccagctg tcctgcagtc tgacctctac


 


 541
accctcagca gctcagtgac tgtaacctcg agcacctggc ccagccagtc catcacctgc


 


 601
aatgtggccc acccggcaag cagcaccaag gtggacaaga aaattgagcc cagagggccc


 


 661
acaatcaagc cctgtcctcc atgcaaatgc ccagcaccta acctcttggg tggaccatcc


 


 721
gtcttcatct tccctccaaa gatcaaggat gtactcatga tctccctgag ccccatagtc


 


 781
acatgtgtgg tggtggatgt gagcgaggat gacccagatg tccagatcag ctggtttgtg


 


 841
aacaacgtgg aagtacacac agctcagaca caaacccata gagaggatta caacagtact


 


 901
ctccgggtgg tcagtgccct ccccatccag caccaggact ggatgagtgg caaggagttc


 


 961
aaatgcaagg tcaacaacaa agacctccca gcgcccatcg agagaaccat ctcaaaaccc


 


1021
aaagggtcag taagagctcc acaggtatat gtcttgcctc caccagaaga agagatgact


 


1081
aagaaacagg tcactctgac ctgcatggtc acagacttca tgcctgaaga catttacgtg


 


1141
gagtggacca acaacgggaa aacagagcta aactacaaga acactgaacc agtcctggac


 


1201
tctgatggtt cttacttcat gtacagcaag ctgagagtgg aaaagaagaa ctgggtggaa


 


1261
agaaatagct actcctgttc agtggtccac gagggtctgc acaatcacca cacgactaag


 


1321
agcttctccc ggactccggg taaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain



Variable Region and IgG2A Constant Region) of 17F06


(SEQ ID NO: 101)










   1
evklvesggg lvkpgaslkl scaasgfifs sygmswvrqt sdkrlewvas issgggttyy



 


  61
ldtvkgrfti srenakdtly lqmsglksed talyyctrgq wllkfaywgq gtlvtvsaak


 


 121
ttapsvypla pvcgdttgss vtlgclvkgy fpepvtltwn sgslssgvht fpavlqsdly


 


 181
tlsssvtvts stwpsqsitc nvahpasstk vdkkieprgp tikpcppckc papnllggps


 


 241
vfifppkikd vlmislspiv tcvvvdvsed dpdvqiswfv nnvevhtaqt qthredynst


 


 301
lrvvsalpiq hqdwmsgkef kckvnnkdlp apiertiskp kgsvrapqvy vlpppeeemt


 


 361
kkqvtltcmv tdfmpediyv ewtnngktel nykntepvld sdgsyfmysk lrvekknwve


 


 421
rnsyscsvvh eglhnhhttk sfsrtpgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Lambda



Chain Variable Region and Constant Region (IGLC2)) of 17F06


(SEQ ID NO: 102)










   1
caacttgtgc tcactcagtc atcttcagcc tctttctccc tgggagcctc agcaaaactc



 


  61
acgtgcacct tgagtagtca gcacactacg tacaccattg aatggtatca gcaactgcca


 


 121
ctcaagcctc ctaagtatgt gatggagctt aagaaagatg gaagccacag cacaggtgtt


 


 181
gggattcctg atcgcttctc tggatccagc tctggtgctg atcgctacct taccatttcc


 


 241
aacatccagc ctgaagatga agcaatatac atctgtggtg tgggtgagac aattgaggac


 


 301
caatttgtgt atgttttcgg cggtggcacc aaggtcactg tcctaggtca gcccaagtcc


 


 361
actcccactc tcaccgtgtt tccaccttcc tctgaggagc tcaaggaaaa caaagccaca


 


 421
ctggtgtgtc tgatttccaa cttttccccg agtggtgtga cagtggcctg gaaggcaaat


 


 481
ggtacaccta tcacccaggg tgtggacact tcaaatccca ccaaagaggg caacaagttc


 


 541
atggccagca gcttcctaca tttgacatcg gaccagtgga gatctcacaa cagttttacc


 


 601
tgtcaagtta cacatgaagg ggacactgtg gagaagagtc tgtctcctgc agaatgtctc


 








Protein Sequence Defining the Full Length Light Chain Sequence (Lambda Chain



Variable Region and Constant Region (IGLC2)) of 17F06


(SEQ ID NO: 103)










   1
qlvltqsssa sfslgasakl tctlssqhtt ytiewyqqlp lkppkyvmel kkdgshstgv



 


  61
gipdrfsgss sgadryltis niqpedeaiy icgvgetied qfvyvfgggt kvtvlgqpks


 


 121
tptltvfpps seelkenkat lvclisnfsp sgvtvawkan gtpitqgvdt snptkegnkf


 


 181
massflhlts dqwrshnsft cqvthegdtv ekslspaecl


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy



Chain Variable Region and IgG1 Constant Region) of 18H09


(SEQ ID NO: 104)










   1
gaggtgcagc ttcaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc



 


  61
acctgttatg tcactggcga ctccatcacc agtgattact ggaattggat ccggaaattc


 


 121
ccaggaaata aacttgagta catgggatat atcagctaca gtggtagcac ttactacaat


 


 181
ccatctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca gttctacctt


 


 241
cggttgaatt ctgtgactac tgaggacaca gccacatatt actgtgcaag aacccatata


 


 301
cttacgattg cttactgggg ccaagggact ctggtcactg tctctgcagc caaaacgaca


 


 361
cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc


 


 421
ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga


 


 481
tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg


 


 541
agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt


 


 601
gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtggttgt


 


 661
aagccttgca tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc


 


 721
aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc


 


 781
aaggatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct


 


 841
cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc


 


 901
atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct


 


 961
ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag


 


1021
gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc


 


1081
atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca


 


1141
gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac


 


1201
agcaagctca atgtgcagaa gagcaactgg gaggcaggaa atactttcac ctgctctgtg


 


1261
ttacatgagg gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain



Variable Region and IgG1 Constant Region) of 18H09


(SEQ ID NO: 105)










   1
evqlqesgps lvkpsqtlsl tcyvtgdsit sdywnwirkf pgnkleymgy isysgstyyn



 


  61
pslksrisit rdtsknqfyl rinsvttedt atyycarthi ltiaywgqgt lvtvsaaktt


 


 121
ppsvyplapg saaqtnsmvt lgclvkgyfp epvtvtwnsg slssgvhtfp avlqsdlytl


 


 181
sssvtvpsst wpsetvtcnv ahpasstkvd kkivprdcgc kpcictvpev ssvfifppkp


 


 241
kdvltitltp kvtcvvvdis kddpevqfsw fvddvevhta qtqpreeqfn stfrsyselp


 


 301
imhqdwlngk efkcrvnsaa fpapiektis ktkgrpkapq vytipppkeq makdkvsltc


 


 361
mitdffpedi tvewqwngqp aenykntqpi mdtdgsyfvy sklnvqksnw eagntftcsv


 


 421
lheglhnhht ekslshspgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Lambda



Chain Variable Region and Constant Region (IGLC1)) of 18H09


(SEQ ID NO: 106)










   1
caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc



 


  61
acttgtcgct caagtgccgg ggctgttaca actagtaact ttgccaactg ggtccaagaa


 


 121
aaaccagatc atttattcac tggtctaata ggtgatacca acatccgagc tccaggtgtt


 


 181
cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca


 


 241
cagactgagg atgaggcaat atatttctgt gctctttggt acagcaacca ttactgggtg


 


 301
ttcggtggag gaaccaaact gactgtccta ggccagccca agtcttcgcc atcagtcacc


 


 361
ctgtttccac cttcctctga agagctcgag actaacaagg ccacactggt gtgtacgatc


 


 421
actgatttct acccaggtgt ggtgacagtg gactggaagg tagatggtac ccctgtcact


 


 481
cagggtatgg agacaaccca gccttccaaa cagagcaaca acaagtacat ggctagcagc


 


 541
tacctgaccc tgacagcaag agcatgggaa aggcatagca gttacagctg ccaggtcact


 


 601
catgaaggtc acactgtgga gaagagtttg tcccgtgctg actgttcc


 








Protein Sequence Defining the Full Length Light Chain Sequence (Lambda Chain



Variable Region and Constant Region (IGLC1)) of 18H09


(SEQ ID NO: 107)










   1
qavvtqesal ttspgetvtl tcrssagavt tsnfanwvqe kpdhlftgli gdtnirapgv



 


  61
parfsgslig dkaaltitga qtedeaiyfc alwysnhywv fgggtkltvl gqpksspsvt


 


 121
lfppsseele tnkatlvcti tdfypgvvtv dwkvdgtpvt qgmettqpsk qsnnkymass


 


 181
yltltarawe rhssyscqvt heghtveksl sradcs


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy



Chain Variable Region and IgG1 Constant Region) of 29B06


(SEQ ID NO: 108)










   1
gaggtgcagc ttcaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc



 


  61
acctgttctg tcactggcga ctccatcacc agtggttact ggaactggat ccggaaattc


 


 121
ccagggaata aacttgagta catggggtac ataagctaca gtggtaaaac ttactacaat


 


 181
ccatctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca ttactacctg


 


 241
cagttgattt ctgtgactgc tgaggacaca gccacatatt actgtgcaag gtctaagtac


 


 301
gactatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc agccaaaacg


 


 361
acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg


 


 421
accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct


 


 481
ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact


 


 541
ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac


 


 601
gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt


 


 661
tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag


 


 721
cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc


 


 781
agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca


 


 841
gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt


 


 901
cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca


 


 961
gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca


 


1021
caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc


 


1081
tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag


 


1141
ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc


 


1201
tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct


 


1261
gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt


 


1321
aaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain



Variable Region and IgG1 Constant Region) of 29B06


(SEQ ID NO: 109)










   1
evqlqesgps lvkpsqtlsl tcsvtgdsit sgywnwirkf pgnkleymgy isysgktyyn



 


  61
pslksrisit rdtsknhyyl qlisvtaedt atyycarsky dyamdywgqg tsvtvssakt


 


 121
tppsvyplap gsaaqtnsmv tlgclvkgyf pepvtvtwns gslssgvhtf pavlqsdlyt


 


 181
lsssvtvpss twpsetvtcn vahpasstkv dkkivprdcg ckpcictvpe vssvfifppk


 


 241
pkdvltitlt pkvtcvvvdi skddpevqfs wfvddvevht aqtqpreeqf nstfrsvsel


 


 301
pimhqdwlng kefkcrvnsa afpapiekti sktkgrpkap qvytipppke qmakdkvslt


 


 361
cmitdffped itvewqwngq paenykntqp imdtdgsyfv ysklnvqksn weagntftcs


 


 421
vlheglhnhh tekslshspg k


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Kappa



Chain Variable Region and Constant Region) of 29B06


(SEQ ID NO: 110)










   1
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctaggaca gagggccacc



 


  61
atctcctgca gagccagcga aattgttgat aattttggca ttagttttat gaactggttc


 


 121
caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc


 


 181
ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat


 


 241
cctgtggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttcctccg


 


 301
acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc


 


 361
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg


 


 421
aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa


 


 481
aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc


 


 541
agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc


 


 601
actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt


 








Protein Sequence Defining the Full Length Light Chain Sequence (Kappa Chain



Variable Region and Constant Region) of 29B06


(SEQ ID NO: 111)










   1
divltqspas lavslgqrat iscraseivd nfgisfmnwf qqkpgqppkl liyaasnqgs



 


  61
gvparfsgsg sgtdfslnih pveeddtamy fcqqskevpp tfgggtklei kradaaptvs


 


 121
ifppsseqlt sggasvvcfl nnfypkdinv kwkidgserq ngvinswtdq dskdstysms


 


 181
stltltkdey erhnsytcea thktstspiv ksfnrnec






Table 4 shows the correspondence between the full-length sequences of the antibodies discussed in this Example with those presented in the Sequence Listing.








TABLE 4





SEQ ID NO.
Nucleic Acid or Protein
















92
07F01 Heavy Variable + IgG1 Constant-nucleic acid


93
07F01 Heavy Variable + IgG1 Constant-protein


94
07F01 Kappa Variable + Constant-nucleic acid


95
07F01 Kappa Variable + Constant-protein


96
12B11 Heavy Variable + IgG1 Constant-nucleic acid


97
12B11 Heavy Variable + IgG1 Constant-protein


98
12B11 Kappa Variable + Constant-nucleic acid


99
12B11 Kappa Variable + Constant-protein


100
17F06 Heavy Variable + IgG2A Constant-nucleic acid


101
17F06 Heavy Variable + IgG2A Constant-protein


102
17F06 Lambda Variable + Constant (IGLC2)-nucleic acid


103
17F06 Lambda Variable + Constant (IGLC2)-protein


104
18H09 Heavy Variable + IgG1 Constant-nucleic acid


105
18H09 Heavy Variable + IgG1 Constant-protein


106
18H09 Lambda Variable + Constant (IGLC1)-nucleic acid


107
18H09 Lambda Variable + Constant (IGLC1)-protein


108
29B06 Heavy Variable + IgG1 Constant-nucleic acid


109
29B06 Heavy Variable + IgG1 Constant-protein


110
29B06 Kappa Variable + Constant-nucleic acid


111
29B06 Kappa Variable + Constant-protein









Example 5
Binding Affinities
The binding affinities and kinetics of binding of antibodies 07F01, 29B06, 17F06, 18H09, and 12B11 to recombinant human RON-ECD/mFc fusion protein (rhRON ECD/mFc) and recombinant human RON SEMA and PSI domains (rhRON SEMA+PSI) (R&D Systems, Inc., Minneapolis, Minn.) were measured by surface plasmon resonance, using a BIACORE® T100 instrument (GE Healthcare, Piscataway, N.J.).
Rabbit anti-mouse IgGs (GE Healthcare) were immobilized on carboxymethylated dextran CM4 sensor chips (GE Healthcare) by amine coupling, according to a standard protocol. Analyses were performed at 25° C. and 37° C., using PBS containing 0.05% surfactant P20 as running buffer. The antibodies were captured in individual flow cells at a flow rate of 10 μl/min. Injection time was varied for each antibody to yield an Rmax between 30 and 60 RU. 250 μg/mL mouse Fc were injected at 30 μl/min for 120 seconds to block non-specific binding of antibodies to Fc portion of the protein when needed. Buffer, rhRon ECD/mFc or rhRON SEMA+PSI diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 300 seconds at 60 μl/minute. The dissociation phase was monitored for up to 3600 seconds. The surface was then regenerated with two 60-second injections of 10 mM Glycine-HCl, pH 1.7, at a flow rate of 60 μl/min. The rhRON ECD/mFc or rhRON SEMA+PSI concentration range tested was 0.625 nM to 20 nM.
Kinetic parameters were determined using the kinetic function of the BIAevaluation software (GE Healthcare) with double reference subtraction. Kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) were determined. Kinetic values of the monoclonal antibodies on rhRON ECD/mFc at 25° C. and 37° C. are summarized in Table 5.







TABLE 5







Antibody Binding to rhRON ECD/mFc












Measurements at 25° C.

Measurements at 37° C.
















