# Patent

## Title
Anti-ErbB3 antibodies

## Abstract

Monoclonal antibodies that bind and inhibit activation of epidermal growth factor receptor related member ErbB3/HER3 are disclosed. The antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with activation of ErbB3/HER3.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 13/082,852, filed Apr. 8, 2011, which claims the benefit of and priority to U.S. provisional patent application Ser. No. 61/322,712, filed Apr. 9, 2010; the contents of each of which are incorporated herein by reference in their entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 17, 2013, is named AVO-009DV_SequenceListing.txt and is 232,709 bytes in size.
FIELD OF THE INVENTION
The field of the invention is molecular biology, immunology and oncology. More particularly, the field is humanized antibodies that bind human ErbB3/HER3.
BACKGROUND
HER3/c-ErbB3 (referred to herein as ErbB3) is a member of the epidermal growth factor receptor (EGFR) family. ErbB3 binds neuregulin/heregulin (NRG/HRG). Receptors in the EGFR family are single transmembrane receptors with an intracellular tyrosine kinase domain. While the other EGFR family members, i.e., EGFR/HER1/ErbB1, HER2/ErbB2, and HER4/ErbB4, each have tyrosine kinase activity, ErbB3 has little or no tyrosine kinase activity, and thus is “kinase-dead.”
The extracellular domain (ECD) of the EGFR family contains four domains. Domains 1 and 3 (also known as domains L1 and L2) are responsible for ligand binding. Cysteine-rich domains 2 and 4 (also known as domains C1 and C2) are involved in dimerization with receptor partners. Upon ligand binding, the ECD undergoes conformational changes. The interaction of domains 2 and 4, which maintains the tethered (inactive) conformation of the receptor, is relieved, and an extended (active) conformation is adopted. The extended conformation favors dimerization with other receptor partners. HER2/ErbB2 is the only exception to this general rule, i.e., Her2-ECD is constitutively in the extended conformation. No ligand for HER2 has been identified thus far.
Because ErbB3 lacks an intrinsic kinase activity, it must dimerize with another active tyrosine kinase receptor to be activated by tyrosine phosphorylation. Dimerization can occur between two different receptors (heterodimerization), e.g., ErbB3 and EGFR/HER1/ErbB1, HER2/ErbB2, or HER4/ErbB4. Recently, ErbB3 was also shown to dimerize with MET. Upon association with another tyrosine kinase receptor, ErbB3 is activated by phosphorylation of at least nine tyrosine residues in the ErbB3 intracellular domain, and then rapidly associates with adaptors or downstream signaling molecules. Six of the ErbB3 phosphorylated tyrosine residues associate directly with the p85 subunit of Phosphatidylinositol 3-Kinase (PIK3), which results in activation of the cellular survival pathway controlled by the PI3K/Akt axis. Constitutive activation of ErbB3 by unregulated dimerization and/or unregulated phosphorylation of ErbB3 can lead to certain cancers.
Overexpression of ErbB3 is associated with poor prognosis in various carcinomas (e.g., breast, ovarian, prostate, colorectal, pancreatic, gastric, and head and neck cancers). Overexpression of ErbB3 also correlates with local to distal metastasis in lung, gastric, and colorectal cancers, and bone invasion in prostate cancer (Sithanandam et al., 2008, CANCER GENE THERAPY 15:413). Overexpression of ErbB3 has been linked to resistance to several cancer treatments, including treatment with EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) and head and neck cancers, treatment with Her2 inhibitor in breast cancers, and treatment with radiotherapy in pancreatic cancers. Moreover, overexpression of NRG, a ligand for ErbB3, was also linked to resistance to EGFR tyrosine kinase inhibitor treatment. Chen et al. describe the use of anti-ErbB3 monoclonal antibodies that inhibit NRG function and show growth inhibitory activity against breast and ovarian cancer cells (Chen et al., 1996, J. BIOL. CHEM. 271: 7620).
There is a need for improved anti-ErbB3 antibodies that can be used as therapeutic agents.
SUMMARY
The invention is based on the discovery of a family of antibodies that specifically bind human ErbB3. The antibodies contain ErbB3 binding sites based on CDRs that specifically bind human ErbB3. 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 disclosed herein prevent or inhibit the activation of human ErbB3. In some embodiments, the antibodies prevent ErbB3 from binding to a ligand, e.g., NRG/HRG, thereby neutralizing the biological activity of ErbB3. In other embodiments, the anti-ErbB3 antibodies inhibit ErbB3 dimerization, thereby neutralizing the biological activity of ErbB3. The antibodies disclosed herein can be used to inhibit the proliferation of tumor cells in vitro or in vivo. When administered to a human cancer patient (or an animal model such as a mouse model), the antibodies inhibit or reduce tumor growth in the human patient (or animal model).
These and other aspects and advantages of the invention are illustrated by 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 antibody.
FIG. 2 is a schematic diagram showing the amino acid sequence of the complete immunoglobulin heavy chain variable region of the antibodies denoted as 04D01, 09D03, 11G01, 12A07, 18H02, 22A02, and 24C05. The amino acid sequences for each antibody 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. 3 is a schematic diagram showing the CDR1, CDR2, and CDR3 sequences (Kabat definition) for each of the immunoglobulin heavy chain variable region sequences in FIG. 2.
FIG. 4 is a schematic diagram showing the amino acid sequence of the complete immunoglobulin light chain variable region of antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02, and 24C05. The amino acid sequences for each antibody 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. 5 is a schematic diagram showing the CDR1, CDR2, and CDR3 sequences (Kabat definition) for each of the immunoglobulin light chain variable region sequences in FIG. 4.
FIGS. 6A and 6B are graphs summarizing results from an experiment to measure the neutralization activity of negative control (murine IgG (Δ)) and anti-ErbB3 monoclonal antibodies 04D01 (▪), 12A07 (◯), 18H02 (⋄), 22A02 (●) and 24C05 (□) to inhibit NRG1-β1 binding to hErbB3 (FIG. 6A) and to measure the enhanced binding of NRG1-β1 to rhErbB3 by the anti-ErbB3 mAb 09D03 (▴) and 11G01 (*) (FIG. 6B).
FIG. 7 is a graph summarizing results from an experiment to measure the neutralization activity of negative control (murine IgG) and anti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 to inhibit NRG1-α1 binding to rhErbB3.
FIG. 8 is a graph summarizing results from an experiment to measure the cell surface recognition of the anti-ErbB3 antibodies of the chimeric protein Her2/3d2 expressed at the surface of CHO cells.
FIG. 9 is a graph summarizing results from an experiment to measure the anti-proliferation activity of negative control IgG (murine IgG (Δ)) and anti-ErbB3 monoclonal antibodies 04D01 (▪), 09D03 (▾), 11G01 (♦), 12A07 (◯), 18H02 (⋄), 22A02 (●) and 24C05 (□) in BaF/3 cells expressing Her2 and ErbB3 in presence of NRG1-β1.
FIG. 10 is a graph summarizing results from an experiment to measure the anti-proliferation activity of anti-ErbB3 monoclonal antibodies 04D01 (▪), 09D03 (▾), 11G01 (♦), 12A07 (◯), 18H02 (⋄), 22A02 (●) and 24C05 (□) in MCF7 cells in the presence of NRG1-β1.
FIG. 11 is a graph summarizing results from an experiment to measure the anti-proliferation activity of negative control (murine IgG) and anti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 in SKBR-3 cells treated with 5 μg/ml of antibodies in the presence of serum.
FIG. 12 is graph summarizing results from an experiment to measure the inhibitory activity of negative control IgG and anti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 on the phosphorylation of ErbB3 induced by NRG in SKBR-3 cells. No antibody/no ligand and no antibody controls are also shown.
FIGS. 13A and 13B are graphs representing results from an experiment to measure the inhibitory activity of anti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 on the phosphorylation of Akt in response to NRG1-β1 in MCF7 cells (FIG. 13A) and in DU145 cells (FIG. 13B) as determined by ELISA. No antibody/no ligand and no antibody controls are also shown.
FIG. 14 is a graph summarizing results from an experiment to measure the tumor inhibitory activity of the anti-ErbB3 antibodies 04D01 (Δ), 09D03 (*), 11G01 (□), 12A07 (▴), 18H02 (●), 22A02 (▪), 24C05 (◯) and a human IgG control (--▪--) dosed at 20 mg/kg in a BxPC3 pancreatic tumor xenograft model in CB17 SCID mice (vehicle control, PBS (♦)).
FIG. 15 is a schematic diagram showing the amino acid sequences of the complete heavy chain variable region of 24C05 and the complete humanized heavy chain variable regions denoted as Sh24C05 Hv3-7, Sh24C05 Hv3-11, Sh24C05 Hv3-11 N62S, Sh24C05 Hv3-21, Sh24C05 Hv3-23, Sh24C05 Hv3-30, and Hu24C05 HvA. The amino acid sequences for each heavy chain variable regions 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. 16 is a schematic diagram showing the amino acid sequences of the complete light chain variable region of 24C05 and the complete humanized light chain variable regions denoted as Sh24C05 Kv1-9, Sh24C05 Kv1-16, Sh24C05 Kv1-17, Sh24C05 Kv1-33, Sh24C05 Kv1-39, and Hu24C05 KvA. The amino acid sequences for each light chain variable regions 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. 17 are Biacore sensorgrams representing results from an experiment to measure the kinetic values of anti-ErbB3 monoclonal antibodies, Sh24C05-31 N62S-IgG1, Ab#6, U1-53, and U1-59.
FIG. 18A is a graph summarizing results from an experiment to measure the neutralization activity of negative control (human IgG (□)) and anti-ErbB3 monoclonal antibodies Sh24C05-25 N62S-IgG1 (▴), Sh24C05-25 N62S-IgG2 (Δ), Sh24C05-31 N62S-IgG1 (●) and Sh24C05-31 N62S-IgG2 (◯). FIG. 18B is a graph summarizing results from an experiment to measure the neutralization activity of human IgG (□) and anti-ErbB3 monoclonal antibodies Ab#6 IgG2 (▾), U1-53 (⋄) and U1-59 (▪).
FIG. 19A is a graph summarizing results from an experiment to measure the inhibitory activity of negative control (human IgG (□)) and anti-ErbB3 monoclonal antibodies Sh24C05-25 N62S-IgG1 (▴), Sh24C05-25 N62S-IgG2 (Δ), Sh24C05-31 N62S-IgG1 (●) and Sh24C05-31 N62S-IgG2 (◯) in BaF/3 cells expressing Her2 and ErbB3 in the presence of NRG1-β1. FIG. 19B is a graph summarizing results from an experiment to measure the inhibitory activity of human IgG (□) and anti-ErbB3 monoclonal antibodies Sh24C05-31 N62S-IgG1 (●), Ab#6 IgG2 (∇), U1-53 (⋄) and U1-59 (▪) in BaF/3 cells expressing Her2 and ErbB3 in the presence of NRG1-β1.
FIG. 20 is a graph summarizing results from an experiment to measure the inhibitory activity of negative control (human IgG) and anti-ErbB3 monoclonal antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 on the steady state phosphorylation of ErbB3 in growing SKBR-3 cells.
FIG. 21 is a graph summarizing results from an experiment to measure the degradation of ErbB3 receptor by negative control (human IgG) and anti-ErbB3 monoclonal antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 in growing SKBR-3 cells.
FIG. 22 is a graph summarizing the results from an experiment to measure tumor inhibitory activity of a human IgG, murine IgG, or anti-ErbB3 monoclonal antibodies dosed at 2 mg/kg in a BxPC3 pancreatic tumor xenograft model in CB17 SCID mice (murine 24C05 (Δ), Sh24C05-31 N62S IgG1 (●), Sh24C05-31 N62S IgG2 (♦), Sh24C05-25 N62S IgG1 (▴), Sh24C05-25 N62S IgG2 (▪), vehicle control (□), murine IgG (x), and human IgG (⋄)).
FIG. 23A is a graph summarizing the results from an experiment to measure tumor inhibitory activity of a murine IgG or anti-ErbB3 monoclonal antibodies dosed at 5 mg/kg in a Calu-3 non-small cell lung cancer xenograft model in NCR nude mice (vehicle control (□), murine IgG (x), Sh24C05-31 N62S IgG1 (▴), Ab#6 IgG2 (●), and U1-59 (▪)).
FIG. 23B is a graph summarizing the results from an experiment to measure tumor inhibitory activity of a murine IgG or anti-ErbB3 monoclonal antibodies dosed at 10 mg/kg in a Calu-3 non-small cell lung cancer xenograft model in NCR nude mice (vehicle control (□), murine IgG (x), Sh24C05-31 N62S IgG1 (▴), Ab#6 IgG2 (●), and U1-59 (▪)).
FIG. 23C is a graph summarizing the results from an experiment to measure tumor inhibitory activity of a murine IgG or anti-ErbB3 monoclonal antibodies dosed at 20 mg/kg in a Calu-3 non-small cell lung cancer xenograft model in NCR nude mice (vehicle control (□), murine IgG (x), Sh24C05-31 N62S IgG1 (▴), Ab#6 IgG2 (●), and U1-59 (▪)).
FIG. 24 is a graph summarizing the results from an experiment to measure tumor inhibitory activity of a human IgG, murine or anti-ErbB3 monoclonal antibodies in a MDA-MB-453 breast cancer xenograft model in NOD SCID mice (vehicle control (□), human IgG (x), Sh24C05-31 N62S IgG1 dosed at 5 mg/kg (⋄), Sh24C05-31 N62S IgG1 dosed at 10 mg/kg (Δ), Sh24C05-31 N62S IgG1 dosed at 20 mg/kg (▴), Ab#6 IgG2 dosed at 10 mg/kg (●), and U1-59 dosed at 10 mg/kg (▪)).



DETAILED DESCRIPTION
The ErbB3 antibodies disclosed herein are based on the antigen binding sites of certain monoclonal antibodies selected for their ability to neutralize the biological activity of human ErbB3 polypeptides. The antibodies contain immunoglobulin variable region CDR sequences that define a binding site for ErbB3. In some embodiments, the antibodies prevent ErbB3 from binding to a ligand, e.g., NRG/HRG, thereby neutralizing the biological activity of ErbB3. In other embodiments, the anti-ErbB3 antibodies inhibit ErbB3 dimerization, thereby neutralizing the biological activity of ErbB3. In still other embodiments, the anti-ErbB3 antibodies inhibit phosphorylation of ErbB3 and downstream signaling.
Because of the neutralizing activity of these antibodies, they are useful for inhibiting the growth and/or proliferation of certain cancer cells and tumors. 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 detectable labels or effector molecules such as small molecule toxins.
I. Antibodies that Bind ErbB3
In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3 and (b) 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 ErbB3. A CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (04D01), SEQ ID NO:15 (09D03), SEQ ID NO: 25 (11G01), SEQ ID NO: 34 (12A07), SEQ ID NO: 41 (18H02), SEQ ID NO: 51 (22A02), SEQ ID NO: 57 (24C05), and SEQ ID NO: 75 (24C05); a CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (04D01), SEQ ID NO:16 (09D03), SEQ ID NO: 26 (11G01), SEQ ID NO: 35 (12A07), SEQ ID NO: 42 (18H02), SEQ ID NO: 52 (22A02), SEQ ID NO: 58 (24C05), and SEQ ID NO: 148 (Sh24C05 Hv3-11 N62S); and a CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 (04D01), SEQ ID NO: 17 (09D03), SEQ ID NO: 27 (11G01), SEQ ID NO: 36 (12A07, 22A02), SEQ ID NO: 43 (18H02), and SEQ ID NO: 59 (24C05). 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 (04D01)” means that SEQ ID NO: 5 comes from antibody 04D01.
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 (04D01), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 6 (04D01), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 7 (04D01).
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 (09D03), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 16 (09D03), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 17 (09D03).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 25 (11G01), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 26 (11G01), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 27 (11G01).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 34 (12A07), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 35 (12A07), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 36 (12A07, 22A02).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 41 (18H02), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 42 (18H02), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 43 (18H02).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 51 (22A02), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 52 (22A02), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 36 (12A07, 22A02).
In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 57 (24C05) or SEQ ID NO: 75 (24C05), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 58 (24C05), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 59 (24C05).
In certain embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 57 (24C05), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 58 (24C05), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 59 (24C05).
In other embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 75 (24C05), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 58 (24C05), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 59 (24C05).
In certain embodiments, the antibody comprises an immunoglobulin heavy chain variable region a CDRH1 comprising the amino acid sequence of SEQ ID NO: 57 (24C05) or SEQ ID NO: 75 (24C05), a CDRH2 comprising the amino acid sequence of SEQ ID NO: 148 (Sh24C05 Hv3-11 N62S), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 59 (24C05).
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 ErbB3. A CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (04D01, 12A07, 22A02), SEQ ID NO: 18 (09D03), SEQ ID NO: 28 (11G01), SEQ ID NO: 44 (18H02), and SEQ ID NO: 60 (24C05); a CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (04D01, 11G01, 12A07, 22A02), SEQ ID NO: 19 (09D03), SEQ ID NO: 45 (18H02), and SEQ ID NO: 61 (24C05); and a CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 (04D01, 12A07, 22A02), SEQ ID NO: 20 (09D03), SEQ ID NO: 29 (11G01), SEQ ID NO: 46 (18H02), and SEQ ID NO: 62 (24C05).
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 (04D01, 12A07, 22A02); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 9 (04D01, 11G01, 12A07, 22A02); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 10 (04D01, 12A07, 22A02).
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 (09D03); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 19 (09D03); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 20 (09D03).
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 (11G01); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 9 (04D01, 11G01, 12A07, 22A02); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 29 (11G01).
In some embodiments, the antibody comprises an immunoglobulin light chain variable region comprising: a CDRL1 comprising the amino acid sequence of SEQ ID NO: 44 (18H02); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 45 (18H02); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 46 (18H02).
In one embodiment, the antibody comprises an immunoglobulin light chain variable region comprising: a CDRL1 comprising the amino acid sequence of SEQ ID NO: 60 (24C05); a CDRL2 comprising the amino acid sequence of SEQ ID NO: 61 (24C05); and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 62 (24C05).
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 IgG heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3 and (b) an IgG 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 ErbB3. The CDRH1 is an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (04D01), SEQ ID NO:15 (09D03), SEQ ID NO: 25 (11G01), SEQ ID NO: 34 (12A07), SEQ ID NO: 41 (18H02), SEQ ID NO: 51 (22A02), SEQ ID NO: 57 (24C05), and SEQ ID NO: 75 (24C05); the CDRH2 is an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (04D01), SEQ ID NO:16 (09D03), SEQ ID NO: 26 (11G01), SEQ ID NO: 35 (12A07), SEQ ID NO: 42 (18H02), SEQ ID NO: 52 (22A02), SEQ ID NO: 58 (24C05), and SEQ ID NO: 148 (Sh24C05 Hv3-11 N62S); and the CDRH3 is an amino acid sequence selected from the group consisting of SEQ ID NO: 7 (04D01), SEQ ID NO: 17 (09D03), SEQ ID NO: 27 (11G01), SEQ ID NO: 36 (12A07, 22A02), SEQ ID NO: 43 (18H02), and SEQ ID NO: 59 (24C05). The CDRL1 is an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (04D01, 12A07, 22A02), SEQ ID NO: 18 (09D03), SEQ ID NO: 28 (11G01), SEQ ID NO: 44 (18H02), and SEQ ID NO: 60 (24C05); the CDRL2 is an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (04D01, 11G01, 12A07, 22A02), SEQ ID NO: 19 (09D03), SEQ ID NO: 45 (18H02), and SEQ ID NO: 61 (24C05); and the CDRL3 is an amino acid sequence selected from the group consisting of SEQ ID NO: 10 (04D01, 12A07, 22A02), SEQ ID NO: 20 (09D03), SEQ ID NO: 29 (11G01), SEQ ID NO: 46 (18H02), and SEQ ID NO: 62 (24C05).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 2 (04D01), SEQ ID NO: 12 (09D03), SEQ ID NO: 22 (11G01), SEQ ID NO: 31 (12A07), SEQ ID NO: 38 (18H02), SEQ ID NO: 48 (22A02), SEQ ID NO: 54 (24C05), and SEQ ID NO: 154 (Sh24C05 Hv3-11 N62S), and an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO: 4 (04D01), SEQ ID NO: 14 (09D03), SEQ ID NO: 24 (11G01), SEQ ID NO: 33 (12A07), SEQ ID NO: 40 (18H02), SEQ ID NO: 50 (22A02), SEQ ID NO: 56 (24C05), SEQ ID NO: 166 (Sh24C05 Kv1-16), and SEQ ID NO: 168 (Sh24C05 Kv1-17).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (04D01), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (04D01).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (09D03), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (09D03).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 (11G01), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 24 (11G01).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 (12A07), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 33 (12A07).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 (18H02), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 40 (18H02).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 (22A02), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 50 (22A02).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 (24C05), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 56 (24C05).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 154 (Sh24C05 Hv3-11 N62S), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 166 (Sh24C05 Kv1-16).
In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 154 (Sh24C05 Hv3-11 N62S), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 168 (Sh24C05 Kv1-17).
In other embodiments, the antibody comprises (i) an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO: 109 (04D01), SEQ ID NO: 113 (09D03), SEQ ID NO: 117 (11G01), SEQ ID NO: 121 (12A07), SEQ ID NO: 125 (18H02), SEQ ID NO: 129 (22A07), SEQ ID NO: 133 (24C05), SEQ ID NO: 190 (Sh24C05 Hv3-11 N62S IgG1), and SEQ ID NO: 192 (Sh24C05 Hv3-11 N62S IgG2), and (ii) an immunoglobulin light chain selected from the group consisting of SEQ ID NO: 111 (04D01), SEQ ID NO: 115 (09D03), SEQ ID NO: 119 (11G01), SEQ ID NO: 123 (12A07), SEQ ID NO: 127 (18H02), SEQ ID NO: 131 (22A07), SEQ ID NO: 135 (24C05), SEQ ID NO: 204 (Sh24C05 Kv1-16 kappa), and SEQ ID NO: 206 (Sh24C05 Kv1-17 kappa).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 109 (04D01), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 111 (04D01).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 113 (09D03), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 115 (09D03).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 117 (11G01), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 119 (11G01).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 121 (12A07), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 123 (12A07).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 125 (18H02), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 127 (18H02).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 129 (22A02), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 131 (22A02).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 133 (24C05), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 135 (24C05).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 190 (Sh24C05 Hv3-11 N62S IgG1), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 204 (Sh24C05 Kv1-16 kappa).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 192 (Sh24C05 Hv3-11 N62S IgG2), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 204 (Sh24C05 Kv1-16 kappa).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 190 (Sh24C05 Hv3-11 N62S IgG1), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 206 (Sh24C05 Kv1-17 kappa).
In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 192 (Sh24C05 Hv3-11 N62S IgG2), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 206 (Sh24C05 Kv1-17 kappa).
As used herein, unless otherwise indicated, the term “antibody” means an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of a 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. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab′, (Fab′)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.
FIG. 1 shows a schematic representation of an intact monoclonal antibody that contains four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin 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 contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity.
In certain embodiments, an isolated antibody that binds human ErbB3 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 (04D01), SEQ ID NO: 12 (09D03), SEQ ID NO: 22 (11G01), SEQ ID NO: 31 (12A07), SEQ ID NO: 38 (18H02), SEQ ID NO: 48 (22A02), SEQ ID NO: 54 (24C05), and SEQ ID NO: 154 (Sh24C05 Hv3-11 N62S).
In certain embodiments, an isolated antibody that binds human ErbB3 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 (04D01), SEQ ID NO: 14 (09D03), SEQ ID NO: 24 (11G01), SEQ ID NO: 33 (12A07), SEQ ID NO: 40 (18H02), SEQ ID NO: 50 (22A02), SEQ ID NO: 56 (24C05), SEQ ID NO: 166 (Sh24C05 Kv1-16), and SEQ ID NO: 168 (Sh24C05 Kv1-17).
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 ErbB3 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 some embodiments, an isolated antibody binds hErbB3 with a KD of 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 20 pM, 10 pM or lower. Unless otherwise specified, KD values are determined by surface plasmon resonance methods. The surface plasmon resonance methods can be performed using the conditions described, for example, in Examples 3 and 12, where the measurements were performed at 25° C. and 37° C., respectively.
In some embodiments, the antibodies inhibit hErbB3 binding to NRG1-β1. For example, the antibodies can have an IC50 (concentration at 50% of maximum inhibition) of about 5 nM, 2 nM or lower, when assayed using the protocols described in Examples 4 and 13.
II. Production of Antibodies
Methods for producing antibodies disclosed herein are known in the art. For example, DNA molecules encoding light chain variable regions and 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 the antibodies disclosed herein 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, 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., Tip 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 a DNA construct encoding an antibody disclosed herein 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 and/or light chain to be expressed. In some embodiments, a single expression vector contains both heavy and light chain variable regions 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 method of producing a polypeptide comprising an immunoglobulin heavy chain variable region or a polypeptide comprising an immunoglobulin light chain variable region may comprise growing a host cell transfected with an expression vector under conditions that permits expression of the polypeptide comprising the immunoglobulin heavy chain variable region or the polypeptide comprising the immunoglobulin light chain variable region. The polypeptide comprising a heavy chain variable region or the polypeptide comprising the light chain variable region then may be purified using techniques well known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) and histidine tags.
A method of producing a monoclonal antibody that binds human ErbB3, or an antigen-binding fragment of the antibody, may comprise 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 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. Modifications to the Antibodies
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, a humanized antibody has 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-ErbB3 antibody are grafted onto 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. Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619.
Any suitable approach, including any of the above approaches, can be used to reduce or eliminate human immunogenicity of an antibody disclosed herein.
Methods of making multispecific antibodies are known in the art. Multi-specific antibodies include bispecific antibodies. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies bind to two different epitopes of the antigen of interest. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)2 bispecific antibodies and diabodies) as described, for example, in Milstein et al., NATURE 305:537-539 (1983), WO 93/08829, Traunecker et al., EMBO J., 10:3655-3659 (1991), WO 94/04690, Suresh et al., METHODS IN ENZYMOLOGY, 121:210 (1986), WO96/27011, Brennan et al., SCIENCE, 229: 81 (1985), Shalaby et al., J. EXP. MED., 175: 217-225 (1992), Kostelny et al., J. IMMUNOL., 148(5):1547-1553 (1992), Hollinger et al., PNAS, 90:6444-6448, Gruber et al., J. IMMUNOL., 152:5368 (1994), Wu et al., NAT. BIOTECHNOL., 25(11): 1290-1297, U.S. Patent Publication No. 2007/0071675, and Bostrom et al., SCIENCE 323:1640-1644 (2009).
In some embodiments, the antibody is 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 the Antibodies
The antibodies disclosed herein can be used to treat various forms of cancer, e.g., breast, ovarian, prostate, cervical, colorectal, lung (e.g., non-small cell lung cancer), pancreatic, gastric, skin, kidney, head and neck, and schwannoma 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 inhibits or reduces proliferation of a tumor cell by inhibiting binding of human ErbB3 to an ErbB3 ligand, e.g., Neuregulin/Heregulin especially NRGβ1/NRG1-β1/NRGβ1/HRGβ1 and NRGα1/NRG1-α1/NRGα1/HRGα1. The antibody can be used in a method to inhibit tumor growth in a human patient. The method comprises administering to the patient a therapeutically effective amount of the antibody.
Cancers associated with ErbB3 overexpression and/or activation include breast cancer, ovarian cancer, prostate cancer, cervical cancer, lung cancer (e.g., non-small cell lung cancer), some forms of brain cancer (e.g., schwannoma), melanomas, skin, kidney, and gastrointestinal cancers (e.g., colorectal, pancreatic, gastric, head and neck).
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; and (c) curing the disease.
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 daily 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. 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 monoclonal 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 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 parabens; 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 Anti-hErbB3 Monoclonal Antibodies
Immunizations, fusions, and primary screens were conducted at Maine Biotechnology Services Inc. following the Repetitive Immunization Multiple Sites (RIMMS) protocol. Three AJ mice and three Balb/c mice were immunized with recombinant human ErbB3/Fc (R&D Systems, Cat. No. 348-RB). Two sets of immunization were performed with either cleaved rhErbB3 (Immunization A) or with cleaved rhErbB3 cross-linked to its ligand, recombinant human NRG1-β1/HRG1-β1-EGF domain (R&D Systems, Cat. No. 396-HB) (Immunization B). Two AJ mice per immunization with sera displaying high anti-ErbB3 activity by Enzyme Linked Immunosorbent Assay (ELISA) were chosen for subsequent fusion. Spleens and lymph nodes from the appropriate mice were harvested. B-cells then were harvested and fused with a myeloma line. Fusion products were serially diluted onto forty 96-well plates to near clonality. A total of 5280 supernatants from the resulting fusions were screened for binding to recombinant rhErbB3/Fc, using ELISA. The same supernatants were also screened for their binding to human ErbB3 overexpressed in CHO cells (by Mesoscale electrochemiluminescence assay). Three hundred supernatants identified as containing antibodies against ErbB3 were further characterized by in vitro biochemical and cell-based assays as discussed below. A panel of hybridomas was selected, and the hybridomas were subcloned and expanded. Hybridoma cell lines were transferred to BioXCell (formerly Bio-Express) for antibody expression and purification by affinity chromatography on Protein G resin under standard conditions.
Anti-hErbB3 monoclonal antibody 04D01 was generated from Immunization A described above. Anti-hErbB3 monoclonal antibodies 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 were generated from Immunization B described above.
Example 2
Sequence Analysis of Anti-hErbB3 Monoclonal Antibodies
The light-chain isotype and heavy chain isotype of each monoclonal antibody in Example 1 was determined using the IsoStripTM Mouse Monoclonal Antibody Isotyping Kit according the manufacturer's instructions (Roche Applied Science). All antibodies were determined to be Kappa light chain and IgG1 or IgG2b IgG 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 vendor's instructions (Qiagen). Full-length first strand cDNA containing 5′ ends was generated using either the GeneRacerTM Kit (Invitrogen) or SMARTerTM RACE cDNA Amplification Kit (Clontech) according to the manufacturer's instructions using random primers for 5′ RACE.
The variable regions of the Kappa and Heavy (IgG1 or IgG2b) IgG chains were amplified by PCR, using KOD Hot Start Polymerase (Novagen) or Advantage 2 Polymerase Mix (Clontech) according to the manufacturer's instructions. For amplification of 5′ cDNA ends in conjunction with the GeneRacerTM Kit, the GeneRacerTM 5′ Primer, 5′ cgactggagcacgaggacactga 3′ (SEQ ID NO: 136) (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: 137) and 5′ CTAATACGACTCACTATAGGGC 3′ (SEQ ID NO: 138), 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, either 5′ TATGCAAGGCTTACAACCACA 3′ (SEQ ID NO: 139) or 5′ GCCAGTGGATAGACAGATGGGGGTGTCG 3′ (SEQ ID NO: 140). IgG2b sequences were amplified with either 5′ AGGACAGGGGTTGATTGTTGA 3′ (SEQ ID NO: 141), 5′ GGCCAGTGGATAGACTGATGGGGGTGTTGT 3′ (SEQ ID NO: 142), or 5′ GGAGGAACCAGTTGTATCTCCACACCCA 3′ (SEQ ID NO: 143). Kappa chain variable regions were amplified with the above 5′ primers and a 3′ Kappa Constant Region specific primer, either 5′ CTCATTCCTGTTGAAGCTCTTGACAAT 3′ (SEQ ID NO: 144) or 5′ CGACTGAGGCACCTCCAGATGTT 3′ (SEQ ID NO: 145).
Individual PCR products were isolated by agarose gel electrophoresis and purified using the Qiaquick® Gel Purification kit according to the manufacturer's instructions (Qiagen). The PCR products were subsequently cloned into the pCR®4Blunt plasmid using the Zero Blunt® TOPO® PCR Cloning Kit according to the manufacturer'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: 146) and M13 Reverse primers (5′ CAGGAAACAGCTATGACC 3′) (SEQ ID NO: 147) by Beckman Genomics, 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 software 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 summarized below (amino terminal signal peptide sequences are not shown). CDR sequences (Kabat definition) are shown in bold/underlined in the amino acid sequences.








