isolated antibody that binds to human erbb3, its uses, its production process and expression vector

专利摘要:
ANTI-ERBB3 ANTIBODIES, THEIR USES AND THEIR PRODUCTION PROCESS, NUCLEIC ACIDS, EXPRESSION VECTORS, HOST CELLS, AS WELL AS A POLYPEPTIDE PRODUCTION PROCESS. The invention relates to monoclonal antibodies that bind and inhibit the activation of the member ErbB3 / HE3 related to the epidermal growth factor receptor. The antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with the activation of ErbB3 / HER3.
公开号:BR112012025730B1
申请号:R112012025730-7
申请日:2011-04-08
公开日:2020-12-08
发明作者:Sylvie Vincent；William M. Jr. Winston；Fang Wang；Solly Weiler；Kristian Meetze；Lyne Breault；Steve Bottega；Ting Chen；Michael DePrima；Christina Fleet；Steven Tyler；Jin-Kyeung Woo；Jeno Gyuris
申请人:Aveo Pharmaceuticals, Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0001] The present invention claims priority of U.S. provisional application, serial number 61 / 322,712, filed on April 9, 2010, the entire content of which is incorporated herein by reference. FIELD OF THE INVENTION
[0002] The field of the invention is molecular biology, immunology and oncology. More particularly, the field is humanized antibodies that bind to human ErbB3 / HER3. BACKGROUND
[0003] HER3 / c-ErbB3 (referred to herein as ErbB3) is a member of the epidermal growth factor (EGFR) receptor family. ErbB3 binds to neuregulin / heregulin (NRG / HRG). The receptors of the EGFR family are individual transmembrane receptors with an intracellular tyrosine kinase domain. While the other members of the EGFR family, ie, EGFR / HER1 / ErbB1, HER2 / ErbB2, and HER4 / ErbB4, each have tyrosine kinase activity, ErbB3 has little or no tyrosine kinase activity and is therefore "kinase- dead."
[0004] The extracellular domain (ECD) of the EGFR family contains four domains. Domains 1 and 3 (also known as L1 and L2 domains) are responsible for ligand binding. Domains rich in cysteine 2 and 4 (also known as C1 and C2 domains) are involved in dimerization with receptor partners. When the ligand is connected, the ECD undergoes conformational changes. The interaction of domains 2 and 4, which maintains the tied (inactive) conformation of the recipient, is relieved and an extended (active) conformation is adopted. The extended conformation favors dimerization with other receiver partners. HER2 / ErbB2 is the only exception to this general rule, that is, HER2-ECD is found constitutively in the extended conformation. A linker for HER2 has not been identified so far.
[0005] As ErbB3 lacks 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), for example, ErbB3 and EGFR / HER1 / ErbB1, HER2 / ErbB2, or HER4 / ErbB4. Recently, ErbB3 has also been shown to dimerize with MET. When combined with another tyrosine kinase receptor, ErbB3 is activated by phosphorylation of at least nine tyrosine residues in the intracellular ErbB3 domain and then quickly associates with adapters or signaling molecules downstream. Six of the phosphorylated tyrosine residues ErbB3 are directly associated with the p85 subunit of phosphatidylinositol-3-kinase (PIK3), which results in the activation of the cell 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.
[0006] Overexpression of ErbB3 is associated with poor prognosis in several carcinomas (for example, cancers of the breast, ovary, prostate, colorectal, pancreas, stomach, head and neck). Overexpression of ErbB3 also correlates with the location for distal metastases in lung, stomach 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 in non-small cell lung cancer (NSCLC) and head and neck cancers, treatment with Her2 inhibitor in cancer breast cancer, and radiotherapy treatment for pancreatic cancers. In addition, overexpression of NRG, a ligand for ErbB3, has also been associated with resistance to treatment with EGFR tyrosine kinase inhibitor. Chen et al. describe the use of anti-ErbB3 monoclonal antibodies that inhibit NRG function and show the growth of inhibitory activity against breast and ovarian cancers (Chen et al., 1996, J. BIOL. CHEM. 271: 7620).
[0007] There is a need to have better anti-ErbB3 antibodies that can be used as therapeutic agents. SUMMARY
[0008] The invention is based on the discovery of a family of antibodies that specifically bind to human ErbB3. The antibodies contain ErbB-binding sites based on CDRs that specifically bind to human ErbB3. When used as therapeutic agents, antibodies are designed, for example, humanized, to reduce or eliminate an immune response when administered to a human patient.
[0009] The antibodies disclosed herein prevent or inhibit the activation of human ErbB3. In some embodiments, antibodies prevent ErbB3 from binding to a ligand, for example, NRG / HRG, thereby neutralizing the biological activity of ErbB3. In other modalities, anti-ErbB3 antibodies inhibit the dimerization of ErbB3, thus neutralizing the biological activity of ErbB3. The antibodies disclosed herein can be used to inhibit tumor cell proliferation in vitro or in vivo. When administered to a human cancer patient (or an animal model, such as a mouse model), antibodies inhibit or reduce tumor growth in the human patient (or in the animal model).
[00010] These and other aspects and advantages of the invention are illustrated by the following figures, detailed description and claims. As used in this document, the term "including" means without limitation and the examples cited are non-limiting. BRIEF DESCRIPTION OF THE DRAWINGS
[00011] The present invention can be better understood by reference to the following figures.
[00012] Figure 1 (state of the art) is a schematic representation of a typical antibody.
[00013] Figure 2 is a schematic diagram showing the amino acid sequence of the variable region of the complete immunoglobulin heavy chain of antibodies, denoted as 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05. The amino acid sequences for each antibody are aligned against each other, and the complementary sequences (CDR) (Kabat definition), CDR1, CDR2 and CDR3, are identified in boxes. Boxless strings represent framework (FR) strings.
[00014] Figure 3 is a schematic diagram showing the sequences CDR1, CDR2 and CDR3 (Kabat definition) for each of the variable region sequences of the immunoglobulin heavy chain in Figure 2.
[00015] Figure 4 is a schematic diagram showing the amino acid sequence of the variable region of the complete immunoglobulin light chain of antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05. The amino acid sequences for each antibody are aligned against each other and the sequences (Kabat definition) CDR1, CDR2 and CDR3 are identified in boxes. Boxless strings represent framework (FR) strings.
[00016] Figure 5 is a schematic diagram showing the sequences CDR1, CDR2 and CDR3 (Kabat definition) for each of the variable region sequences of the immunoglobulin light chain in figure 4.
[00017] Figures 6A and 6B are graphs that summarize the results of an assay to measure the "neutralization" activity of the negative control (murine IgG (Δ)) and anti-ErbB3 monoclonal antibodies 04D01 (■), 12A07 (^) , 18H02 (o), 22A02 (•) and 24C05 (T) to inhibit the binding of NRG1-β1 to hErbB3 (figure 6A) and the measurement of improved binding of NRG1-β1 to rhErbB3 by anti-ErbB3 mAb 09D03 (▲) and 11G01 (♦) (figure 6B).
[00018] Figure 7 is a graph that summarizes the results of an assay to measure the "neutralizing" activity of the negative control (murine IgG) and anti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 to inhibit the binding of NRG1-α1 to rhErbB3.
[00019] Figure 8 is a graph that summarizes the results of an assay to measure cell surface recognition of anti-ErbB3 antibodies to the chimeric Her2 / 3d2 protein expressed on the surface of CHO cells.
[00020] Figure 9 is a graph that summarizes the results of an assay to measure the antiproliferation activity of the negative IgG control (murine IgG (Δ)) and anti-ErbB3 monoclonal antibodies 04D01 (■), 09D03 (▼), 11G01 (♦), 12A07 (<z), 18H02 (o), 22A02 (•) and 24C05 (T) in BaF / 3 cells expressing Her2 and ErbB3 in the presence of NRG1- β1.
[00021] Figure 10 is a graph that summarizes the results of an assay to measure the antiproliferation activity of anti-ErbB3 monoclonal antibodies 04D01 (■), 09D03 (▼), 11G01 (♦), 12A07 (^), 18H02 (o ), 22A02 (•) and 24C05 (T) in MCF7 cells in the presence of NRG1-β1.
[00022] Figure 11 is a graph that summarizes the results of an assay to measure the antiproliferation activity of the negative control (murine IgG) and anti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 in SKBR-3 cells treated with 5Qμg / mL of antibodies in the presence of serum.
[00023] Figure 12 is a graph that summarizes the results of an assay to measure the inhibitory activity of negative IgG control and anti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 in induced ErbB3 phosphorylation by NRG in SKBR-3 cells. No antibody / ligand controls and no antibody controls are also presented.
[00024] Figures 13A and 13B are graphs representing results of an assay to measure the inhibitory activity of anti-ErbB3 monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 on Akt phosphorylation in response to NRG1- β1 in MCF7 cells (figure 13A) and in DU145 cells (figure 13B) as determined by ELISA. No antibody / ligand controls and no antibody controls are also presented.
[00025] Figure 14 is a graph that summarizes the results of an assay to measure tumor inhibitory activity of anti-ErbB3 antibodies 04D01 (Δ), 09D03 (♦), 11G01 (Y), 12A07 (▲), 18H02 ( •), 22A02 (■), 24C05 (^) and the control of human IgG (- - ■ - -) dosed at 20 mg / kg in a model of pancreatic tumor xenograft BxPC3 in CB17 SCID mice (vehicle control, PBS (♦)).
[00026] Figure 15 is a schematic diagram showing the amino acid sequences of the 24C05 full heavy chain variable region 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 the Hu24C05 HvA. The amino acid sequences for each variable region of the heavy chain are aligned against each other, and the complementarity determining sequences (CDR) (Kabat definition), CDR1, CDR2 and CDR3, are identified in boxes. Boxless strings represent framework (FR) strings.
[00027] Figure 16 is a schematic diagram showing the amino acid sequences of the 24C05 complete light chain variable region 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 region are aligned against each other and the CDR1, CDR2 and CDR3 (Kabat definition) sequences are identified in boxes. Boxless strings represent framework (FR) strings.
[00028] Figure 17 are Biacore sensograms that represent results of an assay to measure the kinetic values of anti-ErbB3 monoclonal antibodies, Sh24C05-31 N62S-IgG1, Ab # 6, U1-53 and U1- 59.
[00029] Figure 18A is a graph summarizing the results of an assay to measure the neutralization activity of the negative control (human IgG (Y)) and monoclonal anti-ErbB3 antibodies Sh24C05-25 N62S-IgG1 (▲), Sh24C05- 25 N62S-IgG2 (Δ), Sh24C05-31 N62S-IgG1 (•) and Sh24C05-31 N62S-IgG2 (^). Figure 18B is a graph that summarizes the results of an assay to measure the neutralization activity of human IgG (Y) and monoclonal anti-ErbB3 Ab # 6 IgG2 (▼), U1-53 (o) and U1-59 (■).
[00030] Figure 19A is a graph summarizing the results of an assay to measure the inhibitory activity of the negative control (human IgG (Y)) and monoclonal anti-ErbB3 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. Figure 19B is a graph that summarizes the results of an assay to measure the inhibitory activity of human IgG (Y) and anti-ErbB3 monoclonal antibodies Sh24C05-31 N62S-IgG1 (•), Ab # 6 IgG2 (V), U1 -53 (2) and U1-59 (■) in BaF / 3 cells expressing Her2 and ErbB3 in the presence of NRG1-β1.
[00031] Figure 20 is a graph that summarizes the results of an assay to measure the inhibitory activity of negative control (human IgG) and monoclonal anti-ErbB3 antibodies Sh24C05-25 N62S- IgG1, Sh24C05-25 N62S-IgG2, Sh24C05- 31 N62S-IgG1 and Sh24C05- 31 N62S-IgG2 on steady-state phosphorylation of ErbB3 in growing SKBR-3 cells.
[00032] Figure 21 is a graph that summarizes the results of an assay to measure the degradation of the ErbB3 receptor by negative control (human IgG) and monoclonal anti-ErbB3 antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2 , Sh24C05-31 N62S-IgG1 and Sh24C05-31 N62S-IgG2 in growing SKBR-3 cells.
[00033] Figure 22 is a graph that summarizes the results of an assay to measure the tumor inhibitory activity of a human IgG, murine IgG, or anti-ErbB3 monoclonal antibodies dosed at 2 mg / kg in a pancreatic xenograft model. BxPC3 CB17 in SCID mice (murine 24C05 (Δ), Sh24C05-31 N62S IgG1 (•), Sh24C05-31 N62S IgG2 (♦), Sh24C05-25 N62S IgG1 (▲), Sh24C05-25 256262 IgG2 (■), control vehicle (Y), murine IgG (x) and human IgG (2)).
[00034] Figure 23A is a graph that summarizes the results of an assay to measure tumor inhibitory activity of a murine IgG or anti-ErbB3 monoclonal antibodies dosed at 5 mg / kg, in a cancer Calu-3 xenograft model of non-small cell lungs in nude NCR mice (control vehicle (Y), murine IgG (x), Sh24C05-31 N62S IgG1 (▲), Ab # 6 IgG2 (•), and U1-59 (■)).
[00035] Figure 23B is a graph that summarizes the results of an assay to measure tumor inhibitory activity of a murine IgG or monoclonal anti-ErbB3 antibodies dosed at 10 mg / kg, in a model of cancer Calu-3 xenograft of non-small cell lungs in nude NCR mice (control vehicle (Y), murine IgG (x), Sh24C05-31 N62S IgG1 (▲), Ab # 6 IgG2 (•), and U1-59 (■)).
[00036] Figure 23C is a graph that summarizes the results of an assay to measure tumor inhibitory activity of a murine IgG or anti-ErbB3 monoclonal antibodies dosed at 20 mg / kg, in a model of cancer Calu-3 xenograft of non-small cell lungs in nude NCR mice (control vehicle (Y), murine IgG (x), Sh24C05-31 N62S IgG1 (▲), Ab # 6 IgG2 (•), and U1-59 (■)).
[00037] Figure 24 is a graph that summarizes the results of an assay to measure tumor inhibitory activity of a human IgG, murine, or anti-ErbB3 monoclonal antibodies in a MDA-MB-453 xenograft breast cancer in mice NOD SCID (control vehicle (Y), human IgG (x), Sh24C05-31 N62S IgG1 dosed at 5 mg / kg (o), 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
[00038] The ErbB3 antibodies disclosed here 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 variable regions of CDR immunoglobulins that define an ErbB3 binding site. In some embodiments, antibodies prevent ErbB3 from binding to a ligand, for example, NRG / HRG, thereby neutralizing the biological activity of ErbB3. In other modalities, anti-ErbB3 antibodies inhibit the dimerization of ErbB3, thus neutralizing the biological activity of ErbB3. In yet other embodiments, anti-ErbB3 antibodies inhibit phosphorylation of ErbB3 and downstream signaling.
[00039] Due to the neutralizing activity of these antibodies, they are useful for inhibiting the growth and / or proliferation of certain cancer cells and tumors. Antibodies can be designed to minimize or eliminate an immune response when administered to a human patient. In some embodiments, antibodies are fused or conjugated to other portions, such as detectable markers or effector molecules, such as small molecules of toxins. Antibodies that bind to ErbB3
[00040] In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the CDRH1-CDRH2-CDRH3 structure and (b) an immunoglobulin light chain variable region, wherein the heavy chain variable region and the light chain variable region, together, define a single binding site to bind to human ErbB3. A CDRH1 is composed of a sequence of amino acids selected from a 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 a 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 a 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 description, a given SEQ ID No. is followed, in parentheses, by the antibody that was the origin of that sequence. For example, "SEQ ID NO: 5 (04D01)", SEQ ID NO: 5 comes from antibody 04D01.
[00041] 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 CDR H2 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).
[00042] 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).
[00043] 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).
[00044] 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).
[00045] 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).
[00046] 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).
[00047] 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 amino acid sequence of SEQ ID NO: 58 (24C05), and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 59 (24C05).