Antibody
ka (1/Ms)
kd (1/s)
KD (M)
n
ka (1/Ms)
kd (1/s)
KD (M)
n



















07F01
AVG
4.0E+05
9.3E−05
2.3E−10
4
2.1E+06
3.5E−04
2.1E−10
3



STDEV
7.1E+04
5.5E−06
3.4E−11

2.4E+06
2.8E−04
7.1E−11


29B06
AVG
2.0E+05
1.3E−04
6.5E−10
3
2.3E+06
7.0E−04
2.8E−10
3



STDEV
3.5E+04
1.0E−05
1.2E−10

1.3E+06
4.8E−04
7.8E−11


17F06
AVG
1.7E+05
 4.6E−08*
 2.9E−13*
3
1.4E+05
2.4E−05
2.1E−10
3



STDEV
4.8E+04
3.3E−08
1.7E−13

3.1E+04
2.2E−05
2.4E−10


18H09
AVG
3.3E+05
5.7E−05
2.2E−10
3
1.8E+06
7.0E−04
4.0E−10
1



STDEV
1.5E+05
2.3E−05
1.6E−10


12B11
AVG
1.2E+05
5.9E−05
5.0E−10
3
2.0E+05
2.0E−04
1.1E−09
3



STDEV
2.8E+04
1.7E−05
4.6E−11

1.1E+05
3.8E−05
4.6E−10





*Outside instrument limit of detection






The data in Table 5 demonstrate that antibodies 07F01, 29B06, 17F06, 18H09, and 12B11 bind rhRON ECD/mFc with a KD of about 1 nM or less, 750 pM or less, 650 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 250 pM or less, 200 pM or less, 150 pM or less, 100 pM or less, or 50 pM or less.
Kinetic values of the monoclonal antibodies on rhRON SEMA+PSI at 25° C. and 37° C. are summarized in Table 6.







TABLE 6







Antibody Binding to rhRON SEMA + PSI












Measurements at 25° C.

Measurements at 37° C.
















Antibody
ka (1/Ms)
kd (1/s)
KD (M)
n
ka (1/Ms)
kd (1/s)
KD (M)
n



















07F01
AVG
5.2E+06
3.6E−04
8.8E−11
3
2.0E+06
8.0E−04
4.0E−10
3



STDEV
7.0E+06
4.3E−04
3.3E−11

2.1E+05
7.5E−05
8.3E−12


29B06
AVG
4.2E+05
7.0E−05
1.8E−10
3
5.2E+05
6.9E−04
1.3E−09
3



STDEV
1.2E+05
8.7E−06
6.1E−11

4.7E+04
4.9E−05
9.9E−11


17F06
AVG
1.9E+05
1.4E−06
9.0E−12
4
2.6E+05
2.1E−05
1.3E−10
3



STDEV
3.6E+04
1.7E−06
1.1E−11

1.2E+05
2.9E−05
1.9E−10


18H09
AVG
4.4E+05
3.8E−06
8.6E−12
3
5.8E+05
1.2E−04
2.2E−10
2



STDEV
2.7E+04
6.3E−06
1.4E−11

7.6E+04
5.3E−05
1.2E−10


12B11
AVG
No binding


2
No binding









The data in Table 6 demonstrate that antibodies 07F01, 29B06, 17F06 and 18H09 bind rhRON SEMA+PSI with a KD of about 1 nM or less, 750 pM or less, 650 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 250 pM or less, 200 pM or less, 150 pM or less, 100 pM or less, 75 pM or less, 50 pM or less, or 10 pM or less. Antibody 12B11 did not bind to rhRON SEMA+PSI.
Binding to cell surface human wild-type RON and the delta 160 RON variant by antibodies 29B06 and 07F01 was measured at 4° C., using Fluorescence Activated Cell Sorting (FACS). PC3 cells expressing the human wild-type RON, and HT29 cells expressing the delta 160 variant, were harvested using cell dissociation buffer (Invitrogen), washed twice with FACS buffer (PBS with 0.5% BSA), and treated for 10 minutes with Cyto Q Antibody diluent and FC receptor block (Innovex Biosciences, Richmond, Calif.). Purified antibodies were diluted in FACS buffer over a concentration range from 0.02 nM to 40 nM. Cells were incubated with 100 μl of antibody for one hour, washed with FACS buffer three times, and incubated for 45 minutes with goat anti-mouse PE-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.). Cells were washed three times with FACS buffer, resuspended in 300 μl of FACS buffer, and analyzed using a Beckman Coulter Cytomics FC 500 FACS instrument. Results are summarized in Table 7.










TABLE 7







29B06
07F01




















Human RON - KD (nM)
0.133
0.032



Human RON - KD range (nM)
0.089-0.177
0.025-0.039



Delta 160 RON - KD (nM)
0.146
0.024



Delta 160 RON - KD range (nM)
0.100-0.192
0.020-0.029










The results in Table 7 demonstrate that antibodies 29B06 and 07F01 bind both wild-type RON and the delta 160 RON variant on the cell surface with similar affinity.
Example 6
Cell Surface Binding
Binding to cell surface wild-type RON and delta 160 RON at 4° C. was determined for antibodies 07F01, 12B11, 17F06, 18H09, and 29B06, using FACS. Cells expressing wild-type RON (PC3), and cells expressing delta 160 RON (HT-29), were harvested using cell dissociation buffer (Invitrogen), washed twice with FACS buffer (0.5% BSA PBS) and treated with CytoQ Antibody diluent and FC receptor block (Innovex). Purified antibodies were diluted at a concentration of 10 μg/ml, in FACS buffer. Cells were incubated with 100 μl of antibody mix for one hour, washed with FACS buffer three times, and incubated for 45 minutes with goat anti-mouse PE conjugated antibody (Jackson Immunoresearch Laboratories). Cells were washed three times with FACS buffer, resuspended in 300 μl of FACS buffer and analyzed using a Beckman Coulter Cytomics FC 500 FACS instrument. Percent binding as compared to murine IgG control is shown in Table 8.









TABLE 8





Antibody
PC3 % cell surface binding
HT-29 % cell surface binding

















07F01
99.29
99.08


17F06
99.08
99.00


29B06
99.06
99.04


18H09
99.03
98.33


12B11
94.52
88.64


mIgG
5.50
5.62









The results in Table 8 demonstrate that antibodies 07F01, 29B06, 17F06, 18H09, and 12B11 bind both wild-type RON and the delta 160 RON variant expressed on the surface of cells.
Example 7
Receptor Internalization
Antibody-stimulated receptor internalization was measured using FACS. PC3 cells were used to measure antibody-stimulated internalization of the wild-type RON receptor. HT-29 cells were used for the delta 160 RON receptor variant. Antibodies were first conjugated with R-Phycocerthrin (Prozyme cat. No. PJ31K). All cells were washed with PBS and treated with CytoQ Antibody diluent and FC receptor block (Innovex). Cells were incubated with the antibodies (10 μg/ml) for 2 hours at 37° C. or at 4° C. The cells were transferred to 4° C., washed with an acidic solution (0.5 M NaCl, 0.18 M Acetic Acid, 0.5% Na azide) to strip off the antibody remaining on the cell surface, and fixed using BD CYTOFIX/CYTOPERMTM Plus kit (BD Biosciences, cat. No. 555028) to measure antibodies retained intracellularly due to internalization. At 37° C., cells can undergo antibody-mediated receptor internalization, and the process is inhibited at low temperature of 4° C., thus serving as a baseline (no internalization). The cells were analyzed using a Beckman Coulter Cytomics FC 500 FACS instrument. A lowered anti-RON median fluorescent intensity (MFI) and a left shift of the histograms at 4° C. compared to that obtained at 37° C. indicate antibody-induced receptor internalization. Receptor internalization was quantified by subtracting MFI at 4° C. from that at 37° C. Results are summarized in Table 9.









TABLE 9






MFI @ 37° C. − MFI @
MFI @ 37° C. − MFI @


Antibody
4° C. in PC-3 cells
4° C. in HT-29 cells

















mIgG control
−0.15
−0.07


29B06
0.49
0.00


07F01
0.21
0.22


12B11
0.48
0.81









These results demonstrate that antibodies, 29B06, 07F01 and 12B11 induce receptor internalization in PC-3 cells expressing wild-type RON. Only 07F01 and 12B11 induce receptor internalization in HT-29 cells expressing delta 160 RON variant.
Example 8
Inhibition of MSP-RON Binding
Antibodies 07F01, 12B11, 17F06, 18H09, and 29B06 were tested for inhibition of MSP binding to hRON SEMA+PSI, as measured by electrochemiluminescence (ECL) assay as described in Example 3. The antibodies (concentration range: 0.006-10 μg/mL) were incubated for 45 minutes at room temperature.
The MSP-hRON binding interaction was inhibited by antibodies 07F01, 18H06, and 29B06, but not by antibodies 17F06 and 12B11 (FIG. 6). The IC50 and maximum percent inhibition values for the antibodies (IgG1) are shown in Table 10.











TABLE 10









IC50 (nM)
Maximum Neutralization (%)













Antibody
Average
Std Dev
Average
Std Dev
n















07F01
0.26
0.05
88.3
2.1
3


18H09
0.91
0.15
86.9
6.7
3


29B06
1.11
0.06
87.6
4.7
3


12B11
N/A
N/A
44.8
20
3


17F06
N/A
N/A
7.9
11.2
2









The results in Table 10 demonstrate that antibodies 07F01, 18H09 and 29B06 block MSP binding to hRON SEMA+PSI, while antibodies 12B11 and 17F06 do not.
Example 9
Inhibition of Downstream Signaling by Anti-RON Antibodies
Antibodies 07F01, 12B11, 17F06, 18H09, and 29B06 were tested for inhibition of MSP-dependent phosphorylation of ERK, a RON downstream signaling molecule using the cell-based assay described in Example 3. The antibodies (concentration range: 0.006-10 μg/mL) in RPMI were added to the cells and incubated for one hour at 37° C.
Dose-dependent inhibition of ERK phosphorylation by antibodies 07F01, 12B11, 17F06, 18H09, and 29B06 is shown in Table 11 and FIG. 7.












TABLE 11







Antibody
Mean IC50 (nM)
Std Dev
N





















07F01
0.07
0.02
3



18H09
0.71
0.36
3



29B06
0.44
0.27
3



12B11
5.91
5.92
3



17F06
0.96
0.4
3










The results in Table 11 and FIG. 7 demonstrate that antibodies 07F01, 18H09, 29B06, 12B11 and 17F06 inhibit MSP-induced ERK phosphorylation in T47D breast cancer cell line, even though 12B11 and 17F06 do not effectively block MSP binding to RON (see Examples 3 and 8).
Example 10
Inhibition of MSP-Dependent Cell Migration
Antibodies 07F01, 18H09, 29B06, 12B11 and 17F06 were tested for inhibition of MSP-dependent cell migration. HPAF-II pancreatic cancer cells (ATCC) were incubated overnight under low serum conditions (1% FBS, MEM). Cells were trypsinized, counted, and placed at a concentration of 50,000/well in 45 μl of 1% FBS/MEM in the upper chamber of a BD 96-well FLUOROBLOKTM plate (Becton Dickinson). Antibodies were added at a concentration of 2 μg/ml, and cells were incubated for 2 hours. The bottom chamber contained 1% FBS MEM (200 μl) and 1 nM MSP, and cells were incubated for 24 hours. The number of migrated cells was determined by the addition of Calcien Dye at 4 μg/ml final concentration to the bottom chamber, followed by a one-hour incubation. Fluorescence intensity was measured using a WALLAC 1420 VICTORTM. Baseline fluorescent measurements were done in the absence of MSP. Percent inhibition was determined by comparing antibody-treated and antibody-untreated samples to the baseline using the following formula: 100-(anti-RON antibody treated-baseline)/(control huIgG treated-baseline)*100. Results on inhibition of MSP-induced HPAFII cell migration by antibodies 07F01, 18H09, 29B06, 12B11, and 17F06 are summarized in Table 12 and FIG. 8.