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the 04D01



Antibody


(SEQ ID NO: 1)










1
caggtccaac tgcagcagcc tggggctgaa ctggtgaggc ctgggacttc agtgaagttg



 


61
tcctgcaagg cttctggcta caccttcacc agccactggt tgcactgggt gaagcagagg


 


121
cctggacaag gccttgagtg gatcggagtg cttgatcctt ctgattttta tagtaactac


 


181
aatcaaaact tcaagggcaa ggccacattg actgtagaca catcctccag cacagcctac


 


241
atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc acgaggccta


 


301
ctatccgggg actatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca


 








Protein Sequence Defining the Heavy Chain Variable Region of the 04D01 Antibody



(SEQ ID NO: 2)










1
qvqlqqpgae lvrpgtsvkl sckasgytft shwlhwvkqr pgqglewigv ldpsdfysny



 


61
nqnfkgkatl tvdtssstay mqlssltsed savyycargl lsgdyamdyw gqgtsvtvss


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the 04D01



Antibody


(SEQ ID NO: 3)










1
gatgttttga tgacccaaat tccactctcc ctgcctgtca gtcttggaga tcaagcctcc



 


61
atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg


 


121
tacctgcaga aaccaggcca gtctccaaag tccctgatct acaaagtttc taaccgattt


 


181
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc


 


241
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc atatgttccg


 


301
tggacgttcg gtggaggcac caagctggaa atcaaa


 








Protein Sequence Defining the Kappa Chain Variable Region of the 04D01 Antibody



(SEQ ID NO: 4)










1
dvlmtqipls lpvslgdqas iscrssqsiv hsngntylew ylqkpgqspk sliykvsnrf



 


61
sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgsyvp wtfgggtkle ik


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the 09D03



Antibody


(SEQ ID NO: 11)










1
caggttactc taaaagagtc tggccctggg atattgcggc cctcccagac cctcagtctg



 


61
acttgttctt tctctgggtt ttcactgagc acttttggtt tgagtgtagg ctggattcgt


 


121
cagccttcag ggaagggtct ggagtggctg gcacacattt ggtgggatga tgataagtac


 


181
tataacccag cccttaagag tcggctcaca atctccaagg atacctccaa aaaccaggta


 


241
ttcctcaaga tcgccaatgt ggacactgca gatactgcca catactactg tgctcgaata


 


301
ggggcggacg cccttccttt tgactactgg ggccaaggca ccactctcac agtctcctca


 








Protein Sequence Defining the Heavy Chain Variable Region of the 09D03 Antibody



(SEQ ID NO: 12)










1
qvtlkesgpg ilrpsqtlsl tcsfsgfsls tfglsvgwir qpsgkglewl ahiwwdddky



 


61
ynpalksrlt iskdtsknqv flkianvdta dtatyycari gadalpfdyw gqgttltvss


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the 09D03



Antibody


(SEQ ID NO: 13)










1
gatattgtgt tgactcagac tgcaccctct gtacctgtca ctcctggaga gtcagtatcc



 


61
atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg


 


121
ttcctgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc


 


181
tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc


 


241
agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatct agaatatcct


 


301
ttcacgttcg gctcggggac aaagttggaa ataaaa


 








Protein Sequence Defining the Kappa Chain Variable Region of the 09D03 Antibody



(SEQ ID NO: 14)










1
divltqtaps vpvtpgesys iscrssksll hsngntylyw flqrpgqspq lliyrmsnla



 


61
sgvpdrfsgs gsgtaftlri srveaedvgv yycmqhleyp ftfgsgtkle ik


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the 11G01



Antibody


(SEQ ID NO: 21)










1
caggttcagc tgcaacagtc tgacgctgag ttggtgaaac ctggagcttc agtgaagata



 


61
tcctgcaagg tttctggcta caccttcact gaccatatta ttcactggat gaagcagagg


 


121
cctgaacagg gcctggaatg gattggatat atttatccta gagatggtta tattaagtac


 


181
aatgagaagt tcaagggcaa ggccacattg actgcagaca aatcctccag cacagcctac


 


241
atgcaggtca acagcctgac atctgaggac tctgcagtct atttctgtgc aaggggttac


 


301
tattatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a


 








Protein Sequence Defining the Heavy Chain Variable Region of the 11G01 Antibody



(SEQ ID NO: 22)










1
qvqlqqsdae lvkpgasvki sckvsgytft dhiihwmkqr peqglewigy iyprdgyiky



 


61
nekfkgkatl tadkssstay mqvnsltsed savyfcargy yyamdywgqg tsvtvss


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the 11G01



Antibody


(SEQ ID NO: 23)










1
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc



 


61
atctcttgca gatctagtca gagcattgta catagtattg gaaacaccta tttagaatgg


 


121
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt


 


181
tctggggtcc cagagaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc


 


241
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcca


 


301
ttcacgttcg gctcggggac aaagttggaa ataaaa


 








Protein Sequence Defining the Kappa Chain Variable Region of the 11G01 Antibody



(SEQ ID NO: 24)










1
dvlmtqtpls lpvslgdqas iscrssqsiv hsigntylew ylqkpgqspk lliykvsnrf



 


61
sgvperfsgs gsgtdftlki srveaedlgv yycfqgshvp ftfgsgtkle ik


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the 12A07



Antibody


(SEQ ID NO: 30)










1
caggtccaac tgctgcagcc tggggctgag ctggtgaggc ctgggacttc agtgaagttg



 


61
tcctgcaaga cttctggcta caccttctcc agctactgga tgcactgggt aaagcagagg


 


121
cctggacaag gccttgagtg gatcggaatg attgatcctt ctgatgttta tactaactac


 


181
aatccaaagt tcaagggcaa ggccacattg actgttgaca catcctccag cacagcctac


 


241
atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagaaactac


 


301
tctggggact actggggcca aggcaccact ctcacagtct cctca


 








Protein Sequence Defining the Heavy Chain Variable Region of the 12A07 Antibody



(SEQ ID NO: 31)










1
qvqllqpgae lvrpgtsvkl scktsgytfs sywmhwvkqr pgqglewigm idpsdvytny



 


61
npkfkgkatl tvdtssstay mqlssltsed savyycarny sgdywgqgtt ltvss


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the 12A07



Antibody


(SEQ ID NO: 32)










1
gatgttttga tgacccaaat tccactctcc ctgcctgtca gtcttggaga tcaagcctcc



 


61
atctcttgta gatctagtca gagcattgtc catagtaatg gaaacaccta tttagaatgg


 


121
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt


 


181
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc


 


241
agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc atatgttccg


 


301
tggacgttcg gtggaggcac caagctggaa atcaaa


 








Protein Sequence Defining the Kappa Chain Variable Region of the 12A07 Antibody



(SEQ ID NO: 33)










1
dvlmtqipls lpvslgdqas iscrssqsiv hsngntylew ylqkpgqspk lliykvsnrf



 


61
sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgsyvp wtfgggtkle ik


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the 18H02



Antibody


(SEQ ID NO: 37)










1
cagatccagt tggtacagtc tggacctgaa ctgaagaagc ctggagaggc agtcaagatc



 


61
tcctgcaagt cttctgggta taccttcaca acctatggaa tgagctgggt gaaacaggct


 


121
ccaggaaggg ctttaaagtg gatgggctgg ataaacacct actctggagt gccaacatat


 


181
gctgatgact tcaagggacg gtttgccttc tctttggaat cctctgccag cactgcctat


 


241
ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagagggagg


 


301
gatggttacc aagtggcctg gtttgcttac tggggccaag ggacgctggt cactgtctct


 


361
gca


 








Protein Sequence Defining the Heavy Chain Variable Region of the 18H02



Antibody


(SEQ ID NO: 38)










1
qiqlvqsgpe lkkpgeavki sckssgytft tygmswvkqa pgralkwmgw intysgvpty



 


61
addfkgrfaf slessastay lqinnlkned tatyfcargr dgyqvawfay wgqgtlvtvs


 


121
a


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the 18H02



Antibody


(SEQ ID NO: 39)










1
gaaacaactg tgacccagtc tccagcatcc ctgtccatgg ctataggaga taaagtcacc



 


61
atcagatgca taaccagcac tgatattgat gatgatatga actggttcca gcagaagcca


 


121
ggggaacctc ctaagctcct tatttcagaa ggcaatactc ttcgtcctgg agtcccatcc


 


181
cgattctccg gcagtggcta tggtacagat tttattttta caattgaaaa catgctctct


 


241
gaagatgttg cagattacta ctgtttgcaa agtgataact tgccgtacac gttcggaggg


 


301
gggaccaagc tggaaataaa a


 








Protein Sequence Defining the Kappa Chain Variable Region of the 18H02



Antibody


(SEQ ID NO: 40)










1
ettvtqspas lsmaigdkvt ircitstdid ddmnwfqqkp geppkllise gntlrpgvps



 


61
rfsgsgygtd fiftienmls edvadyyclq sdnlpytfgg gtkleik


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the 22A02



Antibody


(SEQ ID NO: 47)










1
caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctgggacttc agtgaagttg



 


61
tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt aaagcagagg


 


121
cctggacaag gccttgagtg gatcggaatg attgatcctt ctgatagtta tactaactac


 


181
aatccaaagt tcaagggtaa ggccacattg actgtagaca catcctccag cacagcctac


 


241
atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagaaactac


 


301
tctggggact actggggcca aggcaccact ctcacagtct cctca


 








Protein Sequence Defining the Heavy Chain Variable Region of the 22A02 Antibody



(SEQ ID NO: 48)










1
qvqlqqpgae lvrpgtsvkl sckasgytft nywmhwvkqr pgqglewigm idpsdsytny



 


61
npkfkgkatl tvdtssstay mqlssltsed savyycarny sqdywgqgtt ltvss


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the 22A02



Antibody


(SEQ ID NO: 49)










1
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc



 


61
atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg


 


121
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt


 


181
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc


 


241
agcagagtgg aggctgagga tctgggagtt tattattgct ttcaaggttc atatgttccg


 


301
tggacgttcg gtggaggcac caagctggaa atcaaa


 








Protein Sequence Defining the Kappa Chain Variable Region of the 22A02 Antibody



(SEQ ID NO: 50)










1
dvlmtqtpls lpvslgdqas iscrssqsiv hsngntylew ylqkpgqspk lliykvsnrf



 


61
sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgsyvp wtfgggtkle ik


 








Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the 24C05



Antibody


(SEQ ID NO: 53)










1
gaggtgcagc tggtggaatc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc



 


61
tcctgtgcag cctctggatt cactttcagt gactatgcca tgtcttgggt tcgccagact


 


121
ccggaaaaga ggctggagtg ggtcgcaacc attagtgatg gtggtactta cacctactat


 


181
ccagacaatg taaagggccg attcaccatc tccagagaca atgccaagaa caacctgtac


 


241
ctgcaaatga gccatctgaa gtctgaggac acagccatgt attactgtgc aagagaatgg


 


301
ggtgattacg acggatttga ctactggggc caaggcacca ctctcacagt ctcctcg


 








Protein Sequence Defining the Heavy Chain Variable Region of the 24C05 Antibody



(SEQ ID NO: 54)










1
evqlvesggg lvkpggslkl scaasgftfs dyamswvrqt pekrlewvat isdggtytyy



 


61
pdnvkgrfti srdnaknnly lqmshlksed tamyycarew gdydgfdywg qgttltvss


 








Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the 24C05



Antibody


(SEQ ID NO: 55)










1
gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt



 


61
ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcagaaacca


 


121
gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa


 


181
aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcggcag ccttgagtct


 


241
gaagatcttg cagactatta ctgtctacaa tatgatagtt atccgtacac gttcggaggg


 


301
gggaccaagc tggaaataaa a


 








Protein Sequence Defining the Kappa Chain Variable Region of the 24C05 Antibody



(SEQ ID NO: 56)










1
diqmtqspss lsaslgervs ltcrasqeis gylswlqqkp dgtikrliya astldsgvpk



 


61
rfsgsrsgsd ysltigsles edladyyclq ydsypytfgg gtkleik






The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 1 are aligned in FIG. 2. Amino terminal signal peptide sequences (for proper 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 for the antibodies in Example 1 are aligned in FIG. 4. Amino terminal signal peptide sequences (for proper 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 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.








TABLE 1





SEQ. ID



NO.
Nucleic Acid or Protein
















1
04D01 Heavy Chain Variable Region-nucleic acid


2
04D01 Heavy Chain Variable Region-protein


3
04D01 Light (kappa) Chain Variable



Region-nucleic acid


4
04D01 Light (kappa) Chain Variable



Region-protein


5
04D01 Heavy Chain CDR1


6
04D01 Heavy Chain CDR2


7
04D01 Heavy Chain CDR3


8
04D01 Light (kappa) Chain CDR1


9
04D01 Light (kappa) Chain CDR2


10
04D01 Light (kappa) Chain CDR3


11
09D03 Heavy Chain Variable Region-nucleic acid


12
09D03 Heavy Chain Variable Region-protein


13
09D03 Light (kappa) Chain Variable



Region-nucleic acid


14
09D03 Light (kappa) Chain Variable



Region-protein


15
09D03 Heavy Chain CDR1


16
09D03 Heavy Chain CDR2


17
09D03 Heavy Chain CDR3


18
09D03 Light (kappa) Chain CDR1


19
09D03 Light (kappa) Chain CDR2


20
09D03 Light (kappa) Chain CDR3


21
11G01 Heavy Chain Variable Region-nucleic acid


22
11G01 Heavy Chain Variable Region-protein


23
11G01 Light (kappa) Chain Variable



Region-nucleic acid


24
11G01 Light (kappa) Chain Variable



Region-protein


25
11G01 Heavy Chain CDR1


26
11G01 Heavy Chain CDR2


27
11G01 Heavy Chain CDR3


28
11G01 Light (kappa) Chain CDR1


9
11G01 Light (kappa) Chain CDR2


29
11G01 Light (kappa) Chain CDR3


30
12A07 Heavy Chain Variable Region-nucleic acid


31
12A07 Heavy Chain Variable Region-protein


32
12A07 Light (kappa) Chain Variable



Region-nucleic acid


33
12A07 Light (kappa) Chain Variable



Region-protein


34
12A07 Heavy Chain CDR1


35
12A07 Heavy Chain CDR2


36
12A07 Heavy Chain CDR3


8
12A07 Light (kappa) Chain CDR1


9
12A07 Light (kappa) Chain CDR2


10
12A07 Light (kappa) Chain CDR3


37
18H02 Heavy Chain Variable Region-nucleic acid


38
18H02 Heavy Chain Variable Region-protein


39
18H02 Light (kappa) Chain Variable



Region-nucleic acid


40
18H02 Light (kappa) Chain Variable



Region-protein


41
18H02 Heavy Chain CDR1


42
18H02 Heavy Chain CDR2


43
18H02 Heavy Chain CDR3


44
18H02 Light (kappa) Chain CDR1


45
18H02 Light (kappa) Chain CDR2


46
18H02 Light (kappa) Chain CDR3


47
22A02 Heavy Chain Variable Region-nucleic acid


48
22A02 Heavy Chain Variable Region-protein


49
22A02 Light (kappa) Chain Variable



Region-nucleic acid


50
22A02 Light (kappa) Chain Variable



Region-protein


51
22A02 Heavy Chain CDR1


52
22A02 Heavy Chain CDR2


36
22A02 Heavy Chain CDR3


8
22A02 Light (kappa) Chain CDR1


9
22A02 Light (kappa) Chain CDR2


10
22A02 Light (kappa) Chain CDR3


53
24C05 Heavy Chain Variable Region-nucleic acid


54
24C05 Heavy Chain Variable Region-protein


55
24C05 Light (kappa) Chain Variable



Region-nucleic acid


56
24C05 Light (kappa) Chain Variable



Region-protein


57
24C05 Heavy Chain CDR1


58
24C05 Heavy Chain CDR2


59
24C05 Heavy Chain CDR3


60
24C05 Light (kappa) Chain CDR1


61
24C05 Light (kappa) Chain CDR2


62
24C05 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










04D01
SHWLH
VLDPSDFYSNYNQNFKG
GLLSGDYAMDY



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



NO: 5)




09D03
TFGLSVG
HIWWDDDKYYNPALKS
IGADALPFDY



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



NO: 15)




11G01
DHIIH
YIYPRDGYIKYNEKFKG
GYYYAMDY



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



NO: 25)




12A07
SYWMH
MIDPSDVYTNYNPKFKG
NYSGDY



(SEQ ID
(SEQ ID NO: 35)
(SEQ ID NO: 36)



NO: 34)




18H02
TYGMS
WINTYSGVPTYADDFKG
GRDGYQVAWFAY



(SEQ ID
(SEQ ID NO: 42)
(SEQ ID NO: 43)



NO: 41)




22A02
NYWMH
MIDPSDSYTNYNPKFKG
NYSGDY



(SEQ ID
(SEQ ID NO: 52)
(SEQ ID NO: 36)



NO: 51)




24C05
DYAMS
TISDGGTYTYYPDNVKG
EWGDYDGFDY



(SEQ ID
(SEQ ID NO: 58)
(SEQ ID NO: 59)



NO: 57)




 







Chothia










04D01
GYTFTSH
DPSDFY
GLLSGDYAMDY



(SEQ ID
(SEQ ID NO: 64)
(SEQ ID NO: 7)



NO: 63)




09D03
GFSLSTFGL
WWDDD
IGADALPFDY



(SEQ ID
(SEQ ID NO: 66)
(SEQ ID NO: 17)



NO: 65)




11G01
GYTFTDH
YPRDGY
GYYYAMDY



(SEQ ID
(SEQ ID NO: 68)
(SEQ ID NO: 27)



NO: 67)




12A07
GYTFSSY
DPSDVY
NYSGDY



(SEQ ID
(SEQ ID NO: 70)
(SEQ ID NO: 36)



NO: 69)




18H02
GYTFTTY
NTYSGV
GRDGYQVAWFAY



(SEQ ID
(SEQ ID NO: 72)
(SEQ ID NO: 43)



