CN104093742A - Anti-HER3 domain III and domain IV epidermal growth factor receptor 3 (HER3) antibody - Google Patents

Abstract

The present invention relates to antibodies or fragments thereof that bind to a nonlinear epitope within domain 3 of the HER3 receptor and inhibit both ligand-dependent and ligand-independent signal transduction. The present invention also relates to antibodies or fragments thereof that bind to amino acid residues within domains 3-4 of HER3 and inhibit both ligand-dependent and ligand-independent signal transduction; and compositions of such antibodies or fragments thereof and methods of using such antibodies or fragments thereof.

Description

Anti-HER3 domain III and domain IV epidermal growth factor receptor 3 (HER3) antibody

related application

This application claims priority to US Provisional Application No. 61/566,912, filed December 5, 2011, the contents of which are hereby incorporated by reference in their entirety.

technical field

The present invention relates to antibodies or fragments thereof that bind to a non-linear epitope within domain 3 of the HER3 receptor and inhibit both ligand-dependent and ligand-independent signal transduction. The invention also relates to antibodies or fragments thereof that bind to amino acid residues within domains 3-4 of HER3 and inhibit both ligand-dependent and ligand-independent signal transduction; and combinations of such antibodies or fragments thereof and methods of using such antibodies or fragments thereof.

Background technique

Human epidermal growth factor receptor 3 (ErbB3, also known as HER3) is a receptor protein tyrosine kinase and belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes EGFR ( HER1, ErbB1), HER2 (ErbB2, Neu) and HER4 (ErbB4) (Plowman et al., (1990) Proc. Natl. Acad. Sci. U.S.A. 87:4905-4909; Kraus et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86:9193-9197; and Kraus et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90:2900-2904). Like the prototypical EGFR, the transmembrane receptor HER3 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, and an intracellular protein tyrosine kinase-like domain (TKD) and C-terminal phosphorylation domain. Unlike other HER family members, the kinase domain of HER3 displays very low intrinsic kinase activity.

The ligands neuregulin 1 (NRG) or neuregulin 2 bind the extracellular domain of HER3 and activate receptor-mediated signaling pathways by promoting dimerization with other dimerization partners such as HER2 . Heterodimerization leads to activation and transphosphorylation of the intracellular domain of HER3, which is a means not only of signal diversification but also of signal amplification. In addition, HER3 heterodimerization can also occur in the absence of activating ligands, which is often referred to as ligand-independent HER3 activation. For example, when HER2 is expressed at high levels due to gene amplification (eg, in breast, lung, ovarian, or gastric cancer), spontaneous HER2/HER3 dimers can form. In this context, HER2/HER3 is believed to be the most active ErbB signaling dimer, and thus highly transformed.

Increased HER3 has been found in several cancer types, for example in breast, lung, gastrointestinal and pancreatic cancers. Interestingly, a correlation has been shown between expression of HER2/HER3 and progression from non-invasive to invasive stages (Alimandi et al., (1995) Oncogene 10:1813-1821; DeFazio et al., (2000) Cancer 87: 487-498; Naidu et al. (1988) Br. J. Cancer 78:1385-1390). Accordingly, there is a need for drugs that interfere with HER3-mediated signaling.

Summary of the invention

The present invention is based on the ability to bind a non-linear epitope of the HER3 receptor comprising amino acid residues within domain 3 of HER3 and block both ligand-dependent (eg neuregulin) and ligand-independent HER3 signaling pathways The surprising discovery of antibodies or fragments thereof. The invention is also based on the discovery of antibodies or fragments thereof that bind to amino acid residues within domains 3-4 of HER3 and block both ligand-dependent (eg neuregulin) and ligand-independent HER3 signaling pathways.

Accordingly, in one aspect, the invention relates to an isolated antibody or fragment thereof that recognizes a non-linear epitope of the HER3 receptor, wherein the non-linear epitope comprises amino acid residues within domain 3 of the HER3 receptor, wherein the antibody or A fragment thereof binds to binding surface B, and wherein the antibody or fragment thereof blocks both ligand-dependent and ligand-independent signal transduction.

In one embodiment, binding surface B comprises at least one amino acid residue selected from amino acid residues 335-342, 398, 400, 424-428, 431, 433-434 and 455. In another embodiment, the antibody or fragment thereof is further bound to binding surface A. In one embodiment, binding surface A comprises at least one amino acid residue selected from amino acid residues 362-376.

In another aspect, the invention relates to an isolated antibody or fragment thereof that recognizes a non-linear epitope of the HER3 receptor, wherein the non-linear epitope comprises amino acid residues within domain 3 of the HER3 receptor, wherein the antibody or The fragment binds to a binding surface comprising at least one amino acid residue selected from binding surface A and at least one amino acid residue selected from binding surface B, and wherein the antibody or fragment thereof blocks ligand-dependent and ligand-independent Signal transduction both.

In one embodiment, the antibody or fragment thereof blocks binding of a HER3 ligand on the HER3 receptor. In one embodiment, the HER3 ligand is selected from neuregulin 1 (NRG), neuregulin 2, beta animal cellulose, heparin-binding epidermal growth factor and epiregulin. In one embodiment, the antibody or fragment thereof has any one of the following characteristics: binding to the HER3 receptor in an inactive state, preventing HER3 adopts the active conformation, prevents HER3 from adopting the active conformation by reducing the degree of flexibility in domain 3, induces a conformational change in residues 371-377 of domain 3 that prevents HER3 from adopting the active conformation, destabilizes HER3 making it vulnerable Degradation, accelerated downregulation of cell surface HER3, and generation of non-native HER3 dimers that are susceptible to proteolytic degradation or unable to dimerize with other receptor tyrosine kinases. In one embodiment, binding surface A comprises amino acid residues 362-376. In one embodiment, binding surface B comprises amino acid residues 335-342, 398, 400, 424-428, 431, 433-434, and 455.

In one embodiment, the non-linear epitope comprises amino acid residues 335-342, 362-376, 398, 400, 424-428, 431, 433-434, and 455 (within domain 3), or a subset thereof. In one embodiment, the VH of the antibody or fragment thereof binds at least one of the following HER3 residues: Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400, and Lys434. In one embodiment, the VL of the antibody or fragment thereof binds at least one of the following HER3 residues: Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400 , Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455. In one embodiment, binding of the antibody or fragment thereof to the HER3 receptor in the absence of a HER3 ligand reduces ligand-independent formation of a HER2-HER3 protein complex in cells expressing HER2 and HER3. In one embodiment, the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a HER3 ligand-independent phosphorylation assay. In one embodiment, the HER3 ligand-independent phosphorylation assay uses HER2-amplified cells, wherein the HER2-amplified cells are SK-Br-3 cells and BT-474. In one embodiment, binding of the antibody or fragment thereof to the HER3 receptor in the presence of a HER3 ligand reduces ligand-dependent formation of a HER2-HER3 protein complex in cells expressing HER2 and HER3. In one embodiment, the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a HER3 ligand-dependent phosphorylation assay. In one embodiment, the HER3 ligand-dependent phosphorylation assay uses stimulated MCF7 cells in the presence of neuregulin (NRG). In one embodiment, the antibody is selected from monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies and synthetic antibodies.

In another aspect, the invention relates to an isolated antibody or fragment thereof that recognizes an epitope of the HER3 receptor, wherein the epitope comprises amino acid residues within domains 3-4 of the HER3 receptor, and wherein the antibody or fragment thereof Blocks both ligand-dependent and ligand-independent signal transduction.

In one embodiment, the epitope comprises at least one amino acid residue selected from amino acid residues 329-498 (domain 3) of SEQ ID NO:1, and at least one amino acid residue selected from SEQ ID NO:1 Amino acid residues 499-642 (domain 4). In one embodiment, the epitope comprising amino acid residues within domains 3-4 is selected from a linear epitope, a non-linear epitope and a conformational epitope. In one embodiment, binding of the antibody or fragment thereof to the HER3 receptor in the absence of a HER3 ligand reduces ligand-independent formation of a HER2-HER3 protein complex in cells expressing HER2 and HER3. In one embodiment, the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a HER3 ligand-independent phosphorylation assay. In one embodiment, the HER3 ligand-independent phosphorylation assay uses HER2-amplified cells, wherein the HER2-amplified cells are SK-Br-3 cells and BT-474. In one embodiment, binding of the antibody or fragment thereof to the HER3 receptor in the presence of a HER3 ligand reduces ligand-dependent formation of a HER2-HER3 protein complex in cells expressing HER2 and HER3. In one embodiment, the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a HER3 ligand-dependent phosphorylation assay. In one embodiment, the HER3 ligand-dependent phosphorylation assay uses stimulated MCF7 cells in the presence of neuregulin (NRG).

In another aspect, the present invention relates to an isolated antibody or fragment thereof against the HER3 receptor having at least 1 x 10 ⁷ M ⁻¹ , 10 ⁸ M ⁻¹ , 10 ⁹ M ⁻¹ , 10 ¹⁰ M ⁻¹ , 10 ¹¹ M Dissociation (K _D ) of ^-1 , 10 ¹² M ⁻¹ , 10 ¹³ M ⁻¹ , wherein the antibody or fragment thereof blocks both ligand-dependent and ligand-independent signal transduction. In one embodiment, the antibody or fragment thereof inhibits phosphorylation of HER3 as measured by an in vitro phosphorylation assay selected from phospho-HER3 and phospho-Akt.

In another aspect, the invention relates to an isolated antibody or fragment thereof that binds the same non-linear epitope within domain 3 of HER3 as the antibody described in Table 1 .

In another aspect, the invention relates to an isolated antibody or fragment thereof that binds to the same amino acid residues within domains 3-4 of HER3 as the antibodies described in Table 2.

In another aspect, the present invention relates to a HER3-binding antibody fragment selected from the group consisting of Fab, F(ab ₂ )', F(ab) ₂ ', scFv, VHH, VH, VL, dAb, wherein the antibody fragment blocks Both ligand-dependent and ligand-independent signal transduction.

In another aspect, the invention relates to a pharmaceutical composition comprising an antibody or fragment thereof and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises an additional therapeutic agent. In one embodiment, the additional therapeutic agent is selected from a HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an mTOR inhibitor, and a PI3 kinase inhibitor. In one embodiment, the additional therapeutic agent is selected from Matuzumab (EMD72000), Cetuximab, Panitumumab, mAb 806, Nimotuzumab, Gefitinib, CI-1033 (PD183805), Lapatinib (GW-572016), Lapatinib Ditosylate, Erlotinib Hydrochloride (ErlotinibHCL) (OSI-774), PKI-166 and HER1 inhibitors; selected from Pertuzumab, Trastuzumab, MM-111, neratinib, lapatinib, or lapatinib ditosylate HER2 inhibitors; selected from MM-121, MM-111, IB4C3, 2DID12 (U3Pharma AG), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech), MOR10703 (Novartis) and small molecules that inhibit HER3 HER3 inhibitors; and HER4 inhibitors. In one embodiment, the additional therapeutic agent is a HER3 inhibitor, wherein the HER3 inhibitor is MOR10703. In one embodiment, the additional therapeutic agent is selected from Temsirolimus (Temsirolimus) ridaforolimus/rapamycin (Deforolimus), AP23573, MK8669, everolimus (everolimus) mTOR inhibitors. In one embodiment, the additional therapeutic agent is a PI3 kinase inhibitor selected from GDC 0941, BEZ235, BKM120 and BYL719.

In another aspect, the present invention relates to a method of treating cancer comprising selecting an individual suffering from a HER3 expressing cancer, administering to the individual an effective amount of a composition comprising an antibody or fragment thereof disclosed in Table 1 or Table 2. In one embodiment, the individual is human and the cancer is selected from breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, acute myelogenous leukemia, chronic myelogenous leukemia, osteosarcoma, Squamous cell carcinoma, peripheral nerve sheath tumor, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barretts esophagus cancer, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer , melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, and endometriosis. In one embodiment, the cancer is breast cancer.

In one aspect, the invention relates to the use of the antibody or fragment thereof as a medicament. In one aspect, the invention relates to the use of the antibody or fragment thereof for the treatment of cancer mediated by a HER3 ligand-dependent or ligand-independent signal transduction pathway. In one aspect, the present invention relates to the use of the antibody or a fragment thereof in the preparation of a medicament for treating cancer mediated by a HER3 ligand-dependent signal transduction pathway or a ligand-independent signal transduction pathway, the cancer being selected from Breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumor, nerve sheath tumor, head and neck cancer, bladder cancer, esophageal cancer, Barretts esophagus cancer, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia ( BPH), gynacomastica, and endometriosis.

Brief description of the drawings

Figure 1. Representative MOR12615SET curve obtained with human HER3;

Figure 2. SK-Br-3 cell binding assay by FACS titration;

Figure 3. HER3 domain binding ELISA;

Figure 4. (A) Surface representation of the HER3/MOR12604 x-ray crystal structure. HER3 (marked by domains D2, D3 and D4) is in a closed conformation and MOR12604 binds to domain 3. (B) Ca overlay of HER3 (dark grey) and unbound HER3 structure (light grey; Cho et al., (2003) Nature 421:756-760) from the HER3/MOR12064 structure aligned by domain 3. (C) View of the domain 3 epitope recognized by 12604. The highlighted HER3 residues are in the x-ray crystal structure of MOR12604 within. (D) View of domain 3/MOR12604 interaction. The MOR12604 binding surface is highlighted in dark gray, marking surface A (solid line) and surface B (dashed line);

Figure 5. Inhibition of ligand-induced (A) HER3 and (B) Akt phosphorylation in MCF7 cells;

Figure 6. Ligand-dependent inhibition of (A) HER3 and (B) Akt phosphorylation in HER2-amplified SKBr3 cells;

Figure 7. Ligand independent inhibition of (A) HER3 and (B) Akt phosphorylation in HER2 amplified BT474 cells;

Figure 8. Inhibition of ligand-stimulated proliferation of MCF7 cells;

Figure 9. Inhibition of ligand-independent proliferation of SKBr3 cells;

Figure 10. Inhibition of ligand-independent proliferation of BT474 cells;

Figure 11. Data showing inhibition of in vivo tumor growth in BxPC3 (A) and BT474 (B) with MOR12606 and MOR13655; and

Figure 12. Data showing improved inhibition of tumor growth in vivo in BxPC3 with the combination of MOR12606 (DIII binder) and MOR10703 (DII+IV binder).

Detailed description of the invention

definition

In order to make the present invention easier to understand, some terms are first defined. Additional definitions are shown throughout the Detailed Description of the Invention.

As used herein, the phrase "signal transduction" or "signaling activity" refers to a biochemical causal relationship, typically initiated by a protein-protein interaction, such as the binding of a growth factor to a receptor, that results in the transmission of a signal from a part of a cell to a part of the cell another part. For HER3, this transmission involves the specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions leading to signal transduction. The penultimate process generally involves nuclear events, resulting in changes in gene expression.

As used herein, the term "HER3" or "HER3 receptor", also known as "ErbB3", refers to mammalian HER3 protein, and "her3" or "erbB3" refers to mammalian her3 gene. A preferred HER3 protein is human HER3 protein present in the cell membrane of cells. The human her3 gene is described in US Pat. No. 5,480,968 and in Plowman et al., (1990) Proc. Natl. Acad. Sci. USA, 87:4905-4909.

Human HER3 as defined in Accession No. NP_001973 (Human), hereinafter represented by SEQ ID NO: 1. All designations refer to full length, immature HER3 (amino acids 1-1342). Cleavage of immature HER3 between positions 19 and 20 produces the mature HER3 protein (20-1342 amino acids).

mrandalqvl gllfslargs evgnsqavcp gtlnglsvtg daenqyqtly klyercevvm

gnleivltgh nadlsflqwi revtgyvlva mnefstlplp nlrvvrgtqv ydgkfaifvm

lnyntnssha lrqlrltqlt eilsggvuie kndklchmdt idwrdivrdr daeivvkdng

rscppchevc kgrcwgpgse dcqtltktic apqcnghcfg pnpnqcchde caggcsgpqd

tdcfacrhfn dsgacvprcp qplvynkltf qlepnphtky qyggvcvasc phnfvvdqts

cvracppdkm evdknglkmc epcgglcpka cegtgsgsrf qtvdssnidg fvnctkilgn

ldflitglng dpwhkipald peklnvfrtv reitgylniq swpphmhnfs vfsnlttigg

rslynrgfsl limknlnvts lgfrslkeis agriyisanr qlcuhhslnw tkvlrgptee

rldikhnrpr rdcvaegkvc dplcssggcw gpgpgqclsc rnysrggvcv thcnflngep

refaheaecf schpecqpme gtatcngsgs dtcaqcahfr dgphcvsscp hgvlgakgpi

ykypdvqnec rpchenctqg ckgpelqdcl gqtlvligkt hltmaltvia glvvifmmlg

gtflywrgrr iqnkramrry lergesiepl dpsekankvl arifketelr klkvlgsgvf

gtvhkgvwip egesikipvc ikviedksgr qsfqavtdhm laigsldhah ivrllglcpg

sslqlvtqyl plgslldhvr qhrgalgpql llnwgvqiak gmyyleehgm vhrnlaarnv

llkspsqvqv adfgvadllp pddkqllyse aktpikwmal esihfgkyth qsdvwsygvt

vwelmtfgae pyaglrlaev pdllekgerl aqpqictidv ymvmvkcwmi denirptfke

lanetrmar dpprylvikr esgpgiapgp ephgltnkkl eevelepeld ldldleaeed

nlatttlgsa lslpvgtlnr prgsqsllsp ssgympmnqg nlgescqesa vsgssercpr

pvslhpmprg clasessegh vtgseaelqe kvsmcrsrsr srsprprgds ayhsqrhsll

tpvtplsppg leeedvngyv mpdthlkgtp ssregtlssv glssvlgtee ededeeyym

nrrrrhspph pprpssleel gyeymdvgsd lsaslgstqs cplhpvpimp tagttpdedy

eymnrqrdgg gpggdyaamg acpaseqgye emrafqgpgh qaphvhyarl ktlrsleatd

safdnpdywh srlfpkanaq rt (SEQ ID NO: 1)

As used herein, the term "HER3 ligand" refers to a polypeptide that binds and activates HER3. Examples of HER3 ligands include, but are not limited to, neuregulin 1 (NRG) and neuregulin 2, heparin-binding epidermal growth factor, and epiregulin. The term includes biologically active fragments and/or variants of naturally occurring polypeptides.

A "HER2-HER3 protein complex" is a non-covalently bound oligomer comprising a HER2 receptor and a HER3 receptor. This complex can be formed when cells expressing both receptors are exposed to HER3 ligands such as NRG, or when HER2 is active/overexpressed.

The phrase "HER3 activity" or "HER3 activation" as used herein refers to oligomerization (such as an increase in HER3-containing complexes), HER3 phosphorylation, conformational rearrangements (such as those induced by ligands), and HER3-mediated downstream Increased signaling.