[00048] 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).
[00049] 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).
[00050] 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), or SEQ ID NO: 75 (24C05), a CDRH2 comprising 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).
[00051] Preferably, the sequences CDRH1, CDRH2 and CDRH3 interpose between FRs of human or humanized immunoglobulins. The antibody can be an intact antibody or an antibody fragment for binding to the antigen.
[00052] In some embodiments, the antibody comprises (a) an immunoglobulin light chain variable region comprising the CDRL1-CDRL2-CDRL3 structure and (b) an immunoglobulin heavy chain variable region, wherein the light chain variable region IgG cells and the IgG heavy chain variable region together define a single binding site to bind to human ErbB3. A CDRL1 comprises a sequence of amino acids selected from a 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 a 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 a 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).
[00053] 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 and 22A02).
[00054] 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).
[00055] 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).
[00056] 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).
[00057] 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).
[00058] Preferably, the sequences CDRL1, CDRL2 and CDRL3 interpose between FRs of human or humanized immunoglobulins. The antibody can be an intact antibody or an antibody fragment for binding to the antigen.
[00059] In some embodiments, the antibody comprises: (a) an IgG heavy chain variable region comprising the CDRH1-CDRH2-CDRH3 structure and (b) an IgG light chain variable region comprising the CDRL1-CDRL2-CDRL3 structure , wherein the heavy chain variable region and the light chain variable region together define a single binding site to bind to human ErbB3. CDRH1 is a sequence of amino acids selected from a 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 is a sequence of amino acids selected from a 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 CDRH3 is a sequence of amino acids selected from a 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). CDRL1 is a sequence of amino acids selected from a 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 is a sequence of amino acids selected from a 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 CDRL3 is a sequence of amino acids selected from a 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).
[00060] 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 a 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).
[00061] 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).
[00062] 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).
[00063] 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).
[00064] 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).
[00065] 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).
[00066] 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).
[00067] 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).
[00068] 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).
[00069] 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).
[00070] 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 a 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 cover), and SEQ ID NO: 206 (Sh24C05 Kv1-17 cover).
[00071] 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).
[00072] 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).
[00073] 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).
[00074] 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).
[00075] 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).
[00076] 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).
[00077] 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).
[00078] 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 cover).
[00079] 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 cover).
[00080] 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 cover).
[00081] 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 cover).
[00082] As used herein, unless otherwise indicated, the term "antibody" means an intact antibody (for example, an intact monoclonal antibody) or antigen-binding antibody fragment (for example, an antibody fragment monoclonal for antigen binding), including an intact antibody or antigen-binding fragment that has been modified, designed, or chemically conjugated. Examples of antibodies that have been modified or designed include chimeric antibodies, humanized antibodies and multispecific antibodies (for example, bispecific antibodies). Examples of antigen-binding fragments include Fab Fab ', (Fab') 2, Fv, straight chain antibodies (e.g., scFv), minibodies and diabodies. An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.
[00083] Figure 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 linked by an interchain disulfide bond. The heavy chains of immunoglobulins are linked by interchain disulfide bonds. A light chain consists of a variable region (VL in figure 1) and a constant region (CL in figure 1). A heavy chain consists of a variable region (VH in figure 1) and at least three constant regions (CH1, CH2 and CH3 in figure 1). The variable regions determine the specificity of the antibody.
[00084] 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 specificity of antibody binding.
[00085] In certain embodiments, an isolated antibody that binds to 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 sequence of the variable region or framework region 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).
[00086] In certain embodiments, an isolated antibody that binds to 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 sequence of the variable region or framework region 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).
[00087] In each of the previous modalities, it is contemplated in this document that the sequences of the immunoglobulin heavy chain variable region and / or the light chain variable region sequences, which together bind to human ErbB3, may contain changes in amino acids (for example, 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.
[00088] In some embodiments, an isolated antibody joins 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 less. Unless otherwise specified, KD values are determined by surface plasma resonance methods. Surface plasmon resonance methods can be performed using the conditions described, for example, in examples 3 and 12, where measurements were performed at 25 ° C and 37 ° C, respectively.
[00089] In some embodiments, antibodies inhibit the binding of hErbB3 to NRG1-β1. For example, antibodies can have an IC 50 (50% maximum inhibition concentration) of about 5 nM, 2 nM or less, when dosed using the protocols described in examples 4 and 13. II. Antibody production
[00090] Methods for producing antibodies disclosed in this document are known in the art. For example, DNA molecules encoding variable regions of light chain and variable regions of heavy chain can be chemically synthesized using the sequence information provided here. The synthetic DNA molecules can be linked to other suitable nucleotide sequences, including, for example, constant region coding sequences and expression control sequences, to produce constructs with an expression of conventional genes that code for the desired antibodies. The production of the defined gene constructs is included in the routine practices provided for in the technique. Alternatively, the sequences provided herein can be cloned from hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes, whose sequences are based on the sequence information provided herein, or in the sequence information of the prior art considering genes encoding the heavy and light chains of murine antibodies in hybridoma cells.
[00091] Nucleic acids encoding the antibodies disclosed in this document can be incorporated (linked) into expression vectors, which can be introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (eg example, Hep G2) and myeloma cells that do not otherwise produce IgG proteins. The transformed host cells can be grown under conditions that allow the host cells to express the genes encoding the variable regions of light and / or heavy chain.
[00092] The specific conditions of expression and purification will vary, depending on the expression system employed. For example, if a gene is expressed in E. coli, it is first cloned into the expression vector, positioning the modified gene downstream of a suitable bacterial promoter, eg, Trp or Tac, and a prokaryotic signal sequence . The expressed secreted protein accumulates in refractile or inclusion bodies, and can be captured after rupture of cells by the French press or sonication. The refractile bodies are then solubilized, and the proteins rearranged and cleaved by methods known in the art.
[00093] If a DNA construct encoding an antibody disclosed here is expressed in eukaryotic host cells, for example, CHO cells, it is first introduced into an expression vector containing a suitable eukaryotic promoter, a secretion signal, enhancers of IgG, and several introns. This expression vector optionally contains sequences that encode all or part of a constant region, allowing all or part of a heavy and / or light chain to be expressed. In some embodiments, a single expression vector contains the variable regions of both the light and heavy chains to be expressed.
[00094] The gene construct can be introduced into eukaryotic host cells using conventional techniques. Host cells express the VL or VH fragments, the VL-VH heterodimers, the VH-VL or VL-VH linear chain polypeptides, the heavy or light complete immunoglobulin chains, or portions thereof, each of which can be associated with a portion having another function (eg, cytotoxicity). In some embodiments, a host cell is transfected with a single vector expressing a polypeptide expressing all, or part, of a heavy chain (for example, a variable region of the heavy chain) or a light chain (for example, a variable region of light chain). 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) the entire immunoglobulin heavy chain and the entire immunoglobulin light chain. In still other embodiments, a host cell is cotransfected with more than one expression vector (for example, an expression vector expressing a polypeptide comprising all, or a part, of the variable region of the heavy chain or the heavy chain, and another expression vector expressing a polypeptide comprising all or part of the variable region of the light chain or the light chain).
[00095] A method of producing a polypeptide comprising an immunoglobulin heavy chain variable region or a polypeptide comprising an immunoglobulin light chain variable region can comprise the growth of a host cell transfected with an expression vector under conditions that permit 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 can then be purified by techniques well known in the art, for example, affinity markers such as glutathione-S-transferase (GST) and markers of histidine.
[00096] A method of producing a monoclonal antibody that binds to human ErbB3, or an antigen-binding antibody fragment, can include the growth of the transfected host cell with: (a) an expression vector encoding a heavy chain complete or partial immunoglobulin, and a separate expression vector encoding a complete or partial immunoglobulin light chain; or (b) a single expression vector that codes for both strands (for example, complete or partial strings), under conditions that allow the expression of both strands. The intact antibody (or antigen binding fragment) can be captured and purified using techniques well known in the art, for example, Protein A, Protein G, affinity markers such as glutathione-S-transferase (GST) and histidine markers. The expression of the heavy chain and the light chain from a single expression vector or two distinct expression vectors is included in the state of the art. III. Antibody Modifications
[00097] Methods for reducing or eliminating the antigenicity of antibodies and antibody fragments are known in the art. When antibodies are intended to be administered to a human, the antibodies are preferably "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 rat antibody from which it is derived.
[00098] In a humanization approach, chimeric proteins are created in which the rat immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, for example, Morrison et al., 1984, PROC. NAT. ACAD.
[00099] SCI. 81: 6851-6855, Neuberger et al., 1984, NATURE 312: 604-608; U.S. Patent No. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).
[000100] In an approach known as CDR grafting, CDRs from variable regions of light and heavy chains are grafted into frameworks from other species. For example, murine CDRs can be grafted onto human RFs. In some embodiments, CDRs from the variable regions of the light and heavy chain of the anti-ErbB3 antibody are grafted into human FRs or human consensual FRs. To create consensus human FRs, the FRs of various 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. Patent No. 7,022,500 (Queen); 6,982,321 (Winter); 6,180,370 (Queen); 6,054,297 (Carter); 5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332 (Hoogenboom); 5,585,089 (Queen); 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: 9294.
[000101] In an approach called "SUPERHUMANIZA-TION ™," human CDR sequences are selected from human germline genes, based on the structural similarity of human CDRs with those of the rat antibody to be humanized. See, e.g., U.S. Patent No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL 169: 1119-1125.
[000102] Other methods to reduce immunogenicity include "reforming", "hyperquimerization" and "leafing / resurfacing". See, for example, Vaswami et al., 1998, Annals OF ALLERGY, ASTHMA, & IMMUNOL. 81: 105; Roguska et al., 1996, PROT. ENGINEER 9: 895-904; and U.S. Patent No. 6,072,035 (Hardman). In the leafing / resurfacing approach, amino acid residues accessible to the surface of the murine antibody are replaced by amino acid residues most often found in the same positions in a human antibody. This type of antibody resurfacing is described, for example, in U.S. Patent No. 5,639,641 (Pedersen).
[000103] Another approach for converting a mouse antibody into a form suitable for medical use in humans is known as ACTIVMAB TM technology (Vaccinex, Inc., Rochester, NY), which involves a vector based on the vaccinia virus to express antibodies in mammalian cells. High levels of combinatorial diversity of IgG heavy and light chains are said to be produced. See, for example, U.S. Patent No. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer).
[000104] Another approach to converting a mouse antibody into a form suitable for use in humans is the technology commercially practiced by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology involves the use of a proprietary human "acceptor" library to produce an "epitope-focused" library for antibody selection.
[000105] Another approach to modify a mouse antibody in a form suitable for medical use in humans is the HUMAN ENGINEERINGTM technology, which is commercially practiced by XOMA (US) LLC. See, p. , PCT publication No. WO 93/11794 and U.S. Patent No. 5,766,886; 5,770,196; 5,821,123; and 5,869,619.
[000106] Any suitable approach, including any of the above approaches, can be used to reduce or eliminate the human immunogenicity of an antibody disclosed here.
[000107] Methods of making multi-specific antibodies are known in the art. Multi-specific antibodies include bi-specific antibodies. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bi-specific antibodies bind to two different epitopes of the antigen of interest. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (for example, bispecific antibodies and F (ab ') 2 antibodies) 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).
[000108] In some embodiments, the antibody is conjugated to an effector agent such as a small molecule toxin or radionuclide using in vitro standard conjugation chemistries. If the effector agent is a polypeptide, the antibody can be chemically conjugated to the effector or attached to the effector as a fusion protein. The construction of fusion proteins is included in the routine practices provided for in the art. IV. Use of antibodies