NO: 71)




22A02
GYTFTNY
DPSDSY
NYSGDY



(SEQ ID
(SEQ ID NO: 74)
(SEQ ID NO: 36)



NO: 73)




24C05
GFTFSDY
SDGGTY
EWGDYDGFDY



(SEQ ID
(SEQ ID NO: 76)
(SEQ ID NO: 59)



NO: 75)




 







IMGT










04D01
GYTFTSHW
LDPSDFYS
ARGLLSGDYAMDY



(SEQ ID
(SEQ ID NO: 78)
(SEQ ID NO: 79)



NO: 77)




09D03
GFSLSTFGLS
IWWDDDK
ARIGADALPFDY



(SEQ ID
(SEQ ID NO: 81)
(SEQ ID NO: 82)



NO: 80)




11G01
GYTFTDHI
IYPRDGYI
ARGYYYAMDY



(SEQ ID
(SEQ ID NO: 84)
(SEQ ID NO: 85)



NO: 83)




12A07
GYTFSSYW
IDPSDVYT
ARNYSGDY



(SEQ ID
(SEQ ID NO: 87)
(SEQ ID NO: 88)



NO: 86)




18H02
GYTFTTYG
INTYSGVP
ARGRDGYQVAWFAY



(SEQ ID
(SEQ ID NO: 90)
(SEQ ID NO: 91)



NO: 89)




22A02
GYTFTNYW
IDPSDSYT
ARNYSGDY



(SEQ ID
(SEQ ID NO: 93)
(SEQ ID NO: 88)



NO: 92)




24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY



(SEQ ID
(SEQ ID NO: 95)
(SEQ ID NO: 96)



NO: 94)









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










04D01
RSSQSIVHSNGNTYLE
KVSNRFS
FQGSYVPWT



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




NO: 9)



09D03
RSSKSLLHSNGNTYLY
RMSNLAS
MQHLEYPFT



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




NO: 19)



11G01
RSSQSIVHSIGNTYLE
KVSNRFS
FQGSHVPFT



(SEQ ID NO: 28)
(SEQ ID
(SEQ ID NO: 29)




NO: 9)



12A07
RSSQSIVHSNGNTYLE
KVSNRFS
FQGSYVPWT



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




NO: 9)



18H02
ITSTDIDDDMN
EGNTLRP
LQSDNLPYT



(SEQ ID NO: 44)
(SEQ ID
(SEQ ID NO: 46)




NO: 45)



22A02
RSSQSIVHSNGNTYLE
KVSNRFS
FQGSYVPWT



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




NO: 9)



24C05
RASQEISGYLS
AASTLDS
LQYDSYPYT



(SEQ ID NO: 60)
(SEQ ID
(SEQ ID NO: 62)




NO: 61)



 







IMGT










04D01
QSIVHSNGNTY
KVS
FQGSYVPWT



(SEQ ID NO: 97)

(SEQ ID NO: 10)


09D03
KSLLHSNGNTY
RMS
MQHLEYPFT



(SEQ ID NO: 98)

(SEQ ID NO: 20)


11G01
QSIVHSIGNTY
KVS
FQGSHVPFT



(SEQ ID NO: 99)

(SEQ ID NO: 29)


12A07
QSIVHSNGNTY
KVS
FQGSYVPWT



(SEQ ID NO: 97)

(SEQ ID NO: 10)


18H02
TDIDDD
EGN
LQSDNLPYT



(SEQ ID NO: 100)

(SEQ ID NO: 46)


22A02
QSIVHSNGNTY
KVS
FQGSYVPWT



(SEQ ID NO: 97)

(SEQ ID NO: 10)


24C05
QEISGY
AAS
LQYDSYPYT



(SEQ ID NO: 101)

(SEQ ID NO: 62)









In Tables 2 and 3, the longest CDR sequences for the immunoglobulin heavy chain and light chain are shown in bold.
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 IgG2b heavy chain constant sequence and a complete kappa chain comprises a kappa variable sequence followed by the murine kappa light chain constant sequence.








Nucleic Acid Sequence Encoding the Murine IgG1 Heavy Chain Constant Region



(SEQ ID NO: 102)










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 ccagaccgtc


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: 103)










1
akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd



61
lytlsssvtv psstwpsqtv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif


121
ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv


181
selpimhqdw lngkefkory nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv


241
sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf


301
tcsvlheglh nhhtekslsh spgk


 








Nucleic Acid Sequence Encoding the Murine IgG2b Heavy Chain Constant Region



(SEQ ID NO: 104)










1
gccaaaacaa cacccccatc agtctatcca ctggcccctg ggtgtggaga tacaactggt



61
tcctctgtga ctctgggatg cctggtcaag ggctacttcc ctgagtcagt gactgtgact


121
tggaactctg gatccctgtc cagcagtgtg cacaccttcc cagctctcct gcagtctgga


181
ctctacacta tgagcagctc agtgactgtc ccctccagca cctggccaag tcagaccgtc


241
acctgcagcg ttgctcaccc agccagcagc accacggtgg acaaaaaact tgagcccagc


301
gggcccattt caacaatcaa cccctgtcct ccatgcaagg agtgtcacaa atgcccagct


361
cctaacctcg agggtggacc atccgtcttc atcttccctc caaatatcaa ggatgtactc


421
atgatctccc tgacacccaa ggtcacgtgt gtggtggtgg atgtgagcga ggatgaccca


481
gacgtccaga tcagctggtt tgtgaacaac gtggaagtac acacagctca gacacaaacc


541
catagagagg attacaacag tactatccgg gtggtcagca ccctccccat ccagcaccag


601
gactggatga gtggcaagga gttcaaatgc aaggtcaaca acaaagacct cccatcaccc


661
atcgagagaa ccatctcaaa aattaaaggg ctagtcagag ctccacaagt atacatcttg


721
ccgccaccag cagagcagtt gtccaggaaa gatgtcagtc tcacttgcct ggtcgtgggc


781
ttcaaccctg gagacatcag tgtggagtgg accagcaatg ggcatacaga ggagaactac


841
aaggacaccg caccagtcct agactctgac ggttcttact tcatatatag caagctcaat


901
atgaaaacaa gcaagtggga gaaaacagat tccttctcat gcaacgtgag acacgagggt


961
ctgaaaaatt actacctgaa gaagaccatc tcccggtctc cgggtaaa


 








Protein Sequence Defining the Murine IgG2b Heavy Chain Constant Region



(SEQ ID NO: 105)










1
akttppsvyp lapgcgdttg ssvtlgclvk gyfpesvtvt wnsgslsssv htfpallqsg



61
lytmsssvtv psstwpsqtv tcsvahpass ttvdkkleps gpistinpcp pckechkcpa


121
pnleggpsvf ifppnikdvl misltpkvtc vvvdvseddp dvqiswfvnn vevhtaqtqt


181
hredynstir vvstlpiqhq dwmsgkefkc kvnnkdlpsp iertiskikg lvrapqvyil


241
pppaeqlsrk dvsltclvvg fnpgdisvew tsnghteeny kdtapvldsd gsyfiyskln


301
mktskwektd sfscnvrheg lknyylkkti srspgk


 








Nucleic Acid Sequence Encoding the Murine Kappa Light Chain Constant Region



(SEQ ID NO: 106)










1
cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct



61
ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccagagacat caatgtcaag


121
tggaagattg atggcagtga acgacaaaat ggtgtcctga acagttggac tgatcaggac


181
agcaaagaca gcacctacag catgagcagc accctcacat 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: 107)










1
radaaptvsi fppsseqlts ggasvvcfln nfyprdinvk wkidgserqn gvlnswtdqd



61
skdstysmss tltltkdeye rhnsytceat hktstspivk sfnrnec






The following sequences represent the actual or contemplated full length heavy and light chain sequences (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 are also included at the 5′ end of the DNA sequences or the amino terminal end of the protein sequences. 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 04D01


(SEQ ID NO: 108)










1
atgggatgga gctgtatcat tgtcctcttg gtatcaacag ctacaggtgt ccactcccag



61
gtccaactgc agcagcctgg ggctgaactg gtgaggcctg ggacttcagt gaagttgtcc


121
tgcaaggctt ctggctacac cttcaccagc cactggttgc actgggtgaa gcagaggcct


181
ggacaaggcc ttgagtggat cggagtgctt gatccttctg atttttatag taactacaat


241
caaaacttca agggcaaggc cacattgact gtagacacat cctccagcac agcctacatg


301
cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcacg aggcctacta


361
tccggggact atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctcagcc


421
aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc


481
atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg


541
aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc


601
tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcca gaccgtcacc


661
tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat


721
tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc


781
ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta


841
gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg


901
cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt


961
gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac


1021
agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag


1081
gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt


1141
ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat


1201
gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac


1261
ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc


1321
tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct


1381
cctggtaaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 04D01


(SEQ ID NO: 109)










1
mgwsciivll vstatgvhsq vqlqqpgael vrpgtsvkls ckasgytfts hwlhwvkqrp



61
gqglewigvl dpsdfysnyn qnfkgkatlt vdtssstaym qlssltseds avyycargll


121
sgdyamdywg qgtsvtvssa kttppsvypl apgsaaqtns mvtlgclvkg yfpepvtvtw


181
nsgslssgvh tfpavlqsdl ytlsssvtvp sstwpsqtvt cnvahpasst kvdkkivprd


241
cgckpcictv pevssvfifp pkpkdvltit ltpkvtcvvv diskddpevq fswfvddvev


301
htaqtqpree qfnstfrsys elpimhqdwl ngkefkcrvn saafpapiek tisktkgrpk


361
apqvytippp keqmakdkvs ltcmitdffp editvewqwn gqpaenyknt qpimdtdgsy


421
fvysklnvqk snweagntft csvlheglhn hhtekslshs pgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 04D01


(SEQ ID NO: 110)










1
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat



61
gttttgatga cccaaattcc actctccctg cctgtcagtc ttggagatca agcctccatc


121
tcttgcagat ctagtcagag cattgtacat agtaatggaa acacctattt agaatggtac


181
ctgcagaaac caggccagtc tccaaagtcc ctgatctaca aagtttctaa ccgattttct


241
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc


301
agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcata tgttccgtgg


361
acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc


421
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg


481
aacaacttct accccagaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa


541
aatggtgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc


601
agcaccctca cattgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc


661
actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt


 








Protein Sequence Defining the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 04D01


(SEQ ID NO: 111)










1
mklpvrllvl mfwipasssd vlmtqiplsl pvslgdqasi scrssqsivh sngntylewy



61
lqkpgqspks liykvsnrfs gvpdrfsgsg sgtdftlkis rveaedlgvy ycfqgsyvpw


121
tfgggtklei kradaaptvs ifppsseqlt sggasvvcfl nnfyprdinv kwkidgserq


181
ngvinswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG2b Constant Region) of 09D03


(SEQ ID NO: 112)










1
atgggcaggc ttacttcttc attcctgtta ctgattgtcc ctgcatatgt cctgtcccag



61
gttactctaa aagagtctgg ccctgggata ttgcggccct cccagaccct cagtctgact


121
tgttctttct ctgggttttc actgagcact tttggtttga gtgtaggctg gattcgtcag


181
ccttcaggga agggtctgga gtggctggca cacatttggt gggatgatga taagtactat


241
aacccagccc ttaagagtcg gctcacaatc tccaaggata cctccaaaaa ccaggtattc


301
ctcaagatcg ccaatgtgga cactgcagat actgccacat actactgtgc tcgaataggg


361
gcggacgccc ttccttttga ctactggggc caaggcacca ctctcacagt ctcctcagcc


421
aaaacaacac ccccatcagt ctatccactg gcccctgggt gtggagatac aactggttcc


481
tccgtgacct ctgggtgcct ggtcaagggg tacttccctg agccagtgac tgtgacttgg


541
aactctggat ccctgtccag cagtgtgcac accttcccag ctctcctgca gtctggactc


601
tacactatga gcagctcagt gactgtcccc tccagcacct ggccaagtca gaccgtcacc


661
tgcagcgttg ctcacccagc cagcagcacc acggtggaca aaaaacttga gcccagcggg


721
cccatttcaa caatcaaccc ctgtcctcca tgcaaggagt gtcacaaatg cccagctcct


781
aacctcgagg gtggaccatc cgtcttcatc ttccctccaa atatcaagga tgtactcatg


841
atctccctga cacccaaggt cacgtgtgtg gtggtggatg tgagcgagga tgacccagac


901
gtccagatca gctggtttgt gaacaacgtg gaagtacaca cagctcagac acaaacccat


961
agagaggatt acaacagtac tatccgggtg gtcagcaccc tccccatcca gcaccaggac


1021
tggatgagtg gcaaggagtt caaatgcaag gtgaacaaca aagacctccc atcacccatc


1081
gagagaacca tctcaaaaat taaagggcta gtcagagctc cacaagtata cactttgccg


1141
ccaccagcag agcagttgtc caggaaagat gtcagtctca cttgcctggt cgtgggcttc


1201
aaccctggag acatcagtgt ggagtggacc agcaatgggc atacagagga gaactacaag


1261
gacaccgcac cagttcttga ctctgacggt tcttacttca tatatagcaa gctcaatatg


1321
aaaacaagca agtgggagaa aacagattcc ttctcatgca acgtgagaca cgagggtctg


1381
aaaaattact acctgaagaa gaccatctcc cggtctccgg gtaaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG2b Constant Region) of 09D03


(SEQ ID NO: 113)










1
mgrltssfll livpayvlsq vtlkesgpgi lrpsqt1slt csfsgfslst fglsvgwirq



61
psgkglewla hiwwdddkyy npalksrlti skdtsknqvf lkianvdtad tatyycarig


121
adalpfdywg qgttltvssa kttppsvypl apgcgdttgs svtsgclvkg yfpepvtvtw


181
nsgslsssvh tfpallqsgl ytmsssvtvp sstwpsqtvt csvahpasst tvdkklepsg


241
pistinpcpp ckechkcpap nleggpsvfi fppnikdvlm isltpkvtcv vvdvseddpd


301
vqiswfvnnv evhtaqtqth redynstiry vstlpiqhqd wmsgkefkck vnnkdlpspi


361
ertiskikgl vrapqvytlp ppaeqlsrkd vsltclvvgf npgdisvewt snghteenyk


421
dtapvldsdg syfiysklnm ktskwektds fscnvrhegl knyylkktis rspgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 09D03


(SEQ ID NO: 114)










1
atgaggtgcc tagctgagtt cctggggctg cttgtgctct ggatccctgg agccattggg



61
gatattgtgt tgactcagac tgcaccctct gtacctgtca ctcctggaga gtcagtatcc


121
atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg


181
ttcctgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc


241
tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc


301
agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatct agaatatcct


361
ttcacgttcg gctcggggac aaagttggaa ataaaacggg ctgatgctgc accaactgta


421
tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc


481
ttgaacaact tctaccccag agacatcaat gtcaagtgga agattgatgg cagtgaacga


541
caaaatggtg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg


601
agcagcaccc tcacattgac caaggacgag tatgaacgac ataacagcta tacctgtgag


661
gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacaggaa tgagtgt


 








Protein Sequence Defining the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 09D03


(SEQ ID NO: 115)










1
mrclaeflgl lvlwipgaig divltqtaps vpvtpgesys iscrssksll hsngntylyw



61
flqrpgqspq lliyrmsnla sgvpdrfsgs gsgtaftlri srveaedvgv yycmqhleyp


121
ftfgsgtkle ikradaaptv sifppsseql tsggasvvcf lnnfyprdin vkwkidgser


181
qngvinswtd qdskdstysm sstltltkde yerhnsytce athktstspi vksfnrnec


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 11G01


(SEQ ID NO: 116)










1
atggaatgga gctgggtctc tctcttcttc ctgtcagtaa ctacaggtgt ccactcccag



61
gttcagctgc aacagtctga cgctgagttg gtgaaacctg gagcttcagt gaagatatcc


121
tgcaaggttt ctggctacac cttcactgac catattattc actggatgaa gcagaggcct


181
gaacagggcc tggaatggat tggatatatt tatcctagag atggttatat taagtacaat


241
gagaagttca agggcaaggc cacattgact gcagacaaat cctccagcac agcctacatg


301
caggtcaaca gcctgacatc tgaggactct gcagtctatt tctgtgcaag gggttactat


361
tatgctatgg actactgggg tcaaggaacc tcagtcaccg tctcctcagc caaaacgaca


421
cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc


481
ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga


541
tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg


601
agcagctcag tgactgtccc ctccagcacc tggcccagcc agaccgtcac ctgcaacgtt


661
gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtggttgt


721
aagccttgca tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc


781
aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc


841
aaggatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct


901
cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc


961
atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct


1021
ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag


1081
gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc


1141
atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca


1201
gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac


1261
agcaagctca atgtgcagaa gagcaactgg gaggcaggaa atactttcac ctgctctgtg


1321
ttacatgagg gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 11G01


(SEQ ID NO: 117)










1
mewswvslff lsvttgvhsq vqlqqsdael vkpgasvkis ckvsgytftd hiihwmkqrp



61
eqglewigyi yprdgyikyn ekfkgkatlt adkssstaym qvnsltseds avyfcargyy


121
yamdywgqgt svtvssaktt ppsvyplapg saaqtnsmvt lgclvkgyfp epvtvtwnsg


181
slssgvhtfp avlqsdlytl sssvtvpsst wpsqtvtcnv ahpasstkvd kkivprdcgc


241
kpcictvpev ssvfifppkp kdvltitltp kvtcvvvdis kddpevqfsw fvddvevhta


301
qtqpreeqfn stfrsyselp imhqdwlngk efkcrvnsaa fpapiektis ktkgrpkapq


361
vytipppkeq makdkvsltc mitdffpedi tvewqwngqp aenykntqpi mdtdgsyfvy


421
sklnvqksnw eagntftcsv lheglhnhht ekslshspgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 11G01


(SEQ ID NO: 118)










1
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagaagtgat



61
gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc


121
tcttgcagat ctagtcagag cattgtacat agtattggaa acacctattt agaatggtac


181
ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct


241
ggggtcccag agaggttcag tggcagtgga tcagggacag atttcacact caagatcagc


301
agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tgttccattc


361
acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgcacc aactgtatcc


421
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg


481
aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa


541
aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc


601
agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc


661
actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt


 








Protein Sequence Defining the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 11G01


(SEQ ID NO: 119)










1
mklpvrllvl mfwipasrsd vlmtqtplsl pvslgdqasi scrssqsivh signtylewy



61
lqkpgqspkl liykvsnrfs gvperfsgsg sgtdftlkis rveaedlgvy ycfqgshvpf


121
tfgsgtklei kradaaptvs ifppsseqlt sggasvvcfl nnfypkdinv kwkidgserq


181
ngvinswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 12A07


(SEQ ID NO: 120)










1
atgggatgga gctgtatcat tgtcctcttg gtatcaacag ctacatgtgt ccactcccag



61
gtccaactgc tgcagcctgg ggctgagctg gtgaggcctg ggacttcagt gaagttgtcc


121
tgcaagactt ctggctacac cttctccagc tactggatgc actgggtaaa gcagaggcct


181
ggacaaggcc ttgagtggat cggaatgatt gatccttctg atgtttatac taactacaat


241
ccaaagttca agggcaaggc cacattgact gttgacacat cctccagcac agcctacatg


301
cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactactct


361
ggggactact ggggccaagg caccactctc acagtctcct cagccaaaac gacaccccca


421
tctgtctatc cactggcccc tggatctgct gcccaaacta actccatggt gaccctggga


481
tgcctggtca agggctattt ccctgagcca gtgacagtga cctggaactc tggatccctg


541
tccagcggtg tgcacacctt cccagctgtc ctgcagtctg acctctacac tctgagcagc


601
tcagtgactg tcccctccag cacctggccc agccagaccg tcacctgcaa cgttgcccac


661
ccggccagca gcaccaaggt ggacaagaaa attgtgccca gggattgtgg ttgtaagcct


721
tgcatatgta cagtcccaga agtatcatct gtcttcatct tccccccaaa gcccaaggat


781
gtgctcacca ttactctgac tcctaaggtc acgtgtgttg tggtagacat cagcaaggat


841
gatcccgagg tccagttcag ctggtttgta gatgatgtgg aggtgcacac agctcagacg


901
caaccccggg aggagcagtt caacagcact ttccgctcag tcagtgaact tcccatcatg


961
caccaggact ggctcaatgg caaggagttc aaatgcaggg tcaacagtgc agctttccct


1021
gcccccatcg agaaaaccat ctccaaaacc aaaggcagac cgaaggctcc acaggtgtac


1081
accattccac ctcccaagga gcagatggcc aaggataaag tcagtctgac ctgcatgata


1141
acagacttct tccctgaaga cattactgtg gagtggcagt ggaatgggca gccagcggag


1201
aactacaaga acactcagcc catcatggac acagatggct cttacttcgt ctacagcaag


1261
ctcaatgtgc agaagagcaa ctgggaggca ggaaatactt tcacctgctc tgtgttacat


1321
gagggcctgc acaaccacca tactgagaag agcctctccc actctcctgg taaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 12A07


(SEQ ID NO: 121)










1
mgwsciivll vstatcvhsq vqllqpgael vrpgtsvkls cktsgytfss ywmhwvkqrp



61
gqglewigmi dpsdvytnyn pkfkgkatlt vdtssstaym qlssltseds avyycarnys


121
gdywgqgttl tvssakttpp svyplapgsa aqtnsmvtlg clvkgyfpep vtvtwnsgsl


181
ssgvhtfpav lqsdlytlss svtvpsstwp sqtvtcnvah passtkvdkk ivprdcgckp


241
cictvpevss vfifppkpkd vltitltpkv tcvvvdiskd dpevqfswfv ddvevhtaqt


301
qpreeqfnst frsyselpim hqdwlngkef kcrvnsaafp apiektiskt kgrpkapqvy


361
tipppkeqma kdkvsltcmi tdffpeditv ewqwngqpae nykntqpimd tdgsyfvysk


421
lnvqksnwea gntftcsvlh eglhnhhtek slshspgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 12A07


(SEQ ID NO: 122)










1
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat



61
gttttgatga cccaaattcc actctccctg cctgtcagtc ttggagatca agcctccatc


121
tcttgtagat ctagtcagag cattgtccat agtaatggaa acacctattt agaatggtac


181
ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct


241
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc


301
agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcata tgttccgtgg


361
acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc


421
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg


481
aacaacttct accccagaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa


541
aatggtgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc


601
agcaccctca cattgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc


661
actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt


 








Protein Sequence Defining the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 12A07


(SEQ ID NO: 123)










1
mklpvrllvl mfwipasssd vlmtqiplsl pvslgdqasi scrssqsivh sngntylewy



61
lqkpgqspk1 liykvsnrfs gvpdrfsgsg sgtdftlkis rveaedlgvy ycfqgsyvpw


121
tfgggtklei kradaaptvs ifppsseqlt sggasvvcfl nnfyprdinv kwkidgserq


181
ngvinswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 18H02


(SEQ ID NO: 124)










1
atgggttggc tgtggaactt gctattcctg atggcagctg cccaaagtgc ccaagcacag



61
atccagttgg tacagtctgg acctgaactg aagaagcctg gagaggcagt caagatctcc


121
tgcaagtctt ctgggtatac cttcacaacc tatggaatga gctgggtgaa acaggctcca


181
ggaagggctt taaagtggat gggctggata aacacctact ctggagtgcc aacatatgct


241
gatgacttca agggacggtt tgccttctct ttggaatcct ctgccagcac tgcctatttg


301
cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcaag agggagggat


361
ggttaccaag tggcctggtt tgcttactgg ggccaaggga cgctggtcac tgtctctgca


421
gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac


481
tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc


541
tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac


601
ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag ccagaccgtc


661
acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg


721
gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc


781
cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg


841
gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag


901
gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc


961
agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc


1021
aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg


1081
aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc


1141
agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg


1201
aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct


1261
tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc


1321
acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac


1381
tctcctggta aatga


 








Protein Sequence Defining the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 18H02


(SEQ ID NO: 125)










1
mgwlwnllfl maaaqsaqaq iqlvqsgpel kkpgeavkis ckssgytftt ygmswvkqap



61
gralkwmgwi ntysgvptya ddfkgrfafs lessastayl qinnlknedt atyfcargrd


121
gyqvawfayw gqgtivtvsa akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt


181
wnsgslssgv htfpavlqsd lytlsssvtv psstwpsqtv tcnvahpass tkvdkkivpr


241
dcgckpcict vpevssvfif ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve


301
vhtaqtqpre eqfnstfrsv selpimhqdw lngkefkory nsaafpapie ktisktkgrp


361
kapqvytipp pkeqmakdkv sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs


421
yfvysklnvq ksnweagntf tcsvlheglh nhhtekslsh spgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 18H02


(SEQ ID NO: 126)