The term "stabilizing" or "stabilized" as used in the context of HER3 refers to directly maintaining (locking, tethering, maintaining, preferentially binding, favoring) the inactive state or inactive conformation of HER3 without blocking ligand binding to HER3 , so that ligand binding can no longer activate HER3 antibodies or fragments thereof.

The term "ligand-dependent signaling" as used herein refers to activation of HER3 by a ligand. HER3 activation is evidenced by increased heterodimerization and/or HER3 phosphorylation leading to activation of downstream signaling pathways such as PI3K. The antibody or fragment thereof can statistically significantly reduce the amount of phosphorylated HER3 in stimulated cells exposed to the antibody or fragment thereof relative to untreated (control) cells as measured by the assay described in the Examples . A cell expressing HER3 may be a naturally occurring cell line (eg, MCF7), or may be a cell produced recombinantly by introducing a nucleic acid encoding a HER3 protein into a host cell. Cell stimulation can be performed by exogenous addition of activating HER3 ligands or by endogenous expression of activating ligands.

An antibody or fragment thereof that "reduces neuregulin-induced activation of HER3 in cells" is an antibody or fragment thereof that, when measured using the assay described in the Examples, exhibits a greater concentration relative to untreated (control) cells Its fragments statistically significantly reduce HER3 tyrosine phosphorylation. This can be determined based on the level of HER3 phosphorylation following exposure of HER3 to NRG and the antibody of interest. A cell expressing a HER3 protein may be a naturally occurring cell or cell line (eg, MCF7), or may be a recombinantly produced cell or cell line.

As used herein, the term "ligand-independent signaling" refers to cellular HER3 activity (eg, phosphorylation) that does not require ligand binding. For example, ligand-independent HER3 activation can be the result of HER2 overexpression or activating mutations in HER3 heterodimer partners such as EGFR and HER2. The antibody or fragment thereof can statistically significantly reduce the amount of phosphorylated HER3 in cells exposed to the antibody or fragment thereof relative to untreated (control) cells. A cell expressing HER3 may be a naturally occurring cell line (eg, SK-Br-3), or may be a cell produced recombinantly by introducing a nucleic acid encoding a HER3 protein into a host cell.

The term "blocking" as used herein refers to terminating or preventing an interaction or process, eg, terminating ligand-dependent or ligand-independent signaling.

As used herein, the term "recognizes" refers to an antibody or antibody that finds and interacts with (eg binds to) its epitope in Domain 3 of HER3, Domain 4 of HER3, or both Domain 3 and Domain 4 of HER3 its fragment. The epitope may be a linear epitope, a non-linear epitope or a conformational epitope. For example, the antibody or fragment thereof interacts with amino acid residues 335-342, 362-376, 398, 400, 424-428, 431, 433-434, and 455 (within domain 3) or a subset thereof of HER3. In another example, the antibody or fragment thereof interacts with at least one amino acid residue selected from amino acid residues 329-498 (domain 3), or a subset thereof. In another example, the antibody or fragment thereof interacts with at least one amino acid residue selected from amino acid residues 499-642 (domain 4), or a subset thereof. In another example, the antibody or fragment thereof is combined with at least one amino acid residue selected from domain 3 of HER3 (amino acid residues 329-498 of SEQ ID NO: 1) and at least one amino acid residue selected from domain 4 (SEQ ID NO: 1) Amino acid residues 499-642 of NO:1) or a subset thereof interact.

As used herein, the phrase "concurrently binds" refers to a HER3 ligand that can bind to a ligand binding site on the HER3 receptor together with a HER3 antibody or fragment thereof. This means that both the antibody and the ligand can bind the HER3 receptor together. For illustrative purposes only, the HER3 ligand NRG can bind the HER3 receptor in conjunction with a HER3 antibody. Assays to measure simultaneous ligand and antibody binding are described in the Examples section.

As used herein, the term "failure" refers to an antibody or fragment thereof that fails to perform a specified event. For example, an antibody or fragment thereof that "fails to activate signal transduction" is an antibody or fragment thereof that does not trigger signal transduction.

The term "antibody" as used herein refers to a whole antibody that interacts with HER3 epitopes (eg, through binding, steric hindrance, stabilization/destabilization, spatial distribution) and inhibits signal transduction. Naturally occurring "antibodies" are glycoproteins comprising at least 2 heavy (H) chains and 2 light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragments, F( ab') fragments and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. Antibodies can be of any isotype (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass.

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domains of both the light (VL) and heavy (VH) chain portions are responsible for antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental migration, Fc receptor binding, complement fixation, etc. By convention, constant region domains are numbered increasing as they become increasingly distant from the antigen-binding site, or amino-terminus, of the antibody. The N-terminus is the variable region and the C-terminus is the constant region; the CH3 and CL domains actually comprise the carboxy-terminal ends of the heavy and light chains, respectively.

As used herein, the phrase "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically interact with a HER3 epitope (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) and inhibit signal transduction parts. Examples of binding fragments include, but are not limited to, Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; F(ab) ₂ fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds at the hinge region; fragments; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of VH domains composition; and isolated complementarity determining regions (CDRs).

Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by recombinant means through a synthetic linker that allows them to be prepared as a single protein chain in which the VL and VH regions pair to form Monovalent molecules (termed single-chain Fv (scFv); see eg Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened and used in the same manner as intact antibodies.

Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see e.g. Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see US Patent No. 6,703,199, which describes fibronectin polypeptide monoclonal antibodies).

Antibody fragments can be incorporated into single-chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., ( 1995) Protein Eng. 8:1057-1062; and US Patent No. 5,641,870).

The term "epitope" includes a protein determinant capable of specifically binding to an immunoglobulin or otherwise interacting with the molecule. Epitope determinants are generally composed of chemically active surface groups of molecules, such as amino acids or sugars or sugar side chains, and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes may be "linear", "nonlinear" or "conformational" epitopes.

The term "linear epitope" refers to an epitope for which all points of interaction between a protein and an interacting molecule (such as an antibody) occur linearly (contiguously) along the primary amino acid sequence of the protein. Once the desired epitope on the antigen has been identified, it is possible to generate antibodies against that epitope, for example using the techniques described in this invention. Alternatively, during the discovery process, antibody production and characterization can elucidate information about desired epitopes. With this information, it is then possible to competitively screen for antibodies that bind the same epitope. This is achieved by performing cross-competition studies to find antibodies that compete for binding with each other, such as antibodies that compete for binding to an antigen. A high-throughput method for "binning" antibodies based on their cross-competition is described in International Patent Application No. WO 2003/48731. Those skilled in the art will appreciate that virtually anything to which an antibody can specifically bind can be an epitope. An epitope may comprise those residues to which an antibody binds.

The term "non-linear epitope" refers to an epitope having discrete amino acids that form a three-dimensional structure within a particular domain (eg, within domain 1, within domain 2, within domain 3, or within domain 4). In one embodiment, the non-linear epitope is within domain 2. The non-linear epitope may also be present between two or more domains (eg, the interface between domains 3-4). A non-linear epitope also refers to discrete amino acids that are a three-dimensional structure within a particular domain.

The term "conformational epitope" refers to an epitope in which discrete amino acids are brought together in a three-dimensional configuration. In a conformational epitope, the points of interaction occur at amino acid residues on the protein that are separated from each other in sequence. Those skilled in the art will appreciate that the space occupied by the residues or side chains that create the shape of the molecule helps determine what the epitope is.

In one embodiment, the epitope is within domain 3 of HER3. In one embodiment, the epitope is a non-linear epitope comprising amino acid residues within domain 3 of HER3. In one embodiment, the non-linear epitope comprises amino acid residues 335-342, 362-376, 398, 400, 424-428, 431, 433-434 and 455 (within domain 3) of SEQ ID NO: 1 , or a subset thereof.

In another embodiment, the epitope is within domain 4 of HER3. In one embodiment, the epitope comprises at least one amino acid residue selected from domain 4 (amino acid residues 499-642 of SEQ ID NO: 1), or a subset thereof. In one embodiment, the epitope is a linear epitope within domain 4 of HER3. In one embodiment, the epitope is a non-linear epitope within domain 4 of HER3. In another embodiment, the epitope is a conformational epitope within domain 4 of HER3.

In another embodiment, the epitope is within domains 3-4 of HER3. In one embodiment, the epitope comprises at least one amino acid residue selected from domain 3 (amino acid residues 329-498 of SEQ ID NO: 1), or a subset thereof. In one embodiment, the epitope comprises at least one amino acid residue selected from domain 4 (amino acid residues 499-642 of SEQ ID NO: 1), or a subset thereof. In one embodiment, the epitope comprises at least one amino acid residue selected from domain 3 (amino acid residues 329-498 of SEQ ID NO: 1) and at least one amino acid residue selected from domain 4 (amino acid residues 329-498 of SEQ ID NO: 1). bases 499-642) or a subset thereof. In one embodiment, the epitope is a linear epitope. In one embodiment, the epitope is a non-linear epitope. In another embodiment, the epitope is a conformational epitope.

In general, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

The region of a given polypeptide comprising an epitope can be identified using any number of epitope mapping techniques well known in the art. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined by simultaneously synthesizing, for example, large numbers of peptides on a solid support, the peptides corresponding to portions of the protein molecule, reacting the peptides with antibodies while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. No. 4,708,871; Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; USA 82:78-182; Described in Geysen et al., (1986) Mol. Immunol. 23:709-715. Similarly, nonlinear and conformational epitopes are readily identified by determining the spatial conformation of amino acids, eg, by hydrogen/deuterium exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance, for example. See eg Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydrophilicity maps, such as those calculated with, for example, the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program utilizes the Hopp/Woods method (Hopp et al., (1981) Proc. Natl. Acad. Sci USA 78:3824-3828) to determine the antigenic profile, utilizing the Kyte-Doolittle technique (Kyte et al., (1982) J .MoI.Biol.157:105-132) to measure the hydrophilicity figure.

The term "binding surface" as used herein refers to a plurality of continuous or discontinuous surfaces in a 3D configuration on HER3, such as domain 3 of HER3. These surfaces form part of the epitope and interact with the antibody or fragment thereof. For example, the binding surface can comprise at least two surfaces (e.g., Surface A and Surface B, see FIG. 4D ), at least three surfaces (e.g., Surface A, Surface B, and Surface C), at least four surfaces (e.g., Surface A, Surface B , Surface C, and Surface D), at least five surfaces (such as Surface A, Surface B, Surface C, Surface D, and Surface E), at least six surfaces (such as Surface A, Surface B, Surface C, Surface D, Surface E and Surface F), at least seven surfaces (such as Surface A, Surface B, Surface C, Surface D, Surface E, Surface F, and Surface G), at least eight surfaces (such as Surface A, Surface B, Surface C, Surface D , Surface E, Surface F, Surface G, and Surface H), at least nine surfaces (such as Surface A, Surface B, Surface C, Surface D, Surface E, Surface F, Surface G, Surface H, and Surface I) or at least ten Surfaces (such as Surface A, Surface B, Surface C, Surface D, Surface E, Surface F, Surface G, Surface H, Surface I, and Surface J).

The term "binding surface A" as used herein refers to a surface on domain 3 of HER3 comprising at least one amino acid residue selected from amino acid residues 362-376.

The term "binding surface B" as used herein refers to a surface on domain 3 of HER3 comprising at least one amino acid residue selected from amino acid residues 335-342, 398, 400, 424-428, 431, 433-434 and 455 base.

The phrase "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to polypeptides having substantially the same amino acid sequence or derived from the same genetic source, including antibodies, antibody fragments, bispecific antibodies, and the like. The term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

The phrase "human antibody" as used herein includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody comprises a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutated forms of human germline sequences, or contains, for example, from Knappik et al., (2000) J Mol Biol 296: 57-86) antibodies to the consensus framework sequences of the human framework sequence analysis. The structure and position of immunoglobulin variable domains (e.g., CDRs) can be defined using well-known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), Kabat et al., eds.; Lazikani et al., (1997) J.Mol.Bio.273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al- Lazikani et al., (1997) J. Mol. Biol. 273:927-948.

Human antibodies of the invention may include amino acid residues not encoded by human sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation or conservative substitutions in vivo to promote stability or production).

The phrase "human monoclonal antibody" as used herein refers to an antibody displaying a single binding specificity which has variable regions in which both the framework and CDR regions are derived from human sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a human monoclonal antibody obtained from a transgenic non-human animal, such as a transgenic mouse, fused to an immortal cell having a genome comprising a human heavy chain transgene and a light chain transgene. B cells.

As used herein, the phrase "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as from transgenic or transchromosomal animals (such as mice) with human immunoglobulin genes or hybridomas prepared therefrom Isolated antibodies, antibodies isolated from host cells transformed for expression of human antibodies (e.g., from transfectomas), antibodies isolated from recombinant combinatorial human antibody libraries, and antibodies isolated by methods involving all or all of human immunoglobulin genes, sequences Antibodies prepared, expressed, produced or isolated by any other means by which parts are spliced to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using transgenic animals of human Ig sequences, in vivo somatic mutagenesis) such that the _VH and _VL regions of the antibody are recombined The amino acid sequence of is a sequence which, although derived from and related to human germline _VH and _VL sequences, may not naturally occur in the human antibody germline repertoire in vivo.

Specific binding between two entities means at least 10 ² M ⁻¹ , at least 5x10 ² M ⁻¹ , at least 10 ³ M ⁻¹ , at least 5x10 ³ M ⁻¹ , at least 10 ⁴ M ⁻¹ , at least 5x10 ⁴ M ^-1 , at least 10 ⁵ M ^-1 , at least 5x10 ⁵ M ^-1 , at least 10 ⁶ M ^-1 , at least 5x10 ⁶ M ^-1 , at least 10 ⁷ M ^-1 , at least 5x10 ⁷ M ^-1 , at least 10 ⁸ M ^{-1 1} , at least 5x10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 5x10 ⁹ M ^-1 , at least 10 ¹⁰ M ^-1 , at least 5x10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 5x10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , at least 5x10 ¹² M ^-1 , at least 10 ¹³ M ^-1 , at least 5x10 ¹³ M ^-1 , at least 10 ¹⁴ M ^-1 , at least 5x10 ¹⁴ M ^-1 , at least 10 ¹⁵ M ^-1 , Or an equilibrium constant (K _A ) (k _on /k _off ) of at least 5x10 ¹⁵ M ⁻¹ for binding.

The phrase "specifically (or selectively) binds" refers to the binding reaction of HER3-binding antibodies and HER3 receptors in heterogeneous populations of proteins and other biological products. In addition to the equilibrium constant (K _A ) noted above, the HER3-binding antibodies of the invention typically also have less than 5×10 ⁻² M, less than 10 ⁻² M, less than 5×10 ⁻³ M, less than 10 ⁻³ M, Below 5x10 ^-4 M, below 10 ^-4 M, below 5x10 ^-5 M, below 10 ^-5 M, below 5x10 ^{-6 M, below 10 -6 M} , below 5x10 ^-7 M, low at 10 ^-7 M, below 5x10 ^-8 M, below 10 ^-8 M, below 5x10 ^-9 M, below 10 ^-9 M, below 5x10 ^-10 M, below 10 ^-10 M, below 5x10 ^-11 M, below 10 ^-11 M, below 5x10 ^-12 M, below 10 ^-12 M, below 5x10 ^-13 M, below 10 ^-13 M, below 5x10 ^-14 M, below 10 Dissociation rate constant (K _D ) (k _off /k _on ) of ^-14 M, less than 5x10 ^-15 M, or less than 10 ^-15 M or less, and binds non-specific antigens (eg, HSA) at a ratio Binds HER3 with at least 2-fold higher affinity.

In one embodiment, the antibody or fragment thereof has an , less than 100 pM, less than 75 pM, less than 10 pM, less than 1 pM dissociation constant (K _d ), the dissociation constant is estimated by methods described herein or known to those skilled in the art (for example, BIAcore assay, ELISA, FACS , SET) (Biacore International AB, Uppsala, Sweden).

As used herein, the term " _Kassoc " or " _Ka " refers to the on-rate of a particular antibody-antigen interaction, while the term " _Kdis " or " _Kd " as used herein refers to the off-rate of a particular antibody-antigen interaction. As used herein, the term " _KD " refers to the dissociation constant, which is obtained from the ratio of _Kd to _Ka (ie, _Kd / _Ka ) and expressed as molarity (M). _KD values for antibodies can be determined using methods well known in the art. Methods for determining antibody _KD are by using surface plasmon resonance, or using biosensor systems such as system.

The term "affinity" as used herein refers to the strength with which an antibody interacts with an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arm" interact with the antigen through weak non-covalent forces at many sites; the more interactions, the stronger the affinity.

The term "avidity" as used herein refers to an informative measure of the overall stability or strength of an antibody-antigen complex. It is governed by three main factors: antibody epitope affinity; potency of both antigen and antibody; and structural arrangement of interacting moieties. Ultimately, these factors determine antibody specificity, the likelihood that a particular antibody will bind to a precise epitope.

The term "potency" as used herein refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds a target molecule or a specific site (ie, epitope) on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site can specifically bind the same or a different molecule (eg, can bind a different molecule, eg, a different antigen, or a different epitope on the same molecule).

The phrase "inhibiting antibody" as used herein refers to an antibody that binds to HER3 and inhibits the biological activity of HER3 signaling (e.g., for example, reduces, reduces and/or inhibits HER3-induced signaling activity in a phospho-HER3 or phospho-Akt assay). Examples of assays are described in more detail in the Examples below. Accordingly, it is to be understood that an antibody that "inhibits" one or more of these HER3 functional properties (e.g. biochemical, immunochemical, cellular, physiological or other biological activity, etc.) as determined according to methods well known in the art and described herein refers to A statistically significant decrease in a specific activity relative to that observed in the absence of the antibody (eg, or when a control antibody of irrelevant specificity is present). Antibodies that inhibit HER3 activity cause a statistically significant reduction of at least 10%, at least 50%, 80% or 90% of a measured parameter, and in certain embodiments, antibodies of the invention may inhibit more than 95% , 98% or 99% HER3 functional activity, as evidenced by a decrease in cellular HER3 phosphorylation levels.

The phrase "isolated antibody" refers to an antibody that is substantially free of other antibodies having a different antigen specificity (eg, an isolated antibody that specifically binds HER3 is substantially free of antibodies that specifically bind an antigen other than HER3). An isolated antibody that specifically binds HER3 may, however, be cross-reactive with other antigens. Furthermore, an isolated antibody can be substantially free of other cellular material and/or chemicals.

The phrase "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where the codon specifies an alanine, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functional on the same molecule. Accordingly, every silent variation of a nucleic acid which encodes a polypeptide is implicit in every stated sequence.

With respect to a polypeptide sequence, "conservatively modified variants" include each substitution, deletion or addition to a polypeptide sequence which results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues and alleles of the invention. The following 8 groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N) , glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine acid (C), methionine (M) (see eg, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.

The terms "cross-competing" and "cross-competing" are used interchangeably herein to mean the ability of an antibody or fragment thereof to interfere with the binding of another antibody or fragment thereof to HER3 in a standard competitive binding assay.