TABLE 12







Antibody (2 μg/ml)
Percent Inhibition



















07F01
95.63



29B06
96.79



17F06
70.74



18H09
106.96



12B11
98.93










The results in Table 12 demonstrate that antibodies 07F01, 18H09, 29B06, 12B11 and 17F06 inhibit MSP-dependent cell migration in HPAF-II pancreatic cancer cell lines, even though 12B11 and 17F06 do not effectively block MSP binding to RON.
Example 11
Inhibition of Growth of Wild-Type RON-Dependent Tumor Model
Inhibition of tumor growth was tested in a directed complementation model of wild-type RON-driven tumor growth. “Directed complementation” tumors were obtained as described in Robinson et al., U.S. Pat. No. 7,556,796. A cDNA encoding wild-type human RON was introduced into BH3 tumor cells by retroviral transfer. Transfected tumor cells were then implanted subcutaneously into recipient mice. Growth of the BH3 tumors was dependent on expression of an inducible HER2 gene, which was not induced. Therefore, tumors would grow only if the RON gene functionally complemented the uninduced HER2 gene. Growth of the directed complementation tumors was observed. Primary tumors were propagated in vivo to generate sufficient tumor material for drug efficacy studies. Quality control for the directed complemented tumors included RT-PCR for RON expression and immunohistochemistry (IHC) for protein expression. The tumors were stored as frozen archival aliquots of approximately 1.5×105 cells/vial. These tumors were thawed, washed once, resuspended in HBS+matrigel and injected subcutaneously. Tumor measurements were taken twice weekly using vernier calipers. Tumor volume was calculated using the formula: width×width×length/2. When tumors reached approximately 150 mm3, the mice were randomized into five groups of ten mice each. Each group (ten mice each) received one of the following antibody treatments: 07F01, 29B06, 12B11, or 18H09, or murine IgG control, all at 20 mg/kg. Treatment was administered by intra-peritoneal injection, twice weekly, for two weeks. Antibodies 29B06 and 07F01 resulted in tumor growth inhibition (“TGI”) greater than 50% (p<0.001), while antibodies 18H09 and 12B11 exhibited TGI of 25% and 29%, respectively (FIG. 9). All treatments were well-tolerated with no significant loss in body weight.
Pharmacodynamic changes in RON receptor levels after 29B06 and 07F01 treatment were evaluated. Tumors were treated with 20 mg/kg of the following antibodies: mIgG (control), 29B06 or 07F01 and tumors were harvested at 24 or 48 hours. After harvest, the tumors were lysed in standard RIPA buffer (Boston Bioproducts, cat. No. BP-115) containing protease inhibitors (Roche, catalog No. 04693159001) and phosphatase inhibitors I and II (Sigma, cat. Nos. P2350 and P5726). Lysates were cleared and protein concentration was measured. A Western blot for total RON was done using a polyclonal anti-RON antibody (Santa Cruz, cat. No. sc-322). The Western blot analysis showed that antibody 29B06 induced receptor degradation in vivo in RON-DC xenograft at 24 hours, and to a greater extent at 48 hours.
Example 12
Inhibition of Growth of Delta 160 RON-Driven Tumor Model
Inhibition of tumor growth by the antibodies was tested in a directed complementation model of delta 160 RON-driven tumor growth. The model was obtained as described in Example 11, except that the transfected cDNA encoded human delta 160 (oncogenic) form of RON. Growth of the directed complementation tumors was observed. Primary tumors were propagated in vivo to generate sufficient tumor material for drug efficacy studies. Quality control for the directed complimented tumors included RT-PCR for RON expression and IHC for protein expression. The tumors were stored as frozen archival aliquots of approximately 1.5×105 cells/vial. These tumors were thawed, washed once, resuspended in HBS plus matrigel, and injected subcutaneously. Tumor measurements were taken twice weekly. When tumors reached approximately 150 mm3, the mice were randomized into five groups of ten mice each. Each group (ten mice per group) received one of the following treatments: murine IgG control, 07F01, 29B06, 12B11, 17F06, and 18H09, all at 20 mg/kg. Treatment was administered by intra-peritoneal injection, twice weekly, for two weeks. Each treatment group showed similar tumor growth inhibition of greater than 60% (p<0.001) except for 18H09 (TGI 54%) as shown in FIG. 10. All treatments were well-tolerated, with no significant loss in body weight.
Example 13
Inhibition of Growth of NCI-H358 Lung Xenograft Tumor Model
Inhibition of tumor growth by the 29B06 antibody was tested in an NCI-H358 lung xenograft model. The NCI-H358 cells were grown in culture at 37° C. in an atmosphere containing 5% CO2, using RMPI medium (Invitrogen) containing 10% FBS. Cells were inoculated subcutaneously into the flank of 8-week old female CB.17 SCID mice with 5×106 cells per mouse in 50% matrigel. Tumor measurements were taken twice weekly. When tumors reached approximately 150 mm3, the mice were randomized into two groups of ten mice each. Each group received one of the following treatments: murine IgG control or 29B06 at 40 mg/kg. Treatment was administered by intra-peritoneal injection three times per week, for three weeks. Antibody 29B06 treatment resulted in tumor growth inhibition of 70% (p<0.001) (FIG. 11). Treatment was well-tolerated, with no significant loss in body weight.
Example 14
Humanization of Anti-RON Antibodies
A. Construction of Humanized and Chimeric Anti-RON Antibodies
This Example describes the humanization of two murine antibodies, designated 07F01 and 29B06, and the characterization of the resulting humanized antibodies. The humanized anti-RON antibodies were designed using the SUPERHUMANIZATIONTM method (Cephalon, Inc. (Arana Therapeutics Ltd.) and Hwang, W. Y. et al. (2005) METHODS 36:35-42), the CDR grafting method with back mutations (some human framework residues were changed to murine residues) (See e.g., U.S. Pat. Nos. 5,530,101; 5,693,761; 5,693,762; 5,585,089; 6,180,370; 7,022,500), or the HUMAN ENGINEERINGTM method (Studnicka et al., Protein Eng. 1994 June; 7(6):805-14; also see, e.g., PCT Publication No. WO 93/11794 and U.S. Pat. Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619). With the exception of heavy chain CDR1, the Kabat CDR definitions were used for CDR grafting onto human frameworks (SUPERHUMANIZATIONTM and CDR grafting with back mutations). In some cases, a combination of Kabat and Chothia definitions were used for grafting heavy CDR1. In some cases, CDR residues (Kabat or Chothia definitions) were changed to human residues to increase humanness. Models of the murine antibodies were created using the SWISS-MODEL web server (swissmodel.expasy.org). Predicted residue contacts were determined using the Contact Map Analysis web server (ligin.weizmann.ac.il/cma/), and residue surface accessibility was determined using the Accessible Molecular Surface web server (swift.cmbi.ru.nl/servers/html/accessres.html). Residues were selected for back mutation based on predicted surface accessibility, contact with CDR residues, and involvement in the interface between heavy and light chains. Additionally, a cysteine residue present in the heavy chain CDR3 of 07F01 was changed to serine to prevent potential aggregation, and in some examples, a predicted N-linked glycosylation consensus site (N-X-S/T) in 07F01 heavy CDR2 (e.g., N58, Y59, T60) was mutated (e.g., T60A) to prevent any possible glycosylation. The designed amino acid sequences were converted to codon-optimized DNA sequences and synthesized by DNA2.0, Inc. to include (in the following order): 5′ HindIII restriction site, Kozak consensus sequence, amino terminal signal sequence, humanized variable region, human IgG1 or Kappa constant region, stop codon, and a 3′ EcoRI restriction site.
The anti-RON antibody chains humanized according to the SUPERHUMANIZATIONTM method, as described herein, are designated with the prefix “Sh” before the antibody chain name. The anti-RON antibody chains humanized by the CDR grafting method with back mutations, as described herein, are designated with the prefix “Hu” before the antibody chain name. The anti-RON antibody chains humanized by the HUMAN ENGINEERINGTM method, as described herein, are designated with the prefix “HE” before the antibody chain name.
The anti-RON antibody heavy chain 07F01 was humanized according to the SUPERHUMANIZATIONTM method. Human germline sequence IGHV3-48*01 (also referred to herein as Hv3-48) was selected as the human heavy chain framework. In some embodiments, the human Hv3-48 heavy chain framework sequence was mutated at amino acid position 28 (e.g., D28T). Amino acid numbering is based on the Kabat numbering system.
The anti-RON antibody light chain 07F01 was humanized according to the HUMAN ENGINEERINGTM method. Human germline sequence IGKV1-9*01 was selected as the human light chain framework.
The anti-RON antibody heavy chain 29B06 was humanized by the CDR grafting method with back mutations. Human germline sequence IGHV4-59*01 (also referred to herein as Hv4-59) was selected as the human framework. The human framework was back-mutated at amino acid positions 27, 30, 39, 44, 47, 48, 67, 71, and 78 to the murine sequence when the Kabat CDR definitions were used. The back-mutated human Hv4-59 framework sequence was further mutated to comprise at least one amino acid substitution at positions 27, 30, 48, 67, and 78. Amino acid substitutions in the back-mutated Hv4-59 framework sequence (e.g., amino acid substitution from a murine residue to a human residue, e.g., a human residue found in IGHV4-59) may be selected from the group consisting of D27G, T30S, M48I, I67V and Y78F. Amino acid numbering is based on the Kabat numbering system.
The anti-RON antibody light chain 29B06 was humanized according to the SUPERHUMANIZATIONTM method. Human germline sequence IGKV2-28*01 was selected as the human light chain framework.
Chimeric (murine variable region and human constant region) 07F01 and 29B06 heavy (human IgG1) and light (human Kappa) chains were also constructed. The cysteine residue present in the heavy chain CDR3 of 07F01 was changed to serine to prevent potential aggregation. To generate chimeric antibodies, the murine variable regions were fused to the human constant region using overlap extension PCR, including (in the following order): 5′ HindIII restriction site, Kozak consensus sequence, amino terminal signal sequence, mouse variable region, human IgG1 or Kappa constant region, stop codon, and 3′ EcoRI restriction site.
The humanized and chimeric heavy chains were subcloned into pEE6.4 (Lonza, Basel, Switzerland) via HindIII and EcoRI sites using IN-FUSIONTM PCR cloning (Clontech, Mountain View, Calif.). The humanized and chimeric Kappa light chains were subcloned into pEE14.4 (Lonza) via HindIII and EcoRI sites using IN-FUSIONTM PCR cloning.
Humanized antibody chains or chimeric antibody chains were transiently transfected into 293T cells to produce antibody. Antibody was either purified or used in cell culture media supernatant for subsequent in vitro analysis. Binding of the chimeric and humanized antibodies to human RON was measured as described below. The results are summarized in Table 20.
Additionally, some humanized antibody heavy and light chain combinations were stably expressed in CHOK1SV cells using the GS SYSTEMTM (Lonza) in order to produce large quantities of purified humanized antibody. A single expression vector was constructed by combining pEE6.4 and pEE14.4 based vectors. First, pEE6.4 containing full length humanized heavy chain cDNA was digested with NotI and SalI to isolate the hCMV-MIE promoter+full length humanized heavy chain cDNA+SV40 poly A fragment. This fragment was inserted into the pEE14.4 vector already containing full length humanized light chain cDNA via NotI/SalI sites, thus creating an expression vector that simultaneously expresses heavy and light chains. The combined heavy and light chain vector was linearized and transfected into CHOK1SV cells. Stable clones were selected in the presence of methionine sulfoximine.
Each of the possible combinations of the humanized 07F01 immunoglobulin heavy chain and immunoglobulin light chain variable regions are set forth below in Table 13.








TABLE 13





Light Chain Variable Region
Heavy Chain Variable Region







HE L 07F01 Kv1-9 Light
Sh07F01 Hv3-48 Heavy Variable


Variable
(SEQ ID NO: 135)


(SEQ ID NO: 139)


HE L 07F01 Kv1-9 Light
Sh07F01 Hv3-48 D28T T60A


Variable
L63V E65G Heavy Variable


(SEQ ID NO: 139)
(SEQ ID NO: 137)


Sh07F01 Kv1-9 F1 Light
Sh07F01 Hv3-48 Heavy Variable


Variable
(SEQ ID NO: 135)


(SEQ ID NO: 141)


Sh07F01 Kv1-9 F1 Light
Sh07F01 Hv3-48 D28T T60A


Variable
L63V E65G Heavy Variable


(SEQ ID NO: 141)
(SEQ ID NO: 137)









Each of the possible combinations of the humanized 29B06 immunoglobulin heavy chain and immunoglobulin light chain variable regions are set forth below in Table 14.








TABLE 14





Light Chain Variable Region
Heavy Chain Variable Region







Sh29B06 Kv2-28 Kappa Variable
Sh29B06 Hv4-59 Heavy Variable


(SEQ ID NO: 149)
(SEQ ID NO: 143)


Sh29B06_Kv2-28 Kappa Variable
Hu29B06 Hv4-59 Heavy Variable


(SEQ ID NO: 149)
(SEQ ID NO: 145)


Sh29B06 Kv2-28 Kappa Variable
Hu29B06 Hv4-59 D27G T30S M48I


(SEQ ID NO: 149)
I67V Y78F Heavy Variable



(SEQ ID NO: 147)









The nucleic acid sequences encoding and the protein sequences defining variable regions of the humanized 07F01 and 29B06 antibodies are summarized below (amino terminal signal peptide sequences are not shown). Sequences of the modified chimeric 07F01 heavy variable region in which the cysteine in CDR3 is changed to serine are also summarized below. CDR sequences (Kabat definition) are shown in bold and are underlined in the amino acid sequences.








Nucleic Acid Sequence Encoding the Chimeric 07F01 C102S Heavy Chain Variable



Region


(SEQ ID NO: 132)










  1
gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc cgggtggatc cctgaaactc



 


 61
tcctgtgcag cctcaggatt cgattttagt agacactgga tgagttgggt ccggctggct


 


121
ccagggaaag ggctagaatg gatcgcagaa attaatccag atagcagaac gataaactat


 


181
acgccatctc taaaggagaa attcatcatc tccagagaca acgccaaaaa ttcgctgttt


 


241
ctgcaaatga acagagtgag atctgaggac acagcccttt attactgtgc aagacgggta


 


301
agaattcatt actacggcgc tatggacagc tggggtcaag gaacctcagt caccgtctcc


 


361
tca


 








Protein Sequence Defining the Chimeric 07F01 C102S Heavy Chain Variable Region



(SEQ ID NO: 133)










  1
evkllesggg lvqpggslkl scaasgfdfs rhwmswvrla pgkglewiae inpdsrtiny



 


 61
tpslkekfii srdnaknslf lqmnrvrsed talyycarrv rihyygamds wgqgtsvtvs


 


121
s


 








Nucleic Acid Sequence Encoding the Sh07F01 Hv3-48 Heavy Chain Variable Region



(SEQ ID NO: 134)










  1
gaggttcagc tggtagaatc cggaggaggg ttggtccaac ctggtggatc actcagactt



 


 61
tcatgcgccg ccagcggctt tgacttctca cgacattgga tgagctgggt ccggcaggct


 


121
ccaggcaagg gcctcgagtg ggttagcgag atcaatccag acagcagaac cattaactat


 


181
acacccagtc tgaaggagcg gttcaccata agccgtgata atgccaagaa ctccctgtac


 


241
ttgcagatga actccttgcg cgctgaagat acagctgtgt actattgtgc aaggcgcgtg


 


301
cgaatccact attacggggc aatggattct tggggccagg gtactaccgt gactgtgagt


 


361
tct


 








Protein Sequence Defining the Sh07F01 Hv3-48 Heavy Chain Variable Region



(SEQ ID NO: 135)










  1
evqlvesggg lvqpggslrl scaasgfdfs rhwmswvrga pgkglewvse inpdsrtiny



 


 61
tpslkerfti srdnaknsly lqmnslraed tavyycarrv rihyygamds wgqgttvtvs


 


121
s


 








Nucleic Acid Sequence Encoding the Sh07F01 Hv3-48 D28T T60A L63V E65G



Heavy Chain Variable Region


(SEQ ID NO: 136)










  1
gaggttcagc tggtagaatc cggaggaggg ttggtccaac ctggtggatc actcagactt



 


 61
tcatgcgccg ccagcggctt taccttctca cgacattgga tgagctgggt ccggcaggct


 


121
ccaggcaagg gcctcgagtg ggttagcgag atcaatccag acagcagaac cattaactat


 


181
gcccccagtg tgaagggccg gttcaccata agccgtgata atgccaagaa ctccctgtac


 


241
ttgcagatga actccttgcg cgctgaagat acagctgtgt actattgtgc aaggcgcgtg


 


301
cgaatccact attacggggc aatggattct tggggccagg gtactaccgt gactgtgagt


 


361
tct


 








Protein Sequence Defining the Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy



Chain Variable Region


(SEQ ID NO: 137)










  1
evqlvesggg lvqpggslrl scaasgftfs rhwmswvrqa pgkglewvse inpdsrtiny



 


 61
apsvkgrfti srdnaknsly lqmnslraed tavyycarrv rihyygamds wgqgttvtvs


 


121
s


 








Nucleic Acid Sequence Encoding the HE_L 07F01_Kv1-9 Kappa Chain Variable



Region


(SEQ ID NO: 138)










  1
gatatccagt tgactcagtc tcagtccttt gtgagtacat cagtgggcga cagggtcacc



 


 61
gtgacctgcc gagcatcaca gaacgttgga agctctcttg tctggtatca gcaaaagcct


 


121
gggaagagcc ccaaaaccct catctattct gcttcctttc tgtactccgg cgtaccaagt


 


181
agattctctg gtagcggatc cgggacagag ttcactctca caattagcag tgtgcagcct


 


241
gaggatttcg ccgactactt ctgtcagcaa tacaataact atcccctgac ttttggtggc


 


301
ggcaccaaag tggaaatcaa g


 








Protein Sequence Defining the HE L 07F01 Kv1-9 Kappa Chain Variable Region



(SEQ ID NO: 139)










  1
diqltqsqsf vstsvgdrvt vtcrasqnvg sslvwyqqkp gkspktliys asflysgvps



 


 61
rfsgsgsgte ftltissvqp edfadyfcgg ynnypltfgg gtkveik


 








Nucleic Acid Sequence Encoding the sh07F01 Kv1-9 F1 Kappa Chain Variable



Region


(SEQ ID NO: 140)










  1
gacattcagc tgactcagtc gccgtcgttt ttgtcggcgt ccgtgggtga cagagtgact



 