1
atgttctcac tagctcttct cctcagtctt cttctcctct gtgtctctga ttctagggca



61
gaaacaactg tgacccagtc tccagcatcc ctgtccatgg ctataggaga taaagtcacc


121
atcagatgca taaccagcac tgatattgat gatgatatga actggttcca gcagaagcca


181
ggggaacctc ctaagctcct tatttcagaa ggcaatactc ttcgtcctgg agtcccatcc


241
cgattctccg gcagtggcta tggtacagat tttattttta caattgaaaa catgctctct


301
gaagatgttg cagattacta ctgtttgcaa agtgataact tgccgtacac gttcggaggg


361
gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca


421
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac


481
cccagagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggtgtcctg


541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcaca


601
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca


661
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag


 








Protein Sequence Defining the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 18H02


(SEQ ID NO: 127)










1
mfslalllsl lllcvsdsra ettvtqspas lsmaigdkvt ircitstdid ddmnwfqqkp



61
geppkllise gntlrpgvps rfsgsgygtd fiftienmis edvadyyclq sdnlpytfgg


121
gtkleikrad aaptvsifpp sseqltsgga svvcflnnfy prdinvkwki dgserqngvl


181
nswtdqdskd stysmsstlt ltkdeyerhn sytceathkt stspivksfn rnec


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 22A02


(SEQ ID NO: 128)










1
atgggatgga gctgtatcat tgtcctcttg gtatcaacag ctacaggtgt ccactcccag



61
gtccaactgc agcagcctgg ggctgagctg gtgaggcctg ggacttcagt gaagttgtcc


121
tgcaaggctt ctggctacac cttcaccaac tactggatgc actgggtaaa gcagaggcct


181
ggacaaggcc ttgagtggat cggaatgatt gatccttctg atagttatac taactacaat


241
ccaaagttca agggtaaggc cacattgact gtagacacat cctccagcac agcctacatg


301
cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactactct


361
ggggactact ggggccaagg caccactctc acagtctcct cagccaaaac gacaccccca


421
tctgtctatc cactggcccc tggatctgct gcccaaacta actccatggt gaccctggga


481
tgcctggtca agggctattt ccctgagcca gtgacagtga cctggaactc tggatccctg


541
tccagcggtg tgcacacctt cccagctgtc ctgcagtctg acctctacac tctgagcagc


601
tcagtgactg tcccctccag cacctggccc agccagaccg tcacctgcaa cgttgcccac


661
ccggccagca gcaccaaggt ggacaagaaa attgtgccca gggattgtgg ttgtaagcct


721
tgcatatgta cagtcccaga agtatcatct gtcttcatct tccccccaaa gcccaaggat


781
gtgctcacca ttactctgac tcctaaggtc acgtgtgttg tggtagacat cagcaaggat


841
gatcccgagg tccagttcag ctggtttgta gatgatgtgg aggtgcacac agctcagacg


901
caaccccggg aggagcagtt caacagcact ttccgctcag tcagtgaact tcccatcatg


961
caccaggact ggctcaatgg caaggagttc aaatgcaggg tcaacagtgc agctttccct


1021
gcccccatcg agaaaaccat ctccaaaacc aaaggcagac cgaaggctcc acaggtgtac


1081
accattccac ctcccaagga gcagatggcc aaggataaag tcagtctgac ctgcatgata


1141
acagacttct tccctgaaga cattactgtg gagtggcagt ggaatgggca gccagcggag


1201
aactacaaga acactcagcc catcatggac acagatggct cttacttcgt ctacagcaag


1261
ctcaatgtgc agaagagcaa ctgggaggca ggaaatactt tcacctgctc tgtgttacat


1321
gagggcctgc acaaccacca tactgagaag agcctctccc actctcctgg taaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 22A02


(SEQ ID NO: 129)










1
mgwsciivll vstatgvhsq vqlqqpgael vrpgtsvkls ckasgytftn ywmhwvkqrp



61
gqglewigmi dpsdsytnyn pkfkgkatlt vdtssstaym qlssltseds avyycarnys


121
gdywgqgttl tvssakttpp svyplapgsa aqtnsmvtlg clvkgyfpep vtvtwnsgsl


181
ssgvhtfpav lqsdlytlss svtvpsstwp sqtvtcnvah passtkvdkk ivprdcgckp


241
cictvpevss vfifppkpkd vltitltpkv tcvvvdiskd dpevqfswfv ddvevhtaqt


301
qpreeqfnst frsyselpim hqdwlngkef kcrvnsaafp apiektiskt kgrpkapqvy


361
tipppkeqma kdkvsltcmi tdffpeditv ewqwngqpae nykntqpimd tdgsyfvysk


421
lnvqksnwea gntftcsvlh eglhnhhtek slshspgk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 22A02


(SEQ ID NO: 130)










1
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat



61
gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc


121
tcttgcagat ctagtcagag cattgtacat agtaatggaa acacctattt agaatggtac


181
ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct


241
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc


301
agagtggagg ctgaggatct gggagtttat tattgctttc aaggttcata tgttccgtgg


361
acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc


421
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg


481
aacaacttct accccagaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa


541
aatggtgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc


601
agcaccctca cattgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc


661
actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt


 








Protein Sequence Defining the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 22A02


(SEQ ID NO: 131)










1
mklpvrllvl mfwipasssd vlmtqtp1sl pvslgdqasi scrssqsivh sngntylewy



61
lqkpgqspk1 liykvsnrfs gvpdrfsgsg sgtdftlkis rveaedlgvy ycfqgsyvpw


121
tfgggtklei kradaaptvs ifppsseqlt sggasvvcfl nnfyprdinv kwkidgserq


181
ngvinswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec


 








Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 24C05


(SEQ ID NO: 132)










1
atgaacttcg ggctcagctt gatgttcctt gtccttgtct taaaaggtgt ccagtgtgag



61
gtgcagctgg tggaatctgg gggaggctta gtgaagcctg gagggtccct gaaactctcc


121
tgtgcagcct ctggattcac tttcagtgac tatgccatgt cttgggttcg ccagactccg


181
gaaaagaggc tggagtgggt cgcaaccatt agtgatggtg gtacttacac ctactatcca


241
gacaatgtaa agggccgatt caccatctcc agagacaatg ccaagaacaa cctgtacctg


301
caaatgagcc atctgaagtc tgaggacaca gccatgtatt actgtgcaag agaatggggt


361
gattacgacg gatttgacta ctggggccaa ggcaccactc tcacagtctc ctcggccaaa


421
acgacacccc catctgtcta tccactggcc cctggatctg ctgcccaaac taactccatg


481
gtgaccctgg gatgcctggt caagggctat ttccctgagc cagtgacagt gacctggaac


541
tctggatccc tgtccagcgg tgtgcacacc ttcccagctg tcctgcagtc tgacctctac


601
actctgagca gctcagtgac tgtcccctcc agcacctggc ccagccagac cgtcacctgc


661
aacgttgccc acccggccag cagcaccaag gtggacaaga aaattgtgcc cagggattgt


721
ggttgtaagc cttgcatatg tacagtccca gaagtatcat ctgtcttcat cttcccccca


781
aagcccaagg atgtgctcac cattactctg actcctaagg tcacgtgtgt tgtggtagac


841
atcagcaagg atgatcccga ggtccagttc agctggtttg tagatgatgt ggaggtgcac


901
acagctcaga cgcaaccccg ggaggagcag ttcaacagca ctttccgctc agtcagtgaa


961
cttcccatca tgcaccagga ctggctcaat ggcaaggagt tcaaatgcag ggtcaacagt


1021
gcagctttcc ctgcccccat cgagaaaacc atctccaaaa ccaaaggcag accgaaggct


1081
ccacaggtgt acaccattcc acctcccaag gagcagatgg ccaaggataa agtcagtctg


1141
acctgcatga taacagactt cttccctgaa gacattactg tggagtggca gtggaatggg


1201
cagccagcgg agaactacaa gaacactcag cccatcatgg acacagatgg ctcttacttc


1261
gtctacagca agctcaatgt gcagaagagc aactgggagg caggaaatac tttcacctgc


1321
tctgtgttac atgagggcct gcacaaccac catactgaga agagcctctc ccactctcct


1381
ggtaaa


 








Protein Sequence Defining the Full Length Heavy Chain Sequence



(Heavy Chain Variable Region and IgG1 Constant Region) of 24C05


(SEQ ID NO: 133)










1
mnfglslmfl vlvlkgvqce vqlvesgggl vkpggslkls caasgftfsd yamswvrqtp



61
ekrlewvati sdggtytyyp dnvkgrftis rdnaknnlyl qmshlksedt amyycarewg


121
dydgfdywgq gttltvssak ttppsvypla pgsaaqtnsm vtlgclvkgy fpepvtvtwn


181
sgslssgvht fpavlqsdly tlsssvtvps stwpsqtvtc nvahpasstk vdkkivprdc


241
gckpcictvp evssvfifpp kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh


301
taqtqpreeq fnstfrsyse 1pimhqdwln gkefkcrvns aafpapiekt isktkgrpka


361
pqvytipppk eqmakdkvsl tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf


421
vysklnvqks nweagntftc svlheglhnh htekslshsp gk


 








Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 24C05


(SEQ ID NO: 134)










1
atggacatga gggttcctgc tcacgttttt ggcttcttgt tgctctggtt tccaggtacc



61
agatgtgaca tccagatgac ccagtctcca tcctccttat ctgcctctct gggagaaaga


121
gtcagtctca cttgtcgggc aagtcaggaa attagtggtt acttaagctg gcttcagcag


181
aaaccagatg gaactattaa acgcctgatc tacgccgcat ccactttaga ttctggtgtc


241
ccaaaaaggt tcagtggcag taggtctggg tcagattatt ctctcaccat cggcagcctt


301
gagtctgaag atcttgcaga ctattactgt ctacaatatg atagttatcc gtacacgttc


361
ggagggggga ccaagctgga aataaaacgg gctgatgctg caccaactgt atccatcttc


421
ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac


481
ttctacccca gagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggt


541
gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc


601
ctcacattga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac


661
aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtgt


 








Protein Sequence Defining the Full Length Light Chain Sequence



(Kappa Chain Variable Region and Constant Region) of 24C05


(SEQ ID NO: 135)










1
mdmrvpahvf gflllwfpgt rcdiqmtqsp sslsaslger vsltcrasqe isgylswlqq



61
kpdgtikrli yaastldsgv pkrfsgsrsg sdysltigsl esedladyyc lqydsypytf


121
gggtkleikr adaaptvsif ppsseqltsg gasvvcflnn fyprdinvkw kidgserqng


181
vlnswtdqds kdstysmsst ltltkdeyer hnsytceath ktstspivks fnrnec






For convenience, Table 4 provides a concordance chart showing 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







108
04D01 Heavy Variable + IgG1 Constant-nucleic acid


109
04D01 Heavy Variable + IgG1 Constant-protein


110
04D01 Kappa Variable + Constant-nucleic acid


111
04D01 Kappa Variable + Constant-protein


112
09D03 Heavy Variable + IgG2b Constant-nucleic acid


113
09D03 Heavy Variable + IgG2b Constant-protein


114
09D03 Kappa Variable + Constant-nucleic acid


115
09D03 Kappa Variable + Constant-protein


116
11G01 Heavy Variable + IgG1 Constant-nucleic acid


117
11G01 Heavy Variable + IgG1 Constant-protein


118
11G01 Kappa Variable + Constant-nucleic acid


119
11G01 Kappa Variable + Constant-protein


120
12A07 Heavy Variable + IgG1 Constant-nucleic acid


121
12A07 Heavy Variable + IgG1 Constant-protein


122
12A07 Kappa Variable + Constant-nucleic acid


123
12A07 Kappa Variable + Constant-protein


124
18H02 Heavy Variable + IgG1 Constant-nucleic acid


125
18H02 Heavy Variable + IgG1 Constant-protein


126
18H02 Kappa Variable + Constant-nucleic acid


127
18H02 Kappa Variable + Constant-protein


128
22A02 Heavy Variable + IgG1 Constant-nucleic acid


129
22A02 Heavy Variable + IgG1 Constant-protein


130
22A02 Kappa Variable + Constant-nucleic acid


131
22A02 Kappa Variable + Constant-protein


132
24C05 Heavy Variable + IgG1 Constant-nucleic acid


133
24C05 Heavy Variable + IgG1 Constant--protein


134
24C05 Kappa Variable + Constant-nucleic acid


135
24C05 Kappa Variable + Constant-protein









Example 3
Binding Affinities
The binding affinities and kinetics of the binding of monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 to recombinant human ErbB3/Fc fusion protein (rhErbB3-Fc) were measured by surface plasmon resonance using a Biacore® T100 (Biacore) instrument.
Rabbit anti-mouse IgGs (Biacore, Cat. No. BR-1008-38) were immobilized on carboxymethylated dextran CM4 sensor chips (Biacore, Cat. No. BR-1005-34) by amine coupling (BIAcore, Cat. No. BR-1000-50) using a standard coupling protocol according to vendor's instructions. The analyses were performed at 25° C., using PBS (Invitrogen, Cat. No. 14040-133) containing 0.05% surfactant P20 (Biacore, Cat. No. BR-1000-54) as running buffer.
The antibodies were captured in individual flow cells at a flow rate of 10 μl/minute. Injection time was varied for each antibody to yield an Rmax between 30 and 60 RU. Buffer or rhErbB3-Fc 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-seconds injection of 10 mM Glycine-HCl, pH 1.7 (made from Glycine pH 1.5 (Biacore, Cat. No. BR-1003-54) and pH 2.0 (Biacore, Cat. No. BR-1003-55)) at a flow rate of 60 μl/minute. The rhErbB3-Fc concentration range tested was 0.125 nM to 20 nM.
Kinetic parameters were determined using the kinetic function of the BIA evaluation 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. Kinetic values of the monoclonal antibodies on rhErbB3-Fc at 25° C. are summarized in Table 5.














TABLE 5







Standard

Standard

Standard



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







04D01
3.8E+05
3.0E+04
9.3E−05
1.9E−05
2.5E−10
5.6E−11
5


09D03
2.7E+05
3.2E+04
2.0E−05
1.2E−05
8.0E−11
5.5E−11
3


11G01
2.7E+05
9.2E+04
2.2E−05
9.6E−06
9.1E−11
5.5E−11
4


12A07
6.2E+05
8.1E+04
1.9E−04
1.0E−04
3.0E−10
1.4E−10
3


18H02
2.8E+05
3.1E+04
2.5E−05
8.8E−06
9.1E−11
3.7E−11
4


22A02
7.0E+05
8.1E+04
2.2E−04
1.4E−04
3.2E−10
2.4E−10
3


24C05
1.5E+06
2.0E+05
9.2E−06
3.0E−06
6.5E−12
2.8E−12
4









The data in Table 5 demonstrate that the antibodies bind rhErbB3 with a KD of about 350 pM or less, 250 pM or less, 200 pM or less, 150 pM or less, 100 pM or less, 50 pM or less, or 10 pM or less.
Example 4
Neutralization Activity
In this example, the antibodies produced in Example 1 were tested for ability to inhibit rhErbB3 binding to NRG1-β1 and NRG1-α1. The antibodies were tested by electrochemiluminescence (ECL) assay for inhibition of hErbB3 binding to NRG1-β1. MA2400 96-well standard binding plates (Meso Scale Discovery, Cat. No. L15XA-6) were coated with 50 μl of 0.5 μg/mL rhErbB3/Fc (R&D systems, Cat. No. 348-RB) in PBS (Invitrogen, Cat. No. 14040-133) for overnight at 4° C. with no agitation. The plates then were washed 3 times with PBS+0.1% Tween20 (Sigma P5927) and blocked with 200 μl of PBS containing 5% BSA (Sera Care Life Sciences, Cat. No. AP-4510-80) for 1.5 hour at room temperature. After washing the plates 3 times with PBS, 25 μA of the antibody dilutions were added to the plates for another hour at room temperature with agitation. Ligand NRG1-β1 (R&D Systems, Cat. No. 377-HB, 26 kDa) was added to the wells at the final concentration of 0.25 μg/ml. The plates were washed three times with PBS and incubated with 25 μl of 1 μg/mL biotinylated antibody against human NRG1-β1 (R&D systems, Cat. No BAF377) preincubated for one hour with SULTO-TAG Streptavidin (Meso Scale Discovery, Cat. No R32AD-5) for one hour at room temperature with agitation. The plates then were washed 3 times with PBS, and 150 μl of 1× read buffer (Meso Scale Discovery, Cat. No. R92TC-1) was added to each well before the plates were analyzed on a Sector® Imager 2400 (Meso Scale Discovery) instrument.
The interaction of NRG1-β1 with ErbB3 was inhibited by antibodies 04D01, 12A07, 18H02, 22A02 and 24C05 (FIG. 6A). The interaction of NRG1-β1 with rhErbB3 was enhanced by antibody 09D03, but not as well as by antibody 11G01 (FIG. 6B).
The murine anti-human ErbB3 antibody IC50 values for neutralization of NRG1-β1 binding to rhErbB3 for the antibodies (i.e., 04D01, 12A07, 18H02, 22A02 and 24C05) were calculated and are summarized in Table 6.










TABLE 6









IC50 (nM)















Standard




Antibody
Average
Deviation
n







04D01
0.2232
0.0711
4



12A07
0.2351
0.0530
4



18H02
0.3460
0.0873
4



22A02
0.2418
0.0755
4



24C05
0.3367
0.0764
4










The results show that antibodies 04D01, 12A07, 18H02, 22A02, and 24C05 efficiently neutralized NRG1-β1 binding to rhErbB3. Antibodies 09D03 and 11G01 enhanced hNRG1-β1 binding to hErbB3.
The antibodies were tested by ECL assay for inhibition of hErbB3 binding to the second ErbB3 ligand, NRG1-α1. To assay inhibition of binding of NRG1-α1 to rhErbB3, the same method used for NRG1-β1 was used, except for the following changes: concentrations of plated rhErbB3/Fc (R&D 4518-RB) and of ligand NRG1-α1 (Thermo Scientific, RP-317-P1AX) were 1 μg/ml and 1.5 μg/ml, respectively.
The interaction of NRG1-α1 with rhErbB3 was inhibited by 11G01, 12A07, 18H02, 22A02, and 24C05 IgG1, and was enhanced by antibody 09D03 (FIG. 7).
Example 5
Binding to ErbB3 Domain II
In this example, the antibodies produced in Example 1 were tested for binding to the dimerization domain (domain 2) of hErbB3-ECD. Domain 2 of hErbB3 (118 amino acids, position 210-327) was cloned in place of domain 2 of Her2 (119 amino acids, position AA220-338) into the full-length Her2 receptor. The hybrid construct Her2/3d2 was cloned into pLenti6.3 and packaged by transient transfection of 293T cells into a Lentivirus using the ViraPowerTM Lentiviral Support Kit (Invitrogen, Cat. No. K497000). CHO cells were infected with the lentivirus expressing the Her2/3d2 hybrid protein. The binding of the anti-ErbB3 hybridoma supernatants to Her2/3d2 were tested on these engineered CHO cells by ECL with sulfo-tagged anti-mouse antibodies. Data on the binding of the hybridoma supernatants to the chimeric protein Her2/3d2 expressed on the cell surface of CHO cells are summarized in FIG. 8. These results show that antibodies 09D03 and 11G01 bound to the ErbB3 domain 11, AA210-327.
Example 6
Anti-Proliferative Activity
This example describes a characterization of the antibodies produced in Example 1 for their ability to inhibit NRG1-β1 dependent proliferation of cells. Antibodies were tested in the BaF/3 cell system engineered to express both human Her2 and ErbB3 and in the human MCF7 breast cancer cells which naturally express both Her2 and ErbB3 and grow in response to NRG1-β1 stimulation.
BaF/3 cells were infected by two lentiviruses engineered to express human Her2 or human ErbB3. Infected cells were selected with blasticidin (15 μg/ml; Invitrogen, Cat. No. R21001) and individual colonies were isolated and tested for expression of both receptors. Her2/ErbB3 expressing clones were maintained in culture under blasticidin selection with [80% RPMI Medium 1640 (GIBCO, Cat. No. 11875-093), 10% fetal bovine serum (GIBCO, Cat. No. 10438-026) and 10% WEHI cell conditioned media {90% ISCOVE's Modified Dulbecco's Medium (GIBCO, Cat. No. 12440053), 10% fetal bovine serum (GIBCO, Cat. No. 10438-026)+2 mM L-glutamine (GIBCO, Cat. No. 25030-081)+0.0025 mM mercaptoethanol (Invitrogen, Cat. No. 21985-023)}]. To screen for antagonistic ErbB3 antibodies, cells were rinsed with PBS, and grown in the absence of blasticidin and WEHI conditioned media. Assays were conducted in a 96-well plate (5,000 cells/well) in the presence of NRG1-β1 (100 ng/ml) and various concentrations of antibodies (0.018-5000 ng/ml in 100 μl final volume). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were conducted 3-4 days post NRG1-β1 stimulation.
An example of the dose-dependent inhibition of NRG1-β1 dependent cell proliferation of Her2/ErbB3-BaF/3 by murine anti-human ErbB3 antibodies is shown in FIG. 9. Inhibition data of NRG1-β1 dependent Her2/ErbB3-BaF/3 cell line proliferation with monoclonal antibodies (i.e., 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05) are summarized in Table 7.







TABLE 7







Her2/ErbB3-BaF/3, NRG1-β1 dep. Proliferation













IC50 (nM)-





Antibody
Average
Standard deviation
n







04D01
0.373
0.061
3



09D03
1.395
0.268
3



11G01
1.934
0.116
3



12A07
0.854
0.059
3



18H02
1.930
0.276
3



22A02
1.291
0.151
3



24C05
0.145
0.031
3










The results in Table 7 show that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 strongly inhibited NRG1-β1-induced proliferation of BaF/3 cells expressing Her2/ErbB3.
MCF7 cells (ATCC, Cat. No. HTB-22) were maintained as recommended by ATCC. Cells were plated at 5,000 cells/well in a 96-well plate. Cells were starved overnight in the absence of serum. The following day, NRG1-β1 (40 ng/ml) and various concentrations of antibodies (12.8 pg/ml-20 μg/ml in 100 μl final volume) were added to the cells. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were conducted three days post NRG1-β1 stimulation.
An example of the dose-dependent inhibition of NRG1-β1 dependent proliferation of MCF7 cells by murine anti-human ErbB3 antibodies is shown in FIG. 10. Inhibition data of NRG1-β1 dependent MCF7 cell proliferation with antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 are summarized in Table 8.