Standard competition binding assays can be used to determine the ability or extent to which an antibody or fragment thereof is able to interfere with the binding of another antibody or fragment thereof to HER3 to determine whether it can be termed cross-competing according to the invention. One suitable assay involves the use of Biacore technology (for example by using a BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of the interaction with surface plasmon resonance techniques. Another assay for measuring cross-competition uses an ELISA-based method.

As used herein, the term "optimized" refers to a nucleotide sequence that has been altered to encode an amino acid sequence with codons that are specific to a producer cell or organism, typically a eukaryotic cell such as a Pichia cell, wood Preferred among Trichoderma cells, Chinese Hamster Ovary cells (CHO) or human cells. The optimized nucleotide sequence is engineered to preserve all or as much as possible of the amino acid sequence originally encoded by the starting nucleotide sequence (which is also referred to as the "parental" sequence).

Standard assays for evaluating the binding ability of antibodies to HER3 of various species are well known in the art and include, for example, ELISA, Western blot, and RIA. Suitable assays are described in detail in the Examples. Binding kinetics (eg, binding affinity) of antibodies can also be assessed by standard assays well known in the art, eg, by Biacore analysis, or FACS relative affinity (Scatchard). Assays for evaluating the effect of antibodies on functional properties of HER3 (eg, receptor binding assays, modulation of HER3 signaling pathways) are also described in detail in the Examples.

The phrase "percent identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical. When comparing and aligning for maximum correspondence within a comparison window or within a specified region, using the following sequence comparison algorithm or as determined by manual alignment and visual inspection, if two sequences have a specified percentage (i.e., within a specified region, or When not specified, the same amino acid residues of 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity) over the full-length sequence or nucleotides, then the two sequences are "substantially identical". Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 amino acids) in length. or more amino acids) region.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

As used herein, a "comparison window" includes a segment that references any number of contiguous positions selected from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, in which optimal comparison between two sequences can be performed. Alignment compares the sequence to a reference sequence at the same number of adjacent positions. Methods of alignment of sequences for comparison are well known in the art. For example, by the local homology algorithm of Smith and Waterman (1970) Adv.Appl.Math.2:482c, by the homology comparison algorithm of Needleman and Wunsch, (1970) J.Mol.Biol.48:443, by Search for the similarity methods of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementation of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI GAP, BESTFIT, FASTA, and TFASTA), or sequence optimal alignment for comparison by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, respectively described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990 ) J. Mol. Biol. 215:403-410. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score when aligned with a word of the same length in a database sequence T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initial searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. When the cumulative alignment score falls by an X amount from its highest achieved value, the cumulative alignment score reaches zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached, the stop word hits at extension in each direction. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses a default wordlength of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA89:10915) alignment (B) of 50, an expectation of (E) 10, M=5, N=-4 and comparison of the two chains.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, eg, Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci., 4: 11-17) which has been integrated into the ALIGN program (version 2.0) can also be used, using PAM120 weighted residue tables, gap length penalties A score of 12 and a gap penalty of 4 determine the percent identity between two amino acid sequences. In addition, the Needleman and Wunsch (1970) J. Mol, Biol. 48:444-453) algorithm of the GAP program that has been incorporated into the GCG software package (available at www.gcg.com), using the Blossom 62 matrix or PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.

In addition to the percentages of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below . Thus, a polypeptide is typically substantially identical to a second polypeptide, eg, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules, or their complements, hybridize to each other under stringent conditions, as described below. A further indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

The phrase "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, synthetic, naturally occurring and non-naturally occurring, which have similar binding properties to the reference nucleic acid, and which are Metabolized in a similar manner to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methylribonucleotide, peptide nucleic acid (PNA) .

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In particular, as described in detail below, degenerate codon replacement can be accomplished by generating sequences in which the third of one or more selected (or all) codons are replaced with mixed bases and/or deoxyinosine residues. position (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91 -98).

The phrase "operably linked" refers to the functional relationship of two or more polynucleotide (eg, DNA) segments. Generally, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if the promoter or enhancer sequence stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Typically, promoter transcriptional regulatory sequences operably linked to the transcribed sequence are in physical spatial proximity to the transcribed sequence, ie, they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent to or located in the vicinity of coding sequences whose transcription the enhancer enhances.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of a corresponding naturally occurring amino acid, and also to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates such as mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Except where noted, the terms "patient" or "individual" are used interchangeably herein.

The term "anticancer agent" as used herein refers to any drug useful in the treatment of cell proliferative diseases such as cancer, including cytotoxic agents, chemotherapeutic agents, radiation therapy and radiotherapeutic agents, targeted anticancer agents and immunotherapeutic agents.

As used herein, the term "tumor" refers to neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

The term "anti-tumor activity" as used herein refers to a decrease in tumor cell proliferation rate, viability or metastatic activity. A possible way to demonstrate anti-tumor activity is to demonstrate a reduction or decrease in abnormal cell growth rate or tumor size stability that occurs during treatment. Such activity can be assessed using recognized in vitro or in vivo tumor models including, but not limited to, xenograft models, allograft models, MMTV models, and other known models known in the art for studying antitumor activity.

The term "malignancy" as used herein refers to a non-benign tumor or cancer.

The term "cancer" as used herein refers to a malignant tumor characterized by unregulated or uncontrolled cell growth. Exemplary cancers include: carcinoma, sarcoma, leukemia and lymphoma. The term "cancer" includes primary malignancies (e.g., malignancies in which cells have not migrated to a site in an individual other than the site of the original tumor) and secondary malignancies (e.g., malignancies arising by metastasis, tumor cells Migrates to a secondary site different from the original tumor site).

Various aspects of the invention are described in more detail in the following sections and subsections.

Structure and Activation Mechanism of HER Receptor

All four HER receptors possess an extracellular ligand-binding domain, a single transmembrane domain, and a domain containing a cytoplasmic tyrosine kinase. The intracellular tyrosine kinase domain of HER receptors is highly conserved, although the kinase domain of HER3 contains key amino acid substitutions and thus lacks kinase activity (Guy et al., (1994): PNAS 91, 8132-8136). Ligand-induced dimerization of the HER receptor induces kinase activation, receptor transphosphorylation on tyrosine residues in the C-terminal tail, and subsequent recruitment and activation of intracellular signaling effectors (Yarden and Sliwkowski, (2001) Nature Rev 2, 127-137; Jorissen et al., (2003) Exp Cell Res 284, 31-53.

The crystal structure of the extracellular domain of HER provided some information on the process of ligand-induced receptor activation (Schlessinger, (2002) Cell 110, 669-672). The extracellular domain of each HER receptor consists of four subdomains: subdomains I and III cooperate to form the ligand-binding site, while subdomain II (and perhaps subdomain IV) pass the direct receptor -Receptor interactions are involved in receptor dimerization. In the ligand-bound HER1 structure, a β-hairpin in subdomain II (called the dimerization loop) interacts with the dimerization loop of the partner receptor to mediate receptor dimerization (Garrett et al. , (2002) Cell 110, 763-773; Ogiso et al., (2002) Cell 110, 775-787). In contrast, in the structures of inactive HER1, HER3, and HER4, the dimerization loop engages in intramolecular interactions with subdomain IV, which prevents receptor dimerization in the absence of ligand (Cho and Leahy, (2002) Science 297, 1330-1333; Ferguson et al., (2003) Mol Cell 12, 541-552; Bouyan et al., (2005) PNAS 102, 15024-15029). The structure of HER2 is unique among HERs. In the absence of ligand, HER2 has a conformation similar to the ligand-activated state of HER1 with a prominent dimerization loop that can interact with other HER receptors (Cho et al., (2003) Nature 421, 756-760 ; Garrett et al., (2003) Mol Cell 11, 495-505). This could explain the enhanced heterodimerization capacity of HER2.

Although the HER receptor crystal structure provides a model for homo- and heterodimerization of the HER receptor, there is a background in which some HER homo- and heterodimers are more prevalent than others (Franklin et al., (2004 ) Cancer Cell 5,317-328), and the role of each domain in receptor dimerization and autoinhibition (Burgess et al., (2003) Mol Cell 12, 541-552; Mattoon et al., (2004) PNAS101, 923- 928) is still less clear.

HER3 structure and epitope

Conformational epitopes bound by anti-HER3 antibodies have been previously described in PCT/EP2011/064407 and USSN:61/375,408, both filed August 22, 2011, which are hereby incorporated by reference in their entirety. The three-dimensional structure of a truncated form of HER3 in complex with a HER3 antibody fragment shows a conformational epitope comprising domains 2 and 4 of HER3.

The invention provides another class of antibodies or fragments thereof that bind a non-linear epitope within domain 3 of HER3. These antibodies or fragments thereof bind HER3 to inhibit both ligand-dependent and ligand-independent signal transduction.

The present invention also provides a class of antibodies or fragments thereof that bind within domains 3-4 of HER3 to inhibit both ligand-dependent and ligand-independent signal transduction. In one embodiment, such antibodies or fragments thereof bind domain 3 or domain 4 of HER3 to inhibit both ligand-dependent and ligand-independent signal transduction. In another embodiment, such antibodies or fragments thereof bind domain 3 and domain 4 of HER3 to inhibit both ligand-dependent and ligand-independent signal transduction.

This example presents the Crystal structure of HER3 bound to the Fab fragment of MOR12604 determined at resolution.

The three-dimensional structure of a truncated form (residues 20-640) of the extracellular domain of HER3 in complex with an antibody has been shown. exist The resolution of the HER3-MOR12604 Fab complex was determined and shown in Figure 4.

While not necessarily offering a theory, one possible model of the mechanism of action is that HER3 normally exists in an inactive (closed, bound) or active (open) state. Ligand binding induces a conformational change such that HER3 exists in an active (open) state capable of binding heterodimeric partners, resulting in activation of downstream signaling. Antibodies such as MOR12604 bind the inactive (tethered) state of HER3 and apparently block the ligand binding site.

Binding of MOR12604 within domain 3 suggests that MOR12604 may act by a mechanism selected from: blocking HER3 residues required for ligand binding; preventing HER3 from adopting an active conformation due to steric hindrance between the antibody and HER3 domain 3 ; prevents HER3 from adopting an active conformation by reducing the degree of flexibility in the HER3 hinge region (domain 3); induces a conformational change in domain 3 loop 371-377 that prevents HER3 from transitioning to an open conformation; destabilizes HER3, make it susceptible to degradation; act as a partial agonist to accelerate the downregulation of HER3; and bind one molecule of HER3 through each arm of MOR12604, causing the antibody to generate non-native HER3 dimers that are prone to proteolytic degradation or cannot be combined with Other receptor tyrosine kinases dimerize.

To examine the crystal structure of a domain 3 antibody or fragment thereof that binds HER3, by expressing the nucleotide sequence encoding HER3 or a variant thereof in a suitable host cell, followed by purification in the presence of the relevant HER3-targeting Fab Crystallization of the protein to prepare HER3 crystals. Preferably, the HER3 polypeptide contains an extracellular domain (amino acids 20 to 640 of the human polypeptide (SEQ ID NO: 1) or a truncated form thereof, preferably comprising amino acids 20-640), but lacks a transmembrane domain and an intracellular structure area.

HER3 polypeptides can also be produced as fusion proteins, eg, to facilitate extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), histidine (HIS), hexahistidine (6HIS), GAL4 (DNA binding and/or transcriptional activation domain) and β - Galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of the fusion protein sequence.

Following expression, the protein can be purified and/or concentrated, eg, by immobilized metal affinity chromatography, ion exchange chromatography, and/or gel filtration.

Proteins can be crystallized using the techniques described herein. Typically, during crystallization, droplets containing protein solutions are mixed with crystallization buffer and allowed to equilibrate in a sealed vessel. Equilibration can be achieved by well known techniques such as "hanging drop" or "seating drop" methods. In these methods, a droplet is suspended above or seated next to a much larger reservoir of crystallization buffer, equilibrated by vapor diffusion. Alternatively, equilibrium can be reached by other means, such as beneath the oil, through a semi-permeable membrane, or through free interface diffusion (see, eg, Chayen et al., (2008) Nature Methods 5, 147-153).

Once the crystals are obtained, the structure can be resolved by known X-ray diffraction techniques. Many techniques use chemically modified crystals, such as those modified by derivatization of heavy atoms into approximate phases. In practice, soaking the crystal in a solution containing an atomic salt of a heavy metal or an organometallic compound (such as lead chloride, gold thiomalate, thimerosal, or uranyl acetate) that diffuses through the crystal and Bind to the protein surface. The location of bound heavy metal atoms can then be determined by X-ray diffraction analysis of the soaked crystals. The spectrum obtained by diffraction of X-ray monochromatic beams by crystal atoms (scattering centers) can be analyzed by mathematical equations to obtain mathematical coordinates. Diffraction data are used to calculate the electron density map of the repeating unit of the crystal. Another way to obtain phase information is to use a technique called molecular replacement. In this method, a rotation and translation algorithm is applied to a retrieval model derived from a related structure, resulting in an approximate location of the protein of interest (see Rossmann, (1990) Acta Crystals A46, 73-82). Electron density maps are used to determine the position of individual atoms in the unit cell of the crystal (Blundel et al., (1976) Protein Crystallography, Academic Press).

The present disclosure describes the three-dimensional structure of HER3 and the Fab of anti-HER3 antibodies. The rough domain boundaries of the HER3 extracellular domain are as follows: domain 1: amino acids 20-207; domain 2: amino acids 208-328; domain 3: amino acids 329-498; and domain 4: amino acids 499-642. The three-dimensional structure of HER3 and antibodies allows the identification of target binding sites of potential HER3 modulators. Preferred target binding sites are target binding sites involved in HER3 activation. In one embodiment, the target binding site is located within domain 3 of HER3. Thus, an antibody or fragment thereof that binds domain 3 can modulate HER3 activation, for example by altering the relative position of the domain relative to itself or other HER3 domains. Thus, binding of an antibody or fragment thereof to amino acid residues within domain 3 can cause the protein to adopt a conformation that prevents activation.

In some embodiments, the antibody or fragment thereof binds to the binding surface of HER3. This binding surface comprises a plurality of continuous or discontinuous surfaces in a 3D configuration that form part of the epitope that interacts with the antibody or fragment thereof. For example, the binding surface can comprise at least two surfaces (e.g., Surface A and Surface B, see FIG. 4D ), at least three surfaces (e.g., Surface A, Surface B, and Surface C), at least four surfaces (e.g., Surface A, Surface B , Surface C, and Surface D), at least five surfaces (such as Surface A, Surface B, Surface C, Surface D, and Surface E), at least six surfaces (such as Surface A, Surface B, Surface C, Surface D, Surface E and Surface F), at least seven surfaces (such as Surface A, Surface B, Surface C, Surface D, Surface E, Surface F, and Surface G), at least eight surfaces (such as Surface A, Surface B, Surface C, Surface D , Surface E, Surface F, Surface G, and Surface H), at least nine surfaces (such as Surface A, Surface B, Surface C, Surface D, Surface E, Surface F, Surface G, Surface H, and Surface I) or at least ten Surfaces (such as Surface A, Surface B, Surface C, Surface D, Surface E, Surface F, Surface G, Surface H, Surface I, and Surface J).

In one embodiment, an antibody or fragment thereof is bound to binding surface A. In one embodiment, an antibody or fragment thereof is bound to binding surface B. In another embodiment, the antibody or fragment thereof binds to both binding surface A and binding surface B. In one embodiment, binding surface A comprises at least one amino acid residue selected from amino acid residues 362-376. In one embodiment, binding surface B comprises at least one amino acid residue selected from amino acid residues 335-342, 398, 400, 424-428, 431, 433-434 and 455. In another embodiment, the antibody or fragment thereof is bound to the following binding surfaces: binding surface A, wherein at least one amino acid residue is selected from amino acid residues 362-376; binding surface B, wherein at least one amino acid residue is selected from amino acid residues Bases 335-342, 398, 400, 424-428, 431, 433-434 and 455.

In some embodiments, the antibody or fragment thereof is associated with HER3 amino acid residues 335-342, 362-376, 398, 400, 424-428, 431, 433-434, and 455 (domain 3 Human HER3 protein binding to non-linear epitopes within) or a subset thereof. In some embodiments, the antibody or fragment thereof is associated with amino acid residues 335-342, 362-376, 398, 400, 424-428, 431, 433-434, and 455 (within domain 3) of SEQ ID NO: 1 or A combination of amino acids within or overlapping a subset thereof. In some embodiments, the antibody or fragment thereof is associated with amino acids 335-342, 362-376, 398, 400, 424-428, 431, 433-434, and 455 (within domain 3) of SEQ ID NO: 1 or a subunit thereof. Amino acids (and/or amino acid sequences made up of them) within a set.

In some embodiments, the antibody or fragment thereof binds to a human HER3 protein having an epitope (linear, non-linear, conformational) comprising HER3 amino acid residues 499-642 (domain 4) of SEQ ID NO: 1 or a subset thereof combined. In some embodiments, the antibody or fragment thereof binds to amino acids within or overlapping amino acid residues 499-642 (domain 4) of SEQ ID NO: 1, or a subset thereof. In some embodiments, the antibody or fragment thereof binds to amino acids (and/or an amino acid sequence consisting thereof) within amino acid residues 499-642 (domain 4) of SEQ ID NO: 1, or a subset thereof.

In some embodiments, the antibody or fragment thereof binds to a human HER3 protein having HER3 amino acid residues 335-342, 362-376, 398, 400, 424-428, 431, 433 comprising SEQ ID NO: 1 - a non-linear epitope at 434 and 455 (in domain 3) or a subset thereof, and an epitope comprising HER3 amino acid residues 499-642 (domain 4) of SEQ ID NO: 1 or a subset thereof (linear, nonlinearity, conformation). In some embodiments, the antibody or fragment thereof is associated with amino acid residues 335-342, 362-376, 398, 400, 424-428, 431, 433-434, and 455 (within domain 3) of SEQ ID NO: 1 Binding to amino acids within or overlapping with it and epitopes (linear, nonlinear, conformational) comprising HER3 amino acid residues 499-642 (domain 4) of SEQ ID NO: 1 or a subset thereof. In some embodiments, the antibody or fragment thereof is associated with amino acids 335-342, 362-376, 398, 400, 424-428, 431, 433-434, and 455 (within domain 3) of SEQ ID NO: 1 or a subunit thereof. Amino acids within the set (and/or amino acid sequences consisting thereof) and epitopes (linear, non-linear, conformational) comprising amino acid residues 499-642 (domain 4) of SEQ ID NO: 1 or a subset thereof combined.

In some embodiments, the antibody or fragment thereof binds to a human HER3 protein having an epitope (linear, non-linear, conformation) and an epitope (linear, non-linear, conformational) comprising HER3 amino acid residues within domain 4 of SEQ ID NO: 1 or a subset thereof. In some embodiments, the antibody or fragment thereof binds to amino acids within or overlapping amino acid residues of domain 3 and amino acids of domain 4 of SEQ ID NO: 1, or a subset thereof.