 61
atcacatgtc gcgcttcgca aaacgtcgga tcatcgcttg tgtggtatca gcagaaaccc


 


121
ggtaaagccc ctaagaccct catctattca gcgtcatttc tgtatagcgg ggtcccctca


 


181
cggttcagcg gatccggctc cgggaccgag ttcacactca ctatttcgag cttgcagccg


 


241
gaagattttg caacgtacta ctgccagcaa tacaataact acccactcac gttcggaggg


 


301
ggaacgaagg tagagatcaa g


 








Protein Sequence Defining the sh07F01 Kv1-9 F1 Kappa Chain Variable Region



(SEQ ID NO: 141)










  1
diqltqspsf lsasvgdrvt itcrasqnvg sslvwyqqkp gkapktliys asflysgvps



 


 61
rfsgsgsgte ftltisslqp edfatyycqq ynnypltfgg gtkveik


 








Nucleic Acid Sequence Encoding the Sh29B06_Hv4-59 Heavy Chain Variable



Region


(SEQ ID NO: 142)










  1
caagttcagc tgcaagaatc cggaccagga ttggtcaaac cttcagagac actcagcctg



 


 61
acttgcaccg tgagcggtgg cagcatatcc tccggttatt ggaactggat ccggcagcca


 


121
ccaggcaagg gcctcgagtg gattggctac atcagctata gcgggaaaac ctattacaac


 


181
cccagtctga agagccgagt gaccataagc gtcgatacaa gtaagaacca gttctccctg


 


241
aagctctcat ccgtgaccgc cgctgataca gctgtgtact attgtgcaag gtcaaagtat


 


301
gactacgcaa tggactattg gggccagggt actctggtga ctgtgagttc t


 








Protein Sequence Defining the Sh29B06 Hv4-59 Heavy Chain Variable Region



(SEQ ID NO: 143)










  1
qvqlqesgpg lvkpsetlsl tctvsggsis sgvwnwirqp pgkglewigy isysgktyyn



 


 61
pslksrvtis vdtsknqfsl klssvtaadt avyycarsky dyamdywgqg tlvtvss


 








Nucleic Acid Sequence Encoding the Hu29B06_Hv4-59 Heavy Chain Variable



Region


(SEQ ID NO: 144)










  1
caagttcagc tgcaagaatc cggaccagga ttggtcaaac ccagcgaaac actctctctt



 


 61
acatgcaccg tgagcggcga ctctatcacc tcagggtatt ggaattggat tcggaaaccc


 


121
ccaggcaaga agctcgagta catgggttac atcagttaca gcgggaaaac ctactataac


 


181
cccagtctga agagcagaat caccataagc cgtgatacct ctaagaacca gtactccctg


 


241
aagctgagtt ccgtaacagc agctgataca gctgtgtact attgtgcaag gagtaagtat


 


301
gactacgcaa tggactattg gggccagggt actcttgtga ctgtgagttc t


 








Protein Sequence Defining the Hu29B06_Hv4-59 Heavy Chain Variable Region



(SEQ ID NO: 145)










  1
qvqlqesgpg lvkpsetlsl tctvsgdsit sgvwnwirkp pgkkleymgy isysgktyyn



 


 61
pslksritis rdtsknqysl klssvtaadt avyycarsky dyamdywgqg tlvtvss


 








Nucleic Acid Sequence Encoding the Hu29B06 D27G T30S M48I I67V Y78F Heavy



Chain Variable Region


(SEQ ID NO: 146)










  1
caagttcagc tgcaagaatc cggaccagga ttggtcaaac cttcagagac actcagcctg



 


 61
acttgcaccg tgagcggtgg cagcatatcc tccggttatt ggaactggat ccggaagcca


 


121
ccaggcaaga agctcgagta cattggctac atcagctata gcgggaaaac ctattacaac


 


181
cccagtctga agagccgagt gaccataagc agggatacaa gtaagaacca gttctccctg


 


241
aagctctcat ccgtgaccgc cgctgataca gctgtgtact attgtgcaag gtcaaagtat


 


301
gactacgcaa tggactattg gggccagggt actctggtga ctgtgagttc t


 








Protein Sequence Defining the Hu29B06 D27G T30S M48I I67V Y78F Heavy Chain



Variable Region


(SEQ ID NO: 147)










  1
qvqlqesgpg lvkpsetlsl tctvsggsis sgvwnwirkp pgkkleyigy isysgktyyn



 


 61
pslksrvtis rdtsknqfsl klssvtaadt avyycarsky dyamdywgqg tlvtvss


 








Nucleic Acid Sequence Encoding the Sh29B06 Kv2-28 Kappa Chain Variable



Region


(SEQ ID NO: 148)










  1
gatatcgtta tgacccagag cccacttagt ttgcctgtta ctcctggcga gcctgccagt



 


 61
atttcttgcc gtgctagcga aatcgtggat aactttggta tatcattcat gaattggtat


 


121
ctccaaaaac ctggccaaag cccccagctc cttatctacg ccgctagcaa ccaggggtcc


 


181
ggggtacctg atagattttc aggcagcggc tctggaaccg acttcacact gaagatttcc


 


241
cgggtggagg ccgaggacgt gggcgtgtac tattgtcaac agtccaagga agtccctccc


 


301
actttcggcg gtgggacaaa ggttgagatt aag


 








Protein Sequence Defining the Sh29B06 Kv2-28 Kappa Chain Variable Region



(SEQ ID NO: 149)










  1
divmtqspls lpvtpgepas iscraseivd nfgisfmnwy lqkpgqspql liyaasnqgs



 


 61
gvpdrfsgsg sgtdftlkis rveaedvgvy ycqqskevpp tfgggtkvei k






The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 14 are aligned in FIGS. 12A and 12B. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR1, CDR2, and CDR3 (Kabat definition) are identified by boxes. FIGS. 13A and 13B show an alignment of the separate CDR1, CDR2, and CDR3 sequences for each of the variable region sequences shown in FIGS. 12A and 12B, respectively.
The amino acid sequences defining the immunoglobulin light chain variable regions for the antibodies in Example 14 are aligned in FIGS. 14A and 14B. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR1, CDR2 and CDR3 are identified by boxes. FIGS. 15A and 15B show an alignment of the separate CDR1, CDR2, and CDR3 sequences for each of the variable region sequences shown in FIGS. 14A and 14B, respectively.
Table 15 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.








TABLE 15





SEQ. ID NO.
Nucleic Acid or Protein
















132
Chimeric 07F01 C102S Heavy Chain Variable Region-



nucleic acid


133
Chimeric 07F01 C102S Heavy Chain Variable Region-



protein


5
Chimeric 07F01 C102S Heavy Chain CDR1


6
Chimeric 07F01 C102S Heavy Chain CDR2


123
Chimeric 07F01 C102S Heavy Chain CDR3


134
Sh07F01 Hv3-48 Heavy Chain Variable Region-nucleic



acid


135
Sh07F01 Hv3-48 Heavy Chain Variable Region-protein


5
Sh07F01 Hv3-48 Heavy Chain CDR1


6
Sh07F01 Hv3-48 Heavy Chain CDR2


123
Sh07F01 Hv3-48 Heavy Chain CDR3


136
Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy Chain



Variable Region-nucleic acid


137
Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy Chain



Variable Region-protein


5
Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy Chain



CDR1


122
Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy Chain



CDR2


123
Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy Chain



CDR3


138
HE L 07F01 Kv1-9 Light (kappa) Chain Variable



Region-nucleic acid


139
HE L 07F01 Kv1-9 Light (kappa) Chain Variable



Region-protein


130
HE L 07F01 Kv1-9 Light (kappa) Chain CDR1


131
HE L 07F01 Kv1-9 Light (kappa) Chain CDR2


10
HE L 07F01 Kv1-9 Light (kappa) Chain CDR3


140
Sh07F01 Kv1-9 F1 Light (kappa) Chain Variable



Region-nucleic acid


141
Sh07F01 Kv1-9 F1 Light (kappa) Chain Variable



Region-protein


130
Sh07F01 Kv1-9 F1 Light (kappa) Chain CDR1


131
Sh07F01 Kv1-9 F1 Light (kappa) Chain CDR2


10
Sh07F01 Kv1-9 F1 Light (kappa) Chain CDR3


142
Sh29B06 Hv4-59 Heavy Chain Variable Region-nucleic



acid


143
Sh29B06 Hv4-59 Heavy Chain Variable Region-protein


45
Sh29B06 Hv4-59 Heavy Chain CDR1


46
Sh29B06 Hv4-59 Heavy Chain CDR2


47
Sh29B06 Hv4-59 Heavy Chain CDR3


144
Hu29B06 Hv4-59 Heavy Chain Variable Region-nucleic



acid


145
Hu29B06 Hv4-59 Heavy Chain Variable Region-protein


45
Hu29B06 Hv4-59 Heavy Chain CDR1


46
Hu29B06 Hv4-59 Heavy Chain CDR2


47
Hu29B06 Hv4-59 Heavy Chain CDR3


146
Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F Heavy



Chain Variable Region-nucleic acid


147
Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F Heavy



Chain Variable Region-protein


45
Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F Heavy



Chain CDR1


46
Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F Heavy



Chain CDR2


47
Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F Heavy



Chain CDR3


148
Sh29B06 Kv2-28 Light (kappa) Chain Variable



Region-nucleic acid


149
Sh29B06 Kv2-28 Light (kappa) Chain Variable



Region-protein


48
Sh29B06 Kv2-28 Light (kappa) Chain CDR1


49
Sh29B06 Kv2-28 Light (kappa) Chain CDR2


50
Sh29B06 Kv2-28 Light (kappa) Chain CDR3









Humanized monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 16.










TABLE 16






CDR1
CDR2
CDR3















Kabat










07F01
RHWMS
EINPDSRTINYTPSLKE
RVRIHYYGAMDC



(SEQ ID NO: 5)
(SEQ ID NO: 6)
(SEQ ID NO: 7)


 


Chimeric 07F01
RHWMS
EINPDSRTINYTPSLKE
RVRIHYYGAMDS


C102S
(SEQ ID NO: 5)
(SEQ ID NO: 6)
(SEQ ID NO: 123)


 


Sh07F01 Hv3-
RHWMS
EINPDSRTINYTPSLKE
RVRIHYYGAMDS


48
(SEQ ID NO: 5)
(SEQ ID NO: 6)
(SEQ ID NO: 123)


 


Sh07F01 Hv3-
RHWMS
EINPDSRTINYAPSVKG
RVRIHYYGAMDS


48 D28T T60A
(SEQ ID NO: 5)
(SEQ ID NO: 122)
(SEQ ID NO: 123)


L63V E65G





 


29B06
SGYWN
YISYSGKTYYNPSLKS
SKYDYAMDY (SEQ



(SEQ ID NO: 45)
(SEQ ID NO: 46)
ID NO: 47)


 


Sh29B06 Hv4-
SGYWN
YISYSGKTYYNPSLKS
SKYDYAMDY (SEQ


59
(SEQ ID NO: 45)
(SEQ ID NO: 46)
ID NO: 47)


 


Hu29B06 Hv4-
SGYWN
YISYSGKTYYNPSLKS
SKYDYAMDY (SEQ


59
(SEQ ID NO: 45)
(SEQ ID NO: 46)
ID NO: 47)


 


Hu29B06 Hv4-59
SGYWN
YISYSGKTYYNPSLKS
SKYDYAMDY (SEQ


D27G T30S
(SEQ ID NO: 45)
(SEQ ID NO: 46)
ID NO: 47)


M48I I67V Y78F





 







Chothia










07F01
GFDFSRH
NPDSRT
RVRIHYYGAMDC



(SEQ ID NO: 51)
(SEQ ID NO: 52)
(SEQ ID NO: 7)


 


Chimeric 07F01
GFDFSRH
NPDSRT
RVRIHYYGAMDS


C102S
(SEQ ID NO: 51)
(SEQ ID NO: 52)
(SEQ ID NO: 125)


 


Sh07F01 Hv3-
GFDFSRH
NPDSRT
RVRIHYYGAMDS


48
(SEQ ID NO: 51)
(SEQ ID NO: 52)
(SEQ ID NO: 125)


 


Sh07F01 Hv3-
GFTFSRH
NPDSRT
RVRIHYYGAMDS


48 D28T T60A
(SEQ ID NO: 124)
(SEQ ID NO: 52)
(SEQ ID NO: 125)


L63V E65G





 


29B06
GDSITSG
SYSGK
SKYDYAMDY



(SEQ ID NO: 59)
(SEQ ID NO: 60)
(SEQ ID NO: 47)


 


Sh29B06 Hv4-
GGSISSG
SYSGK
SKYDYAMDY


59
(SEQ ID NO: 126)
(SEQ ID NO: 60)
(SEQ ID NO: 47)


 


Hu29B06 Hv4-
GDSITSG
SYSGK
SKYDYAMDY


59
(SEQ ID NO: 59)
(SEQ ID NO: 60)
(SEQ ID NO: 47)


 


Hu29B06 Hv4-
GGSISSG
SYSGK
SKYDYAMDY


59 D27G T30S
(SEQ ID NO: 126)
(SEQ ID NO: 60)
(SEQ ID NO: 47)


M48I I67V





Y78F





 







IMGT










07F01
GFDFSRHW
INPDSRTI
ARRVRIHYYGAMDC



(SEQ ID NO: 61)
(SEQ ID NO: 62)
(SEQ ID NO: 63)


 


Chimeric 07F01
GFDFSRHW
INPDSRTI
ARRVRIHYYGAMDS


C102S
(SEQ ID NO: 61)
(SEQ ID NO: 62)
(SEQ ID NO: 128)


 


Sh07F01 Hv3-
GFDFSRHW
INPDSRTI
ARRVRIHYYGAMDS


48
(SEQ ID NO: 61)
(SEQ ID NO: 62)
(SEQ ID NO: 128)


 


Sh07F01 Hv3-
GFTFSRHW
INPDSRTI
ARRVRIHYYGAMDS


48 D28T T60A
(SEQ ID NO: 127)
(SEQ ID NO: 62)
(SEQ ID NO: 128)


L63V E65G





 


29B06
GDSITSGY
ISYSGKT
ARSKYDYAMDY



(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)


 


Sh29B06 Hv4-
GGSISSGY
ISYSGKT
ARSKYDYAMDY


59
(SEQ ID NO: 129)
(SEQ ID NO: 74)
(SEQ ID NO: 75)


 


Hu29B06 Hv4-
GDSITSGY
ISYSGKT
ARSKYDYAMDY


59
(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)


 


Hu29B06 Hv4-
GGSISSGY
ISYSGKT
ARSKYDYAMDY


59 D27G T30S
(SEQ ID NO: 129)
(SEQ ID NO: 74)
(SEQ ID NO: 75)


M48I I67V





Y78F









Humanized monoclonal antibody Kappa light chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 17.