TABLE 8







MCF7 cells, NRG1-β1 dependent Proliferation













IC50 (nM)-





Antibody
Average
Standard deviation
n







04D01
0.47
0.23
3



09D03
2.28
0.60
3



11G01
1.98
1.34
3



12A07
0.74
0.48
3



18H02
1.00
0.20
3



22A02
1.62
0.60
3



24C05
0.39
0.04
3










The results in Table 8 demonstrate that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02, and 24C05 strongly inhibited NRG1-β1-induced proliferation of MCF7 cells.
The antibodies produced in Example 1 were also tested for their ability to inhibit proliferation of ErbB3 expressing human cancer cells. Breast cancer cells SKBR-3 overexpress Her2 and are sensitive to Her2-specific inhibitory antibodies.
SKBR-3 cells (ATCC, Cat. No. HTB-30) were maintained as recommended by ATCC. Cells were plated at 5,000 cells/well in a 96-well plate in the presence of 5 μg/ml of antibodies but without exogenous NRG1-β1. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were conducted after three days in culture.
An example of inhibition of cell proliferation of SKBR-3 cells by murine anti-human ErbB3 antibodies is shown in FIG. 11. The results in FIG. 11 show that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited proliferation of SKBR-3 cells.
Example 7
Inhibition of Downstream Signaling
This example describes a characterization of the antibodies produced in Example 1 for their ability to inhibit NRG1-β1 dependent phosphorylation of ErbB3 and the downstream kinase Akt, as the readout for PI3K activation. These antibodies were also tested for their ability to inhibit steady state phosphorylation of ErbB3 and Akt in exponentially growing cells.
Breast cancer cells SKBR-3 and MCF7 and prostate cancer cells DU145 were maintained as recommended by ATCC. Cells were starved overnight in 0% FBS, treated for one hour with 5 μg/ml of antibody followed by NRG1-β1 stimulation. Lysates were either analyzed by ELISA with the Phospho-ErbB3 kit from R&D Systems (Cat. No DYC1769) or with the Phospho-Akt ELISA kit from Cell Signaling (Cat. No 7143).
An example of the inhibition of the NRG1-β1 induced phosphorylation of ErbB3 in SKBR-3 cells by murine anti-human ErbB3 antibodies is shown in FIG. 12. The results in FIG. 12 demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited at least 50% of the phosphorylation of ErbB3 induced by NRG1-β1 in SKBR-3 cells.
An example of the inhibition of the NRG1-β1 induced phosphorylation of Akt in MCF7 and DU145 cells by murine anti-human ErbB3 antibodies is shown in FIG. 13A and FIG. 13B, respectively. The results in FIGS. 13A and 13B demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited at least 80% of the phosphorylation of Akt in response to the NRG1-β1 in both MCF7 and DU145 cells.
The capacity of the anti-ErbB3 antibodies to inhibit the steady state phosphorylation status of ErbB3 and Akt in a breast cancer cell line SKBR-3 and a pancreatic cancer cell line BxPC3 were tested by treating these exponentially growing cells for one hour in presence of antibodies at 5 μg/ml.
Western blot analysis of these experiments demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited the steady state level of phosphorylation of Akt and ErbB3 in both SKBR-3 and BxPC3 cells.
Example 8
Inhibition of NRG1-β1-Induced EGFR Phosphorylation
In this example, the antibodies produced in Example 1 were tested for their ability to inhibit NRG1-β1 dependent phosphorylation of EGFR in the ovarian cancer cell line NCI/ADR-RES. NCI/ADR-RES cells (DTP/DCTD NCI tumor repository) were starved overnight in 0% FBS, pre-treated with antibody (5 μg/ml) for one hour followed by NRG1-β1 (20 ng/ml) stimulation for 15 minutes. The phosphorylation of EGFR on tyrosine 1068 was analyzed by Western blot. The results of this experiment demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited the phosphorylation of EGFR in response to the NRG1-β1 in NCI/ADR-RES cells.
Example 9
Inhibition of EGF-Induced ErbB3 Phosphorylation
In this example, the antibodies produced in Example 1 were tested for their ability to inhibit EGF dependent phosphorylation of ErbB3 in the EGFR overexpressing, epidermoid cancer cell line A431. A431 cells (ATCC, Cat. No CRL-1555) were starved overnight in 0% FBS, pre-treated with antibody (5 μg/ml) for one hour followed by EGF (R&D Systems, Cat. No. 236-EG) (50 ng/ml) stimulation for 15 minutes. The phosphorylation of ErbB3 was analyzed by Western blot. The results of this experiment demonstrated that antibodies 04D01, 09D03, 12A07, 18H02, 22A02 and 24C05 inhibited to various extents the phosphorylation of ErbB3 in response to the EGF in A431 cells.
Example 10
Inhibition of NRG1-β1-Induced her2/ErbB3 Heterodimer Formation
This example describes a characterization of the antibodies produced in Example 1 for their ability to inhibit the formation of the Her2/ErbB3 dimer in response to NRG1-β1 in SKBR-3 cells. Breast cancer cells SKBR-3 were starved overnight in 0% FBS, treated for one hour with 5 μg/ml of antibody followed by NRG1-β1 stimulation (30 ng/ml, 30 min). Lysates were immunoprecipitated with anti-Her2 antibody (R&D Systems, Cat. No. BAF1129) and analyzed by Western blot with polyclonal anti-ErbB3 antibody (Santa Cruz, Cat. No. SC285).
The results of this experiment demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited NRG1-β1-induced Her2/ErbB3 dimer formation in SKBR-3 cells.
Example 11
Inhibition of BxPC3 Tumor Xenograft Growth
The ability of murine monoclonal antibodies produced in Example 1 to inhibit tumor growth was tested in a pancreatic BxPC3 xenograft model. Human pancreatic BxPC3 cells were grown in culture in 37° C. in an atmosphere containing 5% CO2, using RMPI medium containing 10% fetal bovine serum. BxPC3 cells were inoculated subcutaneously into the flank of 8-week old female CB.17 SCID mice (Taconic Labs) with 10×106 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were taken twice weekly using vernier calipers. Tumor volume was calculated using the formula: width×width×length/2. When tumors reached approximately 200 mm3, the mice were randomized into 9 groups of 10 mice each. One group received PBS and another received human IgG control (huIgG). Each of the other eight groups received one of the antibody, 04D01, 09D03, 18H02, 11G01, 24C05, 22A02, or 12A07. All antibodies were dosed at 20 mg/kg body weight, twice per week, by intra-peritoneal injection for 6 weeks. Tumor volumes and mouse body weights were recorded twice per week. Tumor growth inhibition was analyzed using ANOVA and is expressed as percent inhibition compared to the PBS control.
The results in FIG. 14 show that antibody 24C05 inhibited tumor growth by 76% in this model (p<0.001). Antibodies 04D01, 18H02 and 11G01 also inhibited tumor growth in this model at 64%, 71%, and 72%, respectively (p<0.001). Antibodies 12A07 and 22A02 demonstrated the least activity, i.e., near 40% tumor growth inhibition, while antibody 09D03 gave 60% tumor growth inhibition in this model.
Example 12
Humanization of Anti-ErbB3 Antibodies
A. Construction of Humanized and Chimeric Anti-ErbB3 Antibodies
This Example describes the humanization of the murine antibody designated 24C05, and the characterization of the resulting humanized antibodies. The humanized anti-ErbB3 antibodies were designed using the SUPERHUMANIZATIONTM method (Arana Therapeutics Ltd. and Hwang, W. Y. et al. (2005) METHODS 36:35-42) or 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). With the exception of heavy chain CDR1, the Kabat CDR definitions were used for CDR grafting onto human frameworks. A combination of Kabat and Chothia definitions were used for grafting heavy CDR1. 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. Additionally, one humanized heavy chain, Sh24C05 Hv3-11 Heavy IgG1, was mutated using overlap extension PCR to enhance humanization, resulting in the Sh24C05 Hv3-11 N62S heavy chain IgG1. A human IgG2 version of the Sh24C05 Hv3-11 N62S heavy chain was also constructed.
The anti-ErbB3 antibody chains humanized according to the SUPERHUMANIZATIONTM method, as described herein, are designated with the prefix “Sh” before the antibody chain name. The anti-ErbB3 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.
Chimeric (murine variable region and human constant region) 24C05 heavy (human IgG1) and light (human Kappa) chains were also constructed. 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 Biologics) via HindIII and EcoRI sites using In-FusionTM PCR cloning (Clontech). The humanized and chimeric Kappa light chains were subcloned into pEE14.4 (Lonza Biologics) 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 ErbB3 was measured as described below. The results are summarized in Table 15.
Additionally, some humanized antibody heavy and light chain combinations were stably expressed in CHOK1 SV cells using the GS SystemTM (Lonza Biologics) 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 polyA 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 immunoglobulin heavy chain and immunoglobulin light chain variable regions are set forth below in Table 9.








TABLE 9





Light Chain Variable Region
Heavy Chain Variable Region







Hu24C05 KvA (SEQ ID NO: 174)
Hu24C05 HvA (SEQ ID NO: 162)


Hu24C05 KvA (SEQ ID NO: 174)
Sh24C05 Hv3-21 (SEQ ID NO: 156)


Hu24C05 KvA (SEQ ID NO: 174)
Sh24C05 Hv3-23 (SEQ ID NO: 158)


Hu24C05 KvA (SEQ ID NO: 174)
Sh24C05 Hv3-30 (SEQ ID NO: 160)


Hu24C05 KvA (SEQ ID NO: 174)
Sh24C05 Hv3-7 (SEQ ID NO: 150)


Hu24C05 KvA (SEQ ID NO: 174)
Sh24C05 Hv3-11 (SEQ ID NO: 152)


Hu24C05 KvA (SEQ ID NO: 174)
Sh24C05 Hv3-11 N62S (SEQ ID NO: 154)


Sh24C05 Kv1-16 (SEQ ID NO: 166)
Hu24C05 HvA (SEQ ID NO: 162)


Sh24C05 Kv1-16 (SEQ ID NO: 166)
Sh24C05 Hv3-21 (SEQ ID NO: 156)


Sh24C05 Kv1-16 (SEQ ID NO: 166)
Sh24C05 Hv3-23 (SEQ ID NO: 158)


Sh24C05 Kv1-16 (SEQ ID NO: 166)
Sh24C05 Hv3-30 (SEQ ID NO: 160)


Sh24C05 Kv1-16 (SEQ ID NO: 166)
Sh24C05 Hv3-7 (SEQ ID NO: 150)


Sh24C05 Kv1-16 (SEQ ID NO: 166)
Sh24C05 Hv3-11 (SEQ ID NO: 152)


Sh24C05 Kv1-16 (SEQ ID NO: 166)
Sh24C05 Hv3-11 N62S (SEQ ID NO: 154)


Sh24C05 Kv1-17 (SEQ ID NO: 168)
Hu24C05 HvA (SEQ ID NO: 162)


Sh24C05 Kv1-17 (SEQ ID NO: 168)
Sh24C05 Hv3-21 (SEQ ID NO: 156)


Sh24C05 Kv1-17 (SEQ ID NO: 168)
Sh24C05 Hv3-23 (SEQ ID NO: 158)


Sh24C05 Kv1-17 (SEQ ID NO: 168)
Sh24C05 Hv3-30 (SEQ ID NO: 160)


Sh24C05 Kv1-17 (SEQ ID NO: 168)
Sh24C05 Hv3-7 (SEQ ID NO: 150)


Sh24C05 Kv1-17 (SEQ ID NO: 168)
Sh24C05 Hv3-11 (SEQ ID NO: 152)


Sh24C05 Kv1-17 (SEQ ID NO: 168)
Sh24C05 Hv3-11 N62S (SEQ ID NO: 154)


Sh24C05 Kv1-33 (SEQ ID NO: 170)
Hu24C05 HvA (SEQ ID NO: 162)


Sh24C05 Kv1-33 (SEQ ID NO: 170)
Sh24C05 Hv3-21 (SEQ ID NO: 156)


Sh24C05 Kv1-33 (SEQ ID NO: 170)
Sh24C05 Hv3-23 (SEQ ID NO: 158)


Sh24C05 Kv1-33 (SEQ ID NO: 170)
Sh24C05 Hv3-30 (SEQ ID NO: 160)


Sh24C05 Kv1-33 (SEQ ID NO: 170)
Sh24C05 Hv3-7 (SEQ ID NO: 150)


Sh24C05 Kv1-33 (SEQ ID NO: 170)
Sh24C05 Hv3-11 (SEQ ID NO: 152)


Sh24C05 Kv1-33 (SEQ ID NO: 170)
Sh24C05 Hv3-11 N62S (SEQ ID NO: 154)


Sh24C05 Kv1-9 (SEQ ID NO: 164)
Hu24C05 HvA (SEQ ID NO: 162)


Sh24C05 Kv1-9 (SEQ ID NO: 164)
Sh24C05 Hv3-21 (SEQ ID NO: 156)


Sh24C05 Kv1-9 (SEQ ID NO: 164)
Sh24C05 Hv3-23 (SEQ ID NO: 158)


Sh24C05 Kv1-9 (SEQ ID NO: 164)
Sh24C05 Hv3-30 (SEQ ID NO: 160)


Sh24C05 Kv1-9 (SEQ ID NO: 164)
Sh24C05 Hv3-7 (SEQ ID NO: 150)


Sh24C05 Kv1-9 (SEQ ID NO: 164)
Sh24C05 Hv3-11 (SEQ ID NO: 152)


Sh24C05 Kv1-9 (SEQ ID NO: 164)
Sh24C05 Hv3-11 N62S (SEQ ID NO: 154)


Sh24C05 Kv1-39 (SEQ ID NO: 172)
Hu24C05 HvA (SEQ ID NO: 162)


Sh24C05 Kv1-39 (SEQ ID NO: 172)
Sh24C05 Hv3-21 (SEQ ID NO: 156)


Sh24C05 Kv1-39 (SEQ ID NO: 172)
Sh24C05 Hv3-23 (SEQ ID NO: 158)


Sh24C05 Kv1-39 (SEQ ID NO: 172)
Sh24C05 Hv3-30 (SEQ ID NO: 160)


Sh24C05 Kv1-39 (SEQ ID NO: 172)
Sh24C05 Hv3-7 (SEQ ID NO: 150)


Sh24C05 Kv1-39 (SEQ ID NO: 172)
Sh24C05 Hv3-11 (SEQ ID NO: 152)


Sh24C05 Kv1-39 (SEQ ID NO: 172)
Sh24C05 Hv3-11 N62S (SEQ ID NO: 154)









The nucleic acid sequences encoding and the protein sequences defining variable regions of the humanized 24C05 antibodies are summarized below (amino terminal signal peptide sequences are not shown). CDR sequences (Kabat definition) are shown in bold and are underlined in the amino acid sequences.








Nucleic Acid Sequence Encoding the Sh24C05 Hv3-7 Heavy Chain Variable Region



(SEQ ID NO: 149)










1
gaggttcagc tggtggaatc tggcggtggg cttgtacaac caggaggctc cctcagactg



61
agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca


121
cccgggaaag gactggagtg ggttgccact atcagcgatg gcggaacgta tacctattac


181
cctgacaatg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctac


241
ctgcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg


301
ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct


 








Protein Sequence Defining the Sh24C05 Hv3-7 Heavy Chain Variable Region



(SEQ ID NO: 150)










1
evqlvesggg lvqpggslrl scaasgftfs dyamswvrga pgkglewvat isdggtytyy



61
pdnvkgrfti srdnaknsly lqmnslraed tavyycarew gdydgfdywg qgtivtvss


 








Nucleic Acid Sequence Encoding the Sh24C05 Hv3-11 Heavy Chain Variable Region



(SEQ ID NO: 151)










1
caagttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg



61
agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatggat caggcaagca


121
cccgggaaag gactggagtg ggttagcact atcagcgatg gcggaacgta tacctattac


181
cctgacaatg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctac


241
cttcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg


301
ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct


 








Protein Sequence Defining the Sh24C05 Hv3-11 Heavy Chain Variable Region



(SEQ ID NO: 152)










1
qvqlvesggg lvkpggslrl scaasgftfs dyamswirga pgkglewvst isdggtytyy



61
pdnvkgrfti srdnaknsly lqmnslraed tavyycarew gdydgfdywg qgtlvtvss


 








Nucleic Acid Sequence Encoding the Sh24C05 Hv3-11 N62S Heavy Chain Variable Region



(SEQ ID NO: 153)










1
caagttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg



61
agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatggat caggcaagca


121
cccgggaaag gactggagtg ggttagcact atcagcgatg gcggaacgta tacctattac


181
cctgactccg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctac


241
cttcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg


301
ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct


 








Protein Sequence Defining the Sh24C05 Hv3-11 N62S Heavy Chain Variable Region



(SEQ ID NO: 154)










1
qvqlvesggg lvkpggslrl scaasgftfs dyamswirga pgkglewvst isdggtytyy



61
pdsvkgrfti srdnaknsly lqmnslraed tavyycarew gdydgfdywg qgtlvtvss


 








Nucleic Acid Sequence Encoding the Sh24C05 Hv3-21 Heavy Chain Variable Region



(SEQ ID NO: 155)










1
gaggttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg



61
agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca


121
cccgggaaag gactggagtg ggttagcact atcagcgatg gcggaacgta tacctattac


181
cctgacaatg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctat


241
ttgcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg


301
ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct


 








Protein Sequence Defining the Sh24C05 Hv3-21 Heavy Chain Variable Region



(SEQ ID NO: 156)










1
evqlvesggg lvkpggslrl scaasgftfs dyamswvrqa pgkglewvst isdggtytyy



61
pdnvkgrfti srdnaknsly lqmnslraed tavyycarew gdydgfdywg qgtlvtvss


 








Nucleic Acid Sequence Encoding the Sh24C05 Hv3-23 Heavy Chain Variable Region



(SEQ ID NO: 157)










1
gaggttcagc ttctggaatc tggcggtggg cttgtacagc caggaggctc cctcagactg



61
agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca


121
cccgggaaag gactggagtg ggtttcaact atcagcgatg gcggaacgta tacctattac


181
cctgacaatg tgaagggtcg gttcaccatt tccagggata acagcaagaa cacactctat


241
ctccagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg


301
ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct


 








Protein Sequence Defining the Sh24C05 Hv3-23 Heavy Chain Variable Region



(SEQ ID NO: 158)










1
evqllesggg lvqpggslrl scaasgftfs dyamswvrqa pgkglewvst isdggtytyy



61
pdnvkgrfti srdnskntly lqmnslraed tavyycarew gdydgfdywg qgtlvtvss


 








Nucleic Acid Sequence Encoding the Sh24C05 Hv3-30 Heavy Chain Variable Region



(SEQ ID NO: 159)










1
caggttcagc tggtggaatc tggcggtggg gtagtacaac caggacggtc cctcagactg



61
agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca


121
cccgggaaag gactggagtg ggttgccact atcagcgatg gcggaacgta tacctattac


181
cctgacaatg tgaagggtcg gttcaccatt tccagggata actcaaagaa caccctctat


241
ctccaaatga gtagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg


301
ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct


 








Protein Sequence Defining the Sh24C05 Hv3-30 Heavy Chain Variable Region



(SEQ ID NO: 160)










1
qvqlvesggg vvqpgrslrl scaasgftfs dyamswvrqa pgkglewvat isdggtytyy



61
pdnvkgrfti srdnskntly lqmsslraed tavyycarew gdydgfdywg qgtlvtvss


 








Nucleic Acid Sequence Encoding the Hu24C05 HvA Heavy Chain Variable Region



(SEQ ID NO: 161)










1
gaggttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg



61
agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca


121
cccgggaaag gactggagtg ggttgccact atcagcgatg gcggaacgta tacctattac


181
cctgacaatg tgaagggtcg gttcaccatt tccagggata acgcaaagaa cagtctctac


241
cttcagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg


301
ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct


 








Protein Sequence Defining the Hu24C05 HvA Heavy Chain Variable Region



(SEQ ID NO: 162)










1
evqlvesggg lvkpggslrl scaasgftfs dyamswvrqa pgkglewvat isdggtytyy



61
pdnvkgrfti srdnaknsly lqmnslraed tavyycarew gdydgfdywg qgtlvtvss


 








Nucleic Acid Sequence Encoding the Sh24C05 Kv1-9 Kappa Chain Variable Region



(SEQ ID NO: 163)










1
gatattcagt tgacccaatc acctagcttc ctctcagctt ccgtgggcga cagagttacc



61
ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtacca acagaagccc


121
ggaaaagccc ctaagctgtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt


181
cgattctccg gttctggctc cggaacagag ttcactctga caatttctag ccttcagcca


241
gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag


301
ggcactaaac tggagatcaa a


 








Protein Sequence Defining the Sh24C05 Kv1-9 Kappa Chain Variable Region



(SEQ ID NO: 164)










1
diqltqspsf lsasvgdrvt itcrasqeis gylswyqqkp gkapklliya astldsgvps



61
rfsgsgsgte ftltisslqp edfatyyclq ydsypytfgq gtkleik


 








Nucleic Acid Sequence Encoding the Sh24C05 Kv1-16 Kappa Chain Variable Region



(SEQ ID NO: 165)










1
gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc



61
ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtttca acagaagccc


121
ggaaaggccc cgaagagctt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt


181
cgattctccg gttctggctc cggaaccgac tttactctga caatttctag ccttcagcca


241
gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag


301
ggcactaaac tggagatcaa a


 








Protein Sequence Defining the Sh24C05 Kv1-16 Kappa Chain Variable Region



(SEQ ID NO: 166)










1
diqmtqspss lsasvgdrvt itcrasqeis gylswfqqkp gkapksliya astldsgvps



61
rfsgsgsgtd ftltisslqp edfatyyclq ydsypytfgq gtkleik


 








Nucleic Acid Sequence Encoding the Sh24C05 Kv1-17 Kappa Chain Variable Region



(SEQ ID NO: 167)










1
gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc



61
ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtatca acagaagccc


121
ggaaaagccc caaagaggtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt


181
cgattctccg gttctggctc cggaaccgag ttcactctga caatttctag ccttcagcca


241
gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag


301
ggcactaaac tggagatcaa a


 








Protein Sequence Defining the Sh24C05 Kv1-17 Kappa Chain Variable Region



(SEQ ID NO: 168)










1
diqmtqspss lsasvgdrvt itcrasqeis gylswyqqkp gkapkrliya astldsgvps



61
rfsgsgsgte ftltisslqp edfatyyclq ydsypytfgq gtkleik


 








Nucleic Acid Sequence Encoding the Sh24C05 Kv1-33 Kappa Chain Variable Region



(SEQ ID NO: 169)










1
gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc



61
ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtacca acagaagccc


121
ggaaaggccc ccaagctgtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt


181
cgattctccg gttctggctc cggaacagac tttactttta caatttctag ccttcagcca


241
gaggacatcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag


301
ggcactaaac tggagatcaa a


 








Protein Sequence Defining the Sh24C05 Kv1-33 Kappa Chain Variable Region



(SEQ ID NO: 170)










1
diqmtqspss lsasvgdrvt itcrasqeis gylswyqqkp gkapklliya astldsgvps



61
rfsgsgsgtd ftftisslqp ediatyyclq ydsypytfgq gtkleik


 








Nucleic Acid Sequence Encoding the Sh24C05 Kv1-39 Kappa Chain Variable Region



(SEQ ID NO: 171)










1
gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc



61
ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggtatca acagaagccc


121
ggaaaagccc ctaagctgtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt


181
cgattctccg gttctggctc cggaactgac ttcactctga caatttctag ccttcagcca


241
gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag


301
ggcactaaac tggagatcaa a


 








Protein Sequence Defining the Sh24C05 Kv1-39 Kappa Chain Variable Region



(SEQ ID NO: 172)










1
diqmtqspss lsasvgdrvt itcrasqeis gylswyqqkp gkapklliya astldsgvps



61
rfsgsgsgtd ftltisslqp edfatyyclq ydsypytfgq gtkleik


 








Nucleic Acid Sequence Encoding the Hu24C05 KvA Kappa Chain Variable Region



(SEQ ID NO: 173)










1
gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc



61
ataacctgtc gggcaagcca ggagatttct gggtacctgt cctggctgca acagaagccc


121
ggaggcgcca tcaagaggtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt


181
cgattctccg gttctggctc cggaagtgac tacactctga caatttctag ccttcagcca


241
gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag


301
ggcactaaac tggagatcaa a


 








Protein Sequence Defining the Hu24C05 KvA Kappa Chain Variable Region



(SEQ ID NO: 174)










1
diqmtqspss lsasvgdrvt itcrasqeis gylswlqqkp ggaikrliya astldsgvps



61
rfsgsgsgsd ytltisslqp edfatyyclq ydsypytfgq gtkleik






The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 12 are aligned in FIG. 15. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR1, CDR2, and CDR3 (Kabat definition) are identified by boxes.
The amino acid sequences defining the immunoglobulin light chain variable regions for the antibodies in Example 12 are aligned in FIG. 16. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR1, CDR2 and CDR3 are identified by boxes.
Table 10 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.








TABLE 10





SEQ. ID



NO.
Nucleic Acid or Protein
















149
Sh24C05 Hv3-7 Heavy Chain Variable Region-nucleic acid


150
Sh24C05 Hv3-7 Heavy Chain Variable Region-protein


57
Sh24C05 Hv3-7 Heavy Chain CDR1


58
Sh24C05 Hv3-7 Heavy Chain CDR2


59
Sh24C05 Hv3-7 Heavy Chain CDR3


151
Sh24C05 Hv3-11 Heavy Chain Variable Region-nucleic acid


152
Sh24C05 Hv3-11 Heavy Chain Variable Region-protein


57
Sh24C05 Hv3-11 Heavy Chain CDR1


58
Sh24C05 Hv3-11 Heavy Chain CDR2


59
Sh24C05 Hv3-11 Heavy Chain CDR3


153
Sh24C05 Hv3-11 N62S Heavy Chain Variable Region-nucleic



acid


154
Sh24C05 Hv3-11 N62S Heavy Chain Variable Region-protein


57
Sh24C05 Hv3-11 N62S Heavy Chain CDR1


148
Sh24C05 Hv3-11 N62S Heavy Chain CDR2


59
Sh24C05 Hv3-11 N62S Heavy Chain CDR3


155
Sh24C05 Hv3-21 Heavy Chain Variable Region-nucleic acid


156
Sh24C05 Hv3-21 Heavy Chain Variable Region-protein


57
Sh24C05 Hv3-21 Heavy Chain CDR1


58
Sh24C05 Hv3-21 Heavy Chain CDR2


59
Sh24C05 Hv3-21 Heavy Chain CDR3


157
Sh24C05 Hv3-23 Heavy Chain Variable Region-nucleic acid


158
Sh24C05 Hv3-23 Heavy Chain Variable Region-protein


57
Sh24C05 Hv3-23 Heavy Chain CDR1


58
Sh24C05 Hv3-23 Heavy Chain CDR2


59
Sh24C05 Hv3-23 Heavy Chain CDR3


159
Sh24C05 Hv3-30 Heavy Chain Variable Region-nucleic acid


160
Sh24C05 Hv3-30 Heavy Chain Variable Region-protein


57
Sh24C05 Hv3-30 Heavy Chain CDR1


58
Sh24C05 Hv3-30 Heavy Chain CDR2


59
Sh24C05 Hv3-30 Heavy Chain CDR3


161
Hu24C05 HvA Heavy Chain Variable Region-nucleic acid


162
Hu24C05 HvA Heavy Chain Variable Region-protein


57
Hu24C05 HvA Heavy Chain CDR1


58
Hu24C05 HvA Heavy Chain CDR2


59
Hu24C05 HvA Heavy Chain CDR3


163
Sh24C05 Kv1-9 Light (kappa) Chain Variable



Region-nucleic acid


164
Sh24C05 Kv1-9 Light (kappa) Chain Variable



Region-protein


60
Sh24C05 Kv1-9 Light (kappa) Chain CDR1


61
Sh24C05 Kv1-9 Light (kappa) Chain CDR2


62
Sh24C05 Kv1-9 Light (kappa) Chain CDR3


165
Sh24C05 Kv1-16 Light (kappa) Chain Variable



Region-nucleic acid


166
Sh24C05 Kv1-16 Light (kappa) Chain Variable



Region-protein


60
Sh24C05 Kv1-16 Light (kappa) Chain CDR1


61
Sh24C05 Kv1-16 Light (kappa) Chain CDR2


62
Sh24C05 Kv1-16 Light (kappa) Chain CDR3


167
Sh24C05 Kv1-17 Light (kappa) Chain Variable



Region-nucleic acid


168
Sh24C05 Kv1-17 Light (kappa) Chain Variable



Region-protein


60
Sh24C05 Kv1-17 Light (kappa) Chain CDR1


61
Sh24C05 Kv1-17 Light (kappa) Chain CDR2


62
Sh24C05 Kv1-17 Light (kappa) Chain CDR3


169
Sh24C05 Kv1-33 Light (kappa) Chain Variable



Region-nucleic acid


170
Sh24C05 Kv1-33 Light (kappa) Chain Variable



Region-protein


60
Sh24C05 Kv1-33 Light (kappa) Chain CDR1


61
Sh24C05 Kv1-33 Light (kappa) Chain CDR2


62
Sh24C05 Kv1-33 Light (kappa) Chain CDR3


171
Sh24C05 Kv1-39 Light (kappa) Chain Variable



Region-nucleic acid


172
Sh24C05 Kv1-39 Light (kappa) Chain Variable



Region-protein


60
Sh24C05 Kv1-39 Light (kappa) Chain CDR1


61
Sh24C05 Kv1-39 Light (kappa) Chain CDR2


62
Sh24C05 Kv1-39 Light (kappa) Chain CDR3


173
Hu24C05 KvA Light (kappa) Chain Variable



Region-nucleic acid


174
Hu24C05 KvA Light (kappa) Chain Variable



Region-protein


60
Hu24C05 KvA Light (kappa) Chain CDR1


61
Hu24C05 KvA Light (kappa) Chain CDR2


62
Hu24C05 KvA Light (kappa) Chain CDR3









Humanized monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 11.