[000109] The antibodies disclosed in this document can be used to treat various forms of cancer, for example, breast, ovarian, prostate, cervical, colorectal, lung (e.g., non-small cell lung cancer), pancreatic, gastric, skin, kidney, head and neck and schwannoma. Cancer cells are exposed to a therapeutically effective amount of the antibody to inhibit or reduce the proliferation of cancer cells. In some modalities, antibodies inhibit cancer cell proliferation by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%.
[000110] In some embodiments, the antibody inhibits or reduces the proliferation of tumor cells by inhibiting the binding of human ErbB3 to an ErbB3 ligand, for example, 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.
[000111] Cancers associated with overexpression and / or activation of ErbB3 include breast cancer, ovarian cancer, prostate cancer, cervical cancer, lung cancer (eg non-small cell cancer), some forms of brain cancer (for example, schwannoma), melanomas, cancers of the skin, kidney and gastrointestinal tract (for example, colorectal, pancreatic, gastric, head and neck).
[000112] As used herein, the terms "treat," treating "and" treatment "mean the treatment of a disease in a mammal, eg, in a human. This includes: (a) inhibition of the disease, ie , suspend its development, and (b) mitigate the disease, that is, cause regression of the disease state, and (c) cure the disease.
[000113] Generally, a therapeutically effective amount of the active component is comprised between 0.1 mg / kg and 100 mg / kg, for example, 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 the disease or the indication to be treated, the general health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation and the route of administration. The starting dose can be increased beyond the upper level in order to quickly reach the desired level in the tissue or tissues. Alternatively, the starting dose may be less than what is considered optimal, and the daily dose may be increased progressively during the course of treatment. The human dose can be optimized, for example, in a conventional Phase I dose escalation study, designed to run from 0.5 mg / kg to 20 mg / kg. The frequency of dosing can vary, depending on factors such as the route of administration, the dosage and the disease to be treated. Exemplary dosing frequencies are once a day, once a week and once every two weeks. A preferred route of administration is the parenteral route, for example, intravenous infusion. The formulation of drugs based on monoclonal antibodies is included in the routine practices provided for in the art. In some embodiments, the monoclonal antibody is lyophilized and reconstituted in saline at the time of administration.
[000114] For therapeutic use, an antibody is preferably combined with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means buffers, vehicles and excipients suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, proportional to a benefit reason. / reasonable risk. The vehicle (s) must be "acceptable" in the sense of being compatible with the other ingredients in the formulations and not harmful to the recipient. Pharmaceutically acceptable vehicles include buffers, solvents, dispersion media, coatings, isotonic and absorbing retardants, and the like, which are compatible with pharmaceutical administration. The use of such means and agents for pharmaceutically active substances is known in the art.
[000115] The pharmaceutical compositions containing antibodies disclosed in this document can be presented in unit dosage form and can be prepared by any suitable method. A pharmaceutical composition must 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 of monoclonal antibodies is IV infusion. Useful formulations can be prepared by methods well known in the art of the pharmaceutical industry. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Suitable components of the formulation for parenteral administration include a sterile diluent, such as water for injection, saline, fixing oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfate; chelating agents, such as EDTA; buffers, such as acetates, citrates or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose.
[000116] For intravenous administration, suitable vehicles include saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or saline phosphate buffer (PBS). The vehicle must be stable under the conditions of manufacture and storage and must 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 polyethylene glycol), and suitable mixtures thereof.
[000117] Pharmaceutical formulations are preferably sterile. Sterilization can be carried out, for example, by filtration through sterile filtration membranes. When the composition is lyophilized, filter sterilization can be carried out before or after lyophilization and reconstitution. EXAMPLES
[000118] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way. Example 1 - Production of Anti-hErbB3 Monoclonal Antibodies
[000119] Immunizations, mergers, and primary analyzes were conducted at Maine Biotechnology Services Inc. in accordance with the Multiple Site Repetitive Immunization (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 immunizations were performed with either cleaved rhErbB3 (immunization A) or with cleaved rhErbB3 cross-linked to its ligand, the human recombinant domain NRG1- 1 / HRG1- 1-EGF (R&D Systems, Cat. No. 396-HB) (Immunization B). Two AJ mice by immunization with sera showing high anti-ErbB3 activity by the enzyme-linked immunosorbent assay (ELISA) were chosen for subsequent fusion. Spleens and lymph nodes from the appropriate rats were collected. The B cells were then harvested and fused with a myeloma line. The fusion products were serially diluted in forty 96-well plates until they approach clonality. A total of 5,280 supernatants from the resulting fusions were evaluated for binding to recombinant rhErbB3 / Fc, using ELISA. The same supernatants were also evaluated for their binding to human ErbB3 overexpressed in CHO cells (by Mesoscale electrochemiluminescence assay). Three hundred supernatants identified as containing antibodies to ErbB3 were further characterized by in vitro biochemical and cellular assays, as discussed below. A hybridoma panel was selected and the hybridomas were subcloned and expanded. The hybridoma cell lines were transferred to BioXCell (formerly BioExpress) for antibody expression and purification by affinity chromatography on G protein resin under standard conditions.
[000120] The anti-hErbB3 monoclonal antibody 04D01 was generated from Immunization A described above. The monoclonal antibodies anti.hErb3 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 were generated by the B immunization described above. Example 2 - Sequence Analysis of Anti-hErbB3 Monoclonal Antibodies
[000121] The light chain isotype and the heavy chain isotype of each monoclonal antibody in Example 1 was determined using the IsoStrip ™ Mouse Monoclonal Antibody Isotyping set according to the manufacturer's instructions (Roche Applied Science). All antibodies were determined to be light chain Capa and heavy chain IgG, IgG1 or IgG2b.
[000122] The light and heavy chain variable regions of 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 set according to the supplier's instructions (Qiagen). A first full length cDNA strand containing 5 'terminations was generated using either the GeneRacerTM kit (Invitrogen) or the SMARTerTM RACE cDNA Amplification kit (Clontech) according to the manufacturer's instructions using random 5' RACE primers.
[000123] The variable regions of the IgG Capa and heavy chains (IgG1or IgG2b) were amplified by PCR, using a polymerase KOD Hot Start (Novagen) or Advantage 2 Polymerase Mix (Clontech) according to the manufacturer's instructions. For amplification of the 5 'cDNA ends in conjunction with the GeneRacerTM set, the GeneRacerTM 5', 5 'cgactggagcacgaggacactga 3' primer (SEQ ID NO: 136) (Invitrogen) was used as the 5 'primer. For amplification of the 5 'terminations of the cDNA in conjunction with the SMARTerTM RACE cDNA amplification set, Primer A from the Universal Primer Mix (Clontech), a mixture of 5'CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAG T 3' (SEQ ID NO: 137) and 5 ' CTAATACGACTCACTATAGGGC 3 '(SEQ ID NO: 138) was used as a 5' primer. The heavy chain variable regions were amplified using the 5 'primers above and a specific 3' IgG1 Constant Region 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) or 5'GGCCAGTGGATAGACTGATGGGGGTGTTGT 3 '(SEQ ID NO: 142) or 5' GGAGGAACCAGTTGTATCTCCACACCA 3 '. The variable regions of the kappa chain were amplified using the primers 5 'above and a specific primer of the Constant Region 3' kappa, either 5 'CTCATTCCTGTTGAAGCTCTTGACAAT 3' (SEQ ID NO: 144) or 5 'CGACTGAGGCACCTCCAGATGTT 3' (SEQ ID NO: 145).
[000124] The individual PCR products were isolated by agarose gel electrophoresis and purified using the Qiaquick® Purification Gel set according to the manufacturer's instructions (Qiagen). The PCR products were subsequently cloned into plasmid pCR®4Blunt using the Zero Blunt® TOPO® PCR Cloning Kit according to the manufacturer's instructions (Invitrogen) and transformed into DH5-α bacteria (Invitrogen) using standardized molecular biology techniques. Plasmid DNA isolated from transformed bacterial clones was sequenced using M13 Direct (5 'GTAAAACGACGGCCAGT 3') (SEQ ID NO: 146) and Inverse M13 (5 'CAGGAAACAGCTATGACC 3') primers (SEQ ID NO: 147) by Beckman Genomics, using standard didesoxy DNA sequencing methods to identify the sequence of variable region sequences. The sequences were analyzed using the Vector NTI program (Invitrogen) and the IMGT / V-Quest program to identify and confirm the sequences of the variable region.
[000125] The nucleic acid sequences that encode and the protein sequences that define the variable regions of the murine monoclonal antibodies are summarized below (the amino acid signal peptide sequences are not shown). The CDR sequences (Kabat definition) are shown in bold / underlined in the amino acid sequences. Sequence of Nucleic Acid encoding the variable region of the heavy 04D01 antibody chain (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 Region Variable Heavy the 04D01 antibody chain (SEQ ID NO: 2) 1 qvqlqqpgae lvrpgtsvkl sckasgytft shwlhwvkqr pgqglewigv Idpsdfysny 61 nqnfkgkatl tvdtssstay mqlssltsed savyycargl Isgdyamdyw gqgtsvtvss Sequence Nuclei Acid Coding for the Variable Region of the Chain Antibody Cover 04D01 (SEQ ID NO: 3) 1 gatgttttga tgacccaaat tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 61 atctcttgca gatctagtca gagcattgta catagat acaccta 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 region Variable Chain Antibody cover 04D01 (SEQ ID NO: 4) 1 dvlmtqipls Ipvslgdqas iscrssqsiv hsngntylew ylqkpgqspk sliykvsnrf 61 sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgsyvp wtfgggtkle ik acid sequence C uclei co encoding the variable region of the heavy 09D03 antibody chain (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 tcgcgggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggtg g ggccaaggca ccactctcac agtctcctca Protein Sequence Defining the region Variable Heavy the 09D03 antibody chain (SEQ ID NO: 12) 1 qvtlkesgpg ilrpsqtlsl tcsfsgfsls tfglsvgwir qpsgkglewl ahiwwdddky 61 ynpalksrlt iskdtsknqv flkianvdta dtatyycari gadalpfdyw gqgttltvss acid sequence C uclei co encoding the variable region of Chain antibody cover 09D03 (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 Variable Region of the Antibody Cover Chain 09D03 (SEQ ID NO: 14) 1 divltqtaps vpvtpgesvs iscrssksll hsngntylyw flqrpgqspq lliyrmsnla 61 sgvpdrfsgs gsgytahri gtkle ik acid sequence C uclei co encoding the variable region of the heavy 11G01 antibody chain (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 Protein Sequence Defining the Region Variable Heavy the 11G01 antibody chain (SEQ ID NO: 22) 1 qvqlqqsdae lvkpgasvki sckvsgytft dhiihwmkqr peqglewigy iyprdgyiky 61 nekfkgkatl tadkssstay mqvnsltsed savyfcargy yyamdywgqg tsvtvss Nucleic Acid Sequence Coding for the Variable Region of the Chain Antibody Cover 11G01 (SEQ ID NO: 23) 1 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcgtaccctcttgtca aaacaccta 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 region Variable Chain Antibody cover 11G01 (SEQ ID NO: 24) 1 dvlmtqtpls Ipvslgdqas iscrssqsiv hsigntylew ylqkpgqspk lliykvsnrf 61 sgvperfsgs gsgtdftlki srveaedlgv yycfqgshvp ftfgsgtkle ik acidon sequence uclei co encoding the variable region of the heavy 12A07 antibody Chain (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 gcagctgac tctgggg tctgggggctgg act ctcacagtct cctca Protein Sequence Defining the region Variable heavy 12A07 Antibody Chain (SEQ ID NO: 31) 1 qvqllqpgae lvrpgtsvkl scktsgytfs sywmhwvkqr pgqglewigm idpsdvytny 61 npkfkgkatl tvdtssstay mqlssltsed savyycarny sgdywgqgtt ltvss acid sequence C uclei co coding for Chain Variable Region Case 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 Variable Region of the Antibody Hood Chain 12A07 (SEQ ID NO: 33) 1 dvlmtqipls Ipvslgdqas iscrssqsiv hsngntylew ylqkpgqspk lliykvsnrf 61 sgvpdrffgsgs gsgtgfggs of acidon uclei co encoding the Region Variable Heavy the 18H02 antibody chain (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 Region Variable Heavy the 18H02 antibody chain (SEQ ID NO: 38) 1 qiqlvqsgpe lkkpgeavki sckssgytft tygmswvkqa pgralkwmgw intysgvpty 61 addfkgrfaf slessast ay lqinnlkned tatyfcargr dgyqvawfaywgqgtlvtvs 121 the Nucleic Acid Sequence Coding for the Variable Region of the Chain Antibody Cover 18H02 (SEQ ID NO: 39) 1 gaaacaactg tgacccagtc tccagcatcc ctgtccatgg ctataggaga taaagtcacc 61 atcagatgca taccagcac tgatatt tatga 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 the defining Protein Sequence Region variable of the 18H02 antibody kappa chain (SEQ ID NO: 40) 1 ettvtqspas Ismaigdkvt ircitstdid ddmnwfqqkp geppkllise gntlrpgvps 61 rfsgsgygtd fiftienmls edvadyyclq sdnlpytfgg gtkleik acid sequence C uclei co encoding the Region Variable heavy 22A02 antibody Chain (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 gcagctgac tctgggg tctggggtg cctca Protein Sequence Defining the Variable Region of the Antibody Heavy Chain 22A02 (SEQ ID NO: 48) 1 qvqlqqpgae lvrpgtsvkl sckasgytft nywmhwvkqr pgqglewigm idpsdsytny String nskwqyqqqggqlqdgtgtgtlg 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 Sequence of Protein Defining the Variable Region of the Chain Antibody Cover 22A02 (SEQ ID NO: 50) 1 dvlmtqtpls Ipvslgdqas iscrssqsiv hsngntylew ylqkpgqspk lliykvsnrf 61 sgvpdrfsgs gsgtdftlkvgged will co encoding the Region Variable Heavy the 24C05 antibody chain (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 region Variable Heavy the 24C05 antibody chain (SEQ ID NO: 54) 1 evqlvesggg lvkpggslkl scaasgftfs dyamswvrqt pekrlewvat isdggtytyy 61 pdnvkgrfti srdnaknnly Iqmshlksed tamyycarew gdydgfdywg qgttltvss sequence of N uclei co Coding for the Variable Region of the Chain Antibody Cover 24C05 (SEQ ID NO: 55) 1 gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 61 ctcacttgtc gggcaagtca ggaatagtca ggaa gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 181 aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcggcag ccttgagtct 241 gaagatcttg cagactatta ctgtctacaa tatgatagtt atccgtacac gttcggaggg 301 gggaccaagc tggaaataaa the defining Protein Sequence Region Variable Chain Antibody cover 24C05 (SEQ ID NO: 56) 1 diqmtqspss Isaslgervs Itcrasqeis gylswlqqkp dgtikrliya astldsgvpk 61 rfsgsrsgsd ysltigsles edladyyclq ydsypytfgg gtkleik
[000126] The amino acid sequences defining the variable regions of the immunoglobulin heavy chain for the antibodies produced in Example 1, are aligned in figure 2. The amino-terminal signal peptide sequences (for the appropriate expression / secretion) are not shown. CDR1, CDR2 and CDR3 (definition of Kabat) are identified by boxes. Figure 3 shows an alignment of the separate CDR1, CDR2 and CDR3 sequences for each antibody.
[000127] The amino acid sequences defining the variable regions of the immunoglobulin light chain for the antibodies produced in Example 1, are aligned in figure 4. The amino-terminal signal peptide sequences (for proper expression / secretion) are not shown. CDR1, CDR2 and CDR3 are identified by boxes. Figure 5 shows an alignment of the separate CDR1, CDR2 and CDR3 sequences for each antibody.
[000128] Table 1 is an agreement graph showing the SEQ ID No. of each sequence discussed in this Example. Table 1