TABLE 17






CDR1
CDR2
CDR3















Kabat/Chothia










07F01
KASQNVGSSLV
SASFRYS
QQYNNYPLT



(SEQ ID NO: 8)
(SEQ ID NO: 9)
(SEQ ID NO: 10)


 


HE L 07F01 Kv1-9
RASQNVGSSLV
SASFLYS
QQYNNYPLT



(SEQ ID NO: 130)
(SEQ ID NO: 131)
(SEQ ID NO: 10)


 


Sh07F01 Kv1-9 F1
RASQNVGSSLV
SASFLYS
QQYNNYPLT



(SEQ ID NO: 130)
(SEQ ID NO: 131)
(SEQ ID NO: 10)


 


29B06
RASEIVDNFGISFMN
AASNQGS
QQSKEVPPT



(SEQ ID NO: 48)
(SEQ ID NO: 49)
(SEQ ID NO: 50)


 


Sh29B06 Kv2-28
RASEIVDNFGISFMN
AASNQGS
QQSKEVPPT



(SEQ ID NO: 48)
(SEQ ID NO: 49)
(SEQ ID NO: 50)


 







IMG










07F01
QNVGSS
SAS
QQYNNYPLT



(SEQ ID NO: 76)

(SEQ ID NO: 10)


 


HE L 07F01 Kv1-9
QNVGSS
SAS
QQYNNYPLT



(SEQ ID NO: 76)

(SEQ ID NO: 10)


 


Sh07F01 Kv1-9 F1
QNVGSS
SAS
QQYNNYPLT



(SEQ ID NO: 76)

(SEQ ID NO: 10)


 


29B06
EIVDNFGISF
AAS
QQSKEVPPT



(SEQ ID NO: 81)

(SEQ ID NO: 50)


 


Sh29B06 Kv2-28
EIVDNFGISF
AAS
QQSKEVPPT



(SEQ ID NO: 81)

(SEQ ID NO: 50)









To create the complete chimeric and humanized heavy or kappa chain antibody sequences, each variable sequence above is combined with its respective human constant region. For example, a complete heavy chain comprises a heavy variable sequence followed by a human IgG1 heavy chain constant sequence. A complete kappa chain comprises a kappa variable sequence followed by the human kappa light chain constant sequence.








Nucleic Acid Sequence Encoding the Human IgG1 Heavy Chain Constant Region



(SEQ ID NO: 150)










  1
gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg



 


 61
ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc


 


121
tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct


 


181
ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc


 


241
tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc


 


301
aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt


 


361
cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc


 


421
gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg


 


481
tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat


 


541
agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa


 


601
gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt


 


661
aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa


 


721
atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc


 


781
gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg


 


841
ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg


 


901
cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc


 


961
cagaagtcac tgagcctgag cccagggaag


 








Protein Sequence Defining the Human IgG1 Heavy Chain Constant Region



(SEQ ID NO: 151)










  1
astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss



 


 61
glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg


 


121
psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn


 


181
styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree


 


241
mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw


 


301
qqgnvfscsv mhealhnhyt qkslslspgk


 








Nucleic Acid Sequence Encoding the Human Kappa Light Chain Constant Region



(used for chimeric antibodies)


(SEQ ID NO: 152)










  1
cgcacagtcg ccgctccctc cgtgttcatc tttccaccaa gtgatgagca actgaagtct



 


 61
ggtactgctt cagtcgtgtg tctgctgaac aatttctacc ctcgagaagc caaagtccaa


 


121
tggaaggtag acaacgcact gcagtccggc aatagccaag aatcagttac cgaacaggat


 


181
tcaaaggaca gtacatattc cctgagcagc actctgaccc tgtcaaaggc cgattacgag


 


241
aaacacaagg tctatgcttg cgaagtgaca catcagggac tgtccagccc agtgacaaaa


 


301
tcttttaacc gtggggagtg t


 








Nucleic Acid Sequence Encoding the Human Kappa Light Chain Constant Region



(used for humanized antibodies)


(SEQ ID NO: 153)










  1
cgcacagttg ctgcccccag cgtgttcatt ttcccaccta gcgatgagca gctgaaaagc



 


 61
ggtactgcct ctgtcgtatg cttgctcaac aacttttacc cacgtgaggc taaggtgcag


 


121
tggaaagtgg ataatgcact tcaatctgga aacagtcaag agtccgtgac agaacaggac


 


181
agcaaagact caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa


 


241
aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc tgtcaccaag


 


301
tccttcaata ggggcgaatg t


 








Protein Sequence Defining the Human Kappa Light Chain Constant Region (used for



chimeric and humanized antibodies)


(SEQ ID NO: 154)










  1
rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd



 


 61
skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec






The following sequences represent the actual or contemplated full length heavy and light chain sequence (i.e., containing both the variable and constant regions sequences) for each antibody described in this Example. Signal sequences for proper secretion of the antibodies (e.g., signal sequences at the 5′ end of the DNA sequences or the amino terminal end of the protein sequences) are not shown in the full length heavy and light chain sequences disclosed herein and are not included in the final secreted protein. Also not shown are stop codons for termination of translation required at the 3′ end of the DNA sequences. It is within ordinary skill in the art to select a signal sequence and/or a stop codon for expression of the disclosed full length IgG heavy chain and light chain sequences. It is also contemplated that the variable region sequences can be ligated to other constant region sequences to produce active full length IgG heavy and light chains.








Nucleic Acid Sequence Encoding the Full Length Chimeric 07F01 C102S Heavy



Chain (Mouse Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 155)










   1
gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc cgggtggatc cctgaaactc



 


  61
tcctgtgcag cctcaggatt cgattttagt agacactgga tgagttgggt ccggctggct


 


 121
ccagggaaag ggctagaatg gatcgcagaa attaatccag atagcagaac gataaactat


 


 181
acgccatctc taaaggagaa attcatcatc tccagagaca acgccaaaaa ttcgctgttt


 


 241
ctgcaaatga acagagtgag atctgaggac acagcccttt attactgtgc aagacgggta


 


 301
agaattcatt actacggcgc tatggacagc tggggtcaag gaacctcagt caccgtctcc


 


 361
tcagcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


 


 421
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


 


 481
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


 


 541
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


 


 601
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


 


 661
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


 


 721
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


 


 781
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


 


 841
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


 


 901
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


 


 961
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


 


1021
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


 


1081
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


 


1141
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


 


1201
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


 


1261
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


 


1321
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Chimeric 07F01 C102S Heavy Chain



(Mouse Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 156)










   1
evkllesggg lvqpggslkl scaasgfdfs rhwmswvrla pgkglewiae inpdsrtiny



 


  61
tpslkekfii srdnaknslf lqmnrvrsed talyycarrv rihyygamds wgqgtsvtvs


 


 121
sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv swnsgaltsg vhtfpavlqs


 


 181
sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve pkscdkthtc ppcpapellg


 


 241
gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn wyvdgvevhn aktkpreeqy


 


 301
nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre


 


 361
emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr


 


 421
wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Chimeric 07F01 Light Chain



(Mouse Kappa Chain Variable Region and Human Kappa Constant Region)


(SEQ ID NO: 157)










   1
gacattgtgt tgacccagtc tcaaaaaatc gtgtccacat cagtaggagc cagggtcagc



 


  61
gtcacctgca aggccagtca gaatgtgggt tctagtttag tctggtatca acagaaacca


 


 121
ggtcaatctc ctaaaacact gatttactcg gcatccttcc ggtacagtgg agtccctgat


 


 181
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct


 


 241
gaagacttgg cagattattt ctgtcaacaa tataataact atccgctcac gttcggtgct


 


 301
gggaccaagc tggagctgaa acgcacagtc gccgctccct ccgtgttcat ctttccacca


 


 361
agtgatgagc aactgaagtc tggtactgct tcagtcgtgt gtctgctgaa caatttctac


 


 421
cctcgagaag ccaaagtcca atggaaggta gacaacgcac tgcagtccgg caatagccaa


 


 481
gaatcagtta ccgaacagga ttcaaaggac agtacatatt ccctgagcag cactctgacc


 


 541
ctgtcaaagg ccgattacga gaaacacaag gtctatgctt gcgaagtgac acatcaggga


 


 601
ctgtccagcc cagtgacaaa atcttttaac cgtggggagt gt


 








Protein Sequence Defining the Full Length Chimeric 07F01 Light Chain (Mouse



Kappa Chain Variable Region and Human Kappa Constant Region)


(SEQ ID NO: 158)










   1
divltqsqki vstsvgarvs vtckasqnvg sslvwyqqkp gqspktliys asfrysgvpd



 


  61
rftgsgsgtd ftltisnvqs edladyfcqq ynnypltfga gtklelkrtv aapsvfifpp


 


 121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt


 


 181
lskadyekhk vyacevthqg lsspvtksfn rgec


 








Nucleic Acid Sequence Encoding the Full Length Chimeric 29B06 Heavy Chain



(Mouse Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 159)










   1
gaggtgcagc ttcaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc



 


  61
acctgttctg tcactggcga ctccatcacc agtggttact ggaactggat ccggaaattc


 


 121
ccagggaata aacttgagta catggggtac ataagctaca gtggtaaaac ttactacaat


 


 181
ccatctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca ttactacctg


 


 241
cagttgattt ctgtgactgc tgaggacaca gccacatatt actgtgcaag gtctaagtac


 


 301
gactatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc agcctcaaca


 


 361
aaaggaccaa gtgtgttccc actcgcccct agcagcaaga gtacatccgg gggcactgca


 


 421
gcactcggct gcctcgtcaa ggattatttt ccagagccag taaccgtgag ctggaacagt


 


 481
ggagcactca cttctggtgt ccatactttt cctgctgtcc tgcaaagctc tggcctgtac


 


 541
tcactcagct ccgtcgtgac cgtgccatct tcatctctgg gcactcagac ctacatctgt


 


 601
aatgtaaacc acaagcctag caatactaag gtcgataagc gggtggaacc caagagctgc


 


 661
gacaagactc acacttgtcc cccatgccct gcccctgaac ttctgggcgg tcccagcgtc


 


 721
tttttgttcc caccaaagcc taaagatact ctgatgataa gtagaacacc cgaggtgaca


 


 781
tgtgttgttg tagacgtttc ccacgaggac ccagaggtta agttcaactg gtacgttgat


 


 841
ggagtcgaag tacataatgc taagaccaag cctagagagg agcagtataa tagtacatac


 


 901
cgtgtagtca gtgttctcac agtgctgcac caagactggc tcaacggcaa agaatacaaa


 


 961
tgcaaagtgt ccaacaaagc actcccagcc cctatcgaga agactattag taaggcaaag


 


1021
gggcagcctc gtgaaccaca ggtgtacact ctgccaccca gtagagagga aatgacaaag


 


1081
aaccaagtct cattgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgttgag


 


1141
tgggagagta acggtcagcc tgagaacaat tacaagacaa cccccccagt gctggatagt


 


1201
gacgggtctt tctttctgta cagtaagctg actgtggaca agtcccgctg gcagcagggt


 


1261
aacgtcttca gctgttccgt gatgcacgag gcattgcaca accactacac ccagaagtca


 


1321
ctgagcctga gcccagggaa g


 








Protein Sequence Defining the Full Length Chimeric 29B06 Heavy Chain (Mouse



Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 160)










   1
evqlqesgps lvkpsqtlsl tcsvtgdsit sgywnwirkf pgnkleymgy isysgktyyn



 


  61
pslksrisit rdtsknhyyl qlisvtaedt atyycarsky dyamdywgqg tsvtvssast


 


 121
kgpsvfplap sskstsggta algclvkdyf pepvtvswns galtsgvhtf pavlqssgly


 


 181
slssvvtvps sslgtqtyic nvnhkpsntk vdkrvepksc dkthtcppcp apellggpsv


 


 241
flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd gvevhnaktk preeqynsty


 


 301
rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak gqprepqvyt lppsreemtk


 


 361
nqvsltclvk gfypsdiave wesngqpenn ykttppvlds dgsfflyskl tvdksrwqqg


 


 421
nvfscsvmhe alhnhytqks lslspgk


 








Nucleic Acid Sequence Encoding the Full Length Chimeric 29B06 Light Chain



(Mouse Kappa Chain Variable Region and Human Kappa Constant Region)


(SEQ ID NO: 161)










   1
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctaggaca gagggccacc



 


  61
atctcctgca gagccagcga aattgttgat aattttggca ttagttttat gaactggttc


 


 121
caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc


 


 181
ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat


 


 241
cctgtggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttcctccg


 


 301
acgttcggtg gaggcaccaa gctggaaatc aaacgcacag tcgccgctcc ctccgtgttc


 


 361
atctttccac caagtgatga gcaactgaag tctggtactg cttcagtcgt gtgtctgctg


 


 421
aacaatttct accctcgaga agccaaagtc caatggaagg tagacaacgc actgcagtcc


 


 481
ggcaatagcc aagaatcagt taccgaacag gattcaaagg acagtacata ttccctgagc


 


 541
agcactctga ccctgtcaaa ggccgattac gagaaacaca aggtctatgc ttgcgaagtg


 


 601
acacatcagg gactgtccag cccagtgaca aaatctttta accgtgggga gtgt


 








Protein Sequence Defining the Full Length Chimeric 29B06 Light Chain (Mouse



Kappa Chain Variable Region and Human Kappa Constant Region)


(SEQ ID NO: 162)










   1
divltqspas layslgqrat iscraseivd nfgisfmnwf qqkpgqppkl liyaasnqgs



 


  61
gvparfsgsg sgtdfslnih pveeddtamy fcqqskevpp tfgggtklei krtvaapsvf


 


 121
ifppsdeqlk sgtasvvcll nnfypreakv qwkvdnalqs gnsqesvteq dskdstysls


 


 181
stltlskady ekhkvyacev thqglsspvt ksfnrgec


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh07F01 Hv3-48



Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 163)










   1
gaggttcagc tggtagaatc cggaggaggg ttggtccaac ctggtggatc actcagactt



 


  61
tcatgcgccg ccagcggctt tgacttctca cgacattgga tgagctgggt ccggcaggct


 


 121
ccaggcaagg gcctcgagtg ggttagcgag atcaatccag acagcagaac cattaactat


 


 181
acacccagtc tgaaggagcg gttcaccata agccgtgata atgccaagaa ctccctgtac


 


 241
ttgcagatga actccttgcg cgctgaagat acagctgtgt actattgtgc aaggcgcgtg


 


 301
cgaatccact attacggggc aatggattct tggggccagg gtactaccgt gactgtgagt


 


 361
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


 


 421
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


 


 481
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


 


 541
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


 


 601
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


 


 661
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


 


 721
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


 


 781
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


 


 841
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


 


 901
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


 


 961
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


 


1021
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


 


1081
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


 


1141
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


 


1201
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


 


1261
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


 


1321
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized Sh07F01 Hv3-48 Heavy



Chain(Humanized Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 164)










   1
evqlvesggg lvqpggslrl scaasgfdfs rhwmswvrqa pgkglewvse inpdsrtiny



 


  61
tpslkerfti srdnaknsly lqmnslraed tavyycarrv rihyygamds wgqgttvtvs


 


 121
sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv swnsgaltsg vhtfpavlqs


 


 181
sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve pkscdkthtc ppcpapellg


 


 241
gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn wyvdgvevhn aktkpreeqy


 


 301
nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre


 


 361
emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr


 


 421
wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh07F01 Hv3-48 D28T



T60A L63V E65G Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1


Constant Region)


(SEQ ID NO: 165)










   1
gaggttcagc tggtagaatc cggaggaggg ttggtccaac ctggtggatc actcagactt



 