In some embodiments, the antibody or fragment thereof binds to the inactive state of the HER3 receptor, thereby preventing HER3 from adopting an active conformation. In some embodiments, the antibody or fragment thereof prevents HER3 from adopting an active conformation due to steric hindrance between the antibody or fragment thereof and a domain of HER3 (eg, for MOR12604, steric interference with domain 1 of HER3). In some embodiments, the antibody or fragment thereof prevents HER3 from adopting an active conformation by reducing the degree of flexibility in domain 3. In some embodiments, the antibody or fragment thereof induces a conformational change in domain 3 loops 371-377 of SEQ ID NO: 1 that prevents HER3 from adopting an active conformation. In some embodiments, the antibody or fragment thereof destabilizes HER3, rendering it susceptible to degradation. In some embodiments, the antibody or fragment thereof accelerates downregulation of cell surface HER3. In some embodiments, the antibody or fragment thereof produces a non-native HER3 dimer that is susceptible to proteolytic degradation or that cannot dimerize with other receptor tyrosine kinases.

In some embodiments, the antibody or fragment thereof can bind either the active or inactive state of HER3. In some embodiments, the antibody or fragment thereof stabilizes the HER3 receptor in an inactive state such that the HER3 receptor fails to dimerize with a co-receptor to form a receptor-receptor complex. Failure to form the receptor-receptor complex prevents activation of both ligand-dependent and ligand-independent signal transduction.

In some embodiments, the antibody or fragment thereof induces dimerization of HER3 with HER3 to form an inactive receptor-receptor complex. The formation of an inactive receptor-receptor complex prevents the activation of HER3-mediated signaling because HER3 is hidden in the inactive receptor-receptor complex.

The structures shown also allow the identification of specific HER3 amino acid residues at the interaction interface of antibodies or fragments thereof (eg MOR12604) with HER3. This is defined as the MOR12604 protein VH chain residues within. The residues are as follows: Ile365, Thr366, Asn369, Gly370, Asp371 , Pro372, Trp373, His374, Lys375, Gln400 and Lys434. The structures shown also allow the identification of specific HER3 amino acid residues at the interaction interface of antibodies or fragments thereof (eg MOR12604) with HER3. This is defined as the MOR12604 protein VL chain residues within. The residues are as follows: Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe45, Leu431, Met433. As can be seen in Tables 5 and 6 (MOR12604), respectively, both light and heavy chains involve the binding of the antigen binding protein to amino acid residues within domain 3 of the epitope.

Likewise, given current teachings, one skilled in the art can predict which residues and regions of an antigen binding protein can be altered without unduly interfering with the ability of the antigen binding protein to bind domains 3 and 4 of HER3.

Core interaction interface amino acids were determined, i.e. having a distance from the HER3 partner protein less than or equal to All amino acid residues with at least one atom of . choose as the core region cutoff distance to allow atoms within one van der Waals radius plus possible water-mediated hydrogen bonding.

In some embodiments, any antigen binding protein that binds, masks or prevents the interaction of MOR012604 with any of the above residues can be used to bind or neutralize HER3. In some embodiments, the antibody or fragment thereof binds to or interacts with at least one of the following HER3 residues (SEQ ID NO: 1): Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400, and Lys434 . In some embodiments, antibodies and fragments thereof bind to or interact with at least one of the following HER3 residues (SEQ ID NO: 1): Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374 , Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455. In some embodiments, antibodies and fragments thereof bind to or interact with at least one of the following HER3 residues (SEQ ID NO: 1): Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400, Lys434 , Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu531, Met4343, Tyr435, Ly. In some embodiments, antibodies and fragments thereof bind to or interact with at least one of the following HER3 residues (SEQ ID NO: 1): Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400, Lys434 , Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu531, Met4343, Tyr435, Ly. In some embodiments, the antibody or fragment thereof binds to or interacts with all of the following HER3 residues (SEQ ID NO: 1): Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400, Lys434, Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu531, Met443, Tyr433. In some embodiments, the antibody or fragment thereof is within 5 Angstroms of one or more of the aforementioned residues. In some embodiments, the antibody or fragment thereof is within 5 to 8 Angstroms of one or more of the aforementioned residues. In some embodiments, the antibody or fragment thereof is associated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 , 45, or 50 of the above residues interact with, block or are within 8 Angstroms of the residue.

For example, the acquisition of the 3D structure of HER3 and the HER3:MOR12604 complex provides a framework for exploring other HER3 antibodies in more detail. The 3D structure of HER3 allows mapping of the epitopes of monoclonal antibodies and speculating on their mode of action, as some inhibit cell growth, some stimulate cell growth, and others have no effect on cell growth. The non-linear epitope of MOR12604 has been mapped to domain 3 of HER3. The availability of the 3D structure of this receptor will facilitate the determination of the precise mechanism of action of these inhibitors and the design of new methods to interfere with HER3 receptor function. In one embodiment, an antibody of the invention binds to the same non-linear epitope as MOR12604.

In some embodiments, non-linear epitopes to which any of the antibodies listed in Table 1 bind are particularly useful. In certain embodiments, non-linear epitopes of HER3 can be used to isolate antibodies or fragments thereof that bind HER3. In certain embodiments, non-linear epitopes of HER3 can be used to generate antibodies or fragments thereof that bind HER3. In certain embodiments, HER3 non-linear epitopes can be used as immunogens to generate antibodies or fragments thereof that bind HER3 non-linear epitopes. In certain embodiments, a non-linear epitope of HER3 can be administered to an animal, and antibodies that bind HER3 can subsequently be obtained from the animal.

The invention also provides a class of antibodies that bind an epitope (linear, non-linear or conformational) within domains 3-4 of HER3. Examples of such antibodies or fragments thereof that bind within domains 3-4 are shown in Table 2. Domain 3-4 antibodies or fragments thereof complexed with HER3 can be produced using the methods described above and in the Examples section below.

In some embodiments, identification can be made by mutating specific residues in HER3 (e.g., wild-type antigen) and determining whether an antibody or fragment thereof can bind the mutated HER3 protein or a variant HER3 protein or measuring the change in affinity relative to wild-type A domain/region containing residues that contact or are obscured by the antibody. By making a number of single mutations, residues that play a direct role in binding, or that are sufficiently close to the antibody that mutations can affect the binding between the antibody and antigen, can be identified. From these amino acid information, antigenic (HER3) domains/regions containing residues that contact or are masked by the antibody can be elucidated. Mutagenesis using known techniques such as alanine scanning can help define functionally related epitopes. Mutagenesis using arginine/glutamate scanning protocols can also be applied (see, e.g., Nanevicz et al., (1995), J. Biol. Chem. 270(37):21619-21625 and Zupnick et al., (2006) , J. Biol. Chem. 281(29):20464-20473). In general, arginine and glutamic acid replace (usually individually) amino acids in the wild-type polypeptide because these amino acids are charged and bulky and thus have the potential to disrupt the antigen-binding protein and antigen in the region of the antigen into which the mutation is introduced. Potential for linkages between. Arginine present in the wild-type antigen was replaced by glutamic acid. A number of such single mutants can be obtained and the collected binding results analyzed to determine which residues affect binding. A series of mutant HER3 antigens can be generated, each having a single mutation. Binding of each mutant HER3 antigen to various HER3 antibodies or fragments thereof can be measured and compared to the ability of the selected antibodies or fragments thereof to bind wild-type HER3 (SEQ ID NO: 1).

As used herein, an alteration (e.g., decrease or increase) in the binding between an antibody or fragment thereof and a mutant or variant HER3 means binding affinity (e.g., by known methods such as Biacore assays or sphere-based assays as described in the Examples below). As measured by assay of beads), changes in _EC50 , and/or changes in the total binding capacity of the antigen-binding protein (e.g., as evidenced by a decrease in _Bmax in a plot of antigen-binding protein concentration versus antigen concentration) (e.g. reduce). A significant change in binding indicates that the mutated residue is involved in binding to the antibody or fragment thereof.

In some embodiments, a significant reduction in binding means the binding affinity between the antibody or fragment thereof and the mutant HER3 antigen relative to the binding between the antibody or fragment thereof and wild-type HER3 (e.g., SEQ ID NO: 1) , _EC50 and/or capacity reduction by more than 10%, more than 20%, more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% %, more than 90% or more than 95%.

In some embodiments, the pair of antibodies or fragments thereof has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutant HER3 protein binding was significantly reduced or increased.

Although the variant forms are referenced to the wild-type sequence shown in SEQ ID NO: 1, it is understood that amino acids may differ in allelic or splice variants of HER3. Also contemplated are antibodies or fragments thereof that exhibit significantly altered binding (eg, lower or higher binding) to such allelic forms of HER3.

In addition to general structural aspects of antibodies, more specific interactions between paratopes and epitopes can be studied by structural methods. In one embodiment, the structure of the CDRs contributes to the paratope by which the antibody is able to bind to the epitope. The shape of such paratopes can be determined in a number of ways. Traditional structural examination methods such as NMR or X-ray crystallography can be used. These methods can study the shape of the paratope alone, or when the paratope is combined with an epitope. Alternatively, molecular models can be generated on a computer. Structures can be generated by assisted homology modeling with commercial software packages such as the InsightII modeling package from Accelrys (San Diego, Calif.). Briefly, the sequence of the antibody to be examined can be searched against a database (eg, Protein DataBank) of proteins of known structure. After identifying homologous proteins with known structures, these homologous proteins were used as modeling templates. Each possible template can be aligned, resulting in a structure-based sequence alignment between the templates. Antibody sequences of unknown structure can then be aligned to these templates to generate a molecular model of the antibody of unknown structure. Those skilled in the art will appreciate that there are many alternative methods for generating such structures in silico, any of which may be used. For example, methods similar to those described by Hardman et al., issued as U.S. Patent No. 5,958,708, using QUANTA (Polygen Corp., Waltham, Mass.) and CHARM (Brooks et al., (1983), J. Comp. Chem. 4:187) (hereby incorporated by reference in its entirety).

Not only is the shape of the paratope important in determining whether and how well a potential paratope will bind the epitope, but the interaction between the epitope and paratope itself provides a rich source of information for designing variant antibodies. Those skilled in the art will appreciate that there are a variety of ways to study this interaction. One way is to use the generated structural model (perhaps as above) and then use a program such as InsightII (Accelrys, San Diego, Calif.), which has a docking module, in particular capable of mapping between a paratope and its epitope. Monte Carlo searches were performed on conformation and orientation space. The result is the ability to estimate where and how epitopes interact with the paratope. In one embodiment, only fragments or variants of the epitope are used to help determine relevant interactions. In one embodiment, the entire epitope is used in modeling the interaction between the paratope and the epitope.

By using these model structures, it is possible to predict which residues are most important in the interaction between the epitope and paratope. Thus, in one embodiment, one can readily choose which residues to alter in order to alter the binding characteristics of the antibody. For example, it is evident from docking models that the side chains of certain residues in the paratope can sterically hinder the binding of the epitope, so changing these residues to residues with smaller side chains may be advantageous. This can be determined in a number of ways. For example, one can simply observe 2 models and estimate interactions based on functional groups and proximity. Alternatively, repeated pairing of epitopes and paratopes can be performed as described above for more favorable energetic interactions. Interactions on various antibody variants can also be assayed to determine in which alternative ways an antibody may bind an epitope. Models can also be combined to determine how antibody structure should be altered to obtain an antibody with desired specific characteristics.

The models identified above can be tested by a variety of techniques. For example, interaction energies can be determined using the procedures discussed above to determine which variants to examine further. In addition, Coulomb and van der Waals interactions were used to determine the interaction energies of epitopes and variant paratopes. Site-directed mutagenesis was also used to see if predicted changes in antibody structure actually resulted in changes in the desired binding characteristics. Alternatively, changes to the epitope can be made to verify that the model is correct, or to determine general binding themes that may exist between the paratope and the epitope.

It will be appreciated by those skilled in the art that while these models will provide the necessary guidance for making the antibodies of the present embodiments and variants thereof, it will be desirable to routinely test the in silico models, perhaps by in vitro studies. Furthermore, it will be obvious to those skilled in the art that any modification may also have additional side effects on the activity of the antibody. For example, while any change predicted to result in stronger binding may induce stronger binding, it may also result in other structural changes that may reduce or alter antibody activity. Determining whether this is the case is routine in the art and can be accomplished in a number of ways. For example, activity can be tested by ELISA assay. Alternatively, the sample can be tested by using a surface plasmon resonance device.

HER3 antibody

The present invention provides a class of antibodies shown in Table 1 that recognize a non-linear epitope within domain 3 of HER3 and inhibit both ligand-dependent and ligand-independent HER3 signaling pathways. The present invention also provides a class of antibodies shown in Table 2 that recognize epitopes (linear, non-linear, conformational) within domains 3-4 of HER3 and inhibit both ligand-dependent and ligand-independent HER3 signaling Both transduction pathways.

Table 1: Examples of HER3 antibodies that bind non-linear epitopes within domain 3 of HER3.

Table 2: Examples of HER3 antibodies that bind amino acids within domains 3-4 of HER3.

In one aspect, the invention provides an antibody that specifically binds domain 3 of a HER3 protein (e.g., human and/or cynomolgus HER3), the antibody comprising a protein having SEQ ID NO: 14, 34, 54, 74, 94, 114 , 134, 154 and 174 amino acid sequences of the VH domain. In another aspect, the invention provides an antibody that specifically binds domain 3 of a HER3 protein (e.g., human and/or cynomolgus HER3), the antibody comprising a protein having SEQ ID NO: 15, 35, 55, 75, 95, VL domains of amino acid sequences of 115, 135, 155 and 175.

In one aspect, the invention provides an antibody that specifically binds domains 3-4 of a HER3 protein (e.g., human and/or cynomolgus HER3), the antibody comprising a protein having SEQ ID NO: 194, 214, 234, 254, 274 , 294, 314, 334, 354, 374, 394, 414, 434, 454, 474, 494, 514, 534, 554, 574, 594, 614, 634, 654, 674, and 694 amino acid sequences of the VH domain. In another aspect, the invention provides an antibody that specifically binds domain 3 of a HER3 protein (e.g., human and/or cynomolgus HER3), the antibody comprising a protein having SEQ ID NO: 195, 215, 235, 255, 275, The VL domain of the amino acid sequence of 295, 315, 335, 355, 375, 395, 415, 435, 455, 475, 495, 515, 535, 555, 575, 595, 615, 635, 655, 675 and 695.

Because each of these antibodies or fragments thereof binds HER3, the VH, VL, full-length light chain, and full-length heavy chain sequences (amino acid sequence and nucleotide sequence encoding the amino acid sequence) can be "mixed and matched" to generate Other HER3 antibodies of the invention. Such "mixed and matched" HER3 antibodies can be tested using binding assays known in the art, such as ELISA and other assays described in the Examples section. When mixing and matching these chains, VH sequences from a particular VH/VL pairing should be replaced with structurally similar VH sequences. Likewise, the full-length heavy chain sequence from a particular full-length heavy chain/full-length light chain pairing should be replaced with a structurally similar full-length heavy chain sequence. Likewise, VL sequences from a particular VH/VL pairing should be replaced with structurally similar VL sequences. Likewise, the full-length light chain sequence from a particular full-length heavy chain/full-length light chain pairing should be replaced with a structurally similar full-length light chain sequence.

Accordingly, in one aspect, the invention provides an isolated monoclonal antibody or fragment thereof having: a heavy antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 34, 54, 74, 94, 114, 134, 154 and 174 chain variable region; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 15, 35, 55, 75, 95, 115, 135, 155 and 175; wherein the antibody specifically binds HER3 (for example, domain 3 of human and/or cynomolgus monkey).

Accordingly, in one aspect, the invention provides an isolated monoclonal antibody or fragment thereof comprising: A heavy chain variable region of the amino acid sequence of 414, 434, 454, 474, 494, 514, 534, 554, 574, 594, 614, 634, 654, 674, and 694; and comprising a sequence selected from the group consisting of SEQ ID NO: 195, 215 , 235, 255, 275, 295, 315, 335, 355, 375, 395, 415, 435, 455, 475, 495, 515, 535, 555, 575, 595, 615, 635, 655, 675, and 695 amino acids sequence; wherein the antibody specifically binds domain 3 of HER3 (eg, human and/or cynomolgus monkey).

In specific embodiments, the antibody that binds domain 3 of HER3 comprises the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15. In specific embodiments, the antibody that binds domain 3 of HER3 comprises the VH of SEQ ID NO:34 and the VL of SEQ ID NO:35. In specific embodiments, the antibody that binds domain 3 of HER3 comprises the VH of SEQ ID NO:54 and the VL of SEQ ID NO:55. In specific embodiments, the antibody that binds domain 3 of HER3 comprises the VH of SEQ ID NO:74 and the VL of SEQ ID NO:75. In specific embodiments, the antibody that binds domain 3 of HER3 comprises the VH of SEQ ID NO:94 and the VL of SEQ ID NO:95. In specific embodiments, the antibody that binds Domain 3 of HER3 comprises the VH of SEQ ID NO: 114 and the VL of SEQ ID NO: 115. In specific embodiments, the antibody that binds domain 3 of HER3 comprises the VH of SEQ ID NO: 134 and the VL of SEQ ID NO: 135. In specific embodiments, the antibody that binds domain 3 of HER3 comprises the VH of SEQ ID NO: 154 and the VL of SEQ ID NO: 155. In specific embodiments, the antibody that binds domain 3 of HER3 comprises the VH of SEQ ID NO: 174 and the VL of SEQ ID NO: 175.

In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO: 194 and the VL of SEQ ID NO: 195. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:214 and the VL of SEQ ID NO:215. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:234 and the VL of SEQ ID NO:235. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:254 and the VL of SEQ ID NO:255.

In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:274 and the VL of SEQ ID NO:275. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:294 and the VL of SEQ ID NO:295. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:314 and the VL of SEQ ID NO:315. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:334 and the VL of SEQ ID NO:335. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:354 and the VL of SEQ ID NO:355. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:374 and the VL of SEQ ID NO:375. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:394 and the VL of SEQ ID NO:395. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:414 and the VL of SEQ ID NO:415. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:434 and the VL of SEQ ID NO:435. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:454 and the VL of SEQ ID NO:255. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:474 and the VL of SEQ ID NO:475. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:494 and the VL of SEQ ID NO:495. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:514 and the VL of SEQ ID NO:515. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:534 and the VL of SEQ ID NO:535. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:554 and the VL of SEQ ID NO:555. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:574 and the VL of SEQ ID NO:575. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:594 and the VL of SEQ ID NO:595. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:614 and the VL of SEQ ID NO:615. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:634 and the VL of SEQ ID NO:635. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:654 and the VL of SEQ ID NO:655. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:674 and the VL of SEQ ID NO:675. In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises the VH of SEQ ID NO:694 and the VL of SEQ ID NO:695.