[000129] Rat monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 2. Table 2


[000130] CDR light chain sequences of the mouse monoclonal antibody (Kabat, Chothia, and IMGT definitions) are shown in Table 3. Table 3

[000131] In Tables 2 and 3, the longest CDR sequences for the immunoglobulin heavy and light chain are shown in bold.
[000132] To create the complete antibody sequences, with heavy chain or cap, each variable sequence above is combined with its respective constant region. For example, a complete heavy chain comprises a variable heavy sequence followed by the murine IgG1 and IgG2b heavy chain constant sequence and a complete kappa chain comprises a variable kappa sequence followed by the murine kappa light chain constant sequence. Sequence of Nucleic Acid encoding the heavy chain constant region of IgG1 murine (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 t tactgtgga 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 region constant heavy chain IgG1 murine (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 lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv 241 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf 301 tcsvlheglh nhhtekslsh spgk Nucleic acid Coding sequence for heavy chain constant region of the IgG2b murine (SEQ ID NO: 104) 1 gccaaaacaa cacccccatc agtctatcca ctggcccctg ggtgtggaga tacaactggt 61 tcctctgtga ctctgggatg cctggtcaag ggctacttcc ctgagtcagt gactgtgact 121 tgga ccctgtc 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 t cccggtctc cgggtaaa Protein Sequence Defining the region constant heavy chain IgG2b murine (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 Region Constant Light Case murine Chain (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 caacttcaccgt catggt agg Protein sequence Defining the Constant Region of the Murine Hood Light Chain (SEQ ID NO: 107) 1 radaaptvsi fppsseqlts ggasvvcfln nfyprdinvk wkidgserqn gvlnswtdqd 61 skdstysmss tltltkdeye rhnsytceat hktstsniv
[000133] The following sequences represent the complete heavy and light chain sequences, real or contemplated (i.e., containing both sequences of the variable and constant regions) for each antibody described in that example. Signal sequences for proper antibody secretion are also included at the 5 'end of the DNA sequences or at the amino terminus of the protein sequences. Sequences from the variable region can be linked to other sequences from the constant region to produce full and active IgG heavy and light chains. Sequence ÁcidoNuclei co encoding the complete sequence of heavy chain (Region Variable Heavy Chain Area Constant IgG1) in 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 aaggtggac agaaatt cagggat 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 Sequence Heavy Chain (Heavy Chain Variable Region and Constant IgG1) from 04D01 (SEQ ID NO: 109) 1 mgwsciivll vstatgvhsq vqlqqpgael vrpgtsvk ls 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 qfnstfrsvs elpimhqdwl ngkefkcrvn saafpapiek tisktkgrpk 361 apqvytippp keqmakdkvs ltcmitdffp editvewqwn gqpaenyknt qpimdtdgsy 421 fvysklnvqk snweagntft csvlheglhn hhtekslshs pgk ÁcidoNuclei Sequence co Coding for Full Sequence Light Chain (Region Variable Area Cover Chain Constant) 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 ca c 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 sequence Defining Protein Chain Complete sequence Light (Region Variable Area Cover chain constant) 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 ngvlnswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec Acid SequenceNuclei co Coding for Complete Heavy Chain Sequence (Va Region riável Heavy Chain Area Constant IgG2b) 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 tgcaaggagtgtcacaaatg 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 Sequence Complete Heavy Chain (Variable Region of the Heavy Chain and Region Constant IgG2b) of 09D03 (SEQ ID NO: 113) 1 mgrltssfll livpayvlsq vtlkesgpgi lrpsqtlslt csfsgfslst fglsvgwirq 61 psgkglewla hiwwdddkyy np alksrlti 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 redynstirv vstlpiqhqd wmsgkefkck vnnkdlpspi 361 ertiskikgl vrapqvytlp ppaeqlsrkd vsltclvvgf npgdisvewt snghteenyk 421 dtapvldsdg syfiysklnm ktskwektds fscnvrhegl knyylkktis rspgk ÁcidoNuclei Sequence co encoding the complete sequence of Light Chain (Region Variable Area Cover chain constant) 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 agggggtg 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 Sequence Complete Light Chain (Variable Region and kappa Constant) of 09D03 (SEQ ID NO: 115) 1 mrclaeflgl lvlwipgaig divltqtaps vpvtpgesvs iscrssksll hsngntylyw 61 flqrpgqspq lliyrmsnla sgvpdrfsgs gsgtaftlri srveaedvgv yycmqhleyp 121 ftfgsgtkle ikradaaptv sifppsseql tsggasvvcf lnnfyprdin vkwkidgser 181 qngvlnswtd qdskdstysm sstltltkde yerhnsytce athktstspi vksfnrnec Acid SequenceNuclei co Coding for the Complete Heavy Chain Sequence (Variable Region of the Heavy Chain and Region Co nstante IgG1) 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 gt gttgtggt 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 Sequence Complete Heavy Chain (Region Variable Heavy Chain Area Constant IgG1) in 11G01 (SEQ ID NO: 117) 1 mewswvslff lsvttgvhsq vqlqqsdael vkpgasvkis ckvsgytftd hiihwmkqrp 61 eqglewigyi yprdgyikyn ekfkgkatlt adkssstaym qvnsltseds avyfcargyy 121 yamdywgqgt svtvssaktt ppsvyplapg saaqtnsmvt l gclvkgyfp epvtvtwnsg 181 slssgvhtfp avlqsdlytl sssvtvpsst wpsqtvtcnv ahpasstkvd kkivprdcgc 241 kpcictvpev ssvfifppkp kdvltitltp kvtcvvvdis kddpevqfsw fvddvevhta 301 qtqpreeqfn stfrsvselp imhqdwlngk efkcrvnsaa fpapiektis ktkgrpkapq 361 vytipppkeq makdkvsltc mitdffpedi tvewqwngqp aenykntqpi mdtdgsyfvy 421 sklnvqksnw eagntftcsv lheglhnhht ekslshspgk ÁcidoNuclei Sequence co Coding for Full Chain Sequence Lite (Region Variable Area Constant kappa chain) 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 aactgtatcc 421 aact tcttcccac 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 Chain Sequence Lite (Variable Region and Constant the kappa chain) 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 ngvlnswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec ÁcidoNuclei Sequence co encoding the complete sequence Heavy Chain (Variable Heavy Chain Region and IgG1 Constant Region) of 12A07 (SEQ ID NO: 120) 1 atgggatgga gctgtatcat tgtcctcttg gtatcaacag ctacatgtgt ccactcccag 6 1 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 aggagca gtt 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 heavy chain sequence (Region Variable Heavy Chain Area Constant IgG1) 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 cictvppk vf vd ltitltpkv tcvvvdiskd dpevqfswfv ddvevhtaqt 301 qpreeqfnst frsvselpim hqdwlngkef kcrvnsaafp apiektiskt kgrpkapqvy 361 tipppkeqma kdkvsltcmi tdffpeditv ewqwngqpae nykntqpimd tdgsyfvysk 421 lnvqksnwea gntftcsvlh eglhnhhtek slshspgk ÁcidoNuclei Sequence co Coding for Full Sequence Light Chain (Variable Region and cover Chain Constant) 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 ttgatgg cag 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 Sequence Complete Light Chain (Variable Region and cover Chain Constant) of 12A07 (SEQ ID NO: 123) 1 mklpvrllvl mfwipasssd vlmtqiplsl pvslgdqasi scrssqsivh sngntylewy 61 lqkpgqspkl liykvsnrfs gvpdrfsgsg sgtdftlkis rveaedlgvy ycfqgsyvpw 121 tfgggtklei kradaaptvs ifppsseqlt sggasvvcfl nnfyprdinv kwkidgserq 181 ngvlnswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec ÁcidoNuclei Sequence co encoding the complete sequence of heavy chain (Region variable heavy and Region Chain 18H02 IgG1 constant) (SEQ ID NO: 124) 1 atgggttggc tgtggaactt gctattcctg atggcagctg cccaaagtgc ccaagcacag 61 atccagttgg tacagtctgg acctgaactg aagaagcctg gagaggcactg gaggggactg ggtgaa 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 10 21 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 Sequence Complete Heavy Chain (Region Variable Heavy Chain Area Constant IgG1) in 18H02 (SEQ ID NO: 125) 1 mgwlwnllfl maaaqsaqaq iqlvqsgpel kkpgeavkis ckssgytftt ygmswvkqap 61 gralkwmgwi ntysgvptya ddfkgrfafs lessastayl qinnlknedt atyfcargrd 121 gyqvawfayw gqgtlvtvsa akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt 181 wnsgslssgv htfpavlqsd lytlsssvtv psstwpsqtv tcnvahpass tkvdkkivpr 241 dcgckpcict vpevssvfif ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve 301 vhtaqtqpre eqfnffrkv lpimhqkng saafpapie ktisktkgrp 361 kapqvytipp pkeqmakdkv sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs 421 yfvysklnvq ksnweagntf tcsvlheglh nhhtekslsh spgk ÁcidoNuclei Sequence co Coding for Full Chain Sequence Lite (Region Variable Area Cover Chain Constant) from 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 Chain Full Sequence Lite (Variable Region and cover Chain Constant) from 18H02 (SEQ ID NO: 127) 1 mfslalllsl lllcvsdsra ettvtqspas lsmaigdkvt ircitstdid ddmnwfqqkp 61 geppkllise gntlrpgvps rfsgsgygtd fiftienmls edvadyyclq sdnlpytfgg 121 gtkleikrad aaptvsifpp sseqltsgga svvcflnnfy prdinvkwki dgserqngvl 181 nswtdqdskd stysmsstlt ltkdeyerhn sytceathkt stspivksfn RNEC Sequence ÁcidoNuclei co encoding the complete sequence of heavy chain (Region variable Heavy Chain Area Constant IgG1) 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 181 ggacaaggcc ttgagtggat gcagaggcct cggaatgatt gatccttctg atagttatac taactacaat 241 ccaaagt TCA 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 gca gatggcc 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 Sequence Heavy Chain (Variable Region Heavy and Region Chain Constant IgG1) 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 frsvselpim hqdwlngkef kcrvnsaafp apiektiskt kgrpkapqvy 361 tipppkeqma kdkvsltcmi tdffpeditv ewqwngqpae nykntqpimd tdgsyfvysk 421 lnvqksnwea gntftcshkte egl slshspgk Nucleic Acid Sequence Coding for the Complete Sequence of the Light Chain (Variable Region and Constant Region of the Chain Cover) of 22A02 (SEQ ID NO: 130) 1 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgtgtggtggtcgtggtggtcgtgtgtggtgtgtgtgtggtgtgtgtgtgtgtgtgtggtgtggtgtgtgtgtggtgtggtgtgctgtgtgtgtgtggtgtggtggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtggtgtgtgtgtggtgtg 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 gtg T Protein Sequence Defining the Chain Full Sequence Lite (Variable Region and cover Chain Constant) of 22A02 (SEQ ID NO: 131) 1 mklpvrllvl mfwipasssd vlmtqtplsl pvslgdqasi scrssqsivh sngntylewy 61 lqkpgqspkl liykvsnrfs gvpdrfsgsg sgtdftlkis rveaedlgvy ycfqgsyvpw 121 tfgggtklei kradaaptvs ifppsseqlt sggasvvcfl nnfyprdinv kwkidgserq 181 ngvlnswtdq dskdstysms stltltkdey erhnsytcea thktstspiv ksfnrnec ÁcidoNuclei Sequence co encoding the complete sequence of heavy chain (Region variable Heavy Chain Area Constant IgG1) of 24C05 (SEQ ID NO: 132) 1 atgaacttcg ggctcagctt gatgttcctt taaaaggtgt ccagtgtgag 61 gtgcagctgg tggaatctgg gggaggctta gagggtccct gaaactctcc 121 tgtgcagcct ctggattcac tttcagtgac cttgggttcg ccagactccg 181 gaaaagaggc tggagtgggt cgcaaccatt gtacttacac ctactatcca 241 gacaatgtaa agggccgatt caccatctcc ccaagaacaa cctgtacctg 301 caaatgagcc atctgaagtc tgaggacaca actgtgcaag agaatggggt 361 gattacgacg gatttgacta ctggggccaa tcacagtctc ctcggcc aaa 421 acgacacccc catctgtcta tccactggcc ctgcccaaac taactccatg 481 gtgaccctgg gatgcctggt caagggctat cagtgacagt gacctggaac 541 tctggatccc tgtccagcgg tgtgcacacc tcctgcagtc tgacctctac 601 actctgagca gctcagtgac tgtcccctcc ccagccagac cgtcacctgc 661 aacgttgccc acccggccag cagcaccaag aaattgtgcc cagggattgt 721 ggttgtaagc cttgcatatg tacagtccca ctgtcttcat cttcccccca 781 aagcccaagg atgtgctcac cattactctg tcacgtgtgt tgtggtagac 841 atcagcaagg atgatcccga ggtccagttc tagatgatgt ggaggtgcac 901 acagctcaga cgcaaccccg ggaggagcag 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 ca tactgaga agagcctctc ccactctcct 1381 ggtaaa Protein Sequence Defining the Sequence Complete Heavy Chain (Region Variable Heavy Chain and Region Constant IgG1) 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 fnstfrsvse lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka 361 pqvytipppk eqmakdkvsl tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf 421 vysklnvqks nweagntftc svlheglhnh htekslshsp gk Sequence ÁcidoNuclei co encoding the complete sequence of the Light Chain (Variable Region and Constant Region of the Capa Chain) of 24C05 (SEQ ID NO: 134) 1 atggacatga gggttcctgc tcacgttttt ggcttcttgt tgctctggtt tccaggtacc 61 agatgtgaca tccagatgac ccagtctcc the 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 Chain Full Sequence Lite (Variable Region and cover Chain Constant) of 24C05 (SEQ ID NO: 135) 1 mdmrvpahvf gflllwfpgt rcdiqmtqsp sslsaslger vsltcrasqe isgylswlqq 61 kpdgtikrli yaastldsgv pkrfsgsrsg sdysltigsl esedladyyc lqydsy pytf 121 gggtkleikr adaaptvsif ppsseqltsg gasvvcflnn fyprdinvkw kidgserqng 181 vlnswtdqds kdstysmsst ltltkdeyer hnsytceath ktstspivks fnrnec
[000134] For convenience, Table 4 provides an agreement graph showing the correspondence between the complete sequences of the antibodies discussed in this Example with those shown in the Sequence Listing. Table 4

Example 3 - Binding affinities
[000135] The affinities and binding kinetics for the binding of monoclonal antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 to the human recombinant fusion protein ErbB3 / Fc (rhErbB3-Fc) were measured by surface plasma resonance using a Biacore® T100 device (Biacore).
[000136] Rabbit anti-rat IgGs (Biacore, Cat. No. BR-1008-38) were immobilized on CM4 sensor chips of carboxymethylated dextran (Biacore, Cat. NO BR-1005-34) by amine bond (BIAcore, Cat. N ° BR-1000-50) using a standard connection protocol according to the supplier's instructions. Analyzes were performed at 25 ° C, using PBS (Invitrogen, Cat. NO 14040-133) containing 0.05% P20 surfactants (Biacore, Cat. No. BR-1000-54) as running buffer.
[000137] Antibodies were captured in individual flow cells at a flow rate of 10 μL / minute. The injection time was varied for each antibody in order to yield an Rmax between 30 and 60 RU. The buffer or rhErbB3-Fc diluted in running buffer was injected sequentially over a reference surface (without captured antibodies) and the active surface (antibody to be tested) for 300 seconds at 60 μL / minute. The dissociation phase was monitored for more than 3,600 seconds. The surface was then regenerated with two 60-second injections with 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 concentration range of the rhErbB3-Fc tested was 0.125 nM to 20 nM.
[000138] The kinetic parameters were determined using the kinetic function of the BIAevaluation software (Biacore) with double reference subtraction. The kinetic parameters for each antibody, ka (constant rate of association), kd (constant rate of dissociation) and KD (constant dissociation at equilibrium) were determined. Kinetic values of monoclonal antibodies in rhErbB3-Fc at 25 ° C are summarized in Table 5. Table 5

[000139] The data in Table 5 demonstrate that the antibodies bind to 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 lower, or 10 pM or lower. Example 4 - Neutralization activity
[000140] In this example, the antibodies produced in Example 1 were tested for their ability to inhibit the binding of rhErbB3 to NRG1-β1 and NRG1-α1. The antibodies were tested in an electrochemiluminescence (ECL) assay to inhibit the binding of hErbB3 to NRG1-β1. Standardized 96-well MA2400 connection 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) overnight at 4 ° C without stirring. The plates were then washed 3 times with PBS + 0.1% Tween 20 (Sigma P5927) and blocked with 200 μL of PBS containing 5% BSA (Sera Care Life Sciences, Cat. NO AP-4510-80) for 1, 5 hours at room temperature. After washing the plates 3 times with PBS, 25 μL of the antibody dilutions were added to the plates for another hour at room temperature, with stirring. The NRG1-β1 ligand (R&D Systems, Cat. No. 377-HB, 26kDa) 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 biotinylated antibody 1 μg / mL against human NRG1- 1 (R&D systems, Cat. NO BAF377) preincubated for one hour with streptavidin SULTO-TAG (Meso Scale Discovery, Cat. NO R32AD-5) for one hour at room temperature, with stirring. The plates were then washed 3 times with PBS and 150 μL of 1X reading buffer (Meso Scale Discovery, Cat. NO R92TC-1) were added to each well before the plates were analyzed on a Sector® Imager 2400 (Meso Scale Discovery) ).
[000141] The interaction of NRG1-β1 with ErbB3 was inhibited by antibodies 04D01, 12A07, 18H02, 22A02 and 24C05 (figure 6A). The interaction of NRG1-β1 with rhErbB3 was enhanced by antibody 09D03, but not as well as by antibodies 11G01 (figure 6B).
[000142] IC50 values of the murine anti-human ErbB3 antibody for neutralizing the binding of NRG1-β1 to rhErbB3 to antibodies (i.e., 04D01, 12A07, 18H02, 22A02 and 24C05) were calculated and are summarized in Table 6. Table 6