  61
tcatgcgccg ccagcggctt taccttctca cgacattgga tgagctgggt ccggcaggct


 


 121
ccaggcaagg gcctcgagtg ggttagcgag atcaatccag acagcagaac cattaactat


 


 181
gcccccagtg tgaagggccg gttcaccata agccgtgata atgccaagaa ctccctgtac


 


 241
ttgcagatga actccttgcg cgctgaagat acagctgtgt actattgtgc aaggcgcgtg


 


 301
cgaatccact attacggggc aatggattct tggggccagg gtactaccgt gactgtgagt


 


 361
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


 


 421
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


 


 481
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


 


 541
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


 


 601
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


 


 661
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


 


 721
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


 


 781
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


 


 841
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


 


 901
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


 


 961
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


 


1021
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


 


1081
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


 


1141
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


 


1201
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


 


1261
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


 


1321
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized Sh07F01 Hv3-48 D28T T60A



L63V E65G Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1


Constant Region)


(SEQ ID NO: 166)










   1
evqlvesggg lvqpggslrl scaasgftfs rhwmswvrqa pgkglewvse inpdsrtiny



 


  61
apsvkgrfti srdnaknsly lqmnslraed tavyycarrv rihyygamds wgqgttvtvs


 


 121
sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv swnsgaltsg vhtfpavlqs


 


 181
sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve pkscdkthtc ppcpapellg


 


 241
gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn wyvdgvevhn aktkpreeqy


 


 301
nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre


 


 361
emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr


 


 421
wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized HE L 07F01 Kv1-9



Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 167)










   1
gatatccagt tgactcagtc tcagtccttt gtgagtacat cagtgggcga cagggtcacc



 


  61
gtgacctgcc gagcatcaca gaacgttgga agctctcttg tctggtatca gcaaaagcct


 


 121
gggaagagcc ccaaaaccct catctattct gcttcctttc tgtactccgg cgtaccaagt


 


 181
agattctctg gtagcggatc cgggacagag ttcactctca caattagcag tgtgcagcct


 


 241
gaggatttcg ccgactactt ctgtcagcaa tacaataact atcccctgac ttttggtggc


 


 301
ggcaccaaag tggaaatcaa gcgcacagtt gctgccccca gcgtgttcat tttcccacct


 


 361
agcgatgagc agctgaaaag cggtactgcc tctgtcgtat gcttgctcaa caacttttac


 


 421
ccacgtgagg ctaaggtgca gtggaaagtg gataatgcac ttcaatctgg aaacagtcaa


 


 481
gagtccgtga cagaacagga cagcaaagac tcaacttatt cactctcttc caccctgact


 


 541
ctgtccaagg cagactatga aaaacacaag gtatacgcct gcgaggttac acaccagggt


 


 601
ttgtctagtc ctgtcaccaa gtccttcaat aggggcgaat gt


 








Protein Sequence Defining the Full Length Humanized HE L 07F01 Kv1-9 Light



Chain (Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 168)










   1
diqltqsqsf vstsvgdrvt vtcrasqnvg sslvwyqqkp gkspktliys asflysgvps



 


  61
rfsgsgsgte ftltissvqp edfadyfcqq ynnypltfgg gtkveikrtv aapsvfifpp


 


 121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt


 


 181
lskadyekhk vyacevthqg lsspvtksfn rgec


 








Nucleic Acid Sequence Encoding the Full Length Humanized sh07F01 Kv1-9 F1



Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 169)










   1
gacattcagc tgactcagtc gccgtcgttt ttgtcggcgt ccgtgggtga cagagtgact



 


  61
atcacatgtc gcgcttcgca aaacgtcgga tcatcgcttg tgtggtatca gcagaaaccc


 


 121
ggtaaagccc ctaagaccct catctattca gcgtcatttc tgtatagcgg ggtcccctca


 


 181
cggttcagcg gatccggctc cgggaccgag ttcacactca ctatttcgag cttgcagccg


 


 241
gaagattttg caacgtacta ctgccagcaa tacaataact acccactcac gttcggaggg


 


 301
ggaacgaagg tagagatcaa gcgcacagtt gctgccccca gcgtgttcat tttcccacct


 


 361
agcgatgagc agctgaaaag cggtactgcc tctgtcgtat gcttgctcaa caacttttac


 


 421
ccacgtgagg ctaaggtgca gtggaaagtg gataatgcac ttcaatctgg aaacagtcaa


 


 481
gagtccgtga cagaacagga cagcaaagac tcaacttatt cactctcttc caccctgact


 


 541
ctgtccaagg cagactatga aaaacacaag gtatacgcct gcgaggttac acaccagggt


 


 601
ttgtctagtc ctgtcaccaa gtccttcaat aggggcgaat gt


 








Protein Sequence Defining the Full Length Humanized sh07F01 Kv1-9 F1 Light



Chain (Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 170)










   1
diqltqspsf lsasvgdrvt itcrasqnvg sslvwyqqkp gkapktliys asflysgvps



 


  61
rfsgsgsgte ftltisslqp edfatyycqq ynnypltfgg gtkveikrtv aapsvfifpp


 


 121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt


 


 181
lskadyekhk vyacevthqg lsspvtksfn rgec


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh29B06 Hv4-59



Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 171)










   1
caagttcagc tgcaagaatc cggaccagga ttggtcaaac cttcagagac actcagcctg



 


  61
acttgcaccg tgagcggtgg cagcatatcc tccggttatt ggaactggat ccggcagcca


 


 121
ccaggcaagg gcctcgagtg gattggctac atcagctata gcgggaaaac ctattacaac


 


 181
cccagtctga agagccgagt gaccataagc gtcgatacaa gtaagaacca gttctccctg


 


 241
aagctctcat ccgtgaccgc cgctgataca gctgtgtact attgtgcaag gtcaaagtat


 


 301
gactacgcaa tggactattg gggccagggt actctggtga ctgtgagttc tgcctcaaca


 


 361
aaaggaccaa gtgtgttccc actcgcccct agcagcaaga gtacatccgg gggcactgca


 


 421
gcactcggct gcctcgtcaa ggattatttt ccagagccag taaccgtgag ctggaacagt


 


 481
ggagcactca cttctggtgt ccatactttt cctgctgtcc tgcaaagctc tggcctgtac


 


 541
tcactcagct ccgtcgtgac cgtgccatct tcatctctgg gcactcagac ctacatctgt


 


 601
aatgtaaacc acaagcctag caatactaag gtcgataagc gggtggaacc caagagctgc


 


 661
gacaagactc acacttgtcc cccatgccct gcccctgaac ttctgggcgg tcccagcgtc


 


 721
tttttgttcc caccaaagcc taaagatact ctgatgataa gtagaacacc cgaggtgaca


 


 781
tgtgttgttg tagacgtttc ccacgaggac ccagaggtta agttcaactg gtacgttgat


 


 841
ggagtcgaag tacataatgc taagaccaag cctagagagg agcagtataa tagtacatac


 


 901
cgtgtagtca gtgttctcac agtgctgcac caagactggc tcaacggcaa agaatacaaa


 


 961
tgcaaagtgt ccaacaaagc actcccagcc cctatcgaga agactattag taaggcaaag


 


1021
gggcagcctc gtgaaccaca ggtgtacact ctgccaccca gtagagagga aatgacaaag


 


1081
aaccaagtct cattgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgttgag


 


1141
tgggagagta acggtcagcc tgagaacaat tacaagacaa cccccccagt gctggatagt


 


1201
gacgggtctt tctttctgta cagtaagctg actgtggaca agtcccgctg gcagcagggt


 


1261
aacgtcttca gctgttccgt gatgcacgag gcattgcaca accactacac ccagaagtca


 


1321
ctgagcctga gcccagggaa g


 








Protein Sequence Defining the Full Length Humanized Sh29B06 Hv4-59 Heavy



Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 172)










   1
qvqlqesgpg lvkpsetlsl tctvsggsis sgywnwirqp pgkglewigy isysgktyyn



 


  61
pslksrvtis vdtsknqfsl klssvtaadt avyycarsky dyamdywgqg tlvtvssast


 


 121
kgpsvfplap sskstsggta algclvkdyf pepvtvswns galtsgvhtf pavlqssgly


 


 181
slssvvtvps sslgtqtyic nvnhkpsntk vdkrvepksc dkthtcppcp apellggpsv


 


 241
flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd gvevhnaktk preeqynsty


 


 301
rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak gqprepqvyt lppsreemtk


 


 361
nqvsltclvk gfypsdiave wesngqpenn ykttppvlds dgsfflyskl tvdksrwqqg


 


 421
nvfscsvmhe alhnhytqks lslspgk


 








Nucleic Acid Sequence Encoding the Full Length Humanized Hu29B06 Hv4-59



Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 173)










   1
caagttcagc tgcaagaatc cggaccagga ttggtcaaac ccagcgaaac actctctctt



 


  61
acatgcaccg tgagcggcga ctctatcacc tcagggtatt ggaattggat tcggaaaccc


 


 121
ccaggcaaga agctcgagta catgggttac atcagttaca gcgggaaaac ctactataac


 


 181
cccagtctga agagcagaat caccataagc cgtgatacct ctaagaacca gtactccctg


 


 241
aagctgagtt ccgtaacagc agctgataca gctgtgtact attgtgcaag gagtaagtat


 


 301
gactacgcaa tggactattg gggccagggt actcttgtga ctgtgagttc tgcctcaaca


 


 361
aaaggaccaa gtgtgttccc actcgcccct agcagcaaga gtacatccgg gggcactgca


 


 421
gcactcggct gcctcgtcaa ggattatttt ccagagccag taaccgtgag ctggaacagt


 


 481
ggagcactca cttctggtgt ccatactttt cctgctgtcc tgcaaagctc tggcctgtac


 


 541
tcactcagct ccgtcgtgac cgtgccatct tcatctctgg gcactcagac ctacatctgt


 


 601
aatgtaaacc acaagcctag caatactaag gtcgataagc gggtggaacc caagagctgc


 


 661
gacaagactc acacttgtcc cccatgccct gcccctgaac ttctgggcgg tcccagcgtc


 


 721
tttttgttcc caccaaagcc taaagatact ctgatgataa gtagaacacc cgaggtgaca


 


 781
tgtgttgttg tagacgtttc ccacgaggac ccagaggtta agttcaactg gtacgttgat


 


 841
ggagtcgaag tacataatgc taagaccaag cctagagagg agcagtataa tagtacatac


 


 901
cgtgtagtca gtgttctcac agtgctgcac caagactggc tcaacggcaa agaatacaaa


 


 961
tgcaaagtgt ccaacaaagc actcccagcc cctatcgaga agactattag taaggcaaag


 


1021
gggcagcctc gtgaaccaca ggtgtacact ctgccaccca gtagagagga aatgacaaag


 


1081
aaccaagtct cattgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgttgag


 


1141
tgggagagta acggtcagcc tgagaacaat tacaagacaa cccccccagt gctggatagt


 


1201
gacgggtctt tctttctgta cagtaagctg actgtggaca agtcccgctg gcagcagggt


 


1261
aacgtcttca gctgttccgt gatgcacgag gcattgcaca accactacac ccagaagtca


 


1321
ctgagcctga gcccagggaa g


 








Protein Sequence Defining the Full Length Humanized Hu29B06 Hv4-59 Heavy



Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 174)










   1
qvqlgesgpg lvkpsetlsl tctvsgdsit sgywnwirkp pgkkleymgy isysgktyyn



 


  61
pslksritis rdtsknqysl klssvtaadt avyycarsky dyamdywgqg tlvtvssast


 


 121
kgpsvfplap sskstsggta algclvkdyf pepvtvswns galtsgvhtf pavlqssgly


 


 181
slssvvtvps sslgtqtyic nvnhkpsntk vdkrvepksc dkthtcppcp apellggpsv


 


 241
flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd gvevhnaktk preeqynsty


 


 301
rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak gqprepqvyt lppsreemtk


 


 361
nqvsltclvk gfypsdiave wesngqpenn ykttppvlds dgsfflyskl tvdksrwqqg


 


 421
nvfscsvmhe alhnhytqks lslspgk


 








Nucleic Acid Sequence Encoding the Full Length Humanized Hu29B06 Hv4-59



D27G T30S M48I I67V Y78F Heavy Chain (Humanized Heavy Chain Variable Region and


Human IgG1 Constant Region)


(SEQ ID NO: 175)










   1
caagttcagc tgcaagaatc cggaccagga ttggtcaaac cttcagagac actcagcctg



 


  61
acttgcaccg tgagcggtgg cagcatatcc tccggttatt ggaactggat ccggaagcca


 


 121
ccaggcaaga agctcgagta cattggctac atcagctata gcgggaaaac ctattacaac


 


 181
cccagtctga agagccgagt gaccataagc agggatacaa gtaagaacca gttctccctg


 


 241
aagctctcat ccgtgaccgc cgctgataca gctgtgtact attgtgcaag gtcaaagtat


 


 301
gactacgcaa tggactattg gggccagggt actctggtga ctgtgagttc tgcctcaaca


 


 361
aaaggaccaa gtgtgttccc actcgcccct agcagcaaga gtacatccgg gggcactgca


 


 421
gcactcggct gcctcgtcaa ggattatttt ccagagccag taaccgtgag ctggaacagt


 


 481
ggagcactca cttctggtgt ccatactttt cctgctgtcc tgcaaagctc tggcctgtac


 


 541
tcactcagct ccgtcgtgac cgtgccatct tcatctctgg gcactcagac ctacatctgt


 


 601
aatgtaaacc acaagcctag caatactaag gtcgataagc gggtggaacc caagagctgc


 


 661
gacaagactc acacttgtcc cccatgccct gcccctgaac ttctgggcgg tcccagcgtc


 


 721
tttttgttcc caccaaagcc taaagatact ctgatgataa gtagaacacc cgaggtgaca


 


 781
tgtgttgttg tagacgtttc ccacgaggac ccagaggtta agttcaactg gtacgttgat


 


 841
ggagtcgaag tacataatgc taagaccaag cctagagagg agcagtataa tagtacatac


 


 901
cgtgtagtca gtgttctcac agtgctgcac caagactggc tcaacggcaa agaatacaaa


 


 961
tgcaaagtgt ccaacaaagc actcccagcc cctatcgaga agactattag taaggcaaag


 


1021
gggcagcctc gtgaaccaca ggtgtacact ctgccaccca gtagagagga aatgacaaag


 


1081
aaccaagtct cattgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgttgag


 


1141
tgggagagta acggtcagcc tgagaacaat tacaagacaa cccccccagt gctggatagt


 


1201
gacgggtctt tctttctgta cagtaagctg actgtggaca agtcccgctg gcagcagggt


 


1261
aacgtcttca gctgttccgt gatgcacgag gcattgcaca accactacac ccagaagtca


 


1321
ctgagcctga gcccagggaa g


 








Protein Sequence Defining the Full Length Humanized Hu29B06 Hv4-59 D27G



T30S M48I I67V Y78F Heavy Chain (Humanized Heavy Chain Variable Region and Human


IgG1 Constant Region)


(SEQ ID NO: 176)










   1
qvglgesgpg lvkpsetlsl tctvsggsis sgywnwirkp pgkkleyigy isysgktyyn



 