In another aspect, the invention provides a domain 3 binding HER3 antibody comprising the heavy and light chain CDR1, CDR2 and CDR3 described in Table 1, or a combination thereof. The amino acid sequence of the VH CDR1 of the antibody is shown in SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 142 and 162. The amino acid sequence of the VH CDR2 of the antibody is shown in SEQ ID NO: 3, 23, 43, 63, 83, 103, 123, 143 and 163. The amino acid sequence of the VH CDR3 of the antibody is shown in SEQ ID NO: 4, 24, 44, 64, 84, 104, 124, 144 and 164. The amino acid sequence of the VL CDR1 of the antibody is shown in SEQ ID NO:8, 28, 48, 68, 88, 108, 128, 148 and 168. The amino acid sequence of the VL CDR2 of the antibody is shown in SEQ ID NO:9, 29, 49, 69, 89, 109, 129, 149 and 169. The amino acid sequence of the VL CDR3 of the antibody is shown in SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 150 and 170. Using the Kabat system (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273, 927-948) describe the CDR regions.

In specific embodiments, the antibody that binds domain 3 of HER3 comprises: the heavy chain variable region CDR1 of SEQ ID NO: 142; the heavy chain variable region CDR2 of SEQ ID NO: 143; the heavy chain of SEQ ID NO: 144 can Variable region CDR3; light chain variable region CDR1 of SEQ ID NO:148; light chain variable region CDR2 of SEQ ID NO:149; and light chain variable region CDR3 of SEQ ID NO:150.

In specific embodiments, the antibody that binds domain 3 of HER3 comprises: the heavy chain variable region CDR1 of SEQ ID NO: 162; the heavy chain variable region CDR2 of SEQ ID NO: 163; the heavy chain of SEQ ID NO: 164 can Variable region CDR3; light chain variable region CDR1 of SEQ ID NO:168; light chain variable region CDR2 of SEQ ID NO:169; and light chain variable region CDR3 of SEQ ID NO:170.

In another aspect, the invention provides a HER3 antibody that binds domains 3-4, comprising the heavy and light chain CDR1, CDR2, and CDR3 described in Table 2, or a combination thereof. The amino acid sequence of the VH CDR1 of the antibody is in SEQ ID NO: 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, 522, 542, 562, 582, 602, 622, 642, 662, and 682 are displayed. The amino acid sequence of the VH CDR2 of the antibody is in SEQ ID NO: 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, 523, 543, 563 , 583, 603, 623, 643, 663, and 683 are displayed. The amino acid sequence of the VH CDR3 of the antibody is in SEQ ID NO: 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, 524, 544, 564, 584, 604, 624, 644, 664, and 684 are displayed. The amino acid sequence of the VL CDR1 of the antibody is in SEQ ID NO: 188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568, 588, 608, 628, 648, 668, and 688 are displayed. The amino acid sequence of the VL CDR2 of the antibody is in SEQ ID NO: 189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, 529, 549, 569, 589, 609, 629, 649, 669, and 689 are displayed. The amino acid sequence of the VL CDR3 of the antibody is in SEQ ID NOs: 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, 530, 550, 570 , 590, 610, 630, 650, 670, and 690 are displayed. Using the Kabat system (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1987) J.Mol.Biol.196: 901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273, 927-948) describe the CDR regions.

In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises: the heavy chain variable region CDR1 of SEQ ID NO:662; the heavy chain variable region CDR2 of SEQ ID NO:663; the heavy chain variable region of SEQ ID NO:664 chain variable region CDR3; light chain variable region CDR1 of SEQ ID NO:668; light chain variable region CDR2 of SEQ ID NO:669; and light chain variable region CDR3 of SEQ ID NO:670.

In specific embodiments, the antibody that binds domains 3-4 of HER3 comprises: heavy chain variable region CDR1 of SEQ ID NO:682; heavy chain variable region CDR2 of SEQ ID NO:683; heavy chain variable region of SEQ ID NO:684 chain variable region CDR3; light chain variable region CDR1 of SEQ ID NO:688; light chain variable region CDR2 of SEQ ID NO:689; and light chain variable region CDR3 of SEQ ID NO:690.

Other antibodies of the invention include amino acids that have been mutated, but at least 50%, 60%, 70%, 80%, 90%, 95% in the CDR regions of the CDR regions shown in the sequences described in Table 1 or Table 2 , 96%, 97%, 98% or 99% identity. In some embodiments, it comprises a mutated amino acid sequence wherein no more than 1, 2, 3, 4 or 5 have been mutated in the CDR regions when compared to the CDR regions shown in the sequences set forth in Table 1 or Table 2 amino acids, but still maintains its specificity for the original antibody epitope.

Other antibodies of the invention include mutated amino acids, but have at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, it comprises a mutated amino acid sequence wherein no more than 1, 2, 3, 4, 5 have been mutated in the framework region when compared to the framework region shown in the sequence set forth in Table 1 or Table 2 , 6 or 7 amino acids, but still maintains its specificity for the original antibody epitope. The invention also provides nucleic acid sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds to a HER3 protein (eg, human and/or cynomolgus HER3).

Other antibodies of the invention include antibodies in which the amino acid or nucleic acid encoding the amino acid has been mutated but at least 50%, 60%, 70%, 80%, 90%, 95% identical to the sequence described in Table 1 or Table 2 , 96%, 97%, 98% or 99% identity. In some embodiments, it comprises a mutated amino acid sequence wherein no more than 1, 2, 3, 4 or 5 amino acids, but retain essentially the same therapeutic activity.

As used herein, a human antibody comprises a heavy or light chain variable region or a full-length heavy or light chain "produced from" or "derived from" a particular germline sequence, if the variable region or full-length chain of the antibody was obtained from using Systematic words of human germline immunoglobulin genes. Such systems include immunization of transgenic mice carrying human immunoglobulin genes with the antigen of interest or screening of human immunoglobulin gene libraries displayed on phage with the antigen of interest. "Can be identified, for example, by comparing the amino acid sequence of a human antibody to that of a human germline immunoglobulin, and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., highest % identity) to the sequence of the human antibody." A human antibody produced from" or "derived from" human germline immunoglobulin sequences. A human antibody "produced from" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences from the germline sequence, for example, resulting from naturally occurring somatic mutations or deliberate introduction of site-directed mutations. However, selected human antibodies generally have at least 90% identity in amino acid sequence with those encoded by human germline immunoglobulin genes in the VH or VL framework regions, and contain amino acid residues that are not identical to other amino acid sequences. These amino acid residues identify a human antibody as human when compared to the germline immunoglobulin amino acid sequences of the species (eg, the murine germline sequence). In certain instances, the human antibody shares at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% in amino acid sequence with that encoded by a germline immunoglobulin gene. % or 99% identity. Typically, recombinant human antibodies differ by no more than 10 amino acids in the VH or VL framework regions from the amino acid sequences encoded by human germline immunoglobulin genes. In certain instances, human antibodies differ from the amino acid sequence encoded by the germline immunoglobulin genes by no more than 5, or even by no more than 4, 3, 2, or 1 amino acid.

The antibodies disclosed herein may be derivatives of single chain antibodies, diabodies, domain antibodies, nanobodies and unibodies. A "single-chain antibody" (scFv) consists of a single polypeptide chain comprising a VL domain linked to a VH domain, wherein the VL and VH domains pair to form a monovalent molecule. Single-chain antibodies can be prepared according to methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) . "Diabodies" consist of two chains, each chain comprising a heavy chain variable region and a light chain variable region connected by a short peptide linker on the same polypeptide chain, wherein the two regions on the same chain do not pair with each other, and are paired with complementary domains on the other chain to form bispecific molecules. Methods for making diabodies are well known in the art (see, e.g., Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448 and Poljak et al., (1994) Structure 2:1121-1123) . Domain antibodies (dAbs) are small functional binding units of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies. Domain antibodies are well expressed in bacterial, yeast and mammalian cell systems. Further details of domain antibodies and methods of producing them are well known in the art (see, e.g., U.S. Patent Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 058821, WO04/003019 and WO03/002609. Nanobodies are derived from the heavy chain of an antibody. Nanobodies generally comprise a single variable domain and 2 constant domains (CH2 and CH3) and retain the antigen-binding ability of the original antibody. Available Nanobodies are prepared by methods known in the art (see, e.g., U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, WO 06/079372). Monobodies are composed of one light chain and one heavy chain of an IgG4 antibody. Can be obtained by removing the IgG4 antibody Hinge Region Preparation of Monoclonal Antibodies Further details of monoantibodies and methods of their preparation can be found in WO2007/059782.

homologous antibody

In another embodiment, the invention provides an antibody or fragment thereof comprising an amino acid sequence homologous to the sequences set forth in Table 1 or Table 2, and which binds to a HER3 protein (e.g., human and/or cynomolgus HER3 ), and retain the desired functional properties of those antibodies described in Table 1 or Table 2.

For example, the invention provides an isolated monoclonal antibody (or a functional fragment thereof) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a compound selected from the group consisting of SEQ ID NO: 14, 34, 54 , 74, 94, 114, 134, 154 and 174 are at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence; An amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15, 35, 55, 75, 95, 115, 135, 155 and 175 ; wherein the antibody binds domain 3 of HER3 (e.g., human and/or cynomolgus HER3) and inhibits HER3 signaling activity that can be measured in a phosphorylation assay or other measurement of HER signaling (e.g., implemented Phospho-HER3 assay, phospho-Akt assay, cell proliferation and ligand blocking assay) described in Examples). Also included within the scope of the invention are the parental nucleotide sequences of the variable heavy and light chains; and full-length heavy and light chain sequences optimized for expression in mammalian cells. Other antibodies of the invention include amino acids or nucleic acids that have been mutated, but have at least 60, 70, 80, 90, 95, 98, or 99% percent identity to the aforementioned sequences. In some embodiments, it includes a mutated amino acid sequence wherein no more than 1, 2, 3 of the variable regions have been mutated by amino acid deletion, insertion or substitution when compared to the variable regions shown in the sequences above. , 4 or 5 amino acids.

For example, the invention provides an isolated monoclonal antibody (or a functional fragment thereof) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a compound selected from ID NO: 194, 214, 234, The amino acid sequence of 254, 274, 294, 314, 334, 354, 374, 394, 414, 434, 454, 474, 494, 514, 534, 554, 574, 594, 614, 634, 654, 674 and 694 has at least 80 amino acid sequences %, 90%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence; the light chain variable region comprises a sequence selected from SEQ ID NO: 195, 215, 235, 255, 275, 295, At least 80%, 90%, 95% of the amino acid sequence of 315, 335, 355, 375, 395, 415, 435, 455, 475, 495, 515, 535, 555, 575, 595, 615, 635, 655, 675, and 695 %, 96%, 97%, 98% or 99% identical amino acid sequence; wherein the antibody binds domains 3-4 of HER3 (e.g., human and/or cynomolgus HER3) and inhibits the signaling activity of HER3 , this activity can be measured in phosphorylation assays or other measures of HER signaling (eg, phospho-HER3 assays, phospho-Akt assays, cell proliferation and ligand blockade assays described in the Examples). Also included within the scope of the invention are the parental nucleotide sequences of the variable heavy and light chains; and full-length heavy and light chain sequences optimized for expression in mammalian cells. Other antibodies of the invention include amino acids or nucleic acids that have been mutated, but have at least 60, 70, 80, 90, 95, 98, or 99% percent identity to the aforementioned sequences. In some embodiments, it includes a mutated amino acid sequence wherein no more than 1, 2, 3 of the variable regions have been mutated by amino acid deletion, insertion or substitution when compared to the variable regions shown in the sequences above. , 4 or 5 amino acids.

In other embodiments, the VH and/or VL amino acid sequences may have 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity. In other embodiments, the VH and/or VL amino acid sequences may be identical with amino acid substitutions at no more than 1, 2, 3, 4 or 5 amino acid positions. Antibodies having high (i.e. 80% or more) identity to the VH and VL regions of the antibodies described in Table 1 or Table 2 can be obtained by mutagenesis (e.g. site-directed mutagenesis or PCR-mediated mutagenesis). The encoded VH and VL regions are then tested for retained function using the functional assays described herein.

In other embodiments, the heavy chain and/or light chain variable region nucleotide sequence has 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% of the sequence shown above % or 99% identity.

As used herein, "percent identity" between two sequences is a function of the number of identical positions shared by the sequences (i.e. % identity equals number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap , this gap needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the invention may further be used as "query sequences" to be searched against public databases, for example to identify related sequences. For example, such searches can be performed with the BLAST program (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences have The amino acid sequence specified for the antibodies described herein, or conservative modifications thereof, and wherein the antibody retains the desired functional properties of the HER3 antibodies of the invention.

Accordingly, the present invention provides isolated HER3 monoclonal antibodies or fragments thereof that bind domain 3 of HER3, consisting of a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences. Region composition, wherein: heavy chain variable region CDR1 amino acid sequence is selected from SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 142 and 162, and conservative modifications thereof; heavy chain variable region CDR2 amino acid sequence Selected from SEQ ID NO:3, 23, 43, 63, 83, 103, 123, 143 and 163, and conservative modifications thereof; heavy chain variable region CDR3 amino acid sequence selected from SEQ ID NO: 4, 24, 44, 64 , 84, 104, 124, 144 and 164, and conservative modifications thereof; the light chain variable region CDR1 amino acid sequence is selected from SEQ ID NO: 8, 28, 48, 68, 88, 108, 128, 148 and 168, and Conservative modification; the light chain variable region CDR2 amino acid sequence is selected from SEQ ID NO:9, 29, 49, 69, 89, 109, 129, 149 and 169, and conservative modifications thereof; the light chain variable region CDR3 amino acid sequence is selected from SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 150 and 170, and conservative modifications thereof; the antibody or its fragment specifically binds to HER3, and inhibits HER3 activity by inhibiting the HER3 signaling pathway, the Activity can be measured in phosphorylation assays or other measures of HER signaling (eg, phospho-HER3 assays, phospho-Akt assays, cell proliferation and ligand blockade assays described in the Examples).

Accordingly, the invention provides isolated HER3 monoclonal antibodies or fragments thereof that bind domains 3-4 of HER3, consisting of a heavy chain variable region comprising CDR1 , CDR2 and CDR3 sequences and a light chain comprising CDR1 , CDR2 and CDR3 sequences Variable region composition, wherein: heavy chain variable region CDR1 amino acid sequence is selected from SEQ ID NO: 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462 , 482, 502, 522, 542, 562, 582, 602, 622, 642, 662 and 682, and conservative modifications thereof; the heavy chain variable region CDR2 amino acid sequence is selected from SEQ ID NO: 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, 523, 543, 563, 583, 603, 623, 643, 663 and 683, and conservative modifications thereof; Chain variable region CDR3 amino acid sequence is selected from SEQID NO:184,204,224,244,264,284,304,324,344,364,384,404,424,444,464,484,504,524,544, 564, 584, 604, 624, 644, 664 and 684, and conservative modifications thereof; the light chain variable region CDR1 amino acid sequence is selected from SEQ ID NO: 188, 208, 228, 248, 268, 288, 308, 328, 348 , 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568, 588, 608, 628, 648, 668 and 688, and conservative modifications thereof; the light chain variable region CDR2 amino acid sequence is selected from SEQ ID NO: 189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, 529, 549, 569, 589, 609, 629, 649, 669 and 689, and conservative modifications thereof; the light chain variable region CDR3 amino acid sequence is selected from SEQ ID NO: 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430 , 450, 470, 490, 510, 530, 550, 570, 590, 610, 630, 650, 670, and 690, and conservative modifications thereof; the antibody or fragment thereof specifically binds to HER3 and inhibits the HER3 signaling pathway to Inhibition of HER3 activity that can be measured in phosphorylation assays or other measures of HER signaling (e.g., phospho-HE as described in the Examples R3 assay, phospho-Akt assay, cell proliferation and ligand blocking assay).

Antibodies that bind the same epitope

The invention provides antibodies that interact (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with epitopes that interact with the HER3 antibodies described in Table 1 or Table 2. Bits are the same. Additional antibodies can thus be identified based on their ability to cross-compete (eg, competitively inhibit binding in a statistically significant manner) other antibodies of the invention in HER3 binding assays. The ability of a test antibody to inhibit binding of an antibody of the invention to a HER3 protein (e.g., human and/or cynomolgus HER3) demonstrates that the test antibody can compete with the antibody for binding to HER3; according to a non-limiting theory, such an antibody can compete with it The antibodies bind the same or related (eg, structurally similar or spatially close) epitopes on the HER3 protein. In a certain embodiment, the antibody that binds to the same epitope on HER3 as an antibody of the invention is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described herein.

In one embodiment, the antibody or fragment thereof binds domain 3 of HER3 and inhibits both ligand-dependent and ligand-independent HER3 signaling. In one embodiment, the antibody or fragment thereof binds domains 3-4 of HER3 and inhibits both ligand-dependent and ligand-independent HER3 signaling.

Antibodies or fragments thereof of the invention inhibit both ligand-dependent and independent activation of HER3 without preventing ligand binding. This is considered advantageous for the following reasons:

(i) Compared to antibodies targeting a single mechanism of HER3 activation (i.e., ligand-dependent or ligand-independent), therapeutic antibodies will have clinical utility in a broad spectrum of tumors, as different tumor types are affected by individual mechanisms drive.

(ii) Therapeutic antibodies will be effective in tumor types involving both mechanisms of HER3 activation. Antibodies targeting a single mechanism of HER3 activation (ie, ligand-dependent or ligand-independent) will exhibit low or no efficacy in these tumor types.

(iii) The potency of antibodies that inhibit ligand-dependent activation of HER3 without preventing ligand binding is less likely to be adversely affected by increasing ligand concentrations. This would translate into increased potency in tumor types driven by very high concentrations of HER3 ligand, or reduced propensity for drug resistance when resistance is mediated by upregulation of HER3 ligand.

(iv) Antibodies that inhibit HER3 activation by stabilizing the inactive form will be less prone to drug resistance driven by alternative mechanisms of HER3 activation.

Accordingly, the antibodies of the invention are useful in the treatment of conditions for which existing therapeutic antibodies are clinically ineffective.

Engineered and Modified Antibodies

Antibodies of the invention can also be prepared using antibodies having one or more of the VH and/or VL sequences shown herein as starting material to engineer modified antibodies that may have altered properties from the starting antibody. Antibodies can be engineered by modifying one or more residues within one or both variable domains (ie, VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues within the constant region, eg, to alter the effector functions of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, amino acid sequences within CDRs are more diverse among antibodies than sequences outside CDRs. Because the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include framework sequences grafted onto different antibodies with different properties. CDR sequences from specific naturally occurring antibodies (see, e.g., Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc.Natl Acad., U.S.A. 86:10029-10033; U.S. Patent No. 5,225,539 to Winter; and U.S. Patent Nos. 5,530,101 to Queen et al.; 5,585,089; 5,693,762 and 6,180,370).

Accordingly, another embodiment of the present invention relates to an isolated HER3 monoclonal antibody or fragment thereof that binds to Domain 3 of HER3, comprising: a heavy chain variable region each comprising A CDR1 sequence having an amino acid sequence of 42, 62, 82, 102, 122, 142 and 162; a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NO: 33, 23, 43, 63, 83, 103, 123, 143 and 163 have the CDR3 sequence of the amino acid sequence of SEQ ID NO:4,24,44,64,84,104,124,144 and 164; A CDR1 sequence having an amino acid sequence of , 48, 68, 88, 108, 128, 148 and 168; a CDR2 having an amino acid sequence selected from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149 and 169 sequence; and a CDR3 sequence having an amino acid sequence selected from SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 150 and 170.