[000143] The results show that antibodies 04D01, 12A07, 18H02, 22A02 and 24C05 efficiently neutralized the binding of NRG1-β1 to rhErbB3. Antibodies 09D03 and 11G01 reinforced the binding of hNRG1-β1 to hErbB3.
[000144] The antibodies were tested in an ECL assay to inhibit the binding of hErbB3 to a second ErbB3 ligand, NRG1- α1. To test the inhibition of the binding of NRG1-α1 to rhErbB3, the same method used for NRG1-β1 was used, except for the following changes: the concentrations of rhErbB3 / Fc plated (R&D 4518-RB) and the ligand NRG1-α1 ( Thermo Scientific, RP-317- P1AX) were 1 μg / mL and 1.5 μg / mL, respectively.
[000145] The interaction of NRG1-α1 with rhErbB3 was inhibited by IgG1 11G01, 12A07, 18H02, 22A02, and 24C05 and was enhanced by the 09D03 antibody (figure 7). Example 5 - Connection to ErbB3 domain II
[000146] In this example, the antibodies produced in Example 1 were tested for their ability to bind 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) in the total length of the Her2 receptor. The hybrid Her2 / 3d2 construct was cloned into pLenti6.3 and packaged by transient transfection of 293T cells into a lentivirus using the ViraPower ™ Lentiviral Support Kit (Invitrogen, Cat. No. K497000). CHO cells were infected with lentiviruses expressing their hybrid protein Her2 / 3d2. The connections of the anti-ErbB3 hybridoma supernatants to Her2 / 3d2 were tested on these CHO cells designed by ECL with sulpho-labeled anti-mouse antibodies. The data on the binding of hybridoma supernatants to the chimeric Her2 / 3d2 protein expressed on the cell surface of CHO cells are summarized in figure 8. These results demonstrate that antibodies 09D03 and 11G01 have bound to domain II ErbB3, AA210-327. Example 6 - Antiproliferative Activity
[000147] This example describes a characterization of the antibodies produced in Example 1, with respect to their ability to inhibit NRG1-β1-dependent proliferation. The antibodies were tested in the BaF / 3 cell system designed to express both human Her2 and ErbB3 and in MCF7 breast cancer cells that naturally express both Her2 and ErbB3 and grow in response to NRG1-β1 stimulation.
[000148] Baf / 3 cells were infected by two lentiviruses designed to express either human Her2 or human ErbB3. The infected cells were selected with blasticidin (15 μg / mL; Invitrogen, Cat. No. R21001) and the individual colonies were isolated and tested for expression of both receptors. Her2 / ErbB3 expression clones were maintained in culture under blasticidin selection with [Medium RPMI 1640 80% (GIBCO, Cat. NO 11875-093), 10% fetal bovine serum (GIBCO, Cat. NO 10438-026) and medium with 10% WEHI cell restriction (ISCOVE 90% Modified Dulbecco 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. 21985023)}]. To analyze ErbB3 antagonist antibodies, the cells were washed with PBS and cultured in the absence of blastidine and WEHI-restricted media. The assays were conducted on 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 a final volume of 100 μL). MTT assays (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) were conducted 3-4 days after NRG1-β1 stimulation.
[000149] An example of dose-dependent inhibition of NRG1-β1-dependent Her2 / ErbB3-BaF / 3 cells proliferation by murine anti-human ErbB3 antibodies is shown in Figure 9. Her2 / cell line proliferation inhibition data NRG1-β1-dependent ErbB3- BaF / 3 with monoclonal antibodies (ie 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05) are summarized in Table 7. Table 7

[000150] The results in Table 7 show that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 strongly inhibited the proliferation of BaF / 3 cells induced by NRG1-β1, expressing Her2 / ErbB3.
[000151] MCF7 cells (ATCC, Cat. NO HTB-22) were maintained as recommended by the ATCC. The cells were plated at 5,000 cells / well in 96-well plates. The cells were deprived of nutrients overnight, in the absence of serum. The next day, NRG1-β1 (40 ng / mL) and various concentrations of antibodies (12.8 pg / mL-20 μg / mL in a final volume of 100 μL) were added to the cells. MTT assays (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) were conducted three days after NRG1- β1 stimulation.
[000152] An example of the dose-dependent inhibition of NRG1-β1-dependent MCF7 cell proliferation by anti-human murine ErbB3 antibodies is shown in figure 10. The inhibition data of the proliferation of the MCF7 cell line dependent on NRG1-β1 with antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05) are summarized in Table 8. Table 8

[000153] The results in Table 8 demonstrate that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 strongly inhibited the proliferation of MCF7 cells induced by NRG1-β1.
[000154] The antibodies produced in Example 1 were also tested for their ability to inhibit the proliferation of human cancer cells expressing ErbB3. SKBR-3 breast cancer cells overexpress Her2 and are sensitive to their Her2-specific inhibitory antibodies.
[000155] SKBR-3 cells (ATCC, Cat. NO HTB-30) were maintained as recommended by the ATCC. The 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 assays (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) were conducted after three days in culture.
[000156] An example of inhibition of SKBR-3 cell proliferation by murine anti-human ErbB3 antibodies is shown in figure 11. The results in figure 11 demonstrate that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited the proliferation of SKBR-3 cells. Example 7 - Inhibition of downstream signaling
[000157] This example describes a characterization of the antibodies produced in Example 1, with respect to their ability to inhibit phosphorylation of NRG1-β1-dependent ErbB3 and downstream Akt kinase, as the reading for PI3K activation. These antibodies were also tested for their ability to inhibit steady-state phosphorylation of ErbB3 and Akt in cells with exponential growth.
[000158] SKBR-3 and MCF7 breast cancer cells and DU145 prostate cancer cells were maintained as recommended by the ATCC. The cells were deprived of nutrients overnight in 0% FBS, treated for one hour with 5 μg / mL of antibody followed by NRG1-β1 stimulation. The lysates were analyzed by ELISA with the Phospho-ErbB3 kit from R&D Systems (Cat. NO DYC1769) or with the Phospho-Akt ELISA Cell Signaling kit (Cat. NO 7143).
[000159] An example of inhibiting phosphorylation of ErbB3 in SKBR-3 cells, induced by NRG1-β1, by murine anti-human ErbB3 antibodies is shown in figure 12. The results shown in Fig. 12 demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited at least 50% of NRG1-β1-induced phosphorylation of ErbB3 in SKBR-3 cells.
[000160] An example of inhibition of Akt phosphorylation in MCF7 and DU145 cells, induced by NRG1-β1, by murine anti-human ErbB3 antibodies is shown in figure 13A and figure 13B, respectively. The results in figures 13A and 13B demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited at least 80% of Akt phosphorylation in response to NRG1-β1 in both MCF7 and DU145 cells.
[000161] The capabilities of anti-ErbB3 antibodies to inhibit the state of steady-state phosphorylation of ErbB3 and Akt in a breast cancer SKBR-3 cell line and in a pancreatic cancer cell BxPC3 cell line were tested by treating these cells growing exponentially for one hour in the presence of antibodies at 5 μg / mL.
[000162] Western blot analysis of these assays showed that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited the steady state phosphorylation of Akt and ErbB3 in both SKBR-3 and BxPC3 cells. Example 8 - Inhibition of EGFR phosphorylation induced by NRG1-β1
[000163] In this example, the antibodies produced in Example 1 were tested for their ability to inhibit phosphorylation of EGFR, dependent on NRG1-β1, in NIC / ADR-RES ovarian cancer cell lines. NIC / ADR-RES cells (DTP / DCTD NIC from the tumor repository) were deprived of nutrients overnight in 0% FBS, pretreated with antibody (5 μg / mL) for one hour followed by NRG1-β1 stimulation ( 20 ng / ml) for 15 minutes. Phosphorylation of EGFR in tyrosine 1068 was analyzed by Western blot. The results of this assay demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited EGFR phosphorylation in response to NRG1-β1 in NIC / ADR-RES cells. Example 9 - Inhibition of EGF-induced ErbB3 phosphorylation
[000164] In this example, the antibodies produced in Example 1 were tested for their ability to inhibit EGF-dependent ErbB3 phosphorylation in A431 epidermoid cancer cell lines, overexpressing EGFR. A431 cells (ATCC, Cat. NO CRL-1555) were deprived of nutrients overnight in 0% FBS, pretreated with antibody (5 μg / mL) for one hour, followed by stimulation by EGF (R&D Systems, Cat NO 236-EG) (50 ng / ml) for 15 minutes. Phosphorylation of ErbB3 was analyzed by Western blot. The results of this assay demonstrated that antibodies 04D01, 09D03, 12A07, 18H02, 22A02 and 24C05 inhibited ErbB3 phosphorylation in response to EGF in A431 cells at various levels. Example 10 - Inhibition of NRG1-β1 induced Her2 / ErbB3 heterodimer formation
[000165] This example describes a characterization of the antibodies produced in Example 1, with regard to their ability to inhibit the formation of the Her2 / ErbB3 dimer in response to NRG1-β1 in SKBR-3 cells. SKBR-3 breast cancer cells were deprived of nutrients overnight in 0% FBS, treated for one hour with 5 μg / ml of antibody followed by stimulation with NRG1-β1 (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).
[000166] The results of this assay demonstrated that antibodies 04D01, 09D03, 11G01, 12A07, 18H02, 22A02 and 24C05 inhibited the formation of the Her2 / ErbB3 dimer induced by NRG1-β1 in SKBR-3 cells. Example 11 - BxPC3 tumor xenograft growth inhibition
[000167] The ability of the murine monoclonal antibodies produced in Example 1 to inhibit tumor growth was tested in a pancreatic BxPC3 xenograft model. BxPC3 human pancreatic cells were cultured in a culture medium at 37 ° C in an atmosphere containing 5% CO2, using the RMPI medium containing 10% fetal bovine serum. BxPC3 cells were inoculated subcutaneously, on the flank of a CB.17 female SCID rat from 8 weeks of age (Taconic Labs) with 10 x 10 6 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were performed twice a week using a caliper. The tumor volume was calculated using the formula: width x width x length / 2. When the tumors reached approximately 200 mm3, the rats were randomly assigned to 9 groups of 10 rats each. One group received PBS and another received human IgG control (huIgG). Each of the other eight groups received one of the antibodies, 04D01, 09D03, 18H02, 11G01, 24C05, 22A02, or 12A07. All antibodies were dosed at 20 mg / kg of body weight, twice a week, by intraperitoneal injection for 6 weeks. Tumor volumes and rat body weights were recorded twice a week. The inhibition of tumor growth was analyzed using ANOVA and is expressed as a percentage of inhibition compared to the PBS control.
[000168] The results in figure 14 showed that the 24C05 antibody inhibited tumor growth by 76% in that model (p <0.001). Antibodies 04D01, 18H02 and 11G01 also inhibited tumor growth in that model at 64%, 71% and 72%, respectively (p <0.001). Antibodies 12A07 and 22A02 demonstrated minimal activity, that is, close to 40% of tumor growth inhibition, while antibody 09D03 provided 60% inhibition of tumor growth in this model. Example 12 - Humanization of Anti-ErbB3 Antibodies Construction of Humanized and Chimeric Anti-ErbB3 Antibodies
[000169] This example describes the humanization of the murine antibody designated 24C05, and the characterization of the resulting humanized antibodies. Humanized anti-ErbB3 antibodies were designed using the SUPERHUMANIZATIONTM method (Arana Therapeutics Ltd. and Hwang, WY et al. (2005) METHODS 36: 35-42) or by the CDR grafting method with subsequent mutations (some human framework residues have been changed to murine residues) (See for example, US Patent No. 5,530,101; 5,693,761; 5,693,762; 5,585,089; 6,180,370; 7,022,500). With the exception of the heavy chain CDR1, Kabat's CDR definitions were used for grafting CDR into human frameworks. A combination of definitions from Kabat and Chothia was used for grafting heavy CDR1. The drawn 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, aminoterminal signal sequence, humanized variable region , human IgG1 or Capa constant region, stop codon and a 3 'EcoRI restriction site. In addition, a humanized heavy chain, the Sh24C05 Hv3-11 Heavy IgG1, was mutated using PCR for overlap extension to improve humanization, resulting in the IgG1 heavy chain Sh24C05 Hv3-11 N62S. A version of the human IgG2 of the heavy chain Sh24C05 Hv3-11 N62S was also constructed.
[000170] Humanized anti-ErbB antibody chains according to the SUPERHUMANIZATIONTM method, as described herein, are designated with the prefix "Sh" before the antibody chain name. Chains of humanized anti-ErbB3 antibody by the reverse mutation CDR grafting method, as described herein, are designated with the prefix "Hu" before the antibody chain name.
[000171] The chimeric (murine variable region and human constant region) 24C05 heavy (human IgG1) and light (human cover) chains were also constructed. The murine variable regions were fused to the human constant region using overlap extension PCR, including (in the following order): The 5 'HindIII restriction site, the Kozak consensus sequence, the aminoterminal signal sequence, the rat variable region, the human IgG1 or Capa constant region, the stop codon and a 3 'EcoRI restriction site.
[000172] 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 Capa light chains were subcloned into pEE14.4 (Lonza Biologics) via HindIII and EcoRI sites using In-FusionTM PCR cloning (Clontech).
[000173] Humanized antibody chains or chimeric antibody chains have been transiently transfected into 293T cells to produce antibodies. The antibody was either purified or used in cell culture media supernatants for further in vitro analysis. The binding of chimeric and humanized antibodies to human ErbB3 was measured as described below. The results are summarized in Table 15.
[000174] In addition, some combinations of humanized antibody heavy and light chains have been stably expressed in CHOK1SV cells using GS SystemTM (Lonza Biologics) in order to produce large amounts of purified humanized antibody. A single expression vector was constructed by combining vectors based on pEE6.4 and pEE14.4. First, the full humanized heavy chain cDNA containing pEE6.4 was digested with NotI and SalI to isolate the hCMV-MIE promoter + full humanized heavy chain cDNA + poly40 SVA fragment. This fragment was inserted in the vector pEE14.4 already containing complete 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. The stable clones were selected in the presence of methionine sulfoximine.
[000175] Each of the possible combinations of the variable regions of the humanized immunoglobulin heavy chain and the immunoglobulin light chain is set out below in Table 9. Table 9