  61
pslksrvtis rdtsknqfsl klssvtaadt avyycarsky dyamdywgqg tlvtvssast


 


 121
kgpsvfplap sskstsggta algclvkdyf pepvtvswns galtsgvhtf pavlqssgly


 


 181
slssvvtvps sslgtqtyic nvnhkpsntk vdkrvepksc dkthtcppcp apellggpsv


 


 241
flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd gvevhnaktk preeqynsty


 


 301
rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak gqprepqvyt lppsreemtk


 


 361
nqvsltclvk gfypsdiave wesnggpenn ykttppvlds dgsfflyskl tvdksrwqqg


 


 421
nvfscsvmhe alhnhytqks lslspgk


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh29B06 Kv2-28 Light



Chain (Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 177)










   1
gatatcgtta tgacccagag cccacttagt ttgcctgtta ctcctggcga gcctgccagt



 


  61
atttcttgcc gtgctagcga aatcgtggat aactttggta tatcattcat gaattggtat


 


 121
ctccaaaaac ctggccaaag cccccagctc cttatctacg ccgctagcaa ccaggggtcc


 


 181
ggggtacctg atagattttc aggcagcggc tctggaaccg acttcacact gaagatttcc


 


 241
cgggtggagg ccgaggacgt gggcgtgtac tattgtcaac agtccaagga agtccctccc


 


 301
actttcggcg gtgggacaaa ggttgagatt aagcgcacag ttgctgcccc cagcgtgttc


 


 361
attttcccac ctagcgatga gcagctgaaa agcggtactg cctctgtcgt atgcttgctc


 


 421
aacaactttt acccacgtga ggctaaggtg cagtggaaag tggataatgc acttcaatct


 


 481
ggaaacagtc aagagtccgt gacagaacag gacagcaaag actcaactta ttcactctct


 


 541
tccaccctga ctctgtccaa ggcagactat gaaaaacaca aggtatacgc ctgcgaggtt


 


 601
acacaccagg gtttgtctag tcctgtcacc aagtccttca ataggggcga atgt


 








Protein Sequence Defining the Full Length Humanized Sh29B06 Kv2-28 Light Chain



(Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 178)










   1
divmtqspls lpvtpgepas iscraseivd nfgisfmnwy lqkpggspql liyaasnqgs



 


  61
gvpdrfsgsg sgtdftlkis rveaedvgvy ycqqskevpp tfgggtkvei krtvaapsvf


 


 121
ifppsdeqlk sgtasvvcll nnfypreakv qwkvdnalqs gnsqesvteq dskdstysls


 


 181
stltlskady ekhkvyacev thqglsspvt ksfnrgec






For convenience, Table 18 provides a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.








TABLE 18





SEQ ID NO.
Nucleic Acid or Protein







150
Human IgG1 constant-nucleic acid


151
Human IgG1 constant-protein


152
Human Kappa constant (used for chimeric antibodies)-nucleic acid


153
Human Kappa constant (used for humanized antibodies)-nucleic acid


154
Human Kappa constant (used for chimeric and humanized antibodies)-protein


155
Chimeric 07F01 C102S Mouse Heavy Chain Variable + Human IgG1 constant-



nucleic acid


156
Chimeric 07F01 C102S Mouse Heavy Chain Variable + Human IgG1 constant-



protein


157
Chimeric 07F01 Mouse Light Chain Variable + Human Kappa constant-nucleic acid


158
Chimeric 07F01 Mouse Light Chain Variable + Human Kappa constant-protein


159
Chimeric 29B06 Mouse Heavy Chain Variable + Human IgG1 constant-nucleic acid


160
Chimeric 29B06 Mouse Heavy Chain Variable + Human IgG1 constant-protein


161
Chimeric 29B06 Mouse Light Chain Variable + Human Kappa constant-nucleic acid


162
Chimeric 29B06 Mouse Light Chain Variable + Human Kappa constant-protein


163
Humanized Sh07F01 Hv3-48 Heavy Human Variable + Human IgG1 constant-



nucleic acid


164
Humanized Sh07F01 Hv3-48 Heavy Human Variable + Human IgG1 constant-



protein


165
Humanized Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy Human Variable +



Human IgG1 constant-nucleic acid


166
Humanized Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy Human Variable +



Human IgG1 constant-protein


167
Humanized HE L 07F01 Kv1-9 Human Variable + Human Kappa constant-nucleic



acid


168
Humanized HE L 07F01 Kv1-9 Human Variable + Human Kappa constant-protein


169
Humanized sh07F01 Kv1-9 F1 Human Variable + Human Kappa constant-nucleic



acid


170
Humanized sh07F01 Kv1-9 F1 Human Variable + Human Kappa constant-protein


171
Humanized Sh29B06 Hv4-59 Heavy Human Variable + Human IgG1 constant-



nucleic acid


172
Humanized Sh29B06 Hv4-59 Heavy Human Variable + Human IgG1 constant-



protein


173
Humanized Hu29B06 Hv4-59 Heavy Human Variable + Human IgG1 constant-



nucleic acid


174
Humanized Hu29B06 Hv4-59 Heavy Human Variable + Human IgG1 constant-



protein


175
Humanized Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F Heavy Human Variable +



Human IgG1 constant-nucleic acid


176
Humanized Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F Heavy Human Variable +



Human IgG1 constant-protein


177
Humanized Sh29B06 Kv2-28 Human Variable + Human Kappa constant-nucleic



acid


178
Humanized Sh29B06 Kv2-28 Human Variable + Human Kappa constant-protein









Table 19 below shows antibodies containing chimeric immunoglobulin heavy and light chains and each of the possible combinations of the full-length chimeric or humanized immunoglobulin heavy and light chains.









TABLE 19





Antibody




Name
Light Chain
Heavy Chain







Sh07F01-2
Chimeric 07F01 Kappa
Chimeric 07F01 C102S



(SEQ ID NO: 158)
Heavy IgG1




(SEQ ID NO: 156)


Sh07F01-43
HE L 07F01 Kv1-9
Sh07F01 Hv3-48 IgG1



Kappa
(SEQ ID NO: 164)



(SEQ ID NO: 168)


Sh07F01-62
HE L 07F01 Kv1-9
Sh07F01 Hv3-48 D28T



Kappa
T60A L63V E65G IgG1



(SEQ ID NO: 168)
(SEQ ID NO: 166)


Sh07F01-69
Sh07F01 Kv1-9 F1
Sh07F01 Hv3-48 IgG1



Kappa
(SEQ ID NO: 164)



(SEQ ID NO: 170)


Sh07F01-83
Sh07F01 Kv1-9 F1
Sh07F01 Hv3-48 D28T



Kappa
T60A L63V E65G IgG1



(SEQ ID NO: 170)
(SEQ ID NO: 166)


Sh07F01-99
Chimeric 07F01 Kappa
Sh07F01 Hv3-48 IgG1



(SEQ ID NO: 158)
(SEQ ID NO: 164)


Sh07F01-100
Chimeric 07F01 Kappa
Sh07F01 Hv3-48 D28T



(SEQ ID NO: 158)
T60A L63V E65G IgG1




(SEQ ID NO: 166)


Sh07F01-101
HE L 07F01 Kv1-9
Chimeric 07F01 C102S



Kappa
Heavy IgG1



(SEQ ID NO: 168)
(SEQ ID NO: 156)


Sh07F01-102
Sh07F01 Kv1-9 F1
Chimeric 07F01 C102S



Kappa
Heavy IgG1



(SEQ ID NO: 170)
(SEQ ID NO: 156)


Sh29B06-1
Chimeric 29B06 Kappa
Chimeric 29B06 Heavy IgG1



(SEQ ID NO: 162)
(SEQ ID NO: 160)


Sh29B06-2
Chimeric 29B06 Kappa
Hu29B06 Hv4-59 IgG1



(SEQ ID NO: 162)
(SEQ ID NO: 174)


Sh29B06-4
Chimeric 29B06 Kappa
Sh29B06 Hv4-59 IgG1



(SEQ ID NO: 162)
(SEQ ID NO: 172)


Sh29B06-9
Sh29B06 Kv2-28 Kappa
Chimeric 29B06 Heavy IgG1



(SEQ ID NO: 178)
(SEQ ID NO: 160)


Sh29B06-23
Sh29B06 Kv2-28 Kappa
Hu29B06 Hv4-59 IgG1



(SEQ ID NO: 178)
(SEQ ID NO: 174)


Sh29B06-25
Sh29B06 Kv2-28 Kappa
Sh29B06 Hv4-59 IgG1



(SEQ ID NO: 178)
(SEQ ID NO: 172)


Sh29B06-78
Sh29B06 Kv2-28 Kappa
Hu29B06 Hv4-59 D27G



(SEQ ID NO: 178)
T30S M48I I67V Y78F IgG1




(SEQ ID NO: 176)


Sh29B06-84
Chimeric 29B06 Kappa
Hu29B06 Hv4-59 D27G



(SEQ ID NO: 162)
T30S M48I I67V Y78F IgG1




(SEQ ID NO: 176)









The antibody constructs containing the full length chimeric heavy and light chains are designated below:

    
    
        Chimeric 07F01 C102S=Full Length Chimeric 07F01 C102S Heavy Chain (Mouse Variable Region with C102S mutation and Human IgG1 Constant Region) (SEQ ID NO: 156) plus Full Length Chimeric 07F01 Light Chain (Mouse Variable Region and Human Kappa Constant Region) (SEQ ID NO: 158)
        Chimeric 29B06=Full Length Chimeric 29B06 Heavy Chain (Mouse Variable Region and Human IgG1 Constant Region) (SEQ ID NO: 160) plus Full Length Chimeric 29B06 Light Chain (Mouse Variable Region and Human Kappa Constant Region) (SEQ ID NO: 162)
    
    


Two of the possible antibody constructs containing the full length immunoglobulin heavy and light chains containing humanized variable regions are designated below:

    
    
        Sh07F01-62=Humanized Sh07F01 Hv3-48 D28T T60A L63V E65G Heavy Chain Variable Region and Human IgG1 Constant Region (SEQ ID NO: 166) plus HE L 07F01 Kv1-9 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 168)
        Sh29B06-78=Humanized Hu29B06 Hv4-59 D27G T30S M48I I67V Y78F Heavy Chain Variable Region and Human IgG1 Constant Region (SEQ ID NO: 176) plus Sh29B06 Kv2-28 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 178)
    
    


B. Binding Affinities of Humanized and Chimeric Anti-RON Monoclonal Antibodies
The binding affinities and kinetics of interaction of monoclonal antibodies produced in Example 14 against recombinant human RON SEMA and PSI domains (rhRON SEMA+PSI) (R&D Systems, Inc., Minneapolis, Minn.) were measured by surface plasmon resonance using a Biacore T100 (Biacore (GE Healthcare), Piscataway, N.J.) instrument.
Goat anti-human IgG Fc (Jackson ImmunoResearch, Catalog No. 109-005-098) was immobilized on carboxymethylated dextran CM4 sensor chips (Biacore) by amine coupling (Biacore) using a standard coupling protocol according to the vendor's instructions. The analyses were performed at 37° C. using PBS (Invitrogen) containing 0.05% surfactant P20 (Biacore) as running buffer.
The antibodies were captured in individual flow cells at a flow rate of 60 μl/minute. Injection time was varied for each antibody to yield an Rmax between 30 and 60 RU. Buffer or rhRON SEMA+PSI diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 300 seconds at 60 μl/minute. The dissociation phase was monitored for up to 1200 seconds. The surface was then regenerated with two 60 second injections of Glycine pH 2.25 (made from Glycine pH 2.0 (Biacore) and pH 2.5 (Biacore)) at 60 μl/minute. For the initial screening, only one or two concentrations of rhRON SEMA+PSI were tested, typically 10.0 and 2.5 nM (results are summarized in Table 20).
Kinetic parameters were determined using the kinetic function of the BIAevaluation software (Biacore) with double reference subtraction. Kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) were determined. Certain monoclonal antibodies were screened using cell culture media supernatant containing secreted antibody, and kinetic values of the monoclonal antibodies on rhRON SEMA+PSI at 37° C. are summarized in Table 20.













TABLE 20







Antibody
ka (1/Ms)
kd (1/s)
KD (M)
n









Sh07F01-2
2.0E+06
7.3E−04
3.8E−10
3



Sh07F01-62
3.9E+06
1.4E−03
3.6E−10
2



Sh07F01-69
2.3E+06
1.2E−03
5.6E−10
2



Sh07F01-76
2.3E+06
1.3E−03
5.7E−10
2



Sh07F01-83
2.6E+06
1.4E−03
5.4E−10
2



Sh29B06-1
6.7E+05
7.6E−04
1.1E−09
3



Sh29B06-9
8.7E+05
2.2E−04
2.6E−10
1



Sh29B06-23
7.8E+05
4.8E−04
6.4E−10
4











Sh29B06-25
No Binding










The results in Table 20 demonstrate that the chimeric and each of the humanized antibodies, except Sh29B06-25, have fast association rates (ka), very slow disassociation rates (kd) and very high affinities (KD). In particular, the antibodies have affinities ranging from about 260 pM to about 1.1 nM. No binding was observed for Sh29B06-25. Because Sh29B06-25 does not bind rhRON SEMA+PSI and Sh29B06-23 does, one or more of the back mutations present in the heavy chain of Sh29B06-23 appear to be required for binding with high affinity.
The binding affinities and kinetics of certain purified monoclonal antibodies were also determined. To further characterize certain antibodies, the surface plasmon resonance experiments described above were conducted using concentrations of rhRON SEMA+PSI between 0.3125 nM and 10.0 nM (a 2-fold serial dilution).
The kinetic values of certain purified monoclonal antibodies (i.e., Sh07F01-62 and Sh29B06-78) on rhRON SEMA+PSI at 25° C. and 37° C. are summarized in Table 21.







TABLE 21







Antibody Binding to rhRON SEMA + PSI










Measurements at 25° C.
Measurements at 37° C.















Antibody
ka (1/Ms)
kd (1/s)
KD (M)
n
ka (1/Ms)
kd (1/s)
KD (M)
n





Sh07F01-2
1.2E+06
9.8E−05
8.2E−11
9
1.7E+06
5.3E−04
3.1E−10
9


Sh07F01-43
1.2E+06
1.1E−04
9.0E−11
3
1.8E+06
5.6E−04
3.0E−10
3


Sh07F01-62
1.8E+06
1.6E−04
8.5E−11
4
2.8E+06
6.9E−04
2.5E−10
4


Sh07F01-69
1.1E+06
1.4E−04
1.2E−10
2
2.5E+06
7.8E−04
3.0E−10
2


Sh07F01-76
9.8E+05
1.3E−04
1.3E−10
2
2.4E+06
7.9E−04
3.3E−10
2


Sh07F01-83
1.6E+06
1.8E−04
1.1E−10
2
3.2E+06
7.9E−04
2.4E−10
2


Sh29B06-1
5.3E+05
2.0E−04
3.6E−10
6
8.2E+05
7.0E−04
8.6E−10
5


Sh29B06-23
6.7E+05
9.5E−05
1.4E−10
4
7.3E+05
3.3E−04
4.6E−10
5


Sh29B06-78
7.5E+05
3.9E−05
5.2E−11
7
1.0E+06
1.1E−04
1.1E−10
9









The results in Table 21 demonstrate the purified antibodies have affinities ranging from about 52 pM to 360 pM when tested at 25° C. or about 110 pM to about 860 pM when tested at 37° C.
Binding to cell surface human wild-type RON and the delta 160 RON variant by antibodies 07F01, Sh07F01-62, 29B06, and Sh29B06-78 was measured at 4° C., using Fluorescence Activated Cell Sorting (FACS). PC3 cells expressing the human wild-type RON, and HT29 cells expressing the delta 160 variant, were harvested using cell dissociation buffer (Invitrogen), washed twice with FACS buffer (PBS with 0.5% BSA), and treated 10 minutes with Cyto Q Antibody diluent and FC receptor block (Innovex Biosciences, Richmond, Calif.). Purified antibodies were diluted in FACS buffer over a concentration range from 0.01 nM to 25 nM. Cells were incubated with 100 μl of antibody for one hour, washed with FACS buffer three times, and incubated for 45 minutes with goat anti-mouse PE-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.) or donkey anti-human PE-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.). Cells were washed three times with FACS buffer, resuspended in 300 μl of FACS buffer, and analyzed using a Beckman Coulter Cytomics FC 500 FACS instrument. All four antibodies were compared in the same experiment. Results are summarized in Table 22.