TABLE 11






CDR1
CDR2
CDR3















Kabat










24C05
DYAMS
TISDGGTYTYYPDNVKG
EWGDYDGFDY



(SEQ ID NO: 57)
(SEQ ID NO: 58)
(SEQ ID NO: 59)


Hv3-7
DYAMS
TISDGGTYTYYPDNVKG
EWGDYDGFDY



(SEQ ID NO: 57)
(SEQ ID NO: 58)
(SEQ ID NO: 59)


Sh24C05
DYAMS
TISDGGTYTYYPDNVKG
EWGDYDGFDY


Hv3-11
(SEQ ID NO: 57)
(SEQ ID NO: 58)
(SEQ ID NO: 59)


Sh24C05
DYAMS
TISDGGTYTYYPDSVKG
EWGDYDGFDY


Hv3-11 N62S
(SEQ ID NO: 57)
(SEQ ID NO: 148)
(SEQ ID NO: 59)


Sh24C05
DYAMS
TISDGGTYTYYPDNVKG
EWGDYDGFDY


Hv3-21
(SEQ ID NO: 57)
(SEQ ID NO: 58)
(SEQ ID NO: 59)


Sh24C05
DYAMS
TISDGGTYTYYPDNVKG
EWGDYDGFDY


Hv3-23
(SEQ ID NO: 57)
(SEQ ID NO: 58)
(SEQ ID NO: 59)


Sh24C05
DYAMS
TISDGGTYTYYPDNVKG
EWGDYDGFDY


Hv3-30
(SEQ ID NO: 57)
(SEQ ID NO: 58)
(SEQ ID NO: 59)


Hu24C05
DYAMS
TISDGGTYTYYPDNVKG
EWGDYDGFDY


HvA
(SEQ ID NO: 57)
(SEQ ID NO: 58)
(SEQ ID NO: 59)


 







Chothia










24C05
GFTFSDY
SDGGTY
EWGDYDGFDY



(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 59)


Sh24C05
GFTFSDY
SDGGTY
EWGDYDGFDY


Hv3-7
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 59)


Sh24C05
GFTFSDY
SDGGTY
EWGDYDGFDY


Hv3-11
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 59)


Sh24C05
GFTFSDY
SDGGTY
EWGDYDGFDY


Hv3-11 N62S
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 59)


Sh24C05
GFTFSDY
SDGGTY
EWGDYDGFDY


Hv3-21
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 59)


Sh24C05
GFTFSDY
SDGGTY
EWGDYDGFDY


Hv3-23
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 59)


Sh24C05
GFTFSDY
SDGGTY
EWGDYDGFDY


Hv3-30
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 59)


Hu24C05
GFTFSDY
SDGGTY
EWGDYDGFDY


HvA
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 59)


 







IMGT










24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY



(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)


Sh24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY


Hv3-7
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)


Sh24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY


Hv3-11
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)


Sh24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY


Hv3-11 N62S
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)


Sh24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY


Hv3-21
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)


Sh24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY


Hv3-23
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)


Sh24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY


Hv3-30
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)


Hu24C05
GFTFSDYA
ISDGGTYT
AREWGDYDGFDY


HvA
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)









Humanized monoclonal antibody Kappa light chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 12.










TABLE 12






CDR1
CDR2
CDR3















Kabat/Chothia










24C05
RASQEISGYLS
AASTLDS
LQYDSYPYT



(SEQ ID NO: 60)
(SEQ ID NO: 61)
(SEQ ID NO: 62)


Sh24C05 Kv1-9
RASQEISGYLS
AASTLDS
LQYDSYPYT



(SEQ ID NO: 60)
(SEQ ID NO: 61)
(SEQ ID NO: 62)


Sh24C05 Kv1-16
RASQEISGYLS
AASTLDS
LQYDSYPYT



(SEQ ID NO: 60)
(SEQ ID NO: 61)
(SEQ ID NO: 62)


Sh24C05 Kv1-17
RASQEISGYLS
AASTLDS
LQYDSYPYT



(SEQ ID NO: 60)
(SEQ ID NO: 61)
(SEQ ID NO: 62)


Sh24C05 Kv1-33
RASQEISGYLS
AASTLDS
LQYDSYPYT



(SEQ ID NO: 60)
(SEQ ID NO: 61)
(SEQ ID NO: 62)


Sh24C05 Kv1-39
RASQEISGYLS
AASTLDS
LQYDSYPYT



(SEQ ID NO: 60)
(SEQ ID NO: 61)
(SEQ ID NO: 62)


Hu24C05 
RASQEISGYLS
AASTLDS
LQYDSYPYT


KvA
(SEQ ID NO: 60)
(SEQ ID NO: 61)
(SEQ ID NO: 62)


 







IMGT










24C05
QEISGY
AAS
LQYDSYPYT



(SEQ ID NO: 101)

(SEQ ID NO: 62)


Sh24C05
QEISGY
AAS
LQYDSYPYT


Kv1-9
(SEQ ID NO: 101)

(SEQ ID NO: 62)


Sh24C05
QEISGY
AAS
LQYDSYPYT


Kv1-16
(SEQ ID NO: 101)

(SEQ ID NO: 62)


Sh24C05
QEISGY
AAS
LQYDSYPYT


Kv1-17
(SEQ ID NO: 101)

(SEQ ID NO: 62)


Sh24C05
QEISGY
AAS
LQYDSYPYT


Kv1-33
(SEQ ID NO: 101)

(SEQ ID NO: 62)


Sh24C05
QEISGY
AAS
LQYDSYPYT


Kv1-39
(SEQ ID NO: 101)

(SEQ ID NO: 62)


Hu24C05
QEISGY
AAS
LQYDSYPYT


KvA
(SEQ ID NO: 101)

(SEQ ID NO: 62)









In Tables 11 and 12, the longest CDR sequences for the immunoglobulin heavy chain and light chain are shown in bold.
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 or a human IgG2 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: 175)










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: 176)










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 1papiektis kakgqprepq vytlppsree


241
mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw


301
qqgnvfscsv mhealhnhyt qkslslspgk


 








Nucleic Acid Sequence Encoding the Human IgG2 Heavy Chain Constant Region



(SEQ ID NO: 177)










1
gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag



61
agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg


121
tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca


181
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc


241
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc


301
aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc


361
ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc


421
gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc


481
gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt


541
gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc


601
aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg


661
cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac


721
caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg


781
gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac


841
ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac


901
gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc


961
tccctgtctc cgggtaaa


 








Protein Sequence Defining the Human IgG2 Heavy Chain Constant Region



(SEQ ID NO: 178)










1
astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss



61
glyslssvvt vpssnfgtqt ytcnvdhkps ntkvdktver kccvecppcp appvagpsvf


121
lfppkpkdtl misrtpevtc vvvdvshedp evqfnwyvdg vevhnaktkp reeqfnstfr


181
vvsvltvvhq dwlngkeykc kvsnkglpap iektisktkg qprepqvytl ppsreemtkn


241
qvsltclvkg fypsdiavew esngqpenny kttppmldsd gsfflysklt vdksrwqqgn


301
vfscsvmhea lhnhytqksl slspgk


 








Nucleic Acid Sequence Encoding the Human Kappa Light Chain Constant Region



(SEQ ID NO: 179)










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



(SEQ ID NO: 180)










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 sequences (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 are also included at the 5′ end of the DNA sequences or the amino terminal end of the protein sequences. It is also contemplated herein 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 24C05 Heavy Chain



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


(SEQ ID NO: 181)










1
atgaacttcg ggctcagctt gatgttcctt gtccttgtct taaaaggtgt ccagtgtgag



61
gtgcagctgg tggaatctgg gggaggctta gtgaagcctg gagggtccct gaaactctcc


121
tgtgcagcct ctggattcac tttcagtgac tatgccatgt cttgggttcg ccagactccg


181
gaaaagaggc tggagtgggt cgcaaccatt agtgatggtg gtacttacac ctactatcca


241
gacaatgtaa agggccgatt caccatctcc agagacaatg ccaagaacaa cctgtacctg


301
caaatgagcc atctgaagtc tgaggacaca gccatgtatt actgtgcaag agaatggggt


361
gattacgacg gatttgacta ctggggccaa ggcaccactc tcacagtctc ctcggcctca


421
acaaaaggac caagtgtgtt cccactcgcc cctagcagca agagtacatc cgggggcact


481
gcagcactcg gctgcctcgt caaggattat tttccagagc cagtaaccgt gagctggaac


541
agtggagcac tcacttctgg tgtccatact tttcctgctg tcctgcaaag ctctggcctg


601
tactcactca gctccgtcgt gaccgtgcca tcttcatctc tgggcactca gacctacatc


661
tgtaatgtaa accacaagcc tagcaatact aaggtcgata agcgggtgga acccaagagc


721
tgcgacaaga ctcacacttg tcccccatgc cctgcccctg aacttctggg cggtcccagc


781
gtctttttgt tcccaccaaa gcctaaagat actctgatga taagtagaac acccgaggtg


841
acatgtgttg ttgtagacgt ttcccacgag gacccagagg ttaagttcaa ctggtacgtt


901
gatggagtcg aagtacataa tgctaagacc aagcctagag aggagcagta taatagtaca


961
taccgtgtag tcagtgttct cacagtgctg caccaagact ggctcaacgg caaagaatac


1021
aaatgcaaag tgtccaacaa agcactccca gcccctatcg agaagactat tagtaaggca


1081
aaggggcagc ctcgtgaacc acaggtgtac actctgccac ccagtagaga ggaaatgaca


1141
aagaaccaag tctcattgac ctgcctggtg aaaggcttct accccagcga catcgccgtt


1201
gagtgggaga gtaacggtca gcctgagaac aattacaaga caaccccccc agtgctggat


1261
agtgacgggt ctttctttct gtacagtaag ctgactgtgg acaagtcccg ctggcagcag


1321
ggtaacgtct tcagctgttc cgtgatgcac gaggcattgc acaaccacta cacccagaag


1381
tcactgagcc tgagcccagg gaag


 








Protein Sequence Defining the Full Length Chimeric 24C05 Heavy Chain (Mouse



Heavy Chain Variable Region and Human IgG1 Constant Region)


(SEQ ID NO: 182)










1
mnfglslmfl vlvlkgvqce vqlvesgggl vkpggslkls caasgftfsd yamswvrqtp



61
ekrlewvati sdggtytyyp dnvkgrftis rdnaknnlyl qmshlksedt amyycarewg


121
dydgfdywgq gttltvssas tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn


181
sgaltsgvht fpavlqssgl yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks


241
cdkthtcppc papellggps vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv


301
dgvevhnakt kpreeqynst yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska


361
kgqprepqvy tlppsreemt knqvsltclv kgfypsdiav ewesngqpen nykttppvld


421
sdgsfflysk ltvdksrwqq gnvfscsvmh ealhnhytqk slslspgk


 








Nucleic Acid Sequence Encoding the Full Length Chimeric 24C05 Light Chain



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


(SEQ ID NO: 183)










1
atggacatga gggttcctgc tcacgttttt ggcttcttgt tgctctggtt tccaggtacc



61
agatgtgaca tccagatgac ccagtctcca tcctccttat ctgcctctct gggagaaaga


121
gtcagtctca cttgtcgggc aagtcaggaa attagtggtt acttaagctg gcttcagcag


181
aaaccagatg gaactattaa acgcctgatc tacgccgcat ccactttaga ttctggtgtc


241
ccaaaaaggt tcagtggcag taggtctggg tcagattatt ctctcaccat cggcagcctt


301
gagtctgaag atcttgcaga ctattactgt ctacaatatg atagttatcc gtacacgttc


361
ggagggggga ccaagctgga aataaaacgc acagtcgccg ctccctccgt gttcatcttt


421
ccaccaagtg atgagcaact gaagtctggt actgcttcag tcgtgtgtct gctgaacaat


481
ttctaccctc gagaagccaa agtccaatgg aaggtagaca acgcactgca gtccggcaat


541
agccaagaat cagttaccga acaggattca aaggacagta catattccct gagcagcact


601
ctgaccctgt caaaggccga ttacgagaaa cacaaggtct atgcttgcga agtgacacat


661
cagggactgt ccagcccagt gacaaaatct tttaaccgtg gggagtgt


 








Protein Sequence Defining the Full Length Chimeric 24C05 Light Chain (Mouse



Kappa Chain Variable Region and Human Kappa Constant Region)


(SEQ ID NO: 184)










1
mdmrvpahvf gflllwfpgt rcdiqmtqsp sslsaslger vsltcrasqe isgylswlqq



61
kpdgtikrli yaastldsgv pkrfsgsrsg sdysltigsl esedladyyc lqydsypytf


121
gggtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn


181
sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec


 








Nucleic Acid Sequence Encoding the Full Length Humanized sh24C05 Hv3-7 Heavy



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


(SEQ ID NO: 185)










1
atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct



61
aggtgcgagg ttcagctggt ggaatctggc ggtgggcttg tacaaccagg aggctccctc


121
agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc


181
caagcacccg ggaaaggact ggagtgggtt gccactatca gcgatggcgg aacgtatacc


241
tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt


301
ctctacctgc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga


361
gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt


421
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


481
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


541
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


601
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


661
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


721
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


781
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


841
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


901
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


961
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


1021
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


1081
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


1141
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


1201
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


1261
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


1321
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


1381
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-7 Heavy Chain



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


(SEQ ID NO: 186)










1
mdmrvpaqll gllllwlrga rcevqlvesg gglvqpggsl rlscaasgft fsdyamswvr



61
qapgkglewv atisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar


121
ewgdydgfdy wgqgtivtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv


181
swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve


241
pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn


301
wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti


361
skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp


421
vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-11



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


(SEQ ID NO: 187)










1
atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct



61
aggtgccaag ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc


121
agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atggatcagg


181
caagcacccg ggaaaggact ggagtgggtt agcactatca gcgatggcgg aacgtatacc


241
tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt


301
ctctaccttc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga


361
gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt


421
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


481
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


541
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


601
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


661
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


721
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


781
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


841
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


901
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


961
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


1021
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


1081
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


1141
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


1201
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


1261
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


1321
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


1381
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-11 Heavy



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


(SEQ ID NO: 188)










1
mdmrvpaqll gllllwlrga rcqvqlvesg gglvkpggsl rlscaasgft fsdyamswir



61
qapgkglewv stisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar


121
ewgdydgfdy wgqgtivtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv


181
swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve


241
pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn


301
wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti


361
skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp


421
vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-11



N62S IgG1 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant


Region)


(SEQ ID NO: 189)










1
atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct



61
aggtgccaag ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc


121
agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atggatcagg


181
caagcacccg ggaaaggact ggagtgggtt agcactatca gcgatggcgg aacgtatacc


241
tattaccctg actccgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt


301
ctctaccttc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga


361
gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt


421
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


481
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


541
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


601
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


661
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


721
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


781
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


841
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


901
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


961
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


1021
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


1081
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


1141
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


1201
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


1261
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


1321
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


1381
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-11 N62S IgG1



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


(SEQ ID NO: 190)










1
mdmrvpaqll gllllwlrga rcqvqlvesg gglvkpggsl rlscaasgft fsdyamswir



61
qapgkglewv stisdggtyt yypdsvkgrf tisrdnakns lylqmnslra edtavyycar


121
ewgdydgfdy wgqgtivtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv


181
swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve


241
pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn


301
wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti


361
skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp


421
vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-11



N62S IgG2 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG2


Constant Region)


(SEQ ID NO: 191)










1
atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct



61
aggtgccaag ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc


121
agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atggatcagg


181
caagcacccg ggaaaggact ggagtgggtt agcactatca gcgatggcgg aacgtatacc


241
tattaccctg actccgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt


301
ctctaccttc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga


361
gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt


421
tctgcctcca ccaagggccc atcggtcttc cccctggcgc cctgctccag gagcacctcc


481
gagagcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg


541
tcgtggaact caggcgctct gaccagcggc gtgcacacct tcccagctgt cctacagtcc


601
tcaggactct actccctcag cagcgtggtg accgtgccct ccagcaactt cggcacccag


661
acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa gacagttgag


721
cgcaaatgtt gtgtcgagtg cccaccgtgc ccagcaccac ctgtggcagg accgtcagtc


781
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcacg


841
tgcgtggtgg tggacgtgag ccacgaagac cccgaggtcc agttcaactg gtacgtggac


901
ggcgtggagg tgcataatgc caagacaaag ccacgggagg agcagttcaa cagcacgttc


961
cgtgtggtca gcgtcctcac cgttgtgcac caggactggc tgaacggcaa ggagtacaag


1021
tgcaaggtct ccaacaaagg cctcccagcc cccatcgaga aaaccatctc caaaaccaaa


1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag


1141
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag


1201
tgggagagca atgggcagcc ggagaacaac tacaagacca cacctcccat gctggactcc


1261
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg


1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc


1381
ctctccctgt ctccgggtaa a


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-11 N62S IgG2



Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG2 Constant Region)


(SEQ ID NO: 192)










1
mdmrvpaqll gllllwlrga rcqvqlvesg gglvkpggsl rlscaasgft fsdyamswir



61
qapgkglewv stisdggtyt yypdsvkgrf tisrdnakns lylqmnslra edtavyycar


121
ewgdydgfdy wgqgtivtvs sastkgpsvf plapcsrsts estaalgclv kdyfpepvtv


181
swnsgaltsg vhtfpavlqs sglyslssvv tvpssnfgtq tytcnvdhkp sntkvdktve


241
rkccvecppc pappvagpsv flfppkpkdt lmisrtpevt cvvvdvshed pevqfnwyvd


301
gvevhnaktk preeqfnstf rvvsvltvvh qdwlngkeyk ckvsnkglpa piektisktk


361
gqprepqvyt lppsreemtk nqvsltclvk gfypsdiave wesngqpenn ykttppmlds


421
dgsfflyskl tvdksrwqqg nvfscsvmhe alhnhytqks lslspgk


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-21



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


(SEQ ID NO: 193)










1
atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct



61
aggtgcgagg ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc


121
agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc


181
caagcacccg ggaaaggact ggagtgggtt agcactatca gcgatggcgg aacgtatacc


241
tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt


301
ctctatttgc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga


361
gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt


421
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


481
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


541
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


601
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


661
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


721
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


781
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


841
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


901
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


961
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


1021
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


1081
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


1141
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


1201
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


1261
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


1321
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


1381
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized 24C05 Hv3-21 Heavy Chain



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


(SEQ ID NO: 194)










1
mdmrvpaqll gllllwlrga rcevqlvesg gglvkpggsl rlscaasgft fsdyamswvr



61
qapgkglewv stisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar


121
ewgdydgfdy wgqgtivtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv


181
swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve


241
pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn


301
wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti


361
skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp


421
vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-23



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


(SEQ ID NO: 195)










1
atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct



61
aggtgcgagg ttcagcttct ggaatctggc ggtgggcttg tacagccagg aggctccctc


121
agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc


181
caagcacccg ggaaaggact ggagtgggtt tcaactatca gcgatggcgg aacgtatacc


241
tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacag caagaacaca


301
ctctatctcc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga


361
gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt


421
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


481
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


541
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


601
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


661
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


721
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


781
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


841
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


901
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


961
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


1021
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


1081
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


1141
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


1201
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


1261
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


1321
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


1381
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Hv3-23 Heavy



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


(SEQ ID NO: 196)










1
mdmrvpaqll gllllwlrga rcevqllesg gglvqpggsl rlscaasgft fsdyamswvr



61
qapgkglewv stisdggtyt yypdnvkgrf tisrdnsknt lylqmnslra edtavyycar


121
ewgdydgfdy wgqgtivtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv


181
swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve


241
pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn


301
wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti


361
skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp


421
vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Hv3-30



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


(SEQ ID NO: 197)










1
atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct



61
aggtgccagg ttcagctggt ggaatctggc ggtggggtag tacaaccagg acggtccctc


121
agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc


181
caagcacccg ggaaaggact ggagtgggtt gccactatca gcgatggcgg aacgtatacc


241
tattaccctg acaatgtgaa gggtcggttc accatttcca gggataactc aaagaacacc


301
ctctatctcc aaatgagtag cctgagggct gaggacaccg ccgtctacta ctgcgcccga


361
gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt


421
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


481
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


541
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


601
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


661
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


721
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


781
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


841
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


901
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


961
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


1021
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


1081
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


1141
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


1201
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


1261
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


1321
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


1381
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized of Sh24C05 Hv3-30 Heavy



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


(SEQ ID NO: 198)










1
mdmrvpaqll gllllwlrga rcqvqlvesg ggvvqpgrsl rlscaasgft fsdyamswvr



61
qapgkglewv atisdggtyt yypdnvkgrf tisrdnsknt lylqmsslra edtavyycar


121
ewgdydgfdy wgqgtivtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv


181
swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve


241
pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn


301
wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti


361
skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp


421
vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized Hu24C05 HvA Heavy



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


(SEQ ID NO: 199)










1
atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct



61
aggtgcgagg ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc


121
agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atgggtgcgc


181
caagcacccg ggaaaggact ggagtgggtt gccactatca gcgatggcgg aacgtatacc


241
tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt


301
ctctaccttc agatgaacag cctgagggct gaggacaccg ccgtctacta ctgcgcccga


361
gaatggggag attatgatgg gtttgactat tggggccagg gcactttggt gacagtcagt


421
tctgcctcaa caaaaggacc aagtgtgttc ccactcgccc ctagcagcaa gagtacatcc


481
gggggcactg cagcactcgg ctgcctcgtc aaggattatt ttccagagcc agtaaccgtg


541
agctggaaca gtggagcact cacttctggt gtccatactt ttcctgctgt cctgcaaagc


601
tctggcctgt actcactcag ctccgtcgtg accgtgccat cttcatctct gggcactcag


661
acctacatct gtaatgtaaa ccacaagcct agcaatacta aggtcgataa gcgggtggaa


721
cccaagagct gcgacaagac tcacacttgt cccccatgcc ctgcccctga acttctgggc


781
ggtcccagcg tctttttgtt cccaccaaag cctaaagata ctctgatgat aagtagaaca


841
cccgaggtga catgtgttgt tgtagacgtt tcccacgagg acccagaggt taagttcaac


901
tggtacgttg atggagtcga agtacataat gctaagacca agcctagaga ggagcagtat


961
aatagtacat accgtgtagt cagtgttctc acagtgctgc accaagactg gctcaacggc


1021
aaagaataca aatgcaaagt gtccaacaaa gcactcccag cccctatcga gaagactatt


1081
agtaaggcaa aggggcagcc tcgtgaacca caggtgtaca ctctgccacc cagtagagag


1141
gaaatgacaa agaaccaagt ctcattgacc tgcctggtga aaggcttcta ccccagcgac


1201
atcgccgttg agtgggagag taacggtcag cctgagaaca attacaagac aaccccccca


1261
gtgctggata gtgacgggtc tttctttctg tacagtaagc tgactgtgga caagtcccgc


1321
tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac


1381
acccagaagt cactgagcct gagcccaggg aag


 