[000176] The nucleic acid sequences encoding and the protein sequences that define the variable regions of the humanized 24C05 antibodies are summarized below (the amino acid sequences of the signal peptide are not shown). The CDR sequences (Kabat definition) are shown in bold and are underlined in the amino acid sequences. Nucleic Acid Sequence Coding for Variable Region of the Heavy Chain Sh24C05 Hv3-7 (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 Variable Region of the Heavy Chain Sh24C05 Hv3-7 (SEQ ID NO: 150) 1 evqlvesggg lvqpggslrl scaasgftfs dyamswvrqa pgkglewvat isdggtytyy 61 pdnvkgrfti srdnaknsly Iqmnslraed tavyycarew gdydgfdywg qgtlvtvss Nucleic Acid Sequence Coding for the Heavy Chain Variable Region Sh24C05 Hv3-11 (SEQ ID NO: 151) 1 caagttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagggg cttctggggctttgg 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 Variable Region of the Heavy Chain Sh24C05 Hv3-11 (SEQ ID NO: 152) 1 qvqlvesggg lvkpggslrl scaasgftfs dyamswirqa pgkglewvst isdggtytyy 61 pdnvkgrfti srdnaknsly Iqmnslraed tavyycarew gdydgfdywg qgtlvtvss Nucleic acid Sequence Coding for the heavy Chain Variable Region Sh24C05 Hv3-11 N62S (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 ggagattat g atgggtttga ctattggggc cagggcactt tggtgacagt cagttct Protein Sequence Defining the Variable Region of the Heavy Chain Sh24C05 Hv3-11 N62S (SEQ ID NO: 154) 1 qvqlvesggg lvkpggslrl scaasgftfs dyamswirqa pgkglewvst isdggtytyy 61 pdsvkgrfti srdnaknsly Iqmnslraed tavyycarew gdydgfdywg qgtlvtvss Nucleic Acid Sequence Coding Region for Variable Heavy Chain Sh24C05 Hv3-21 (SEQ ID NO: 155) 1 gaggttcagc tggtggaatc tggcggtggg cttgtaaagc caggaggctc cctcagactg 61 agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca 121 gactggagtg ggttagcact atcagcgatg cccgggaaag 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 Variable Region of the Heavy Chain Sh24C05 Hv3-21 (SEQ ID NO: 156) 1 evqlvesggg lvkpggslrv scaasggggw srdnaknsly Iqmnslraed tavyycarew gdydgfdywg qgtlvtvss Nucleic Acid Sequence coding for the variable region of the heavy chain Sh24C05 Hv3-23 (SEQ ID NO: 157) 1 gaggttcagc ttctggaatc tggcggtggg cttgtacagc caggaggctc cctcagactg 61 agttgtgccg cttcagggtt cacattctcc gactatgcga tgtcatgggt gcgccaagca 121 cccgggaaag gactggagtg ggtttcaact atcagcgatg gcggaacgta 181 tacctattac cctgacaatg tgaagggtcg gttcaccatt tccagggata acagcaagaa cacactctat 241 ctccagatga acagcctgag ggctgaggac accgccgtct actactgcgc ccgagaatgg 301 ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct Protein Sequence Defining the Variable Region of the Heavy Chain Sh24C05 Hv3-23 (SEQ ID NO: 158) 1 evqllesggg lvqpggslrl scaasgftfs dyamswvrqa pgkglewvst isdggtytyy 61 pdnvkgrfti srdnskntly Iqmnslraed tavyycarew gdydgfdywg qgtlvtvss Nucleic Acid Sequence Coding for the Heavy Chain Variable Region Sh24C05 Hv3-30 (SEQ ID NO: 159) 1 caggttcagc tggtggaatc tggggggggggggggggggggggggggggggggggggg 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 Variable Region of the Heavy Chain Sh24C05 Hv3-30 (SEQ ID NO: 160) 1 qvqlvesggg vvqpgrslrl scaasgftfs dyamswvrqa pgkglewvat isdggtytyy 61 pdnvkgrfti srdnskntly Iqmsslraed tavyycarew gdydgfdywg qgtlvtvss nucleic acid sequence encoding the variable region of the heavy Hu24C05 HVA chain (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 tccagggg acga c accgccgtct actactgcgc ccgagaatgg 301 ggagattatg atgggtttga ctattggggc cagggcactt tggtgacagt cagttct Protein Sequence Defining the region Variable Heavy Hu24C05 HVA chain (SEQ ID NO: 162) 1 evqlvesggg lvkpggslrl scaasgftfs dyamswvrqa pgkglewvat isdggtytyy 61 pdnvkgrfti srdnaknsly Iqmnslraed tavyycarew gdydgfdywg qgtlvtvss nucleic acid sequence coding for the variable region of the kappa chain Sh24C05 Kv1-9 (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 The Protein Sequence Defining the Variable Region of the Chain Cover Sh24C05 Kv1-9 (SEQ ID NO: 164) 1 diqltqspsf Isasvgdrvt itcrasqeis gylswyqqkp gkapkllgt as 61 isslqp edfatyyclq ydsypytfgq gtkleik Nucleic Acid Sequence coding for the variable region kappa chain Sh24C05 Kv1-16 (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 Protein Sequence Defining the Region Variable Chain Cover Sh24C05 Kv1-16 (SEQ ID NO: 166) 1 diqmtqspss Isasvgdrvt itcrasqeis gylswfqqkp gkapksliya astldsgvps 61 rfsgsgsgtd ftltisslqp edfatyyclq ydsypytfgq gtkleik Nucleic Acid Sequence Coding for the Variable Region of the Capa Chain Sh24C05 Kv1-17 (SEQ ID NO: 167) 1 gatattcaga tgacccaatc acctagcagt ctctcagctt ccgtgggcga cagagttacc 61 ataacctgtc gggcaagcgggtggtggtg ccc caaagaggtt gatctatgct gcgtcaacct tggatagcgg tgtcccgagt 181 cgattctccg gttctggctc cggaaccgag ttcactctga caatttctag ccttcagcca 241 gaagatttcg ccacgtacta ttgcctccag tacgacagct atccctatac atttgggcag 301 ggcactaaac tggagatcaa Protein Sequence Defining the Region Variable Chain Cover Sh24C05 Kv1-17 (SEQ ID NO: 168) 1 diqmtqspss Isasvgdrvt itcrasqeis gylswyqqkp gkapkrliya astldsgvps 61 rfsgsgsgte ftltisslqp edfatyyclq ydsypytfgq gtkleik Nucleic acid Sequence Coding for the chain variable region Case Sh24C05 Kv1-33 (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 Atttgggacaga Region Tac 4C05 Kv1-33 (SEQ ID NO: 170) 1 diqmtqspss Isasvgdrvt itcrasqeis gylswyqqkp gkapklliya astldsgvps 61 rfsgsgsgtd ftftisslqp ediatyyclq ydsypytfgq gtkleic Sequence of the acidic code for a 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 Protein Sequence Defining the Region Variable Chain Cover Sh24C05 Kv1-39 (SEQ ID NO: 172) 1 diqmtqspss Isasvgdrvt itcrasqeis gylswyqqkp gkapklliya astldsgvps 61 rfsgsgsgtd ftltisslqp edfatyyclq ydsypytfgq gtkleik Sequence of the ACC Accelerated code for the region tctcagctt ccgtgggcga cagagttacc 61 gggcaagcca ggagatttct gggtacctgt ataacctgtc 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 Protein Sequence Defining the Region Variable Chain Cover Hu24C05 KvA (SEQ ID NO: 174) 1 diqmtqspss Isasvgdrvt itcrasqeis gylswlqqkp ggaikrliya astldsgvps 61 rfsgsgsgsd ytltisslqp edfatyyclq ydsypytfgq gtkleik
[000177] The amino acid sequences defining the variable regions of the immunoglobulin heavy chain for the antibodies produced in Example 12, are aligned in figure 15. The amino-terminal signal peptide sequences (for proper expression / secretion) are not shown. CDR1, CDR2 and CDR3 (definition of Kabat) are identified by boxes.
[000178] The amino acid sequences defining the variable regions of the immunoglobulin light chain for the antibodies produced in Example 12, are aligned in figure 16. The amino-terminal signal peptide sequences (for proper expression / secretion) are not shown. CDR1, CDR2 and CDR3 are identified by boxes.
[000179] Table 10 is an agreement graph showing the SEQ ID NO of each sequence discussed in this Example. Table 10



[000180] Humanized monoclonal antibody heavy chain CDR sequences (Kabat, Chothia and IMGT definitions) are shown in Table 11. Table 11



[000181] CDR sequences of the humanized monoclonal antibody kappa light chain (Kabat, Chothia and IMGT definitions) are shown in Table 12. Table 12


[000182] In Tables 11 and 12, the longest CDR sequences for the immunoglobulin heavy and light chain are shown in bold.
[000183] To create the complete antibody sequences, with heavy chain or cap, chimeric and humanized, each variable sequence above is combined with its respective human constant region. For example, a complete heavy chain comprises a variable heavy sequence followed by a human IgG1 heavy chain constant sequence or a human IgG2 heavy chain constant sequence. A complete kappa chain comprises a variable kappa sequence followed by the human kappa light chain constant sequence. Sequence of Nucleic Acid encoding the Region constant heavy chain human IgG1 (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 gc ttctaccc 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 Region Constant Guy Human IgG1 chain (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 lpapiektis kakgqprepq vytlppsree 241 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 301 qqgnvfscsv mhealhnhyt qkslslspgk Nucleic acid Sequence Coding for Region Constant Heavy IgG2 chain Human (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 tcctctacag caagctcacc gtggacaaga ggctccttct gcaggtggca gcaggggaac 901 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 961 tccctgtctc cggg taaa Protein Sequence Defining the Region Constant Heavy Chain Human IgG2 (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 Coding Sequences for Region Constant Light Chain human kappa (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 30g tccttcaata gg the Constant Region of the Light Chain Human Cover (SEQ ID NO: 180) 1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd 61 skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec
[000184] The following sequences represent the complete heavy and light chain sequences, real or contemplated (i.e., containing both sequences of the variable and constant regions) for each antibody described in that example. Signal sequences for proper antibody secretion are also included at the 5 'end of the DNA sequences or at the amino terminus of the protein sequences. It is also contemplated that sequences from the variable region can be linked to other sequences from the constant region, to produce full and active IgG heavy and light chains. Sequence ÁcidoNuclei co encoding the heavy chain 24C05 Chimeric Complete (variable region of the Heavy Chain Mouse Area Constant Human IgG1) (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 aagggtcgatac gtgga 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 heavy Chain 24C05 Chimeric Full (variable region of the heavy Chain Mouse and Human IgG1 Constant Region) (SEQ ID NO: 182) 1 mnfgls lmfl 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 ÁcidoNuclei Sequence co Coding for the Light Chain 24C05 Chimeric Full (variable region Chain Mouse Cover Area Constant Human Cover) (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 ccaaaaagg 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 Light Chain 24C05 Chimeric Full (Region Variable Chain Mouse Cover Area Constant Human Cover) (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 Acid SequenceNuclei co Codifi cating for the Humanized Heavy Chain Complete sh24C05 Hv3-7 (Region Heavy Chain Humanized Variable Region and Human IgG1 Constant) (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 gcgggtgga 7 ccaagagct 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 Chain Heavy Sh24C05 Hv3-7 Humanized Full (Variable Region Heavy Chain Humanized and Human IgG1 Constant Region) (SEQ ID NO: 186) 1 mdmrvp aqll gllllwlrga rcevqlvesg gglvqpggsl rlscaasgft fsdyamswvr 61 qapgkglewv atisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar 121 ewgdydgfdy wgqgtlvtvs 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 Coding for the Humanized Heavy Chain Sh24C05 Hv3-11 (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region) (SEQ ID NO: 187) 1 atggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg ttcagctggt ggaatctggc ggtgggcttg taaagccagg aggctccctc 121 agactgagtt gtgccgcttc agggttcaca ttctccgact atgcgatgtc atggatcagg 181 caagcacccg ggaaatgggggggggggggggggggggg acgtatacc 241 tattaccctg acaatgtgaa gggtcggttc accatttcca gggataacgc aaagaacagt 301 agatgaacag cctgagggct gaggacaccg ctctaccttc 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 agta aggcaa 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 Heavy Chain Sh24C05 Hv3-11 humanized Full (heavy chain variable region humanized Area Constant Human IgG1) (SEQ ID NO: 188) 1 mdmrvpaqll gllllwlrga rcqvqlvesg gglvkpggsl rlscaasgft fsdyamswir 61 qapgkglewv stisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar 121 ewgdydgfdy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv 181 swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkrve 241 pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn 301 wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiek 36ap qvytlppsre emtknqvslt clvkgfypsd iavewesngq pennykttpp 421 vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k Nucleic Acid Sequence Coding for the Human IgG1 Heavy Chain (H24C05 HG3H Noise) ) 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 agctgtg gtggtg gt ctgctgt 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 Sequence Protein D efinindo IgG1 Heavy Chain Humanized Full Sh24C05 Hv3-11 N62S (Region Heavy Chain Humanized Variable Region and Human IgG1 Constant) (SEQ ID NO: 190) 1 mdmrvpaqll gllllwlrga rcqvqlvesg gglvkpggsl rlscaasgft fsdyamswir 61 qapgkglewv stisdggtyt yypdsvkgrf tisrdnakns lylqmnslra edtavyycar 121 ewgdydgfdy wgqgtlvtvs 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 Coding Sequences for heavy chain IgG2 Sh24C05 Hv3-11 N62S Humanized Complete (Humanized Heavy Chain Variable Region and Human IgG2 Constant Region) (SEQ ID NO: 191) 1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61 ccaag 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 agatgaacag cctgagggct gaggacaccg ctctaccttc 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 fall gacaaag 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 The Protein Sequence Defining the IgG2 Heavy Chain Sh24C05 Hv3-11 N62S Complete Humanized (Humanized Heavy Chain Variable Region and Human IgG2 Constant Region) (mqq lgg rg lg lg lg lg rl lg rl ls fsdyamswir 61 qapgkglewv stisdggtyt yypdsvkgrf tisrdnakns lylqmnslra edtavyycar 121 ewgdydgfdy wgqgtlvtvs sastkgpsvf plapcsrsts estaalgclv kdyfpepvtv 181 swnsgalts vv 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 Coding Sequences for the Heavy Chain Humanized Sh24C05 Hv3-21 Full (Variable Region heavy chain is humanized and Region Constant Human IgG1) (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 gaccactggt 42 aaggacc 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 tttcttt ctg tacagtaagc tgactgtgga caagtcccgc 1321 tggcagcagg gtaacgtctt cagctgttcc gtgatgcacg aggcattgca caaccactac 1381 acccagaagt cactgagcct gagcccagg aG Humanized Region 1 1 mdmrvpaqll gllllwlrga rcevqlvesg gglvkpggsl rlscaasgft fsdyamswvr 61 qapgkglewv stisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar 121 ewgdydgfdy wgqgtlvtvs 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 Coding for the Heavy Chain Humanized Sh24C05 Hv3-23 Complete (Variable Region of the P Chain ESADA Humanized Area Constant Human IgG1) (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 tctttttgt t 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 Humanized heavy Chain Sh24C05 Hv3-23 Full (Variable Region heavy Chain Humanized and Human IgG1 Constant Region) (SEQ ID NO: 196) 1 mdmrvpaqll gllllwlrga rcevqllesg gglvqpggsl rlscaasgft fsdyamswvr 61 qapgkglewv stisdggtyt yyp dnvkgrf tisrdnsknt lylqmnslra edtavyycar 121 ewgdydgfdy wgqgtlvtvs 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 coding for the heavy Chain Humanized Sh24C05 Hv3-30 Complete (Region heavy Chain Humanized Variable Region and Human IgG1 Constant) (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 accactcca gg atctcc 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 Humanized Heavy Chain Sh24C05 Hv3-30 Full (Variable Region Heavy Chain Humanized Area Constant Human IgG1) (SEQ ID NO: 198) 1 mdmrvpaqll gllllwlrga rcqvqlvesg ggvvqpgrsl rlscaasgft fsdyamswvr 61 qapgkglewv atisdggtyt yypdnvkgrf tisrdnsknt lylqmsslra edtavyycar 121 ewgdydgfdy wgqgtlvtvs 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 vldsdgsrq yskltvd scs vmhealhnhy tqkslslspg k ÁcidoNuclei Sequence co Coding for the Heavy Chain Hu24C05 HVA Humanized Full (Variable Region Heavy Chain Humanized Area Constant Human IgG1) (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 acctatt 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 heavy Chain Hu24C05 HVA Humanized Full (Region Humanized Heavy Chain Variable and Region Constant Human IgG1) (SEQ ID NO: 200) 1 mdmrvpaqll gllllwlrga rcevqlvesg gglvkpggsl rlscaasgft fsdyamswvr 61 qapgkglewv atisdggtyt yypdnvkgrf tisrdnakns lylqmnslra edtavyycar 121 ewgdydgfdy wgqgtlvtvs 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 Sequence Nucleic acid encoding for Light Chain Sh24C05 Kv1-9 Humanized Full (Region Humane Chain Variable Region and Human Constant Cover) (SEQ ID NO : 201) 1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61 cgttgcgata ttcagttgac ccaatcacct agcttcctct cagcttccgt gggcggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg 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 Humanized Light Chain Sh24C05 Kv1-9 Full (Variable Chain Humanized Cape Region and Human Constant) (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 kdstyslss t ltlskadyek hkvyacevth qglsspvtks fnrgec Nucleic Acid Sequence Coding for the Complete Humanized Sh24C05 Kv1-16 Light Chain (Humanized Cover Variable Region and Human Constant Region) (SEQ ID NO: 203) 1 atggacatga gggtgggggggggggggggggggggggg 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 ggttacact 661 cagggacac TTGT ctagtcctgt caccaagtcc ttcaataggg gcgaatgt Protein Sequence Defining Humanized Light Chain Sh24C05 Kv1-16 Full (Variable Chain Humanized Cape Region and Human Constant) (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 Human-language ACC Sequencing (CVE) and ACE Sequential Locking System 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 ctctga CAAT 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 Light ChainSh24C05 Complete Humanized Kv1-17 (Variable Region of the Humanized Cape Chain and Human Constant Region) (SEQ ID NO: 206) Humanized Cover Chain Variable and Human Constant Region) (SEQ ID NO: 206) 1 mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswyqq 61 kpgkapkrli yaastldsgv psrfsgsgsg teftltissl qpedfatyyc lqydsypytf 121 gqgtkleikr tvaapsvfn kpsvsnn vtks fnrgec Nucleic Acid Sequence Coding for the Light Chain sh24C05 Kv1-33 Humanized Full (Variable Chain Humanized Cape Region and Human Constant) (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 ctagtctgt cact aataggg gcgaatgt Protein Sequence Defining Humanized Light Chain Complete Sh24C05 Kv1-33 (Variable Region chain humanized cover 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 Coding for the Humanized Humanized Humanized Humanized Humanized Humanized Human Gene (ACG) 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 Humanized Light Chain Complete Sh24C05 Kv1-39 (Chain Humanized Variable Region and Human Case Constant) (SEQ ID NO: 210) 1 mdmrvpaqll gllllwlrga rcdiqmtqsp sslsasvgdr vtitcrasqe isgylswyqq 61 kpgkapklli yaastldsgv psrfsgsgsg tdftltissl qpedfatyyc lqydsypytf 121 gqgtkleikr tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw 181 kvdnalqsgn sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks fnrgec Nucleic Acid Sequence Coding for the Hu24C05 Humanized KvA Light Chain Complete (Regi the varying the chain humanized Case Area Human Constant) (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 Humanized Hu24C05 Light Chain Complete Human KvA (Variable Region Case Chain Humanized 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
[000185] For convenience, Table 13 provides an agreement graph showing the SEQ ID NO of each sequence discussed in this Example. Table 13