Accordingly, another embodiment of the present invention relates to an isolated HER3 monoclonal antibody or fragment thereof that binds to domains 3-4 of HER3, comprising: a heavy chain variable region comprising a protein selected from the group consisting of SEQ ID NO: 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, 522, 542, 562, 582, 602, 622, 642, 662, and 682 The CDR1 sequence of the amino acid sequence; , 563, 583, 603, 623, 643, 663, and 683 amino acid sequences; having SEQ ID NO: 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, The CDR3 sequence of the amino acid sequence of 424, 444, 464, 484, 504, 524, 544, 564, 584, 604, 624, 644, 664 and 684; :188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568, 588, 608, 628, 648, 668 The CDR1 sequence of the amino acid sequence of and 688; Have and be selected from SEQ ID NO:189,209,229,249,269,289,309,329,349,369,389,409,429,449,469,489,509, The CDR2 sequence of the amino acid sequence of 529, 549, 569, 589, 609, 629, 649, 669 and 689; , 390, 410, 430, 450, 470, 490, 510, 530, 550, 570, 590, 610, 630, 650, 670 and 690 the CDR3 sequence of the amino acid sequence.

Such antibodies contain the VH and VL CDR sequences of monoclonal antibodies, but may also contain different framework sequences from these antibodies. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "Vbase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), and the Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1987) J.Mol.Biol.196:901-917; Chothia et al. et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J.Mol.Biol.273:927-948; Tomlinson et al., (1992) J.fol.Biol.227: 776-798; and Cox et al., (1994) Eur. J Immunol. 24:827-836; the contents of each are expressly incorporated herein by reference.

Examples of framework sequences for antibodies of the invention are those framework sequences that are structurally similar to framework sequences used by selected antibodies of the invention (eg, consensus sequences and/or framework sequences used by monoclonal antibodies of the invention). The VH CDR1, 2 and 3 sequences and the VL CDR1, 2 and 3 sequences may be grafted onto the framework regions having the same sequence as that found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences may be grafted onto In framework regions that contain one or more mutations compared to the germline sequence. For example, it has been found that in some cases it may be advantageous to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, eg, Queen et al., US Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Another type of variable region modification is to mutate amino acid residues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest, referred to as "Affinity maturity". Mutations can be introduced by site-directed or PCR-mediated mutagenesis and assessed for effects on antibody binding or other functional properties of interest in in vitro or in vivo assays as described herein and provided in the Examples. Conservative modifications (as discussed above) may be introduced. The mutation may be an amino acid substitution, addition or deletion. In addition, typically no more than 1, 2, 3, 4 or 5 residues within a CDR region are altered.

Accordingly, in another embodiment, the invention provides an isolated HER3 monoclonal antibody or fragment thereof that binds domain 3 of HER3, consisting of a heavy chain variable region having: The amino acid sequence of ID NO: 22, 22, 42, 62, 82, 102, 122, 142 and 162, or having the A VH CDR1 region consisting of 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions of amino acid sequences; Amino acid sequence, or VH having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions to the amino acid sequence compared to SEQ ID NO: 3, 23, 43, 63, 83, 103, 123, 143 and 163 CDR2 region; having an amino acid sequence selected from SEQ ID NO:4, 24, 44, 64, 84, 104, 124, 144 and 164, or with SEQ ID NO: 4, 24, 44, 64, 84, 104, 124 , 144, 164 and 370 compared to the VH CDR3 region with 1, 2, 3, 4 or 5 amino acid substitutions, deletions or added amino acid sequences; The amino acid sequence of 108, 128, 148 and 168, or having 1, 2, 3, 4 or 5 amino acid substitutions compared to SEQ ID NO: 8, 28, 48, 68, 88, 108, 128, 148 and 168, The VL CDR1 region of the amino acid sequence of deletion or addition; Have the aminoacid sequence selected from SEQ ID NO:9,29,49,69,89,109,129,149 and 169, or with SEQ ID NO:9,29,49 , 69, 89, 109, 129, 149 and 169 compared to the VL CDR2 region with 1, 2, 3, 4 or 5 amino acid substitutions, deletions or added amino acid sequences; and having a sequence selected from SEQ ID NO: 10, 30, The amino acid sequence of 50, 70, 90, 110, 130, 150, 170 and 373, or having 1, 2, 3 compared to SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 150 and 170 , 4 or 5 amino acid substitutions, deletions or additions to the VL CDR3 region of the amino acid sequence.

Accordingly, in another embodiment, the invention provides an isolated HER3 monoclonal antibody or fragment thereof that binds domains 3-4 of HER3, consisting of a heavy chain variable region having: From SEQ ID NO: 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, 522, 542, 562, 582, 602, 622 , 642, 662 and 682 amino acid sequences, or with SEQ ID NO: 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, 522, 542, 562, 582, 602, 622, 642, 662 and 682 have 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the VH CDR1 region of the amino acid sequence; NO: 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, 523, 543, 563, 583, 603, 623, 643, The amino acid sequence of 663 and 683, or with SEQ ID NO: 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, 523, 543 , 563, 583, 603, 623, 643, 663 and 683 compared to the VH CDR2 region having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or added amino acid sequences; having a sequence selected from SEQ ID NO: 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, 524, 544, 564, 584, 604, 624, 644, 664, and 684 Amino acid sequence, or sequence with SEQ ID NO: 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, 524, 544, 564, 584 , 604, 624, 644, 664 and 684 compared to the VH CDR3 region with 1, 2, 3, 4 or 5 amino acid substitutions, deletions or added amino acid sequences; , 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568 , 588, 608, 628, 648, 668 and 688 amino acid sequences, or with SEQ ID NO: 188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568, 588, 608, 628, 648, 668, and 688 have a VL CDR1 region of 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions to the amino acid sequence; Having the selected from SEQ ID NO:189,209,229,249,269,289,309,329,349,369,389,409,429,449,469,489,509,529,549,569,589,609 , 629, 649, 669 and 689 amino acid sequences, or with SEQ ID NO: 189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, 529, 549, 569, 589, 609, 629, 649, 669, and 689 VL CDR2 regions having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions to the amino acid sequence; SEQ ID NO: 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, 530, 550, 570, 590, 610, 630, The amino acid sequence of 650, 670 and 690, or with SEQ ID NO: 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, 530 , 550, 570, 590, 610, 630, 650, 670, and 690 compared to the VL CDR3 region with 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or added amino acid sequences.

Grafting of antibody fragments into alternative frameworks or scaffolds

A variety of antibody/immunoglobulin frameworks or scaffolds can be used so long as the resulting polypeptide includes at least one binding region that specifically binds HER3. Such frameworks or scaffolds include the 5 major idiotypes of human immunoglobulins or fragments thereof, and include immunoglobulins of other animal species, preferably with humanized aspects. Those skilled in the art are constantly discovering and developing new frameworks, scaffolds and fragments.

In one aspect, the invention relates to the production of non-immunoglobulin-based antibodies using non-immunoglobulin scaffolds onto which the CDRs of the invention can be grafted. Known or unknown non-immunoglobulin frameworks and scaffolds can be utilized so long as they contain binding regions specific for the target HER3 protein (eg, human and/or cynomolgus HER3). Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small module immune drugs (Trubion Pharmaceuticals Inc., Seattle, WA), giant antibodies (Avidia, Inc., Mountain View, CA), protein A (Affibody AG, Sweden) and affilin (γ-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

Fibronectin scaffolds are based on type III fibronectin domains (eg, the tenth module of type III fibronectin ( ¹⁰ Fn3 domain)). Type III fibronectin domains have 7 or 8 β-strands distributed between two β-sheets that wrap themselves around each other to form the core of the protein and also contain the The ring in (similar to CDR). There are at least three such loops at each edge of the beta-sheet sandwich, where the edge is the protein boundary perpendicular to the beta-strand orientation (see US 6,818,418). These fibronectin-based scaffolds are not immunoglobulins, although the overall fold closely resembles the minimal functional antibody fragment (heavy chain variable region comprising the entire antigen recognition unit) in camelid and llama IgG. Because of this structure, non-immunoglobulin antibodies mimic antigen-binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in an in vitro loop randomization and shuffling strategy that is similar to the affinity maturation process of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds in which the loop regions of the molecule are replaced with the CDRs of the invention using standard cloning techniques.

Ankyrin technology is based on using proteins with ankyrin-derived repeat modules as scaffolds carrying variable regions that can be used to bind different targets. The ankyrin repeat module is a 33-amino acid polypeptide composed of two antiparallel α-helices and β-turns. Binding of variable regions was maximally optimized by using ribosome display.

Avimers are derived from proteins containing native A domains, such as HER3. These domains are used naturally in protein-protein interactions, and in humans over 250 proteins are structurally based on A domains. Avimers consist of a large number of different "A domain" monomers (2-10) linked by amino acid linkers. Avimers that can bind a target antigen can be generated using methods described, for example, in US Patent Application Publication Nos. 20040175756, 20050053973, 20050048512, and 20060008844.

Affibody affinity ligands are small simple proteins composed of three-helical bundles based on a scaffold of one IgG-binding domain of protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. The scaffold domain consists of 58 amino acids, 13 of which were randomized to generate an affibody library with a large number of ligand variants (see eg US5,831,012). Affibody molecules mimic antibodies and they have a molecular weight of 6kDa compared to the molecular weight of antibodies of 150kDa. Despite its small size, the binding site of the affibody molecule is similar to that of an antibody.

Anticalins are products developed by Pieris ProteoLab AG. They are derived from lipocalins, a large class of small, robust proteins that are often involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins are present in human tissues or body fluids. The protein structure indicated a hypervariable loop immunoglobulin on a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins consist of a single polypeptide chain of 160 to 180 amino acid residues and are only slightly larger than a single immunoglobulin domain. The tetracyclic group constituting the binding pocket shows marked structural plasticity and tolerates a wide variety of side chains. Binding sites are thus remodelable in proprietary methods to recognize defined target molecules of different shapes with high affinity and specificity. A protein of the lipocalin family, the bilin binding protein (BBP) of the European white butterfly (Pieris Brassicae), has been used to develop anticalins by mutagenizing the tetracyclic group. An example of a patent application describing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins designed for specific affinity for proteins and small molecules. New affilin molecules can be selected very quickly from two libraries, each based on a different scaffold protein from humans. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are utilized, one of which is gamma crystallin, a human structural lens protein, and the other is a protein of the "ubiquitin" superfamily. Both human scaffolds are very small, exhibit high temperature stability, and are virtually resistant to pH changes and denaturants. This high stability is mainly due to the protein's extended beta-sheet structure. Examples of gamma-crystallin-derived proteins are described in WO200104144 and examples of "ubiquitin-like" proteins are described in WO2004106368.

Protein epitope mimics (PEMs) are medium-sized, circular, peptide-like molecules (MW 1-2kDa) that mimic the secondary structure of protein beta hairpins, the main secondary structure involved in protein-protein interactions. level structure.

In some embodiments, the Fab is converted to a silent IgGl form by altering the Fc region. For example, antibodies in Table 1 or Table 2 can be converted to IgG form.

Human Antibody or Humanized Antibody

The invention provides fully human antibodies that specifically bind to a HER3 protein (eg, human and/or cynomolgus/mouse/rat HER3). A human HER3 antibody or fragment thereof has further reduced antigenicity when administered to a human individual compared to a chimeric antibody or a humanized antibody.

Human HER3 antibodies or fragments thereof can be produced using methods known in the art. For example, human engineering techniques are used to convert non-human antibodies into engineered human antibodies. US Patent Publication No. 20050008625 describes an in vivo method for replacing the variable regions of a non-human antibody with human variable regions in an antibody while maintaining the same or providing better binding characteristics than those of the non-human antibody. This method relies on the epitope-directed replacement of the variable region of a non-human reference antibody with a fully human antibody. The resulting human antibody is generally not structurally related to the reference non-human antibody, but binds to the same epitope on the same antigen as the reference antibody. Briefly, in the presence of a reporter system responsive to the binding of the test antibody to the antigen, a limited amount of antigen is bound by establishing in cells a library of multiple hybrids of "competitors" and reference antibodies ("test antibodies"). competition, enabling a sequential epitope-guided complementarity replacement approach. The competitor may be a reference antibody or a derivative thereof, such as a single chain Fv fragment. The competitor can also be a natural or artificial antigen ligand that binds to the same epitope as the reference antibody. The only requirement for this competitor is that it binds to the same epitope as the reference antibody and that it competes with the reference antibody for antigen binding. The test antibodies have one common antigen-binding V region from a non-human reference antibody, and other V regions randomly selected from a variety of sources, such as a repertoire library of human antibodies. The common V region from the reference antibody serves as a guide to position the test antibody on the same epitope on the antigen, and in the same orientation, biasing the selection towards the highest antigen binding fidelity to the reference antibody.

Many types of reporter systems can be used to detect the desired interaction between the test antibody and the antigen. For example, complementary reporter fragments can be linked to antigen and test antibody separately such that reporter activation by fragment complementarity occurs only when the test antibody binds the antigen. When the test antibody and the antigen-reporter fragment fusion are co-expressed with the competitor, reporter activation becomes dependent on the ability of the test antibody to compete with the competitor, which is proportional to the affinity of the test antibody for the antigen. Other reporter systems that can be used include reactivators of the autoinhibited reporter reactivation system (RAIR) as disclosed in U.S. Patent Application Serial No. 10/208,730 (Publication No. 20030198971), or in U.S. Patent Application Serial No. 10/076,845 Competition activation system disclosed in (publication number 20030157579).

Using a sequential epitope-directed complementarity replacement system, selection is performed to identify cells expressing individual test antibodies together with competitor, antigen and reporter components. In these cells, each test antibody competes one-to-one with competitors for binding a limited amount of antigen. The activity of the reporter is proportional to the amount of antigen bound to the test antibody, which in turn is proportional to the affinity of the test antibody for the antigen and the stability of the test antibody. When expressed as a test antibody, the test antibody is initially selected on the basis of its activity relative to the reference antibody. The result of the first round of selection is a panel of "hybrid" antibodies, in which each antibody comprises the same non-human V region from the reference antibody and a human V region from the library, and in which each antibody binds to the same expression on the antigen as the reference antibody. bit. The one or more hybrid antibodies selected in the first round will have an affinity for the antigen that is comparable to or higher than the affinity of the reference antibody for the antigen.

In the second V region replacement step, the human V region selected in the first step is used as a guide for selecting a human replacement that replaces the remaining non-human reference antibody V region with a diverse library of cognate human V regions. Hybrid antibodies selected in the first round can also be used as competitors for the second round of selection. The result of the second round of selection is a panel of fully human antibodies that differ structurally from, but compete with, the reference antibody for binding to the same antigen. Some selected human antibodies bind to the same epitope on the same antigen as the reference antibody. Among these selected human antibodies, one or more antibodies bind the same epitope with an affinity comparable to or greater than that of the reference antibody.

Using one of the mouse or chimeric HER3 antibodies or fragments thereof described above as a reference antibody, this method can be readily exploited to generate human antibodies that bind human HER3 with the same binding specificity and with the same or better binding affinity . In addition, such human HER3 antibodies or fragments thereof are also commercially available from companies that generally produce human antibodies, such as KaloBios, Inc. (Mountain View, CA).

camelid antibody

From members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) families, including New World members such as llama species (Lama paccos, llama (Lama glama) and lean camel (Lama vicugna)) Antibody proteins have been characterized with respect to size, structural complexity, and antigenicity to human subjects. Certain IgG antibodies from this family of mammals found in nature lack light chains and thus differ structurally from the typical four-chain quaternary structure of antibodies from other animals with two heavy and two light chains. See PCT/EP93/02214 (WO 94/04678 published March 3, 1994).

The regions of camelid antibodies identified as small single variable domains of VHH can be genetically engineered to generate small proteins with high affinity for targets, resulting in low molecular weight antibody-derived proteins known as "camelid nanobodies". See U.S. Patent No. 5,759,808 issued June 2, 1998; see also Stijlemans et al., (2004) J Biol Chem 279:1256-1261; Dumoulin et al., (2003) Nature 424:783-788; Pleschberger et al., (2003) Bioconjugate Chem 14:440-448; Cortez-Retamozo et al., (2002) Int J Cancer 89:456-62; and Lauwereys et al., (1998) EMBO J 17:3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. (For example, US20060115470; Domantis (US20070065440, US20090148434). Like other antibodies of non-human origin, the amino acid sequence of the camelid antibody can be recombinantly changed to obtain a sequence more similar to the human sequence, that is, the nanobody can be "humanized". Therefore, the natural low antigenicity of camelid antibodies to humans can be further reduced.

Camelid nanobodies have a molecular weight roughly one-tenth that of a human IgG molecule, and the protein is only a few nanometers in physical diameter. One consequence of the small size is that camelid nanobodies bind antigenic sites that are not functionally visible to larger antibody proteins, i.e. camelid nanobodies can be used as detection reagents for antigens that cannot be detected using classical immunological techniques and may Used as a therapeutic agent. Thus, another consequence of the small size is that camelid nanobodies can thus inhibit binding to specific heterosites in the groove or slit of the target protein, and thus can have the ability to behave more like classical low molecular weight drugs than classical antibodies function.

The low molecular weight and compact size also lead to camelid nanobodies being extremely thermostable, stable to extreme pH and proteolytic digestion, and weakly antigenic. Another consequence is that camelid nanobodies readily transfer from the circulatory system to tissues, and even cross the blood-brain barrier, potentially treating disorders affecting nervous tissue. Nanobodies can further facilitate drug transport across the blood-brain barrier. See US Patent Application 20040161738 published August 19, 2004. These features combined with low antigenicity to humans indicate great therapeutic potential. In addition, these molecules can be fully expressed in prokaryotic cells, such as E. coli, and expressed as fusion proteins using phage, and are functional.

Thus, the invention features camelid antibodies or nanobodies with high affinity for HER3. In certain embodiments herein, the camelid antibody or Nanobody is produced naturally in a camelid animal by immunizing a camelid with HER3 or a peptide fragment thereof using techniques described herein for other antibodies. Alternatively, camelid nanobodies engineered to bind HER3, i.e. panning methods using HER3 as a target as described in the Examples herein, generate HER3-binding nanobodies by, for example, selection from a phage display library of appropriately mutagenized camelid nanobody proteins. camelid nanobody. Engineered Nanobodies can be further tailored by genetic engineering to have a half-life in recipient individuals ranging from 45 minutes to two weeks. In a specific embodiment, camelid antibodies or nanobodies are obtained by grafting the CDR sequences of the heavy or light chains of a human antibody of the invention into a Nanobody or single domain antibody framework sequence, as described, for example, in PCT/EP93/02214. Antibody. In one embodiment, the camelid antibody or Nanobody binds to at least one amino acid residue in domain 3 of HER3 selected from the following amino acid residues: 335-342, 362-376, 398, 400, 424-428, 431 , 433-434 and 455 (within domain 3), or a subset thereof. In one embodiment, the camelid antibody or Nanobody binds to at least one residue in domain 3 of HER3 selected from the following amino acid residues: 571, 582-584, 596-597, 600-602, 609-615 ( Domain 4), or a subset thereof.