Protein Sequence Defining the Full Length Humanized Hu24C05 HvA Heavy Chain



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


(SEQ ID NO: 200)










1
mdmrvpaqll gllllwlrga rcevqlvesg gglvkpggsl rlscaasgft fsdyamswvr



61
qapgkglewv atisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar


121
ewgdydgfdy wgqgtivtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv


181
swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve


241
pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn


301
wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti


361
skakgqprep qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp


421
vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Kv1-9 Light



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


(SEQ ID NO: 201)










1
atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct



61
cgttgcgata ttcagttgac ccaatcacct agcttcctct cagcttccgt gggcgacaga


121
gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtaccaacag


181
aagcccggaa aagcccctaa gctgttgatc tatgctgcgt caaccttgga tagcggtgtc


241
ccgagtcgat tctccggttc tggctccgga acagagttca ctctgacaat ttctagcctt


301
cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt


361
gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc


421
ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac


481
ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac


541
agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc


601
ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac


661
cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-9 Light Chain



(Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 202)










1
mdmrvpaqll gllllwlrga rcdiqltqsp sflsasvgdr vtitcrasqe isgylswyqq



61
kpgkapklli yaastldsgv psrfsgsgsg teftltissl qpedfatyyc lqydsypytf


121
gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn


181
sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Kv1-16 Light



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


(SEQ ID NO: 203)










1
atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct



61
cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga


121
gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtttcaacag


181
aagcccggaa aggccccgaa gagcttgatc tatgctgcgt caaccttgga tagcggtgtc


241
ccgagtcgat tctccggttc tggctccgga accgacttta ctctgacaat ttctagcctt


301
cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt


361
gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc


421
ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac


481
ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac


541
agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc


601
ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac


661
cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-16 Light Chain



(Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 204)










1
mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswfqq



61
kpgkapksli yaastldsgv psrfsgsgsg tdftltissl qpedfatyyc lqydsypytf


121
gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn


181
sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec


 








Nucleic Acid Sequence Encoding the Full Length Humanized Sh24C05 Kv1-17 Light



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


(SEQ ID NO: 205)










1
atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct



61
cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga


121
gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtatcaacag


181
aagcccggaa aagccccaaa gaggttgatc tatgctgcgt caaccttgga tagcggtgtc


241
ccgagtcgat tctccggttc tggctccgga accgagttca ctctgacaat ttctagcctt


301
cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt


361
gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc


421
ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac


481
ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac


541
agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc


601
ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac


661
cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-17 Light Chain



(Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 206)










1
mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswyqq



61
kpgkapkrli yaastldsgv psrfsgsgsg teftltissl qpedfatyyc lqydsypytf


121
gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn


181
sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec


 








Nucleic Acid Sequence Encoding the Full Length Humanized sh24C05 Kv1-33 Light



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


(SEQ ID NO: 207)










1
atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct



61
cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga


121
gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtaccaacag


181
aagcccggaa aggcccccaa gctgttgatc tatgctgcgt caaccttgga tagcggtgtc


241
ccgagtcgat tctccggttc tggctccgga acagacttta cttttacaat ttctagcctt


301
cagccagagg acatcgccac gtactattgc ctccagtacg acagctatcc ctatacattt


361
gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc


421
ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac


481
ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac


541
agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc


601
ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac


661
cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-33 Light Chain



(Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 208)










1
mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswyqq



61
kpgkapklli yaastldsgv psrfsgsgsg tdftftissl qpediatyyc lqydsypytf


121
gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn


181
sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec


 








Nucleic Acid Sequence Encoding the Full Length Humanzied Sh24C05 Kv1-39 Light



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


(SEQ ID NO: 209)










1
atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct



61
cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga


121
gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gtatcaacag


181
aagcccggaa aagcccctaa gctgttgatc tatgctgcgt caaccttgga tagcggtgtc


241
ccgagtcgat tctccggttc tggctccgga actgacttca ctctgacaat ttctagcctt


301
cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt


361
gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc


421
ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac


481
ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac


541
agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc


601
ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac


661
cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt


 








Protein Sequence Defining the Full Length Humanized Sh24C05 Kv1-39 Light Chain



(Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 210)










1
mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswyqq



61
kpgkapklli yaastldsgv psrfsgsgsg tdftltissl qpedfatyyc lqydsypytf


121
gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn


181
sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec


 








Nucleic Acid Sequence Encoding the Full Length Humanized Hu24C05 KvA Light



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


(SEQ ID NO: 211)










1
atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct



61
cgttgcgata ttcagatgac ccaatcacct agcagtctct cagcttccgt gggcgacaga


121
gttaccataa cctgtcgggc aagccaggag atttctgggt acctgtcctg gctgcaacag


181
aagcccggag gcgccatcaa gaggttgatc tatgctgcgt caaccttgga tagcggtgtc


241
ccgagtcgat tctccggttc tggctccgga agtgactaca ctctgacaat ttctagcctt


301
cagccagaag atttcgccac gtactattgc ctccagtacg acagctatcc ctatacattt


361
gggcagggca ctaaactgga gatcaaacgc acagttgctg cccccagcgt gttcattttc


421
ccacctagcg atgagcagct gaaaagcggt actgcctctg tcgtatgctt gctcaacaac


481
ttttacccac gtgaggctaa ggtgcagtgg aaagtggata atgcacttca atctggaaac


541
agtcaagagt ccgtgacaga acaggacagc aaagactcaa cttattcact ctcttccacc


601
ctgactctgt ccaaggcaga ctatgaaaaa cacaaggtat acgcctgcga ggttacacac


661
cagggtttgt ctagtcctgt caccaagtcc ttcaataggg gcgaatgt


 








Protein Sequence Defining the Full Length Humanized Hu24C05 KvA Light Chain



(Humanized Kappa Chain Variable Region and Human Constant Region)


(SEQ ID NO: 212)










1
mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswlqq



61
kpggaikrli yaastldsgv psrfsgsgsg sdytltissl qpedfatyyc lqydsypytf


121
gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn


181
sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec






For convenience, Table 13 provides a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.








TABLE 13





SEQ ID



NO.
Nucleic Acid or Protein







175
Human IgG1 constant-nucleic acid


176
Human IgG1 constant-protein


177
Human IgG2 constant-nucleic acid


178
Human IgG2 constant-protein


179
Human Kappa constant-nucleic acid


180
Human Kappa constant-protein


181
Chimeric 24C05 Mouse Heavy Chain Variable + Human IgG1



constant-nucleic acid


182
Chimeric 24C05 Mouse Heavy Chain Variable + Human IgG1



constant-protein


183
Chimeric 24C05 Mouse Light Chain Variable + Human Kappa



constant-nucleic acid


184
Chimeric 24C05 Mouse Light Chain Variable + Human Kappa



constant-protein


185
Humanized Sh24C05 Hv3-7 Heavy Human Variable + Human



IgG1 constant-nucleic acid


186
Humanized Sh24C05 Hv3-7 Heavy Human Variable + Human



IgG1 constant-protein


187
Humanized Sh24C05 Hv3-11 Heavy Human Variable + Human



IgG1 constant-nucleic acid


188
Humanized Sh24C05 Hv3-11 Heavy Human Variable + Human



IgG1 constant-protein


189
Humanized Sh24C05 Hv3-11 N62S IgG1 Heavy Human



Variable + Human IgG1 constant-nucleic acid


190
Humanized Sh24C05 Hv3-11 N62S IgG1 Heavy Human



Variable + Human IgG1 constant-protein


191
Humanized Sh24C05 Hv3-11 N62S IgG2 Heavy Human



Variable + Human IgG2 constant-nucleic acid


192
Humanized Sh24C05 Hv3-11 N62S IgG2 Heavy Human



Variable + Human IgG2 constant-protein


193
Humanized Sh24C05 Hv3-21 Heavy Human Variable + Human



IgG1 constant-nucleic acid


194
Humanized Sh24C05 Hv3-21 Heavy Human Variable + Human



IgG1 constant-protein


195
Humanized Sh24C05 Hv3-23 Heavy Human Variable + Human



IgG1 constant-nucleic acid


196
Humanized Sh24C05 Hv3-23 Heavy Human Variable + Human



IgG1 constant-protein


197
Humanized Sh24C05 Hv3-30 Heavy Human Variable + Human



IgG1 constant-nucleic acid


198
Humanized Sh24C05 Hv3-30 Heavy Human Variable + Human



IgG1 constant-protein


199
Humanized Hu24C05 HvA Heavy Human Variable + Human



IgG1 constant-nucleic acid


200
Humanized Hu24C05 HvA Heavy Human Variable + Human



IgG1 constant-protein


201
Humanized Sh24C05 Kv1-9 Human Variable + Human Kappa



constant-nucleic acid


202
Humanized Sh24C05 Kv1-9 Human Variable + Human Kappa



constant-protein


203
Humanized Sh24C05 Kv1-16 Human Variable + Human Kappa



constant-nucleic acid


204
Humanized Sh24C05 Kv1-16 Human Variable + Human Kappa



constant-protein


205
Humanized Sh24C05 Kv1-17 Human Variable + Human Kappa



constant-nucleic acid


206
Humanized Sh24C05 Kv1-17 Human Variable + Human Kappa



constant-protein


207
Humanized Sh24C05 Kv1-33 Human Variable + Human Kappa



constant-nucleic acid


208
Humanized Sh24C05 Kv1-33 Human Variable + Human Kappa



constant-protein


209
Humanized Sh24C05 Kv1-39 Human Variable + Human Kappa



constant-nucleic acid


210
Humanized Sh24C05 Kv1-39 Human Variable + Human Kappa



constant-protein


211
Humanized Hu24C05 KvA Human Variable + Human Kappa



constant-nucleic acid


212
Humanized Hu24C05 KvA Human Variable + Human Kappa



constant-protein









Table 14 below shows antibodies containing chimeric immunoglobulin heavy and light chains and each of the possible combinations of the full-length humanized immunoglobulin heavy and light chains.









TABLE 14





Antibody




Name
Light Chain
Heavy Chain







Sh24C05-1
24C05 Chimeric Kappa
GP203 24C05 Chimeric



(SEQ ID NO: 184)
Heavy IgG1




(SEQ ID NO: 182)


Sh24C05-14
Hu24C05 KvA Kappa
Hu24C05 HvA IgG1



(SEQ ID NO: 212)
(SEQ ID NO: 200)


Sh24C05-15
Hu24C05 KvA Kappa
Sh24C05 Hv3-21 Heavy IgG1



(SEQ ID NO: 212)
(SEQ ID NO: 194)


Sh24C05-16
Hu24C05 KvA Kappa
Sh24C05 Hv3-23 Heavy IgG1



(SEQ ID NO: 212)
(SEQ ID NO: 196)


Sh24C05-17
Hu24C05 KvA Kappa
Sh24C05 Hv3-30 Heavy IgG1



(SEQ ID NO: 212)
(SEQ ID NO: 198)


Sh24C05-18
Hu24C05 KvA Kappa
Sh24C05 Hv3-7 Heavy IgG1



(SEQ ID NO: 212)
(SEQ ID NO: 186)


Sh24C05-19
Hu24C05 KvA Kappa
Sh24C05 Hv3-11 Heavy IgG1



(SEQ ID NO: 212)
(SEQ ID NO: 188)


Sh24C05-19
Hu24C05 KvA Kappa
Sh24C05 Hv3-11 N62S


N62S IgG1
(SEQ ID NO: 212)
Heavy IgG1




(SEQ ID NO: 190)


Sh24C05-19
Hu24C05 KvA Kappa
Sh24C05 Hv3-11 N62S


N62S IgG2
(SEQ ID NO: 212)
Heavy IgG2




(SEQ ID NO: 192)


Sh24C05-20
Sh24C05 Kv1-16 Kappa
Hu24C05 HvA IgG1



(SEQ ID NO: 204)
(SEQ ID NO: 200)


Sh24C05-21
Sh24C05 Kv1-16 Kappa
Sh24C05 Hv3-21 Heavy IgG1



(SEQ ID NO: 204)
(SEQ ID NO: 194)


Sh24C05-22
Sh24C05 Kv1-16 Kappa
Sh24C05 Hv3-23 Heavy IgG1



(SEQ ID NO: 204)
(SEQ ID NO: 196)


Sh24C05-23
Sh24C05 Kv1-16 Kappa
Sh24C05 Hv3-30 Heavy IgG1



(SEQ ID NO: 204)
(SEQ ID NO: 198)


Sh24C05-24
Sh24C05 Kv1-16 Kappa
Sh24C05 Hv3-7 Heavy IgG1



(SEQ ID NO: 204)
(SEQ ID NO: 186)


Sh24C05-25
Sh24C05 Kv1-16 Kappa
Sh24C05 Hv3-11 Heavy IgG1



(SEQ ID NO: 204)
(SEQ ID NO: 188)


Sh24C05-25
Sh24C05 Kv1-16 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG1
(SEQ ID NO: 204)
Heavy IgG1




(SEQ ID NO: 190)


Sh24C05-25
Sh24C05 Kv1-16 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG2
(SEQ ID NO: 204)
Heavy IgG2




(SEQ ID NO: 192)


Sh24C05-26
Sh24C05 Kv1-17 Kappa
Hu24C05 HvA IgG1



(SEQ ID NO: 206)
(SEQ ID NO: 200)


Sh24C05-27
Sh24C05 Kv1-17 Kappa
Sh24C05 Hv3-21 Heavy IgG1



(SEQ ID NO: 206)
(SEQ ID NO: 194)


Sh24C05-28
Sh24C05 Kv1-17 Kappa
Sh24C05 Hv3-23 Heavy IgG1



(SEQ ID NO: 206)
(SEQ ID NO: 196)


Sh24C05-29
Sh24C05 Kv1-17 Kappa
Sh24C05 Hv3-30 Heavy IgG1



(SEQ ID NO: 206)
(SEQ ID NO: 198)


Sh24C05-30
Sh24C05 Kv1-17 Kappa
Sh24C05 Hv3-7 Heavy IgG1



(SEQ ID NO: 206)
(SEQ ID NO: 186)


Sh24C05-31
Sh24C05 Kv1-17 Kappa
Sh24C05 Hv3-11 Heavy IgG1



(SEQ ID NO: 206)
(SEQ ID NO: 188)


Sh24C05-31
Sh24C05 Kv1-17 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG1
(SEQ ID NO: 206)
Heavy IgG1




(SEQ ID NO: 190)


Sh24C05-31
Sh24C05 Kv1-17 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG2
(SEQ ID NO: 206)
Heavy IgG2




(SEQ ID NO: 192)


Sh24C05-32
Sh24C05 Kv1-33 Kappa
Hu24C05 HvA IgG1



(SEQ ID NO: 208)
(SEQ ID NO: 200)


Sh24C05-33
Sh24C05 Kv1-33 Kappa
Sh24C05 Hv3-21 Heavy IgG1



(SEQ ID NO: 208)
(SEQ ID NO: 194)


Sh24C05-34
Sh24C05 Kv1-33 Kappa
Sh24C05 Hv3-23 Heavy IgG1



(SEQ ID NO: 208)
(SEQ ID NO: 196)


Sh24C05-35
Sh24C05 Kv1-33 Kappa
Sh24C05 Hv3-30 Heavy IgG1



(SEQ ID NO: 208)
(SEQ ID NO: 198)


Sh24C05-36
Sh24C05 Kv1-33 Kappa
Sh24C05 Hv3-7 Heavy IgG1



(SEQ ID NO: 208)
(SEQ ID NO: 186)


Sh24C05-37
Sh24C05 Kv1-33 Kappa
Sh24C05 Hv3-11 Heavy IgG1



(SEQ ID NO: 208)
(SEQ ID NO: 188)


Sh24C05-37
Sh24C05 Kv1-33 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG1
(SEQ ID NO: 208)
Heavy IgG1




(SEQ ID NO: 190)


Sh24C05-37
Sh24C05 Kv1-33 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG2
(SEQ ID NO: 208)
Heavy IgG2




(SEQ ID NO: 192)


Sh24C05-38
Sh24C05 Kv1-9 Kappa
Hu24C05 HvA IgG1



(SEQ ID NO: 202)
(SEQ ID NO: 200)


Sh24C05-39
Sh24C05 Kv1-9 Kappa
Sh24C05 Hv3-21 Heavy IgG1



(SEQ ID NO: 202)
(SEQ ID NO: 194)


Sh24C05-40
Sh24C05 Kv1-9 Kappa
Sh24C05 Hv3-23 Heavy IgG1



(SEQ ID NO: 202)
(SEQ ID NO: 196)


Sh24C05-41
Sh24C05 Kv1-9 Kappa
Sh24C05 Hv3-30 Heavy IgG1



(SEQ ID NO: 202)
(SEQ ID NO: 198)


Sh24C05-42
Sh24C05 Kv1-9 Kappa
Sh24C05 Hv3-7 Heavy IgG1



(SEQ ID NO: 202)
(SEQ ID NO: 186)


Sh24C05-43
Sh24C05 Kv1-9 Kappa
Sh24C05 Hv3-11 Heavy IgG1



(SEQ ID NO: 202)
(SEQ ID NO: 188)


Sh24C05-43
Sh24C05 Kv1-9 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG1
(SEQ ID NO: 202)
Heavy IgG1




(SEQ ID NO: 190)


Sh24C05-43
Sh24C05 Kv1-9 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG2
(SEQ ID NO: 202)
Heavy IgG2




(SEQ ID NO: 192)


Sh24C05-44
Sh24C05 Kv1-39 Kappa
Hu24C05 HvA IgG1



(SEQ ID NO: 210)
(SEQ ID NO: 200)


Sh24C05-45
Sh24C05 Kv1-39 Kappa
Sh24C05 Hv3-21 Heavy IgG1



(SEQ ID NO: 210)
(SEQ ID NO: 194)


Sh24C05-46
Sh24C05 Kv1-39 Kappa
Sh24C05 Hv3-23 Heavy IgG1



(SEQ ID NO: 210)
(SEQ ID NO: 196)


Sh24C05-47
Sh24C05 Kv1-39 Kappa
Sh24C05 Hv3-30 Heavy IgG1



(SEQ ID NO: 210)
(SEQ ID NO: 198)


Sh24C05-48
Sh24C05 Kv1-39 Kappa
Sh24C05 Hv3-7 Heavy IgG1



(SEQ ID NO: 210)
(SEQ ID NO: 186)


Sh24C05-49
Sh24C05 Kv1-39 Kappa
Sh24C05 Hv3-11 Heavy IgG1



(SEQ ID NO: 210)
(SEQ ID NO: 188)


Sh24C05-49
Sh24C05 Kv1-39 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG1
(SEQ ID NO: 210)
Heavy IgG1




(SEQ ID NO: 190)


Sh24C05-49
Sh24C05 Kv1-39 Kappa
Sh24C05 Hv3-11 N62S


N62S IgG2
(SEQ ID NO: 210)
Heavy IgG2




(SEQ ID NO: 192)









The antibody construct containing the full length chimeric heavy and light chains is designated below:

    
    
        Chimeric 24C05=Full Length Chimeric 24C05 Heavy Chain (Mouse Variable Region and Human IgG1 Constant Region) (SEQ ID NO: 182) plus Full Length Chimeric 24C05 Light Chain (Mouse Variable Region and Human Kappa Constant Region) (SEQ ID NO: 184)
    
    


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

    
    
        Sh24C05-25 N62S IgG1=Humanized Sh24C05 Hv3-11 N62S Heavy Chain Variable Region and Human IgG1 Constant Region (SEQ ID NO: 190) plus Sh24C05 Kv1-16 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 204)
        Sh24C05-25 N62S IgG2=Humanized Sh24C05 Hv3-11 N62S Heavy Chain Variable Region and Human IgG2 Constant Region (SEQ ID NO: 192) plus Sh24C05 Kv1-16 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 204)
        Sh24C05-31 N62S IgG1=Humanized Sh24C05 Hv3-11 N62S Heavy Chain Variable Region and Human IgG1 Constant Region (SEQ ID NO: 190) plus Sh24C05 Kv1-17 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 206)
        Sh24C05-31 N62S IgG2=Humanized Sh24C05 Hv3-11 N62S Heavy Chain Variable Region and Human IgG2 Constant Region (SEQ ID NO: 192) plus Sh24C05 Kv1-17 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 206)
    
    


B. Binding Affinities of Humanized and Chimeric Anti-ErbB3 Monoclonal Antibodies
The binding affinities and kinetics of interaction of monoclonal antibodies produced in Example 12 against recombinant human ErbB3 monomeric protein (cleaved rhErbB3) were measured by surface plasmon resonance using a Biacore® T100 (Biacore) instrument. Monomeric ErbB3 was obtained by protease cleavage of rhErbB3-Fc (R&D Systems, Cat. No. 348-RB).
Goat anti-human IgG Fc (Jackson ImmunoResearch, Catalog No. 109-005-098) was immobilized on carboxymethylated dextran CM4 sensor chips (Biacore, Catalog No. BR-1005-34) by amine coupling (Biacore, Catalog No. BR-1000-50) using a standard coupling protocol according to the vendor's instructions. The analyses were performed at 37° C. using PBS (Invitrogen, Catalog No. 14040-133) containing 0.05% surfactant P20 (Biacore, Catalog No. BR-1000-54) 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 cleaved rhErbB3 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, Catalog No. BR-1003-55) and pH 2.5 (Biacore, Catalog No. BR-1003-56)) at 60 μl/minute. For the initial screening, only one or two concentrations of cleaved rhErbB3 were tested, typically 5.0 and 1.25 nM (results are summarized in Table 15).
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. The initial monoclonal antibodies were screened using cell culture media supernatant containing secreted antibody, and kinetic values of the monoclonal antibodies on cleaved rhErbB3 at 37° C. are summarized in Table 15.













TABLE 15







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









Sh24C05-1
2.52E+06
4.48E−04
1.78E−10
3



Sh24C05-14
2.88E+06
4.98E−04
1.73E−10
2



Sh24C05-15
2.67E+06
4.99E−04
1.87E−10
2



Sh24C05-16
2.75E+06
4.04E−04
1.47E−10
2



Sh24C05-17
2.79E+06
4.17E−04
1.50E−10
2



Sh24C05-18
2.88E+06
4.63E−04
1.61E−10
2



Sh24C05-19
3.00E+06
2.55E−04
8.55E−11
2



Sh24C05-20
2.67E+06
5.91E−04
2.21E−10
2



Sh24C05-21
3.11E+06
6.62E−04
2.20E−10
2



Sh24C05-22
2.79E+06
6.01E−04
2.16E−10
2



Sh24C05-23
2.79E+06
7.21E−04
2.63E−10
2



Sh24C05-24
2.90E+06
6.28E−04
2.18E−10
2



Sh24C05-25
2.63E+06
4.59E−04
1.75E−10
2



Sh24C05-26
3.36E+06
7.39E−04
2.20E−10
2



Sh24C05-27
3.34E+06
7.98E−04
2.40E−10
2



Sh24C05-28
3.26E+06
6.14E−04
1.89E−10
2



Sh24C05-29
3.25E+06
5.88E−04
1.82E−10
2



Sh24C05-30
4.48E+06
7.87E−04
1.90E−10
2



Sh24C05-31
3.47E+06
2.92E−04
8.65E−11
2



Sh24C05-32
9.98E+06
6.02E−03
6.03E−10
1



Sh24C05-33
4.02E+06
4.33E−03
1.08E−09
1



Sh24C05-34
1.09E+07
6.00E−03
5.52E−10
1



Sh24C05-35
8.44E+06
5.53E−03
6.55E−10
1



Sh24C05-36
5.18E+06
4.34E−03
8.37E−10
1



Sh24C05-37
5.94E+06
2.00E−03
3.74E−10
2



Sh24C05-38
2.71E+07
1.54E−02
5.67E−10
1



Sh24C05-39
1.18E+07
9.67E−03
8.19E−10
1



Sh24C05-40
2.11E+07
1.06E−02
5.03E−10
1



Sh24C05-41
1.81E+07
1.21E−02
6.69E−10
1



Sh24C05-42
7.35E+06
6.82E−03
9.27E−10
1



Sh24C05-43
6.16E+06
3.58E−03
5.82E−10
1



Sh24C05-44
7.96E+06
5.12E−03
6.44E−10
1



Sh24C05-45
8.57E+06
6.06E−03
7.07E−10
1



Sh24C05-46
7.99E+06
4.40E−03
5.51E−10
1



Sh24C05-47
7.98E+06
4.41E−03
5.53E−10
1



Sh24C05-48
8.72E+06
4.90E−03
5.62E−10
1



Sh24C05-49
4.08E+06
1.70E−03
4.16E−10
2










The results in Table 15 demonstrate that the chimeric and each of the humanized 24C05 antibodies have fast association rates (ka), very slow disassociation rates (kd) and very high affinities (KD). In particular, the antibodies have affinities ranging from about 87 pM to about 1 nM.
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 cleaved rhErbB3 between 0.3125 nM and 5.0 nM (a 2-fold serial dilution).
The kinetic values of certain purified monoclonal antibodies (i.e., Sh24C05-1, Sh24C05-25, Sh24C05-25 N62S IgG1, Sh24C05-25 N62S IgG2, Sh24C05-31, Sh24C05-31 N62S IgG1, and Sh24C05-31 N62S IgG2) on cleaved rhErbB3 at 37° C. are summarized in Table 16.