[000186] Table 14 below shows the antibodies containing heavy and light chains of chimeric immunoglobulins and each of the possible combinations of the heavy and light chains of the complete humanized immunoglobulins. Table 14



[000187] The antibody construct containing the complete chimeric heavy and light chains is designated below:
[000188] 24C05 Chimeric = Heavy Chain 24C05 Chimeric Complete (Rat Variable region and Human IgG1 Constant Region) (SEQ ID NO: 182) plus 24C05 Chimeric Complete Chain (Rat Variable region and Constant Human Cover Region) (SEQ ID NO: 184)
[000189] Four of the possible antibody constructs containing the heavy and light chains of complete immunoglobulin, containing humanized variable regions are designated below:
[000190] Sh24C05-25 N62S IgG1 = Heavy Chain Variable Region Sh24C05 Hv3-11 N62S Humanized and Human IgG1 Constant Region (SEQ ID NO: 190) plus the Sh24C05 Kv1-16 Light Chain Variable Region and the Human Cover Constant Region ) (SEQ ID NO: 204)
[000191] Sh24C05-25 N62S IgG2 = Heavy Chain Variable Region Sh24C05 Hv3-11 N62S Humanized and Human IgG2 Constant Region (SEQ ID NO: 192) plus the Sh24C05 Kv1-16 Light Chain Variable Region and the Human Cover Constant Region ) (SEQ ID NO: 204)
[000192] Sh24C05-31 N62S IgG1 = Heavy Chain Variable Region Sh24C05 Hv3-11 N62S Humanized and Human IgG1 Constant Region (SEQ ID NO: 190) plus the Sh24C05 Kv1-17 Light Chain Variable Region and the Human Cover Constant Region ) (SEQ ID NO: 206)
[000193] Sh24C05-31 N62S IgG2 = Heavy Chain Variable Region Sh24C05 Hv3-11 N62S Humanized and Human IgG2 Constant Region (SEQ ID NO: 192) plus the Sh24C05 Kv1-17 Light Chain Variable Region and the Human Cover Constant Region ) (SEQ ID NO: 206) B. Binding Affinities of Humanized and Chimeric AntiErb B3 Monoclonal Antibodies
[000194] The binding affinities and interaction kinetics of the monoclonal antibodies produced in Example 12 against the recombinant human monomeric protein ErbB3 (cleaved rhErbB3) were measured by surface plasmon resonance using a Biacore® T100 (Biacore) equipment. Monomeric ErbB3 was obtained by cleavage with rhErbB3-Fc proteases (R&D Systems, Cat. No. 348-RB).
[000195] A goat anti-human IgG Fc (Jackson ImmunoResearch, Catalog Number 109-005-098) was immobilized on CM4 carboxymethylated dextran sensor chips (Biacore, Catalog NO BR-1005-34) by amine coupling (Biacore, Catalog NO BR-1000-50) using a standardized coupling protocol according to the trader's instructions. Analyzes were performed at 37 ° C using PBS (Invitrogen, Catalog NO 14040-133) containing 0.05% P20 surfactant (Biacore, Catalog NO BR-1000-54) as running buffer.
[000196] Antibodies were captured in individual flow cells at a flow rate of 60 μL / minute. The injection time was varied for each antibody in order to yield an Rmax between 30 and 60 RU. The buffer or cleaved rhErbB3 diluted in running buffer was injected sequentially across a reference surface (without captured antibodies) and the active surface (antibody to be tested) for 300 seconds at 60 μL / minute. The dissociation phase was monitored for more than 1,200 seconds. The surface was then regenerated with two 60-second injections of glycine pH 2.25 (made of glycine pH 2.0 (Biacore, NO from Catalog BR-1003-55) and pH 2.5 (Biacore, NO from Catalog BR- 1003-56)) at 60 μL / minute. For the initial screening, only one or two concentrations of rhErbB3 were tested, typically 5.0 and 1.25 nM (the results are summarized in Table 15).
[000197] The kinetic parameters were determined using the kinetic function of the BIAevaluation software (Biacore) with double reference subtraction. The kinetic parameters for each antibody, ka (constant rate of association), kd (constant rate of dissociation) and KD (constant dissociation at equilibrium) were determined. The initial monoclonal antibodies were screened using the cell culture medium supernatant containing secreted antibodies and kinetic values of monoclonal antibodies in rhErbB3 cleaved at 37 ° C are summarized in Table 15. Table 15



[000198] The results in Table 15 demonstrate that the chimeric antibody and each of the humanized antibodies 24C05 have fast association rates (ka), very slow dissociation rates (kd) and very high affinities (KD). In particular, antibodies have affinities ranging from about 87 pM to about 1 nM.
[000199] The binding affinities and kinetics of certain purified monoclonal antibodies have also been determined. To better characterize certain antibodies, the surface plasmon resonance assays described above were conducted using concentrations of rhErbB3 cleaved between 0.3125 nM and 5.0 nM (a 2X serial dilution).
[000200] 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) in rhErbB3 cleaved at 37 ° C are summarized in Table 16. Table 16

[000201] The results shown in Table 16 demonstrate that the purified antibodies have affinities ranging from about 63 pM to about 160 pM when tested at 37 ° C. C. Comparison of other Anti-ErbB3 Antibodies
[000202] Three human antibodies that inhibit human ErbB3 function have been constructed and expressed using the published information. An antibody, referred to as Ab # 6, was constructed as a human IgG2 / Lambda antibody based on the disclosure by 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 / Capa antibodies based on the disclosure by Rothe et al., US 2008/0124345 (U3 Pharma AG and Amgen Inc.).
[000203] The kinetic parameters for 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. Monoclonal Antibody Binding Affinities) -ErbB3 Humanized and Chimeric). Both Biacore sensograms (figure 17) and kinetic values (Table 17) are shown for each antibody. Table 17

[000204] The results in Table 17 demonstrate that the global dissociation constant at equilibrium (KD) for Sh24C05-31 N62S IgG1 (76 pM) was lower (ie, greater affinity) than KD for Ab # 6 antibodies and U1-59 (230 pmM (p <0.01) and 530 pM (p <0.0005), respectively). The equilibrium dissociation constant (KD) for U1-53 could not be determined because the curve adjustment is insufficient (see figure 17, which has a faster Koff speed than U1-53). The KD of antibodies Ab # 6, U1-53 and U1-59 can also be compared with other humanized 24C05 variants, comparing Tables 16 and 17.
[000205] Therefore, the affinity of IgG1 Sh24C05-31 N62S is significantly greater than the affinity of Ab # 6 and U1-59 as disclosed herein. Example 13 - Neutralization Activity of Humanized Anti-ErbB3 Antibodies
[000206] In this example, the humanized antibodies produced in Example 12 were tested for their ability to inhibit the binding of rhErbB3 to NRG1-β1 by an ECL assay. Standardized 96-well multi-array connection plates (Meso Scale Discovery, Cat. No. L15XA-6) were coated with 50 μL of rhErbB3 / Fc 0.5 μg / mL (R&D systems, Cat. No. 348-RB) in PBS (Invitrogen, Cat. NO 14040-133) for one hour at room temperature, without stirring. The plates were then washed three times with PBS + 0.1% Tween 20 (Sigma P5927) and blocked with 200 μL of 100% Horse Whey, inactivated by heat (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 stirring. The NRG1-β1 ligand (R&D Systems, Cat. No. 377-HB, 26kDa) was added to the wells at the final concentration of 0.25 μg / mL. The plates were washed three times with PBS + 0.1% Tween and incubated with 25 μL of biotinylated antibody 1 μg / mL against human NRG1-β1 (R&D systems, Cat. NO BAF377) preincubated for one hour with streptavidin SULTO- TAG (Meso Scale Discovery, Cat. NO R32AD-5) for one hour at room temperature, with stirring. The plates were then washed three times with PBS + 0.1% Tween and 150 μL of 1X reading buffer (Meso Scale Discovery, Cat. NO R92TC-1) were added to each well before the plates were analyzed in Sector® equipment Imager 2400 (Meso Scale Discovery).
[000207] The interaction of NRG1-β1 with rhErbB3 was inhibited by the antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 (figure 18A). The Ab # 6 IgG2 antibody as described in Schoeberl et al. (above) and antibodies U1-53 and U1-59 as described in Rothe et al. (above) were also tested for their ability to inhibit the binding of ErbB3 to NRG1-β1. As shown in figure 18B, each of the Ab # 6 IgG2, U1-53, and U1-59 antibodies inhibited ErbB3 binding to NRG1-β1.
[000208] IC50 values for neutralizing the binding of NRG1-β1 to hErbB3 for humanized antibodies 24C05 (ie, 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. IC50 values for the NRG1-β1 neutralizing activity of human anti-ErbB3 Ab # 6 IgG2, U1-53 and U1-59 antibodies are also shown in Table 18. Table 18

[000209] The results shown in Table 18 demonstrate that the antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 effectively neutralized the binding of NRG1-β1 to rhErbB3. While human anti-ErbB3 Ab # 6 IgG2, U1-53 and U1-59 antibodies also showed neutralizing activity, humanized antibodies Sh24C05 (ie, Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2) had a higher neutralization capacity than U1-59 or Ab # 6 IgG2. Example 14 - Antiproliferative Activity
[000210] 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 designed to express both human Her2 and ErbB3.
[000211] BaF / 3 cells expressing Her2 and ErbB3 receptors are described in Example 6 as having been treated with anti-ErbB3 antibodies in the absence of WEHI-restricted means but in the presence of NRG1-β1 (100 ng / ml). The assays were conducted on 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 a final volume of 100 μL). MTT assays (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenylthetrazolium bromide) were conducted 3-4 days after NRG1-β1 stimulation.
[000212] The results demonstrate that Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 inhibited NRG-induced cell proliferation in Her2 / ErbB3-BaF cells / 3 in a dose-dependent manner (figure 19A).
[000213] IC50 values for the inhibition of NRG1-β1-dependent proliferation of the Her2 / ErbB3-BaF / 3 cell line with humanized antibodies 24C05 (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

[000214] The results in Table 19 demonstrate that the antibodies Sh24C05-25 N62S-IgG1, Sh24C05-25 N62S-IgG2, Sh24C05-31 N62S-IgG1, and Sh24C05-31 N62S-IgG2 strongly inhibited the proliferation induced by NRG1-β1 of BaF / 3 cells, expressing Her2 / ErbB3.
[000215] The inhibitory activities of IgG2 Ab # 6 antibodies anti-ErbB3, U1-53 and U1-59 were also tested in the NRG1-β1 dependent Her2 / ErbB3-BaF / 3 cell proliferation assay. As shown in figure 19B, the results demonstrate that the antibodies Ab # 6 IgG2, U1-53 and U1-59 inhibited NRG-induced cell proliferation in Her2 / ErbB3-BaF / 3 cells in a dose dependent manner. The proliferation inhibition data for the NRG1-β1-dependent Her2 / ErbB3-BaF / 3 cell line with monoclonal antibodies Ab # 6 IgG2, U1-53 and U1-59, are summarized in Table 19. The results in Table 19 demonstrate that Ab # 6 IgG2, U1-53 and U1-59 antibodies 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 proliferation assay of Her2 / ErbB3-BaF / 3 cells indicates that the inhibitory activity of humanized Sh24C05 antibodies is superior to the inhibitory activity of Ab # 6 antibodies IgG2, U1-53 and U1-59 antibodies (for example, 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
[000216] This example describes a characterization of the humanized antibodies produced in Example 12 with respect to their ability to degrade total ErbB3 and inhibit phosphorylation of ErbB3 in the exponential growth phase of SKBR-3 cells.
[000217] SKBR-3 breast cancer cells were maintained as recommended by the ATCC. The cells maintained in full with serum 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 analyzed by ELISA with Total-ErbB3 and the Phospho-ErbB3 set from R&D Systems (Cat. NO DYC234 and Cat. NO DYC1769, respectively).
[000218] 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 ErbB3 phosphorylation in exponential growth phase of SKBR-3 cells (figure 20).
[000219] The results also demonstrate that the 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 in the exponential growth phase of SKBR-3 cells (figure 21). Example 16 - BxPC3 Tumor Xenograft Growth Inhibition
[000220] The ability of the humanized monoclonal antibodies produced in Example 12 to inhibit tumor growth was tested in a pancreatic BxPC3 xenograft model. BxPC3 human pancreatic cells were cultured in a culture medium at 37 ° C in an atmosphere containing 5% CO2, using the RMPI medium containing 10% fetal bovine serum. BxPC3 cells were inoculated subcutaneously, on the flank of a CB.17 female SCID rat from 8 weeks of age (Taconic Labs) with 10 x 10 6 cells per rat in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were performed twice a week using a caliper. The tumor volume was calculated using the formula: width x width x length / 2. When the tumors reached approximately 200 mm3, the rats were randomly assigned to 8 groups of 10 rats 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 antibodies were dosed at 2 mg / kg of body weight, twice a week, by intraperitoneal injection for 7 weeks. Tumor volumes and rat body weights were recorded twice a week. The inhibition of tumor growth was analyzed using ANOVA and is expressed as a percentage of inhibition compared to the PBS control.
[000221] The humanized antibodies tested remained 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% inhibition of tumor growth (p <0.001) (ie, 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 (figure 22). The murine antibody demonstrated 65% inhibition of tumor growth in the present study (p <0.05). These results suggest similar potency and activity of the four humanized antibodies in this model.
[000222] The capabilities of 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 rats. When the tumors reached approximately 200 mm3, the rats were randomly assigned to 11 groups of 10 rats 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). All antibodies were assayed at 0.5 mg / kg, 1 mg / kg, or 5 mg / kg of body weight, twice a week, by intraperitoneal injection for 7 weeks. Tumor volumes and rat body weights were recorded twice a week. The inhibition of tumor growth was analyzed using ANOVA and is expressed as a percentage of inhibition compared to the PBS control.
[000223] The tumor growth inhibition data determined on day 29 after 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