Bispecific Molecules and Multivalent Antibodies

In another aspect, the invention relates to a biparatopic, bispecific or multispecific molecule comprising an antibody or fragment thereof that binds a non-linear or conformational epitope within domain 3 of HER3. In another aspect, the biparatopic, bispecific or multispecific molecule comprises an antibody or fragment thereof that binds an epitope within domain 4 of HER3. The antibody or fragment thereof can be derivatized or linked to another functional molecule (e.g., another peptide or protein (e.g., another antibody or ligand for a receptor)) to generate an antibody that binds at least two different binding sites or target molecules. bispecific molecules. The antibody or fragment thereof may actually be derivatized or linked to more than one other functional molecule to create a biparatopic or multispecific molecule that binds more than two different binding sites and/or target molecules; such biparatopic or multispecific molecules. To generate bispecific molecules, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment , peptides or binding mimetics, allowing the generation of bispecific molecules.

Additional clinical benefit may be provided by binding two or more antigens within one antibody (Coloma et al., (1997); Merchant et al., (1998); Alt et al., (1999); Zuo et al., (2000) ; Lu et al., (2004); Lu et al., (2005); Marvin et al., (2005); Marvin et al., (2006); Shen et al., (2007); Wu et al., (2007); et al., (2009); Michaelson et al., (2009)). (Morrison et al., (1997) Nature Biotech.15:159-163; Alt et al. (1999) FEBS Letters 454:90-94; Zuo et al. , (2000) Protein Engineering 13:361-367; Lu et al., (2004) JBC 279:2856-2865; Lu et al., (2005) JBC 280:19665-19672; Marvin et al., (2005) Acta Pharmacologica Sinica 26: 649-658; Marvin et al., (2006) Curr Opin Drug Disc Develop 9:184-193; Shen et al., (2007) J Immun Methods 218:65-74; Wu et al., (2007) Nat Biotechnol.11:1290 -1297; Dimasi et al., (2009) J Mol Biol. 393:672-692; and Michaelson et al., (2009) mAbs 1:128-141.

Bispecific molecules can be prepared by conjugating components with binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be produced separately and then conjugated to each other, eg, covalently, using a variety of coupling or cross-linking agents. Examples of crosslinkers include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrophenyl Formic acid) (DTNB), o-phenylene dimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and sulfosuccinate Imido 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., (1984) J. Exp. Med. 160:1686; Liu et al., (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those in Paulus (1985) Behring Ins. Mitt. No. 78:118-132; Brennan et al., (1985) Science 229:81-83), and Glennie et al., (1987) J. Immunol.139: 2367-2375). The conjugating agents were SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

In the case of antibodies, they can be conjugated via sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains. In specific embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, eg, 1, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector, expressed and assembled in the same host cell. This approach is especially useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab') ₂ or Ligand x Fab fusion protein. A bispecific molecule of the invention may be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. A bispecific molecule may comprise at least two single chain molecules. US Patent No. 5,260,203; US Patent No. 5,455,030; US Patent No. 4,881,175; US Patent No. 5,132,405; US Patent No. 5,091,513; Methods for making bispecific molecules.

Binding of bispecific molecules to their specific targets can be verified by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, biological assay (eg, growth inhibition), or Western blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by the use of labeled reagents (eg, antibodies) specific for the complexes of interest.

In another aspect, the invention provides a multivalent compound comprising at least two identical or different fragments of an antibody that binds HER3. The antibody fragments can be linked together by protein fusion or covalent or non-covalent linkages. Tetravalent compounds are obtained, for example, by cross-linking an antibody of an antibody of the invention with an antibody that binds a constant region (eg, Fc or hinge region) of an antibody of the invention. Trimerization domains are described eg in Borean patent EP 1012280B1. Pentameric modules are described eg in PCT/EP97/05897.

In one embodiment, the biparatopic/bispecific antibody binds at least amino acid residues in domain 3 of HER3 selected from amino acid residues: 335-342, 362-376, 398, 400, 424-428 , 431, 433-434, and 455 (within domain 3), or a subset thereof. In one embodiment, the biparatopic/bispecific antibody binds at least amino acid residues in domain 3 of HER3 selected from amino acid residues: 571, 582-584, 596-597, 600-602, 609 -615 (domain 4), or a subset thereof.

In another embodiment, the invention relates to bifunctional antibodies, wherein a single monoclonal antibody is modified such that the antigen binding site binds more than one antigen, such as one that binds both HER3 and another antigen (e.g., HER1, HER2, and HER4). Bifunctional antibody. In another embodiment, the invention relates to diabodies that target an antigen that has the same conformation, eg, an antigen that has the same conformation as HER3 in the "blocked" or "inactive" state. Examples of antigens that have the same conformation as HER3 in the "blocked" or "inactive" state include, but are not limited to, HER1 and HER4. Thus, a diabody can bind both HER3 and HER1; both HER3 and HER4; or both HER1 and HER4. The dual binding specificities of diabodies can be further translated into dual activity, or dual activity inhibition. (See eg, Jenny Bostrom et al., (2009) Science: 323; 1610-1614).

Antibodies with extended half-life

The present invention provides antibodies or fragments thereof that specifically bind a non-linear or conformational epitope within domain 3 of HER3 having an extended half-life in vivo. The present invention also provides antibodies that specifically bind an epitope within domain 4 of HER3 that have an extended half-life in vivo.

Many factors can affect the half-life of a protein in the body. For example, renal filtration, metabolism in the liver, degradation by proteolytic enzymes (proteases), and immunogenic responses (eg, protein neutralization by antibodies and uptake by macrophages and dendritic cells). Various strategies can be used to extend the half-life of the antibodies of the invention. For example, by chemically linking polyethylene glycol (PEG), reCODEPEG, antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and a protective carbohydrate layer; by binding to serum proteins ( Proteins such as albumin, IgG, FcRn) are genetically fused and transferred; by conjugation (genetically or chemically) to other binding moieties that bind serum proteins such as Nanobodies, Fab, DARPin, avimer, affibody and anticalin ; by genetic fusion of rPEG, albumin, domains of albumin, albumin binding protein and Fc; or by incorporation into nanocarriers, sustained release formulations or medical devices.

To prolong the serum circulation of antibodies in vivo, the inert polymeric molecule can be inertly polymerized by site-specific conjugation of PEG to the N- or C-terminus of the antibody or via the epsilon amino groups present on lysine residues, with or without the use of a multifunctional linker. (such as high molecular weight PEG) attached to antibodies or fragments thereof. To pegylate an antibody, the antibody or fragment thereof is typically combined with polyethylene glycol (PEG) (such as an active ester or aldehyde derivative of PEG) under conditions in which one or more PEG groups become attached to the antibody or antibody fragment react below. PEGylation can be performed with reactive PEG molecules (or similar reactive water-soluble polymers) by acylation or alkylation. The term "polyethylene glycol" as used herein is intended to include any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10) alkoxy or aryloxy-polyethylene glycol or polyethylene glycol Alcohol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Use linear or branched polymer derivatizations that result in minimal loss of biological activity. The extent of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion chromatography or by ion exchange chromatography. PEG-derived antibodies can be tested for binding activity and in vivo efficacy using methods well known to those skilled in the art, eg, by immunoassays described herein. Methods of PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401384 by Ishikawa et al.

Other modified PEGylation techniques include Reconstitution Chemical Orthogonal Directed Engineering (ReCODE PEG), which incorporates chemically specified side chains into biosynthetic proteins through a remodeling system involving tRNA synthetase and tRNA. This technique enables the incorporation of more than 30 novel amino acids into biosynthetic proteins in E. coli, yeast and mammalian cells. This tRNA incorporates the unnatural amino acid wherever the amber codon is located, converting the amber codon from a stop codon to a codon that signals the incorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) can also be used for serum half-life extension. The technology involves the genetic fusion of unstructured protein tails of 300-600 amino acids to existing drug proteins. Since the apparent molecular weight of this unstructured protein chain is about 15 times larger than its actual molecular weight, the serum half-life of the protein is greatly increased. Unlike traditional PEGylation, which requires chemical conjugation and repurification, the preparation method is extremely simplified and the product is homogeneous.

Polysialylation is another technology that uses the natural polymer polysialic acid (PSA) to extend the lifespan and increase the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment for conjugation. This increases the active period of the therapeutic protein in circulation and prevents it from being recognized by the immune system. PSA polymers are naturally found in the human body. It is adopted by certain bacteria, which have evolved over millions of years, to coat their walls with this PSA polymer. Through molecular mimicry, these naturally polysialylated bacteria are then able to thwart the body's defense system. PSA is nature's ultimate stealth technology, readily produced in large quantities from such bacteria and possessing predetermined physical characteristics. Even when conjugated to proteins, bacterial PSA is completely non-immunogenic because it is chemically identical to PSA in humans.

Another technique involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy cornstarch and is metabolized by the body's enzymes. HES solutions are generally administered to replace deficient blood volume and to improve the rheological properties of blood. Hesylation of antibodies enables prolonged circulatory half-life by increasing the stability of the molecule, as well as by reducing renal clearance, resulting in enhanced biological activity. A wide range of HES antibody conjugates can be customized by varying various parameters, such as the molecular weight of HES.

Antibodies with increased in vivo half-life may also be produced by introducing one or more amino acid modifications (ie, substitutions, insertions or deletions) into an IgG constant domain or FcRn-binding fragment thereof (preferably an Fc or hinge Fc domain fragment). See, eg, International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Patent No. 6,277,375.

Additionally, antibodies can be conjugated to albumin to make the antibody or antibody fragment more stable in vivo or have a longer half-life in vivo. The technique is well known in the art, see for example International Publication Nos. WO 93/15199, WO 93/15200 and WO 01/77137; and European Patent No. EP 413,622.

HER3 antibodies or fragments thereof can also be fused to one or more human serum albumin (HSA) polypeptides or portions thereof. The mature form of HSA is a 585 amino acid protein responsible for the majority of serum osmolarity and also serves as a carrier for endogenous and exogenous ligands. The function of albumin as a carrier molecule and its inert nature are ideal properties for use as a carrier and transporter of polypeptides in vivo. The use of albumin as a carrier for various proteins as a component of albumin fusion proteins has been proposed in WO 93/15199, WO 93/15200 and EP 413 622. It has also been proposed to fuse an N-terminal fragment of HSA to a polypeptide (EP399 666). Thus, by genetically or chemically fusing or conjugating antibodies or fragments thereof to albumin, it is possible to stabilize or extend shelf-life, and/or maintain molecular activity in solution, in vitro, and/or in vivo for extended periods of time.

Fusion of albumin to another protein can be achieved by genetic manipulation such that the DNA encoding HSA or a fragment thereof is linked to the DNA encoding the protein. A suitable host is then transformed or transfected with the fusion nucleotide sequence, which is arrayed on a suitable plasmid to express the fusion polypeptide. Expression can be in vitro, eg from prokaryotic or eukaryotic cells, or in vivo from transgenic organisms. Other methods involving HSA fusions can be found, for example, in WO 2001077137 and WO 200306007, incorporated herein by reference. In specific embodiments, expression of the fusion protein is performed in a mammalian cell line, such as a CHO cell line. Altered differential binding of antibodies to receptors at low or high pH is also contemplated within the scope of the present invention. For example, by modifying the antibody by including additional amino acids (such as histidine) in its CDRs, the affinity of the antibody can be modified so that it remains bound to its receptor at low pH (such as in lysosomes) (See eg, Tomoyuki Igawa et al. (2010) Nature Biotechnology; 28, 1203-1207).

antibody conjugate

The present invention provides an antibody or fragment thereof that specifically binds HER3, which antibody or fragment thereof is recombinantly fused or chemically conjugated (including covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, preferably at least 10, A polypeptide of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to produce a fusion protein. In particular, the invention provides antibody fragments (e.g., Fab fragments, Fd fragments, Fv fragments, F(ab)2 fragments, VH domains, VH CDRs, VL domains, or VL CDRs) and heterologous proteins comprising antibody fragments described herein. , polypeptide or peptide fusion protein. Methods for fusing or conjugating proteins, polypeptides or peptides to antibodies or antibody fragments are known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., (1991) .Acad.Sci.USA 88:10535-10539; Zheng et al., (1995), J.Immunol.154:5590-5600; and Vil et al., (1992), Proc.Natl.Acad.Sci.USA 89:11337- 11341.

Other fusion proteins can be produced by the techniques of gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of an antibody or fragment thereof of the invention (eg, an antibody or fragment thereof with higher affinity and lower off-rate). See generally U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., (1997), Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998), Trends Biotechnol. 16(2):76-82 ; Hansson et al., (1999), J.Mol.Biol.287:265-76; and Lorenzo and Blasco, (1998), Biotechniques24(2):308-313 (each of these patents and publications in their entirety incorporated by reference). Antibodies or fragments thereof, or encoded antibodies or fragments thereof, may be altered prior to recombination by random mutagenesis by error-prone PCR, random nucleotide insertion or other methods. A polynucleotide encoding an antibody or fragment thereof that specifically binds a HER3 protein may be recombined with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

In addition, antibodies or fragments thereof can be fused to marker sequences (eg, peptides) to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. As described in Gentz et al., (1989), Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine facilitates the purification of fusion proteins. Other peptide tags that can be used for purification include, but are not limited to, hemagglutinin ("HA") tags corresponding to epitopes derived from the influenza hemagglutinin protein (Wilson et al., (1984), Cell 37:767) and "flag" tag.

In other embodiments, an antibody or fragment thereof of the invention is conjugated to a diagnostic or detection agent. Such antibodies may be used as part of a clinical testing program (eg, to determine the efficacy of a particular therapy) for monitoring or prognosing the onset, development, progression and/or severity of a disease or disorder. Such diagnosis and detection can be accomplished by conjugating antibodies to detectable substances including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galact Glycosidase, or acetylcholinesterase; prosthetic groups such as but not limited to streptavidin-biotin and avidin/biotin; fluorescent substances such as but not limited to umbelliferone, fluorescein, isothiocyanate Fluorescein, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent substances such as but not limited to luminol; bioluminescent substances such as but not limited to luciferase, luciferin and Aequorin; radioactive substances such as but not limited to iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I and ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ In, ¹¹³ In, ¹¹² In and ¹¹¹ In,), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, 47Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, and ¹¹⁷ Tin; and various positron-emitting metals using positron emission tomography, and nonradioactive Paramagnetic metal ions.

The invention also encompasses the use of antibodies or fragments thereof conjugated to therapeutic moieties. Antibodies or fragments thereof may be conjugated to therapeutic moieties, such as cytotoxins (eg, cytostatic or cytocidal agents), therapeutic agents, or radioactive metal ions, eg, alpha particle emitters. A cytotoxin or cytotoxic agent includes any substance that is harmful to cells.

Additionally, antibodies or fragments thereof may be conjugated to therapeutic or drug moieties that modify a given biological response. Therapeutic or drug moieties are not to be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein, peptide or polypeptide having the desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon , nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agent, anti-angiogenic agent; or a biological response modifier such as a lymphokine. In one embodiment, the HER3 antibody or fragment thereof is conjugated to a therapeutic moiety, such as a cytotoxin, drug (eg, immunosuppressant), or radiotoxin. Such conjugates are referred to herein as "immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any substance that is harmful to (eg, kills) cells. Examples include taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin Daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, Lidocaine, propranolol, and puromycin and their analogs or homologues. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, arabinoside, 5-fluorouracil dacarbazine), ablating agents ( For example, mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and rolomustine (CCNU), cyclophosphamide, busulfan, bromomannitol, streptozotocin , mitomycin C and cis-dichlorodiamminoplatinum(II) (DDP) cisplatin, anthracyclines (eg, daunorubicin (formerly known as normycin) and doxorubicin), antibiotics (eg, , dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthranimycin (AMC)) and antimitotic agents (eg, vincristine and vinblastine). (See eg , Seattle Genetics US20090304721).

Other examples of therapeutic cytotoxins that can be conjugated to the antibodies or fragments thereof of the invention include duocarmycin, calicheamicin, maytansine, and auristatin, and derivatives thereof. Examples of calicheamicin antibody conjugates are commercially available (MylotargTm; Wyeth-Ayerst).

Cytotoxins can be conjugated to antibodies or fragments thereof of the invention using linker techniques available in the art. Examples of linker types that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. Linkers can be chosen that are readily cleavable, for example, by low pH within the lysosomal compartment, or by proteases (such as proteases that are preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsins B, C, D)) cutting.

For further discussion of cytotoxins, types of linkers, and methods for conjugating therapeutic agents to antibodies see also Saito et al., (2003) Adv.Drug Deliv.Rev.55:199-215; Trail et al., (2003 ) Cancer Immunol.Immunother.52:328-337; Payne, (2003) Cancer Cell 3:207-212; Allen, (2002) Nat.Rev.Cancer 2:750-763; Pastan and Kreitman, (2002) Curr.Opin Investig. Drugs 3:1089-1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev. 53:247-264.

Antibodies or fragments thereof of the invention may also be conjugated to radioactive isotopes to produce cytotoxic radiopharmaceuticals, also known as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ⁹⁰ , and lutetium ¹⁷⁷ . Methods for preparing radioimmunoconjugates are well known in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin ^™ (DEC Pharmaceuticals) and Bexxar ^™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), which can be Molecules attach to antibodies. Such linker molecules are well known in the art and described in Denardo et al., (1998), Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999), Bioconjug.Chem.10(4):553-90; 7; and Zimmerman et al., (1999), Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference in its entirety.

Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", Controlled Drug Delivery (2nd Ed. Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Lab Of The Therapeutic Antibodies Use Ofed Cancer Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al (eds), pp. 303-16 (Academic Press 1985); and Thorpe et al, (1982), Immunol. Rev. 62:119-58.

Antibodies can also be attached to solid supports, especially for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Antibody combination

In another aspect, the invention relates to the use of a HER3 antibody or fragment thereof of the invention in combination with other therapeutic agents, such as another antibody, a small molecule inhibitor, an mTOR inhibitor, or a PI3 kinase inhibitor. Examples include but are not limited to the following:

HER1 Inhibitors: HER3 antibodies or fragments thereof may be used with HER1 inhibitors including, but not limited to, Matuzumab (EMD72000), Cetuximab (Imclone), Panitumumab (Amgen), mAb 806 and Nimotuzumab (TheraCIM), Gefitinib (Astrazeneca); CI-1033 (PD183805) (Pfizer), lapatinib (GW-572016) (GlaxoSmithKline), lapatinib ditosylate (SmithKlineBeecham), Erlotinib hydrochloride (OSI-774) (OSI Pharma), PKI-166 (Novartis) and N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3″S ")-tetrahydro-3-furyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide (provided by Boehringer Ingelheim under the trade name sell).