TABLE 22







07F01
Sh07F01-62
29B06
Sh29B06-78




















Human RON - KD
0.053
0.043
0.136
0.090


(nM)


Human RON - KD
0.036 to
0.026 to
0.083 to
0.063 to


range (nM)
0.069
0.060
0.190
0.117


Delta 160 RON - KD
0.100
0.118
0.167
0.239


(nM)


Delta 160 RON - KD
0.071 to
0.045 to
0.066 to
0.202 to


range (nM)
0.129
0.191
0.267
0.277









The results in Table 22 demonstrate that the humanized antibodies Sh07F01-62 and Sh29B06-78 retain their ability to bind both wild-type RON and the delta 160 RON variant on the cell surface with affinities equivalent to their murine antibody counterparts (i.e., 07F01 and 29B06, respectively).
C. Comparison of Other Anti-RON Antibodies
Three antibodies that inhibit the function of human RON were constructed and expressed using published information. One antibody, referred to as 1P3B2-BIIB Ab, was constructed based on the disclosure of Huet et al., U.S. Patent Publication No. 2009/0226442 (Biogen Idec, Inc.). Two additional antibodies, referred to as RON6 and RON8, were constructed based on the disclosure of Pereira et al., U.S. Patent Publication No. 2009/0136510 (Imclone Systems, Inc.).
Kinetic parameters for the 1P3B2-BIIB Ab, RON6, and RON8 antibodies on rhRON SEMA+PSI at 25° C. and 37° C. were determined by Biacore as described above (See Section B. Binding Affinities of Humanized and Chimeric Anti-RON Monoclonal Antibodies). The kinetic values for each antibody are summarized in Table 23.







TABLE 23







Antibody Binding to rhRON SEMA + PSI










Measurements at 25° C.
Measurements at 37° C.















Antibody
ka (1/Ms)
kd (1/s)
KD (M)
n
ka (1/Ms)
kd (1/s)
KD (M)
n





Sh29B06-78
6.8E+05
3.1E−05
4.8E−11
6
9.6E+05
1.0E−04
1.1E−10
8


Sh07F01-62
1.8E+06
1.6E−04
8.5E−11
4
2.8E+06
6.9E−04
2.5E−10
4


1P3B2-BIIB
1.5E+06
1.2E−03
8.0E−10
1
2.2E+07
2.6E−02
1.2E−09
1


RON6
2.3E+06
2.6E−03
1.1E−09
1
1.9E+10
1.9E−01
1.0E−09
1


RON8
1.2E+06
6.8E−04
6.7E−10
3
7.0E+06
2.5E−03
9.2E−10
3









The results in Table 23 demonstrate that the overall equilibrium dissociation constant (KD) for Sh29B06-78 and Sh07F01-62 were smaller (i.e., higher affinity) than the KD for 1P3B2-BIIB, RON6, and RON8 at both 25° C. and 37° C. The KD of 1P3B2-BIIB, RON6, and RON8 antibodies can also be compared with other humanized 29B06 or 07F01 variants by comparing Tables 21 and 23.
Therefore, the binding affinities of Sh29B06-78 and Sh07F01-62 are significantly higher than the affinities of 1P3B2-BIIB, RON6, and RON8 antibodies as disclosed herein.
Example 15
Inhibition of MSP-RON Binding
The chimeric and humanized antibodies produced in Example 14 were tested for inhibition of MSP binding to hRON SEMA+PSI, as measured by electrochemiluminescence (ECL) assay as described in Example 3. The antibodies (concentration range: 0.006-10 μg/mL) were incubated for 45 minutes at room temperature.
The MSP-hRON binding interaction was inhibited by the chimeric and humanized antibodies listed in Table 24, which were tested in this assay. The IC50 for the antibodies (IgG1) are shown in Table 24.












TABLE 24







Antibody
Mean IC50
Std Dev of IC50
N









Sh29B06-1
1.73
1.24
8



Sh29B06-23
1.24
1.57
9



Sh29B06-78
0.41
0.24
8



Sh07F01-2
0.91
1.42
8



Sh07F01-43
0.22
0.09
2



Sh07F01-62
0.32
0.12
6



Sh07F01-69
0.28
0.18
2



Sh07F01-76
0.38
0.33
2



Sh07F01-83
0.33
0.24
2










The results in Table 24 demonstrate that the chimeric and humanized anti-RON antibodies listed in Table 24 (i.e., Sh29B06-1, Sh29B06-23, Sh29B06-78, Sh07F01-2, Sh07F01-43, Sh07F01-62, Sh07F01-69, Sh07F01-76, and Sh07F01-83) retain the ability to block MSP binding to hRON SEMA+PSI with high potency.
Example 16
Inhibition of Downstream Signaling by Anti-RON Antibodies
The chimeric and humanized anti-RON antibodies produced in Example 14 were tested for their ability to inhibit MSP-induced phosphorylation of ERK, a RON downstream signaling molecule, using the cell-based assay described in Example 3. The antibodies (concentration range: 0.006-10 μg/mL) in RPMI were added to the cells and incubated for one hour at 37° C. The IC50s of ERK phosphorylation inhibition by the chimeric and humanized anti-RON antibodies tested in this assay are shown in Table 25.












TABLE 25







Antibody
Mean IC50
Std Dev of IC50
N





















Sh29B06-1
0.10
0.10
6



Sh29B06-23
0.11
0.08
10



Sh29B06-78
0.13
0.08
5



Sh07F01-2
0.06
0.06
7



Sh07F01-43
0.02
0.00
3



Sh07F01-62
0.03
0.03
2



Sh07F01-69
0.05
0.02
2



Sh07F01-76
0.10
0.03
2



Sh07F01-83
0.03
0.02
2










The results in Table 25 demonstrate that the chimeric and humanized anti-RON antibodies listed in Table 25 (i.e., Sh29B06-1, Sh29B06-23, Sh29B06-78, Sh07F01-2, Sh07F01-43, Sh07F01-62, Sh07F01-69, Sh07F01-76, and Sh07F01-83) inhibit MSP-induced ERK phosphorylation in T47D breast cancer cell line with high potency.
Example 17
Inhibition of MSP-Dependent Cell Migration
Humanized antibodies sh29B06-78 and sh07F01-62 as produced in Example 14 were tested for their ability to inhibit MSP-induced cell migration as described in Example 10. In this example, antibodies were added at a concentration of 1 μg/ml and serially diluted at a 1:5 dilution, and cells were incubated for 2 hours. Percent inhibition was determined by the following formula: 100−(anti-RON antibody treated-baseline)/(control huIgG treated-baseline)*100. Results on inhibition of MSP-induced HPAF-II cell migration by anti-RON antibodies, sh29B06-78 and sh07F01-62, are summarized in Table 26 and FIG. 16.








TABLE 26







AB












concentration
sh29B06-78

sh07F01-62












ng/ml
AVG
Std DEV
AVG
Std DEV














1000.00
94.82
3.34
98.96
3.79


200.00
90.67
2.37
97.80
1.12


40.00
59.85
12.50
67.18
7.67


8.00
59.71
2.87
37.22
4.16


1.60
63.95
20.15
38.91
13.79


0.32
42.03
39.88
43.27
5.76


0.06
60.37
11.92
34.40
2.31









The results in Table 26 demonstrate that humanized anti-RON antibodies, sh29B06-78 and sh07F01-62, potently inhibit MSP-induced cell migration in HPAF-II pancreatic cancer cell lines.
Example 18
Inhibition of MSP-Induced Cell Invasion
Humanized antibodies sh29B06-78 and sh07F01-62 as produced in Example 14 were tested for their ability to inhibit MSP-induced cell invasion. HPAF-II pancreatic cancer cells were trypsinized, counted, and placed at a concentration of 50,000/well in 45 μl of 10% FBS/MEM in the upper chamber of a BD 96-well BD BIOCOATTM MATRIGELTM invasion FLUOROBLOKTM plate (Becton Dickinson). Antibodies were added at a concentration of 30 μg/ml and cells were incubated for 2 hours. The bottom chamber contained 10% FBS MEM (2000 and 1 nM MSP, and cells were incubated for 24 hours. The number of cells that underwent invasion through the membrane was determined by the addition of Calcien Dye at 4 μg/ml final concentration to the bottom chamber, followed by a one-hour incubation. Fluorescence intensity was measured using a WALLAC 1420 VICTORTM instrument. Results on inhibition of MSP-induced HPAFII cell invasion by anti-RON antibodies are summarized in FIG. 17.
The results in FIG. 17 demonstrate that humanized anti-RON antibodies sh29B06-78 and sh07F01-6 potently inhibit MSP-dependent cell invasion in HPAF-II pancreatic cancer cell line.
Example 19
Inhibition of Growth of NCI-H358 Lung Xenograft Tumor Model
Inhibition of tumor growth by the humanized anti-RON antibodies was tested in an NCI-H358 lung xenograft model. The NCI-H358 cells (ATCC) were grown in culture at 37° C. in an atmosphere containing 5% CO2, using RMPI medium (Invitrogen) containing 10% FBS. Cells were inoculated subcutaneously into the flank of 8-week old female CB.17 SCID mice (Taconic Labs) with 5×106 cells per mouse in 50% matrigel (Becton Dickinson). Tumor measurements were taken twice weekly using vernier calipers. When tumors reached approximately 150 mm3, the mice were randomized into six groups of ten mice each. Each group received one of the following treatments: human IgG (huIgG) control, mu29B06, sh29B06-78, mu07F01, sh07F01-62 and RON8. Treatment was administered by intra-peritoneal injection two times per week at 10 mg/kg for seven weeks. Treatment was well-tolerated, with no significant loss in body weight. Tumor growth inhibition is expressed as percent inhibition (baseline subtracted) to the huIgG control and statistical analysis was conducted using ANOVA. Results for tumor growth inhibition on day 41 in the NCI-H358 model are shown in FIG. 18 and Table 27.











TABLE 27









ANOVA



Treatment
TGI %
(compared to huIgG)









mu29B06
88.93
P < 0.01



sh29B06-78
89.02
P < 0.01



mu07F01
34.15
P > 0.05



sh07F01-62
39.05
P > 0.05



RON8
37.99
P > 0.05










Anti-RON antibody treatments resulted in tumor growth inhibition compared to huIgG control. Specifically, mu29B06 antibody treatment resulted in tumor growth inhibition of 89% (P<0.01); sh29B06-78 antibody treatment resulted in tumor growth inhibition of 89% (P<0.01); mu07F01 antibody treatment resulted in tumor growth inhibition of 34% (P>0.05); sh07F01-62 antibody treatment resulted in tumor growth inhibition of 39% (P>0.05); and RON8 antibody treatment resulted in tumor growth inhibition of 38% (P>0.05). These results demonstrate that sh29B06-78 and mu29B06 inhibit tumor growth in a NCI-H358 xenograft model (P<0.01), whereas the mu07F01, sh07F01-62, and RON8 antibodies did not inhibit tumor growth in this model (P>0.05, which is not statically significant).
Example 20
RON Receptor Degradation
Western blots were performed to determine total levels of RON receptor at the end of treatment. Four tumor samples from each of the treatment groups were weighed, lysed in RIPA buffer (Boston Bioproducts), 1 mM EDTA (Boston Bioproducts), 1 mM Sodium OrthoVandadate (Sigma), 1× protease inhibitor (Sigma) and 1× Phosphatase Inhibitor I and II (Sigma). The samples were homogenized using a hand-held electric homogenizer and incubated for 10 minutes on ice. Samples are spun down at 11,000 RPM for 30 minutes at 4° C. Supernatants were collected and protein concentrations were determined using Pierce BCATM assay kit according to the manufacturers protocol. The C-20 (Santa Cruz) antibody was used to detect total RON protein. β-tubulin (Cell Signaling Technologies) was blotted as loading control. The Western blots were blocked for one hour in 5% Milk in 1×TBST (TBS-0.1% TWEENTM) (Sigma), followed by primary antibody incubation over night at 4° C. in 5% BSA 1×TBST at 1:1000 for both antibodies. Western blots were washed three times with 1×TBST, incubated with anti-rabbit HRP conjugated secondary antibody (Cell Signaling Technologies), for one hour at room temperature. Western blots were washed three times with 1×TBST and then developed using Dura Signal (Pierce).
The results in FIG. 19 demonstrate RON receptor degradation in the mu29B06 and sh29B06-78 treated samples and to a lesser extent in the mu07F01 and sh07F01-62 treated samples. RON receptor degradation was not observed in the RON8 treated samples.
INCORPORATION BY REFERENCE
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
EQUIVALENTS
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and the range of equivalency of the claims are intended to be embraced therein.

## Claims

1. An isolated antibody that binds human RON (Recepteur d'Origine Natais), comprising:
(i) an immunoglobulin heavy chain variable region comprising a CDRH1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 124, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 122, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 123; and
(ii) an immunoglobulin light chain variable region comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 130, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 131, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 10.

2. The antibody of claim 1, wherein the immunoglobulin heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 5, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 122, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 123; and
the immunoglobulin light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 130, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 131, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 10.

3. The antibody of claim 1, wherein the CDR sequences are interposed between human framework sequences.

4. The antibody of claim 1, wherein the immunoglobulin heavy chain variable region comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 124, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 122, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 123; and
the immunoglobulin light chain variable region comprises a CDRL1 comprising the amino acid sequence of SEQ ID NO: 130, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 131, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 10.

5. The antibody of claim 1, wherein the antibody is an antigen-binding fragment.

6. The antibody of claim 1, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 137 and the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO: 139.

7. The antibody of claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 166, and the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO: 168.

8. The antibody of claim 1, wherein the antibody binds human RON with a KD of 900 pM or lower as measured by surface plasmon resonance.

9. The antibody of claim 8, wherein the antibody binds human RON with a KD of 500 pM or lower as measured by surface plasmon resonance.

10. The antibody of claim 9, wherein the antibody binds human RON with a KD of 250 pM or lower as measured by surface plasmon resonance.

11. The antibody of claim 1, wherein the CDR sequences are interposed between humanized framework sequences.