[000224] The results demonstrate that Sh24C05-31 N62S-IgG1 showed the greatest inhibition of tumor growth at day 29 (76.5%, p <0.001) for a dose of 5 mg / kg in the BxPC3 pancreatic xenograft model. Antibodies U1-59 and Ab # 6 IgG2 demonstrated approximately 60% and 41% inhibition of tumor growth at a dose of 5 mg / kg in the BxPC3 model, respectively (P <0.001).
[000225] The results also demonstrate that Sh24C05-31 N62S-IgG1 showed the greatest inhibition of tumor growth at day 29 for a dose of 0.5 mg / kg and (63.3%, p <0.001) for a dose of 1 mg / kg (75.0%, p <0.001) in the BxPC3 pancreatic xenograft model. The antibodies U1-59 and Ab # 6 IgG2 demonstrated approximately 33% (p <0.01) and 31% (p <0.05) of tumor growth inhibition at a dose of 0.5 mg / kg in the BxPC3 model, respectively. The antibodies U1-59 and Ab # 6 IgG2 demonstrated approximately 53% (p <0.001) and 2% (not significant) of tumor growth inhibition at a dose of 1.0 mg / kg in the BxPC3 model, respectively. Example 17 - Calu-3 Tumor Xenograft Growth Inhibition
[000226] The ability of the humanized monoclonal antibodies produced in Example 12 to inhibit tumor growth was tested in a non-small cell lung cancer Calu-3 xenograft model. The ability of 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.
[000227] Human non-small cell lung cancer Calu-3 cells were cultured in culture medium at 37 ° C in an atmosphere containing 5% CO2 using EMEM medium containing 10% fetal bovine serum . Calu-3 cells were inoculated subcutaneously, on the flank of an 8 week old NCR female rat (Taconic Labs) with 10 x 10 6 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were performed twice a week using a caliper. The tumor volume was calculated using the formula: width x width x length / 2.
[000228] When the tumors reached approximately 200 mm3, the rats were randomly assigned to 11 groups of 10 rats each. One group received PBS and another received muIgG control. Each of the other nine groups received one of the humanized antibodies (ie, Sh24C05-31 N62S-IgG1, U1-59, or Ab # 6 IgG2) at a dose of 5 mg / kg, 10 mg / kg or 20 mg / kg of body weight, twice a week, by intraperitoneal injection for 4 weeks. Tumor volumes and rat body weights were recorded twice a week. The inhibition of tumor growth was analyzed using ANOVA and is expressed as a percentage of inhibition compared to the PBS control.
[000229] The tumor growth inhibition data determined on day 26 after treatment with one of the humanized antibodies (ie., Sh24C05-31 N62S-IgG1, U1-59, or Ab # 6 IgG2) is shown in Table 21. Table 21


[000230] Results using the Calu-3 xenograft model of non-small cell lung cancer demonstrate that Sh24C05-31 N62S-IgG1 showed the greatest inhibition of tumor growth at day 26 for all tested doses (ie, 5 mg / kg, 10mg / kg and 20 mg / kg of body weight).
[000231] For example, for the 10mg / kg dose, Sh24C05-31 N62S-IgG1 showed the greatest inhibition of tumor growth at day 26 (62%, P <0.001) when compared to Ab # 6 IgG2 (36% , NS) or U1-59 (57%, P <0.001). For the dose of 20 mg / kg, Sh24C05-31 N62S-IgG1 showed the greatest inhibition of tumor growth at 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 - MDA-MB-453 Tumor Xenograft Growth Inhibition
[000232] The ability of the humanized monoclonal antibodies produced in Example 12 to inhibit tumor growth was tested in an MDA-MB-453 breast cancer xenograft model (which is an HER2 positive breast cancer model). The ability of 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.
[000233] Human breast cancer MDA-MB-453 cells were cultured in culture medium at 37 ° C in an atmosphere containing 0% CO2, using Leibovitz ATCC medium (NO Cat. 30-2008) containing 10% fetal bovine serum. MDA-MB-453 cells were inoculated subcutaneously, on the flank of an 8 week old NOD SCID female rat (Taconic Labs) with 20 x 10 6 cells per rat in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were performed twice a week using a caliper. The tumor volume was calculated using the formula: width x width x length / 2.
[000234] When the tumors reached approximately 200 mm3, the rats were randomly assigned to 7 groups of 10 rats 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). 24C05-31 N62S-IgG1 was dosed at either 5 mg / kg, 10 mg / kg or 20 mg / kg of body weight, twice a week, by intraperitoneal injection for more than 10 weeks; U1-59, or Ab # 6 were dosed at 10 mg / kg with the same frequency. Tumor volumes and rat body weights were recorded twice a week. The inhibition of tumor growth was analyzed using ANOVA and is expressed as a percentage of inhibition compared to the PBS control.
[000235] The tumor growth inhibition data determined on day 71 after 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


[000236] The results using the MDA-MB-453 xenograft model demonstrate that the Sh24C05-31 N62S-IgG1 showed a potent inhibition of tumor growth at day 71 for all tested doses (ie, 5 mg / kg, 10 mg / kg) kg and 20 mg / kg body weight).
[000237] The results also demonstrated that for the 10mg / kg dose, Sh24C05-31 N62S-IgG1 showed the greatest inhibition of tumor growth at day 71 (84%, P <0.001) when compared to Ab # 6 IgG2 ( 62%, P <0.001). Sh24C05-31 N62S-IgG1 showed a tumor growth inhibition equivalent to that of U1-59 for the same dose. INCORPORATION BY REFERENCE
[000238] The complete disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. EQUIVALENTS
[000239] The invention can be carried out in other specific forms without leaving them outside its scope or its essential characteristics. The foregoing embodiments should therefore be considered in all respects as illustrative rather than limiting the invention described in this document. The scope of the invention is thus indicated by the appended claims rather than by the preceding description, and any changes that are provided for in the meaning and scope of the equivalence of the claims are intended to be encompassed therein.

权利要求:
Claims (20)
[0001]
1. Isolated antibody that binds to human ErbB3, characterized by the fact that it comprises: (i) an immunoglobulin heavy chain variable region comprising a CDRH1 consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 57 and SEQ ID NO: 75; a CDRH2 consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 58 and SEQ ID NO: 148, and a CDRH3 consisting of the amino acid sequence of SEQ ID NO: 59; and (ii) an immunoglobulin light chain variable region comprising a CDRL1 consisting of the amino acid sequence of SEQ ID NO: 60, a CDRL2 consisting of the amino acid sequence of SEQ ID NO: 61 and a CDRL3 consisting of the amino acid sequence of SEQ ID NO: 62.
[0002]
2. Antibody according to claim 1, characterized in that the immunoglobulin heavy chain variable region comprises a CDRH1 consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 75, a CDRH2 consisting of the amino acid sequence of SEQ ID NO: 148, and a CDRH3 consisting of the amino acid sequence of SEQ ID NO: 59; and wherein the immunoglobulin light chain variable region comprises a CDRL1 consisting of the amino acid sequence of SEQ ID NO: 60, a CDRL2 consisting of the amino acid sequence of SEQ ID NO: 61 and a CDRL3 consisting of the amino acid sequence of SEQ ID NO: 62.
[0003]
3. Antibody according to claim 1, characterized by the fact that the immunoglobulin heavy chain variable region comprises a CDRH1 consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 75, a CDRH2 consisting of the amino acid sequence of SEQ ID NO: 58, and a CDRH3 consisting of the amino acid sequence of SEQ ID NO: 59; and wherein the immunoglobulin light chain variable region comprises a CDRL1 consisting of the amino acid sequence of SEQ ID NO: 60, a CDRL2 consisting of the amino acid sequence of SEQ ID NO: 61 and a CDRL3 consisting of the amino acid sequence of SEQ ID NO: 62.
[0004]
Antibody according to any one of claims 1 to 3, characterized by the fact that the CDR sequences are interposed between human or humanized framework sequences.
[0005]
5. Isolated antibody that binds to human ErbB3, characterized by the fact that it comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, selected from a group consisting of: (a) a variable region an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 154, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 168; and (b) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 154, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 166.
[0006]
Antibody according to claim 5, characterized in that the immunoglobulin heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 154, and the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO: 168.
[0007]
Antibody according to claim 2, characterized in that it comprises an immunoglobulin heavy chain and an immunoglobulin light chain, selected from a group consisting of: (a) an immunoglobulin heavy chain comprising the amino acid sequence SEQ ID NO: 190, and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 206; and (b) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 190, and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 204.
[0008]
Antibody according to claim 7, characterized in that the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO: 190, and the immunoglobulin light chain comprises the amino acid sequence of SEQ ID NO: 206.
[0009]
9. Antibody according to claim 3, characterized by the fact that it comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, selected from a group consisting of: (a) a variable region of immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 152, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 168; and (b) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 152, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 166.
[0010]
Antibody according to claim 3, characterized in that it comprises an immunoglobulin heavy chain and an immunoglobulin light chain, selected from a group consisting of: (a) an immunoglobulin heavy chain comprising the amino acid sequence SEQ ID NO: 188, and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 206; and (b) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 188, and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 204.
[0011]
Antibody according to any one of claims 1 to 10, characterized in that the antibody is a monoclonal antibody or an antigen-binding fragment thereof.
[0012]
Antibody according to any one of claims 1 to 11, characterized by the fact that the antibody has a KD of 200 pM or less, when measured by surface plasmon resonance.
[0013]
13. Use of an effective amount of the antibody, as defined in any one of claims 1 to 12, characterized by the fact that it is for the preparation of a pharmaceutical composition to inhibit or reduce the proliferation of a tumor cell.
[0014]
14. Use of an effective amount of the antibody, as defined in any one of claims 1 to 12, characterized by the fact that it is for the preparation of a pharmaceutical composition to inhibit or reduce tumor growth in a mammal.
[0015]
15. Use of an effective amount of the antibody, as defined in any of claims 1 to 12, characterized by the fact that it is for the preparation of a pharmaceutical composition for treating cancer in a mammal.
[0016]
16. Use according to claim 15, characterized by the fact that the cancer is selected from the group consisting of cancers of the breast, ovary, prostate, cervical, colorectal, lung, pancreas, stomach, skin, kidney, and head and neck and schwannoma.
[0017]
17. Expression vector, characterized by the fact that it comprises a nucleotide sequence that encodes an immunoglobulin heavy chain variable region and a nucleotide sequence that encodes an immunoglobulin light chain variable region selected from the group consisting of: (a ) a nucleotide sequence that encodes a variable region of the immunoglobulin heavy chain comprising the nucleotide sequence of SEQ ID NO: 153, and a nucleotide sequence that encodes a variable region of the immunoglobulin light chain comprising the nucleotide sequence of SEQ ID NO: 167; (b) a nucleotide sequence encoding an immunoglobulin heavy chain variable region comprising the nucleotide sequence of SEQ ID NO: 153, and a nucleotide sequence encoding an immunoglobulin light chain variable region comprising the nucleotide sequence of SEQ ID NO: 165; (c) a nucleotide sequence that encodes an immunoglobulin heavy chain variable region comprising the nucleotide sequence of SEQ ID NO: 151, and a nucleotide sequence that encodes an immunoglobulin light chain variable region comprising the nucleotide sequence of SEQ ID NO: 165; and (d) a nucleotide sequence that encodes an immunoglobulin heavy chain variable region comprising the nucleotide sequence of SEQ ID NO: 151, and a nucleotide sequence that encodes an immunoglobulin light chain variable region comprising the nucleotide sequence SEQ ID NO: 167.
[0018]
18. Expression vector, characterized by the fact that it comprises a nucleotide sequence that encodes an immunoglobulin heavy chain and a nucleotide sequence that encodes an immunoglobulin light chain selected from the group consisting of: (a) a nucleotide sequence that encodes an immunoglobulin heavy chain comprising the nucleotide sequence of SEQ ID NO: 189, and a nucleotide sequence encoding an immunoglobulin light chain comprising the nucleotide sequence of SEQ ID NO: 203; (b) a nucleotide sequence that encodes an immunoglobulin heavy chain comprising the nucleotide sequence of SEQ ID NO: 189, and a nucleotide sequence that encodes an immunoglobulin light chain comprising the nucleotide sequence of SEQ ID NO: 205; (c) a nucleotide sequence encoding an immunoglobulin heavy chain comprising the nucleotide sequence of SEQ ID NO: 187, and a nucleotide sequence encoding an immunoglobulin light chain comprising the nucleotide sequence of SEQ ID NO: 203; and (d) a nucleotide sequence encoding an immunoglobulin heavy chain comprising the nucleotide sequence of SEQ ID NO: 187, and a nucleotide sequence encoding an immunoglobulin light chain comprising the nucleotide sequence of SEQ ID NO: 205 .
[0019]
19. Process of producing an antibody that binds to human ErbB3 or an antigen-binding fragment of the antibody, characterized by the fact that it comprises: (a) cultivating a host cell comprising an expression vector comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable region as defined in any of claims 1 to 10 and a nucleotide sequence encoding an immunoglobulin light chain variable region of any of claims 1 to 10 under conditions where the host cell expresses the polypeptide comprising the variable region of the immunoglobulin heavy chain and / or a polypeptide comprising the variable region of the immunoglobulin light chain, thereby producing the antibody or antibody fragment for binding to the antigen; and (b) purifying the antibody or antigen-binding fragment of the antibody.
[0020]
20. Method for producing an antibody that binds human ErbB3 or an antigen-binding fragment of the antibody, characterized in that it comprises: (a) cultivating a host cell comprising (i) an expression vector comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable region as defined in any one of claims 1 to 10 and (ii) an expression vector comprising a nucleotide sequence encoding an immunoglobulin light chain variable region, as defined in any of the claims 1 to 10, under conditions under which the host cell expresses a polypeptide comprising the variable region of the immunoglobulin heavy chain and a polypeptide comprising the variable region of the immunoglobulin light chain, thereby producing the antibody or antigen binding fragment of the antibody ; and (b) purifying the antibody or antigen-binding fragment of the antibody.

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KR20200045520A|2017-09-07|2020-05-04|오거스타 유니버시티 리서치 인스티튜트, 인크.|Antibodies to programmed cell death protein 1|

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TW201916890A|2017-10-17|2019-05-01|大陸商江蘇恆瑞醫藥股份有限公司|Combination use of anti-PD-1 antibody and anti-LAG-3 antibody in the preparation of a medicament for the treatment of tumor|

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GB201804094D0|2018-03-14|2018-04-25|Ultrahuman Thirteen Ltd|ERBB3 Binding agents|

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CN112912109A|2018-09-20|2021-06-04|第一三共株式会社|Treatment of HER 3-mutated cancer by administration of anti-HER 3 antibody-drug conjugates|

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WO2020163646A1|2019-02-08|2020-08-13|Igm Biosciences, Inc.|Anti-gitr antigen-binding domains and uses thereof|

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WO2020214957A1|2019-04-19|2020-10-22|Tcrcure Biopharma Corp.|Anti-pd-1 antibodies and uses thereof|

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法律状态:
2017-02-07| B08F| Application fees: dismissal - article 86 of industrial property law|

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2017-05-30| B08H| Application fees: decision cancelled|

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2017-07-04| B15V| Prolongation of time limit allowed|

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2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-07-02| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-03-17| B07A| Technical examination (opinion): publication of technical examination (opinion)|

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2020-08-11| B09A| Decision: intention to grant|

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2020-12-08| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 08/04/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US32271210P| true| 2010-04-09|2010-04-09|

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US61/322,712|2010-04-09|

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PCT/US2011/031829|WO2011136911A2|2010-04-09|2011-04-08|Anti-erbb3 antibodies|

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