TABLE 16





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







Sh24C05-1
3.5E+06
4.4E−04
1.4E−10
3


Sh24C05-25
4.0E+06
5.0E−04
1.3E−10
4


Sh24C05-25 N62S IgG1
2.9E+06
4.5E−04
1.6E−10
4


Sh24C05-25 N62S IgG2
2.7E+06
3.4E−04
1.2E−10
4


Sh24C05-31
4.7E+06
2.8E−04
6.3E−11
3


Sh24C05-31 N62S IgG1
3.5E+06
2.7E−04
7.6E−11
6


Sh24C05-31 N62S IgG2
3.2E+06
2.4E−04
7.4E−11
3









The results in Table 16 demonstrate the purified antibodies have a have affinities ranging from about 63 pM to about 160 pM when tested at 37° C.
C. Comparison of Other Anti-ErbB3 Antibodies
Three human antibodies that inhibit the function of human ErbB3 were constructed and expressed using published information. One antibody, referred to as Ab #6, was constructed as a human IgG2/Lambda antibody based the disclosure of Schoeberl et al., US 2009/0291085 (Merrimack Pharmaceuticals, Inc.). Two additional antibodies, referred to as U1-53 and U1-59, were constructed as human IgG1/Kappa antibodies based on the disclosure of Rothe et al., US 2008/0124345 (U3 Pharma AG and Amgen, Inc.).
Kinetic parameters for the Ab#6, U1-53, and U1-59 antibodies were determined by Biacore at 37° C. using cleaved rhErbB3 (monomer) as described above (See Section B. Binding Affinities of Humanized and Chimeric Anti-ErbB3 Monoclonal Antibodies). Both Biacore sensorgrams (FIG. 17) and kinetic values (Table 17) are displayed for each antibody.











TABLE 17





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







Sh24C05-31 N62S IgG1
 3.5+06
2.7E−04
7.6E−11
6


Ab#6
9.3E+05
1.9E−04
2.3E−10
3


U1-59
1.8E+06
9.4E−04
5.3E−10
3


U1-53
—
—
—
—









The results in Table 17 demonstrate that the overall equilibrium dissociation constant (KD) for the Sh24C05-31 N62S IgG1 (76 pM) was smaller (i.e., higher affinity) than the KD for the Ab#6 and U1-59 antibodies (230 pM (p<0.01) and 530 pM (p<0.0005), respectively). The equilibrium dissociation constant (KD) for U1-53 could not determined because of poor curve fits (see FIG. 17, which shows a fast Koff rate of U1-53). The KD of Ab #6, U1-53, and U1-59 antibodies can also be compared with other humanized 24C05 variants by comparing Tables 16 and 17.
Therefore, the affinity for Sh24C05-31 N62S IgG1 is significantly higher than the affinity of Ab#6 and U1-59 as disclosed herein.
Example 13
Neutralization Activity of the Humanized Anti-ErbB3 Antibodies
In this example, the humanized antibodies produced in Example 12 were tested for their ability to inhibit rhErbB3 binding to NRG1-β1 by ECL assay. Multi-array 96-well standard binding plates (Meso Scale Discovery, Cat. No. L15XA-3) were coated with 50 μl of 0.5 μg/mL rhErbB3/Fc (R&D systems, Cat. No. 348-RB) in PBS (Invitrogen, Cat. No. 14040-133) for one hour at room temperature with no agitation. The plates then were washed three times with PBS+0.1% Tween20 (Sigma P5927) and blocked with 200 μl of 100% Horse Serum, heat inactivated (GIBCO, Cat. No. 26050-088) for 1.5 hours at room temperature. After washing the plates three times with PBS+0.1% Tween, 25 μl of the antibody dilutions were added to the plates for another hour at room temperature with agitation. Ligand NRG1-β1 (R&D Systems, Cat. No. 377-HB, 26 kDa) was added to the wells at a final concentration of 0.25 μg/ml. The plates were washed three times with PBS+0.1% Tween and incubated with 25 μl of 1 μg/mL biotinylated antibody against human NRG1-β1 (R&D systems, Cat. No BAF377) preincubated for one hour with SULTO-TAG Streptavidin (Meso Scale Discovery, Cat. No R32AD-5) for one hour at room temperature with agitation. The plates then were washed three times with PBS+0.1% Tween, and 150 μl of 1× read buffer (Meso Scale Discovery, Cat. No. R92TC-1) was added to each well before the plates were analyzed on a Sector® Imager 2400 (Meso Scale Discovery) instrument.
The interaction of NRG1-β1 with rhErbB3 was inhibited by antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 (FIG. 18A). The Ab#6 IgG2 antibody as described in Schoeberl et al. (supra) and the U1-53 and U1-59 antibodies as described in Rothe et al. (supra) were also tested for their ability to inhibit ErbB3 binding to NRG1-β1. As shown in FIG. 18B, each of the Ab#6 IgG2, U1-53, and U1-59 antibodies inhibited ErbB3 binding to NRG1-β1.
The IC50 values for neutralization of NRG1-β1 binding to hErbB3 for the humanized 24C05 antibodies (i.e., Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2) were calculated and are summarized in Table 18. The IC50 values for the NRG1-β1 neutralization activity of the anti-ErbB3 human antibodies Ab#6 IgG2, U1-53 and U1-59 are also shown in Table 18.










TABLE 18









IC50 (nM)











Antibody
Average
Standard Deviation
n





Sh24C05-25 N62S-IgG1
0.1219
0.0173
4


Sh24C05-25 N62S-IgG2
0.1117
0.0154
4


Sh24C05-31 N62S-IgG1
0.1242
0.0391
5


Sh24C05-31 N62S-IgG2
0.0860
0.0588
4


U1-53
0.1128
0.0615
3


U1-59
0.3181
0.0274
3


Ab#6 IgG2
1.5161
0.5883
5









The results in Table 18 demonstrate that antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 efficiently neutralized NRG1-β1 binding to rhErbB3. While the anti-ErbB3 human antibodies Ab#6 IgG2, U1-53 and U1-59 also showed neutralization activity, the humanized Sh24C05 antibodies (i.e., Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2) had superior neutralization capacity than U1-59 or Ab#6 IgG2.
Example 14
Anti-Proliferative Activity
In this example, the humanized antibodies produced in Example 12 were tested for their ability to inhibit NRG1-β1 dependent proliferation of cells in the BaF/3 cell system engineered to express both human Her2 and ErbB3.
BaF/3 cells expressing Her2 and ErbB3 receptors as described in Example 6 were treated with anti-ErbB3 antibodies in the absence of WEHI conditioned media but in the presence of NRG1-β1 (100 ng/ml). Assays were conducted in a 96-well plate (5,000 cells/well) in the presence of NRG1-β1 (100 ng/ml) and various concentrations of antibodies (0.018-5000 ng/ml in 100 μl of final volume). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were conducted 3-4 days post NRG1-β1 stimulation.
The results demonstrate that Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 inhibited NRG induced Her2/ErbB3-BaF/3 cell proliferation in a dose dependent manner (FIG. 19A).
The IC50 values for the inhibition of NRG1-β1 dependent Her2/ErbB3-BaF/3 cell line proliferation with the humanized 24C05 antibodies (i.e., Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, Sh24C05-31 N62S-IgG2) were calculated and are summarized in Table 19.







TABLE 19







Her2/ErbB3-BaF/3, NRG1-β1 Dependent Proliferation











IC50 (nM)-




Antibody
Average
Standard deviation
n





Sh24C05-25 N62S-IgG1
0.0981
0.0187
2


Sh24C05-25 N62S-IgG2
0.2482
0.0124
2


Sh24C05-31 N62S-IgG1
0.1245
0.0181
5


Sh24C05-31 N62S-IgG2
0.2392
0.0217
2


U1-53
0.8128
0.0268
3


U1-59
0.8364
0.0434
5


Ab#6 IgG2
6.3015
0.8577
2









The results in Table 19 demonstrate that antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 strongly inhibited NRG1-β1-induced proliferation of BaF/3 cells expressing Her2/ErbB3.
The inhibitory activity of anti-ErbB3 Ab#6 IgG2, U1-53 and U1-59 antibodies were also tested in the NRG1-β1 dependent Her2/ErbB3-BaF/3 cells proliferation assay. As shown in FIG. 19B, the results demonstrate that the Ab#6 IgG2, U1-53 and U1-59 antibodies inhibited NRG induced Her2/ErbB3-BaF/3 cell proliferation in a dose dependent manner. Inhibition data of NRG1-β1 dependent Her2/ErbB3-BaF/3 cell proliferation with antibodies Ab#6 IgG2, U1-53 and U1-59 are summarized in Table 19. The results in Table 19 demonstrate that antibodies Ab#6 IgG2, U1-53, and U1-59 inhibited NRG1-β1-induced proliferation of Her2/ErbB3-BaF/3 cells. A comparison of the inhibitory activity of the tested anti-ErbB3 antibodies in the NRG1-β1 dependent Her2/ErbB3-BaF/3 cells proliferation assay indicates that the inhibitory activity of the humanized Sh24C05 antibodies is superior to the inhibitory activity of the Ab#6 IgG2, U1-53 and U1-59 antibodies (e.g., the IC50 was 0.1245 nM for Sh24C05-31 N62S-IgG1 compared to 0.8128 nM for U1-53).
Example 15
Inhibition of Downstream Signaling in SKBR-3 Cells
This example describes a characterization of the humanized antibodies produced in Example 12 for their ability to degrade total ErbB3 and inhibit phosphorylation of ErbB3 in exponentially growing SKBR-3 cells.
The breast cancer SKBR-3 cells were maintained as recommended by ATCC. Cells maintained in full serum condition were treated for 1, 2, 4 or 6 hours with 40 μg/ml of anti-ErbB3 antibody (i.e., Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2). Lysates were either analyzed by ELISA with the Total-ErbB3 and the Phospho-ErbB3 kit from R&D Systems (Cat. No DYC234 and Cat. No DYC1769, respectively).
The results demonstrate that anti-ErbB3 antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 inhibit at least 50% of the phosphorylation of ErbB3 in exponentially growing SKBR-3 cells (FIG. 20).
The results also demonstrate that anti-ErbB3 antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 degraded at least 50% of the total ErbB3 receptor present in exponentially growing SKBR-3 cells (FIG. 21).
Example 16
Inhibition of BxPC3 Tumor Xenograft Growth
The ability of the humanized monoclonal antibodies produced in Example 12 to inhibit tumor growth were tested in a BxPC3 pancreatic xenograft model. Human pancreatic BxPC3 cells were grown in culture in 37° C. in an atmosphere containing 5% CO2, using RMPI medium containing 10% fetal bovine serum. BxPC3 cells were inoculated subcutaneously into the flank of 8-week old female CB.17 SCID mice (Taconic Labs) with 10×106 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were taken twice weekly using vernier calipers. Tumor volume was calculated using the formula: width×width×length/2. When tumors reached approximately 200 mm3, the mice were randomized into 8 groups of 10 mice each. One group received PBS, another received huIgG control, and another received muIgG control. Each of the remaining five groups received one of the antibodies (i.e., murine 24C05, Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1 or Sh24C05-31 N62S-IgG2). All of the antibodies were dosed at 2 mg/kg body weight, twice per week, by intra-peritoneal injection for 7 weeks. Tumor volumes and mouse body weights were recorded twice per week. Tumor growth inhibition was analyzed using ANOVA and is expressed as percent inhibition compared to the PBS control.
The tested humanized antibodies were active in vivo. All four humanized anti-ErbB3 antibodies had similar efficacy in the BxPC3 model when dosed at 2 mg/kg, ranging from 75-80% tumor growth inhibition (p<0.001) (i.e., Sh24C05-25 N62S-IgG1, 75%; Sh24C05-25 N62S-IgG2, 76%; Sh24C05-31 N62S-IgG1, 79%; and Sh24C05-31 N62S-IgG2, 80%) at day 28 of the study (FIG. 22). The murine antibody demonstrated 65% tumor growth inhibition in this study (p<0.05). These results suggest similar potency and activity of the four humanized antibodies in this model.
The ability of the humanized monoclonal antibodies U1-53, U1-59, and Ab#6 IgG2 to inhibit tumor growth were also tested in a BxPC3 xenograft model. Using the protocol described above, BxPC3 tumors were generated in CB.17 SCID mice. When tumors reached approximately 200 mm3, the mice were randomized into 11 groups of 10 mice each. One group received PBS and another received huIgG control. Each of the other nine groups received one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-53, U1-59, or Ab#6 IgG2). The antibodies were dosed either at 0.5 mg/kg, 1 mg/kg, or 5 mg/kg body weight, twice per week, by intra-peritoneal injection for 7 weeks. Tumor volumes and mouse body weights were recorded twice per week. Tumor growth inhibition was analyzed using ANOVA and is expressed as percent inhibition compared to the PBS control.
Tumor growth inhibition data determined at day 29 following treatment with one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59, or Ab#6 IgG2) is shown in Table 20.











TABLE 20









Tumor
ANOVA
ANOVA



Growth
Analysis
Analysis












Treatment
Inhibition
(Compared
(Compared












Gr.
Agent
mg/kg
(%)
to PBS)
to hIgG)















1
PBS
—
NA
NA
NA


2
hIgG
5
29.2
NS
NS


3
Sh24C05-31
0.5
63.3
P < 0.001
P < 0.01 



N62S-IgG1


4
Sh24C05-31
1
75.0
P < 0.001
P < 0.001



N62S-IgG1


5
Sh24C05-31
5
76.5
P < 0.001
P < 0.001



N62S-IgG1


6
Ab#6 IgG2
0.5
31.5
P < 0.05 
NS


7
Ab#6 IgG2
1
2.1
NS
NS


8
Ab#6 IgG2
5
40.6
P < 0.001
NS


9
U1-59
0.5
32.6
P < 0.01 
NS


10
U1-59
1
52.9
P < 0.001
NS


11
U1-59
5
60.3
P < 0.001
P < 0.05 









The results demonstrate that Sh24C05-31 N62S-IgG1 showed the greatest tumor growth inhibition by day 29 (76.5%, p<0.001) at a dose of 5 mg/kg in the BxPC3 pancreatic xenograft model. The U1-59 and Ab#6 IgG2 antibodies demonstrated approximately 60% and 41% tumor growth inhibition at a dose of 5 mg/kg in the BxPC3 model, respectively (P<0.001).
The results also demonstrate that Sh24C05-31 N62S-IgG1 showed the greatest tumor growth inhibition by day 29 at a dose of 0.5 mg/kg (63.3%, p<0.001) and at a dose of 1 mg/kg (75.0%, p<0.001) in the BxPC3 pancreatic xenograft model. The U1-59 and AB#6 IgG2 antibodies demonstrate approximately 33% (p<0.01) and 31% (p<0.05) tumor growth inhibition at a dose of 0.5 mg/kg in the BxPC3 model, respectively. The U1-59 and AB#6 IgG2 antibodies demonstrated approximately 53% (p<0.001) and 2% (not significant) tumor growth inhibition at a dose of 1.0 mg/kg in the BxPC3 model, respectively.
Example 17
Inhibition of Calu-3 Tumor Xenograft Growth
The ability of the humanized monoclonal antibodies produced in Example 12 to inhibit tumor growth was tested in a Calu-3 non-small cell lung cancer xenograft model. The ability of the humanized monoclonal antibodies U1-59 and Ab#6 IgG2, as described in Example 12, to inhibit tumor growth were also tested in the same model.
Human Non-Small Cell Lung Cancer Calu-3 cells were grown in culture in 37° C. in an atmosphere containing 5% CO2, using EMEM medium containing 10% fetal bovine serum. Calu-3 cells were inoculated subcutaneously into the flank of 8-week old female NCR nude mice (Taconic Labs) with 10×106 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were taken twice weekly using vernier calipers. Tumor volume was calculated using the formula: width×width×length/2.
When tumors reached approximately 200 mm3, the mice were randomized into 11 groups of 10 mice each. One group received PBS and another received muIgG control. Each of the other nine groups received one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59, or Ab#6 IgG2) at a dose of either 5 mg/kg, 10 mg/kg or 20 mg/kg body weight, twice per week, by intra-peritoneal injection for 4 weeks. Tumor volumes and mouse body weights were recorded twice per week. Tumor growth inhibition was analyzed using ANOVA and is expressed as percent inhibition compared to the PBS control.
Tumor growth inhibition data determined at day 26 following treatment with one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59, or Ab#6 IgG2) is shown in Table 21.











TABLE 21









Tumor
ANOVA
ANOVA



Growth
Analysis
Analysis












Treatment
Inhibition
(Compared
(Compared












Gr.
Agent
mg/kg
(%)
to PBS)
to hIgG)















1
PBS
—
NA
NA
NA


2
muIgG
20
−1.2
NS
NA


3
Sh24C05-31
5
62.3
P < 0.001
P < 0.001



N62S-IgG1


4
Sh24C05-31
10
62.0
P < 0.001
P < 0.001



N62S-IgG1


5
Sh24C05-31
20
69.0
P < 0.001
P < 0.001



N62S-IgG1


6
Ab#6 IgG2
5
24.7
NS
NS


7
Ab#6 IgG2
10
35.9
P < 0.01 
P < 0.01 


8
Ab#6 IgG2
20
48.4
P < 0.001
P < 0.001


9
U1-59
5
47.8
P < 0.001
P < 0.001


10
U1-59
10
56.7
P < 0.001
P < 0.001


11
U1-59
20
57.7
P < 0.001
P < 0.001









The results using the Calu-3 non-small cell lung cancer xenograft model demonstrate that Sh24C05-31 N62S-IgG1 showed the greatest tumor growth inhibition by day 26 at all doses tested (i.e., 5 mg/kg, 10 mg/kg, and 20 mg/kg of body weight).
For example, at the 10 mg/kg dose, Sh24C05-31 N62S-IgG1 showed the greatest tumor growth inhibition by day 26 (62%, P<0.001) when compared to Ab#6 IgG2 (36%, NS) or U1-59 (57%, P<0.001). At the 20 mg/kg dose, Sh24C05-31 N62S-IgG1 also showed the greatest tumor growth inhibition by day 26 (69%, P<0.001) when compared to Ab#6 IgG2 (48%, P<0.001) or U1-59 (58%, P<0.001).
Example 18
Inhibition of MDA-MB-453 Tumor Xenograft Growth
The ability of the humanized monoclonal antibodies produced in Example 12 to inhibit tumor growth were tested in a MDA-MB-453 breast xenograft model (which is a HER2 positive breast model). The ability of the humanized monoclonal antibodies U1-59 and Ab#6 IgG2, as described in Example 12, to inhibit tumor growth were also tested in the same model.
Human Breast MDA-MB-453 cells were grown in culture in 37° C. in an atmosphere containing 0% CO2, using Leibovitz ATCC medium (Cat No. 30-2008) containing 10% fetal bovine serum. MDA-MB-453 cells were inoculated subcutaneously into the flank of 8-week old female NOD SCID mice (Taconic Labs) with 20×106 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were taken twice weekly using vernier calipers. Tumor volume was calculated using the formula: width×width×length/2.
When tumors reached approximately 200 mm3, the mice were randomized into 7 groups of 10 mice each. One group received PBS and another received huIgG control. Each of the other nine groups received one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59, or Ab#6 IgG2). Sh24C05-31 N62S-IgG1 was dosed either at 5 mg/kg, 10 mg/kg, or 20 mg/kg body weight, twice per week, by intra-peritoneal injection for more than 10 weeks; U1-59, or Ab#6 were dosed at 10 mg/kg with the same frequency. Tumor volumes and mouse body weights were recorded twice per week. Tumor growth inhibition was analyzed using ANOVA and is expressed as percent inhibition compared to the PBS control.
Tumor growth inhibition data determined at day 71 following treatment with one of the humanized antibodies (i.e., Sh24C05-31 N62S-IgG1, U1-59, or Ab#6 IgG2) is shown in Table 22.












TABLE 22










Tumor
ANOVA
ANOVA




Growth
Analysis
Analysis



Treatment
Inhibition
(Compared
(Compared












Gr.
Agent
mg/kg
(%)
to PBS)
to hIgG)





1
PBS
—
NA
NA
NA


2
hIgG
20
28.87
p < 0.001
p < 0.001


3
Sh24C05-31
5
86.57
p < 0.001
p < 0.001



N62S-IgG1


4
Sh24C05-31
10
84.09
p < 0.001
p < 0.001



N62S-IgG1


5
Sh24C05-31
20
85.26
p < 0.001
p < 0.001



N62S-IgG1


6
Ab#6 IgG2
10
62.48
p < 0.001
p < 0.001


7
U1-59
10
83.93
p < 0.001
p < 0.001









The results using the MDA-MB-453 xenograft model demonstrate that Sh24C05-31 N62S-IgG1 showed potent tumor growth inhibition by day 71 at all doses tested (i.e., 5 mg/kg, 10 mg/kg, and 20 mg/kg of body weight).
The results also demonstrate that at the 10 mg/kg dose, Sh24C05-31 N62S-IgG1 showed greater tumor growth inhibition by day 71 (84%, P<0.001) when compared to Ab#6 IgG2 (62%, P<0.001). Sh24C05-31 N62S-IgG1 showed equivalent tumor growth inhibition as U1-59 at the same dose.
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 nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region comprising a CDRH1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 75, a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 58 and SEQ ID NO: 148, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 59.

2. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable region comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 60, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 62.

3. An expression vector comprising the nucleic acid of claim 1.

4. An expression vector comprising the nucleic acid of claim 2.

5. The expression vector of claim 4, further comprising the nucleic acid of claim 1.

6. A host cell comprising the expression vector of claim 3.

7. A host cell comprising the expression vector of claim 4.

8. A host cell comprising the expression vector of claim 5.

9. The host cell comprising of claim 7, further comprising the expression vector of claim 3.

10. A method of producing a polypeptide comprising an immunoglobulin heavy chain variable region or an immunoglobulin light chain variable region, the method comprising:
(a) growing the host cell of claim 6 or 7 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region; and
(b) purifying the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region.

11. A method of producing an antibody that binds human ErbB3 or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 8 or 9 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain variable region and/or the immunoglobulin light chain variable region, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.

12. The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 154.

13. The isolated nucleic acid of claim 2, wherein the nucleotide sequence encodes an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 168 or SEQ ID NO: 166.

14. An expression vector containing the nucleic acid of claim 12.

15. An expression vector containing the nucleic acid of claim 13.

16. The expression vector of claim 15, further comprising the nucleic acid of claim 12.

17. A host cell comprising the expression vector of claim 14.

18. A host cell comprising the expression vector of claim 15.

19. A host cell comprising the expression vector of claim 16.

20. The host of claim 18, further comprising the expression vector of claim 14.

21. A method of producing a polypeptide comprising an immunoglobulin heavy chain variable region or an immunoglobulin light chain variable region, the method comprising:
(a) growing the host cell of claim 17 or 18 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region; and
(b) purifying the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region.

22. A method of producing an antibody that binds human ErbB3 or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 19 or 20 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.

23. The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 190.

24. The isolated nucleic acid of claim 2, wherein the nucleotide sequence encodes an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 206 or SEQ ID NO: 204.

25. An expression vector containing the nucleic acid of claim 23.

26. An expression vector containing the nucleic acid of claim 24.

27. The expression vector of claim 26, further comprising the nucleic acid of claim 23.

28. A host cell comprising the expression vector of claim 25.

29. A host cell comprising the expression vector of claim 26.

30. A host cell comprising the expression vector of claim 27.

31. The host cell of claim 29, further comprising the expression vector of claim 25.

32. A method of producing a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain, the method comprising:
(a) growing the host cell of claim 28 or 29 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain or the immunoglobulin light chain; and
(b) purifying the polypeptide comprising the immunoglobulin heavy chain or the immunoglobulin light chain.

33. A method of producing an antibody that binds human ErbB3 or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 30 or 31 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain and the immunoglobulin light chain, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.

34. The isolated nucleic acid of claim 1, wherein the immunoglobulin heavy chain variable region comprises a CDRH1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 75, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 148, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 59.

35. The isolated nucleic acid of claim 1, wherein the immunoglobulin heavy chain variable region comprises a CDRH1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 75, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 58, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 59.

36. The isolated nucleic acid of claim 13, wherein the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO: 168.

37. The isolated nucleic acid of claim 24, wherein the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO: 206.

38. The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 152.

39. The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 188.