HER2 Inhibitors: HER3 antibodies or fragments thereof may be used with HER2 inhibitors including, but not limited to, Pertuzumab (provided by Genentech under the trademark sold), trastuzumab (trademarked by Genentech/Roche sold), MM-111, neratinib (also known as HKI-272, (2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl] Amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide, described in PCT Publication No. WO 05/028443), lapatide Nicilin or lapatinib ditosylate (trademarked by GlaxoSmithKline sell).

HER3 Inhibitors: HER3 antibodies or fragments thereof may be used with HER3 inhibitors including, but not limited to, MM-121, MM-111, IB4C3, 2DID12 (U3Pharma AG), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech) and small molecules that inhibit HER3.

HER4 Inhibitors: HER3 antibodies or fragments thereof may be used with HER4 inhibitors.

PI3K inhibitors: HER3 antibodies or fragments thereof may be used with PI3 kinase inhibitors including, but not limited to, 4-[2-(1H-indol-4-yl)-6-[[4- (Methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941, in PCT Publication Nos. WO09/036082 and WO 09/055730 described in), 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c ]quinolin-1-yl]phenyl]propionitrile (also known as BEZ 235 or NVP-BEZ235, described in PCT Publication No. WO 06/122806), BKM120 and BYL719.

mTOR Inhibitors: HER3 antibodies or fragments thereof may be used with mTOR inhibitors including, but not limited to, temsirolimus (produced by Pfizer under the trade name sold), ridaforolimus (formerly deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E, 28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20 -Pentoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexadecane-16,24,26,28-tetraen-12-yl]propyl]- 2-Methoxycyclohexyldimethylphosphonate, also known as rapamycin, AP23573 and MK8669 (Ariad Pharm.), described in PCT Publication No. WO 03/064383), everolimus (RAD001) ( by Novartis under the trade name sell). One or more therapeutic agents can be administered simultaneously with, or before or after administration of, a HER3 antibody or fragment thereof of the invention.

Methods of Producing Antibodies of the Invention

(i) nucleic acid encoding an antibody

The invention provides substantially purified nucleic acid molecules encoding polypeptides comprising segments or domains of the HER3 antibody chains described above. Some nucleic acids of the invention comprise a nucleotide sequence encoding a heavy chain variable region of a HER3 antibody, and/or a nucleotide sequence encoding a light chain variable region. In specific embodiments, the nucleic acid molecules are those identified in Table 1 or Table 2. Some other nucleic acid molecules of the invention comprise nucleotide sequences that are substantially identical (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%) to those nucleic acid molecules identified in Table 1 or Table 2. ) nucleotide sequence. The polypeptides encoded by these polynucleotides are capable of displaying HER3 antigen binding ability when expressed from an appropriate expression vector.

The invention also provides polynucleotides encoding at least one CDR region and typically all three CDR regions from the heavy or light chain of the antibodies or fragments thereof set forth above. Some other polynucleotides encode all or substantially all of the variable region sequences of the heavy and/or light chains of the antibodies or fragments thereof set forth above. Due to codon degeneracy, multiple nucleic acid sequences will encode each immunoglobulin amino acid sequence.

Nucleic acid molecules of the invention may encode both variable and constant regions of antibodies. Some nucleic acid sequences of the invention comprise nucleotides encoding a mature heavy chain variable region sequence substantially identical to the mature heavy chain variable region sequence of a HER3 antibody shown in Table 1 or Table 2 (eg, at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%). Some other nucleic acid sequences comprise nucleotides encoding a mature light chain variable region sequence that is substantially identical to the mature light chain variable region sequence of the HER3 antibody shown in Table 1 or Table 2 (e.g. , at least 80%, 90%, 95%, 96%, 97%, 98% or 99%).

Polynucleotide sequences can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of existing sequences encoding antibodies or fragments thereof. Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., (1979), Meth.Enzymol.68:90; Brown et al., (1979) Meth.Enzymol.68:109 the phosphodiester method; the diethylphosphoramidite method of Beaucage et al., (1981) Tetra. Lett., 22:1859; and the solid support method of US Patent No. 4,458,066. According to PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (editor), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al (editors), Academic Press, San Diego, CA, 1990; Introduction of mutations into polynucleotide sequences by PCR is performed as described in Mattila et al., (1991) Nucleic Acids Res. 19:967; and Eckert et al., (1991) PCR Methods and Applications 1:17.

The invention also provides expression vectors and host cells for producing antibodies or fragments thereof. A variety of expression vectors can be used to express polynucleotides encoding HER3 antibody chains or fragments thereof. Both viral-based and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (often with an expression cassette for expression of protein or RNA), and artificial chromosomes (see, eg, Harrington et al., (1997) Nat Genet 15:345). For example, non-viral vectors for expressing HER3 polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B&C, pcDNA3.1/His, pEBVHis A, B&C (Invitrogen, San Diego, CA), MPSV vector and many others known in the art for expressing other proteins. Useful viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus based vectors, SV40, papillomavirus, HBP Epstein-Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV) based vectors. See, Brent et al., (1995) supra; Smith, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., (1992) Cell 68:143.

The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (eg, enhancers) operably linked to the polynucleotide encoding the antibody chain or fragment thereof. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. The culture of transformed organisms can be expanded under non-inducing conditions without favoring the population of coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of the antibody chain or fragment thereof. These elements typically include the ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression can be enhanced by including an enhancer appropriate to the cell system used (see, e.g., Scharf et al., (1994) Results Probl. Cell Differ. 20:125; and Bittner et al., (1987) Meth. Enzymol ., 153:516). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

The expression vector may also provide a location for a secretion signal sequence to form a fusion protein with the inserted polypeptide encoded by the antibody or fragment. More often, the inserted antibody or fragment sequence is linked to a signal sequence prior to inclusion into the vector. Vectors to be used to receive sequences encoding the antibody or fragment light and heavy chain variable domains sometimes also encode constant regions or portions thereof. Such vectors allow expression of the variable regions as fusion proteins with constant regions, thereby resulting in the production of intact antibodies or fragments thereof. Typically, such constant regions are human constant regions.

Host cells for containing and expressing the antibody or fragment chains can be prokaryotic or eukaryotic. E. coli is a prokaryotic host for cloning and expressing the polynucleotides of the invention. Other microbial hosts suitable for use include Bacillus, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which generally contain expression control sequences (eg, origins of replication) compatible with the host cell. In addition, any number of various well-known promoters will be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from lambda phage. The promoter typically controls expression, optionally with an operator sequence, and has ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express antibodies or fragments thereof. Insect cells can also be used in combination with baculovirus vectors.

In some preferred embodiments, mammalian host cells are used to express and produce antibodies or fragments thereof. For example, they may be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines containing exogenous expression vectors. These include any normally dead or normally or abnormally immortal animal or human cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures for expression of polypeptides is generally discussed in, eg, Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for use in mammalian host cells can include expression control sequences, such as origins of replication, promoters, and enhancers (see, e.g., Queen et al., (1986) Immunol. Rev. 89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors generally contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific, and/or adjustable or regulatable. Useful promoters include, but are not limited to, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone-inducible MMTV promoter, SV40 promoter, MRP polIII promoter, constitutive MPSV promoter, tetracycline-inducible CMV promoters (such as the human immediate early CMV promoter), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

The method for introducing an expression vector containing a polynucleotide sequence of interest differs depending on the type of cellular host. For example, calcium chloride transfection is commonly used in prokaryotic cells, while calcium phosphate treatment or electroporation can be used in other cellular hosts. (See generally Sambrook, et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, biolistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, Artificial virions, fusions to the herpesvirus structural protein VP22 (Elliot and O'Hare, (1997) Cell 88:223), active agents of DNA enhance uptake and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will generally be desired. For example, cell lines stably expressing antibody chains or fragments can be prepared using expression vectors of the invention containing viral origins of replication or endogenous expression elements and selectable marker genes. Following vector introduction, cells can be grown in enriched media for 1-2 days before switching to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows cells that successfully express the introduced sequence to grow in selective media. Resistant stably transfected cells can be propagated using tissue culture techniques appropriate to the cell type.

(ii) Production of monoclonal antibodies of the present invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methods such as the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256:495. Many techniques for producing monoclonal antibodies are available, eg, viral or oncogenic transformation of B lymphocytes.

The animal system used to prepare hybridomas is the murine system. Generation of hybridomas in mice is a well established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest using standard molecular biology techniques and engineered to contain non-murine (eg, human) immunoglobulin sequences. For example, to generate chimeric antibodies, murine variable regions can be linked to human constant regions using methods known in the art (see, eg, US Patent No. 4,816,567 to Cabilly et al.). To generate humanized antibodies, the murine CDR regions can be inserted into a human framework by methods known in the art. See, eg, US Patent No. 5,225,539 to Winter and US Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

In a certain embodiment, an antibody of the invention is a human monoclonal antibody. Such human monoclonal antibodies directed against HER3 can be produced in transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

HuMAb mice (Medarex, Inc.) human immunoglobulin gene miniloci (miniloci) containing the immunoglobulin sequences encoding unrearranged human heavy (μ and γ) and κ light chains, and inactivated endogenous μ and κ chains Targeted mutagenesis of genetic loci (see eg, Lonberg, et al., (1994) Nature 368(6474):856-859). Thus, the mice display reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high affinity human IgG kappa monoclonal antibodies (Lonberg, N. et al., (1994) supra; reviewed in Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg and Huszar, (1995) Intern. Rev. Immunol. 13:65-93; and Harding and Lonberg , (1995) Ann.NYAcad.Sci.764:536-546). The preparation and use of HuMAb mice and the genomic modifications carried by the mice are further described in Taylor et al., (1992) Nucleic Acids Research 20:6287-6295; Chen et al., (1993) International Immunology 5:647-656; Tuaillon et al., (1993) Proc.Natl.Acad.Sci.USA94:3720-3724; Choi et al., (1993) Nature Genetics 4:117-123; Chen et al., (1993) EMBO J.12:821-830; Tuaillon et al., ( 1994) J. Immunol.152:2912-2920; Taylor et al., (1994) International Immunology 579-591; and Fishwild et al., (1996) Nature Biotechnology 14:845-851, the contents of all references are hereby incorporated in their entirety expressly incorporated by reference.进一步参阅均为Lonberg和Kay的美国专利号5,545,806、5,569,825、5,625,126、5,633,425、5,789,650、5,877,397、5,661,016、5,814,318、5,874,299和5,770,429；Surani等的美国专利号5,545,807；均为Lonberg和Kay的PCT公开号WO 92103918 , WO 93/12227, WO 94/25585, WO 97113852, WO 98/24884, and WO 99/45962; and PCT Publication No. WO 01/14424 by Korman et al.

In another embodiment, human antibodies of the invention can be produced using mice that carry human immunoglobulin sequences on transgenes and transchromosomes, such as mice that carry a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice," are described in detail in PCT Publication WO 02/43478 by Ishida et al.

Additionally, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to prepare the HER3 antibodies of the invention. For example, an alternative transgenic system known as Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in US Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

In addition, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to prepare the HER3 antibodies of the invention. For example, a mouse carrying both a human heavy chain transchromosome and a human light chain transchromosome called a "TC mouse" can be used; Tomizuka et al., (2000) Proc. Natl. Acad. Sci. USA 97:722-727 Such mice are described in . In addition, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., (2002) Nature Biotechnology 20:889-894) and can be used to prepare HER3 antibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared by phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art or are described in the Examples below.参阅例如：Ladner等的美国专利号5,223,409、5,403,484和5,571,698；Dower等的美国专利号5,427,908和5,580,717；McCafferty等的美国专利号5,969,108和6,172,197；及Griffiths等的美国专利号5,885,793、6,521,404、6,544,731、6,555,313、 6,582,915 and 6,593,081.

Human monoclonal antibodies of the invention can also be prepared using SCID mice in which human immune cells have been reconstituted such that a human antibody response can be generated following immunization. Such mice are described, for example, in US Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

(iii) Architecture or Fc modification

Engineered antibodies of the invention include those in which modifications have been made to framework residues within the VH and/or VL, eg, to improve the properties of the antibody. Such framework modifications are often made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequence from which the antibody is derived. To restore the framework region sequences to their germline configuration, somatic mutations can be "backmutated" into the germline sequences, eg, by site-directed mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

Another type of framework modification involves mutating one or more residues within the framework regions or even one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is described in further detail in US Patent Publication No. 20030153043 by Carr et al.

In addition to or as an alternative to making modifications within the framework or CDR regions, antibodies of the invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cytotoxicity. In addition, an antibody of the invention can be chemically modified (eg, one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, also to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of Kabat's EU index.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, eg increased or decreased. This method is further described in US Patent No. 5,677,425 to Bodmer et al. Altering the number of cysteine residues in the hinge region of CH1 is used, for example, to facilitate light and heavy chain assembly, or to increase or decrease antibody stability.

In another embodiment, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the antibody has impaired staphylococcal protein A (SpA) binding relative to native Fc hinge domain SpA binding. This method is described in further detail in US Patent No. 6,165,745 to Ward et al.

In still other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids may be substituted with a different amino acid residue such that the antibody has an altered affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This method is described in further detail in both US Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

In another embodiment, one or more amino acids selected from amino acid residues may be substituted with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or eliminated complement dependent cytotoxicity (CDC). This method is described in further detail in US Patent No. 6,194,551 to Idusogie et al.

In another embodiment, one or more amino acid residues are altered, thereby altering the ability of the antibody to fix complement. This method is described in further detail in PCT Publication WO 94/29351 by Bodmer et al.

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. This method is described in further detail in PCT Publication WO 00/42072 by Presta. Furthermore, the FcγR1, FcγRII, FcγRIII and FcRn binding sites on human IgG1 have been mapped and variants with improved binding have been described (see Shields et al., (2001) J. Biol. Chen. 276:6591-6604 ).

In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (ie, the antibody lacks glycosylation) can be prepared. Glycosylation can be altered, for example, to increase the affinity of the antibody for the "antigen". Such carbohydrate modifications can be achieved, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This deglycosylation increases the affinity of the antibody for the antigen. This method is described in further detail in US Patent Nos. 5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, antibodies can be prepared with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such carbohydrate modifications are achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in the loss of antibodies expressed in this host cell. Hypofucosylation (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycosyltransferases that modify glycoproteins (e.g., β(1,4)-N-acetylglucosaminyltransferase III ( GnTIII)), such that antibodies expressed in engineered cell lines display increased bisecting GlcNac structure, which leads to increased ADCC activity of the antibodies (see also Umana et al., (1999) Nat. Biotech. 17:176-180).

In another embodiment, the antibody is modified to increase its biological half-life. Various methods can be used. For example, as described in US Patent No. 6,277,375 to Ward, one or more of the following mutations can be introduced: T252L, T254S, T256F. Alternatively, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al., to increase biological half-life, antibodies can be altered within the CH1 or CL region to contain two loops taken from the CH2 domain of the Fc region of IgG. Salvage receptor binding epitope.

(iv) Methods of Engineering Altered Antibodies

HER3 antibodies or fragments thereof of the invention having the VH and VL sequences or full-length heavy and light chain sequences shown herein can be used by modifying the full-length heavy and/or light chain sequences, VH and/or VL sequences, or attaching to the constant region on it to generate new HER3-binding antibodies. Accordingly, in another aspect of the invention, structural features of HER3 antibodies or fragments thereof are used to generate structurally related HER3 antibodies that retain at least one functional property of the antibodies of the invention, such as binding to human HER3, and inhibiting One or more functional properties of HER3. For example, one or more CDR regions of an antibody of the invention, or mutations thereof, can be combined recombinantly with known framework regions and/or other CDRs to generate other recombinantly engineered HER3 antibodies of the invention discussed above. Other types of modifications include those described in the previous sections. The starting material for the engineering method is one or more VH and/or VL sequences provided herein, or one or more CDR regions thereof. In order to generate an engineered antibody, it is not necessary to actually prepare (ie, express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein, or one or more of its CDR regions. Instead, the information contained in the sequence is used as starting material to generate a "second generation" sequence derived from the original sequence, which is then prepared and expressed as a protein.

Accordingly, in another embodiment, the present invention provides a method for preparing an antibody that binds domain 3 of HER3 consisting of a heavy chain variable region antibody sequence and a light chain variable region antibody sequence; the heavy chain variable region The amino acid sequence has a CDR1 sequence selected from SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 142 and 162, selected from SEQ ID NO: 3, 23, 43, 63, 83, 103, 123, The CDR2 sequence of 143 and 163 and/or the CDR3 sequence selected from SEQ ID NO:4, 24, 44, 64, 84, 104, 124, 144 and 164, the light chain variable region antibody sequence has a sequence selected from SEQ ID NO : a CDR1 sequence of 8, 28, 48, 68, 88, 108, 128, 148 and 168, a CDR2 sequence selected from SEQ ID NO: 9, 29, 49, 69, 89, 109, 129, 149 and 169 and/or Or be selected from the CDR3 sequence of SEQ ID NO:10, 30, 50, 70, 90, 110, 130, 150 and 170; Change at least in the heavy chain variable region antibody sequence and/or the light chain variable region antibody sequence one amino acid residue to generate at least one altered antibody sequence; and expressing the altered antibody sequence as a protein. The altered antibody sequences can also be prepared by screening antibody libraries with fixed CDR3 sequences or minimal essential binding determinants and diversity in CDR1 and CDR2 sequences as described in US20050255552. Screening can be performed according to any screening technique suitable for screening antibodies from antibody libraries, such as phage display technology.

Accordingly, in another embodiment, the present invention provides a method for making an antibody that binds domains 3-4 of HER3 consisting of a heavy chain variable region antibody sequence and a light chain variable region antibody sequence; the heavy chain can be The variable region amino acid sequence has a sequence selected from SEQ ID NO: 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, 522, 542, 562 , 582, 602, 622, 642, 662 and 682 CDR1 sequences selected from SEQ ID NO: 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463 , 483, 503, 523, 543, 563, 583, 603, 623, 643, 663 and 683 CDR2 sequences and/or selected from SEQ ID NO: 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, 524, 544, 564, 584, 604, 624, 644, 664 and 684, the light chain variable region antibody sequence having a sequence selected from SEQ ID NO: 188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568, 588, 608, 628, The CDR1 sequence of 648, 668 and 688 selected from the group consisting of SEQ ID NO: 189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, 529 , 549, 569, 589, 609, 629, 649, 669 and 689 CDR2 sequences and/or selected from SEQ ID NO: 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, CDR3 sequences of 410, 430, 450, 470, 490, 510, 530, 550, 570, 590, 610, 630, 650, 670, and 690; altering the heavy chain variable region antibody sequence and/or the light chain variable region at least one amino acid residue within the antibody sequence to produce at least one altered antibody sequence; and expressing the altered antibody sequence as a protein. The altered antibody sequences can also be prepared by screening antibody libraries with fixed CDR3 sequences or minimal essential binding determinants and diversity in CDR1 and CDR2 sequences as described in US20050255552. Screening can be performed according to any screening technique suitable for screening antibodies from antibody libraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and express altered antibody sequences. The antibody encoded by the altered antibody sequence is an antibody that retains one, some or all of the functional properties of the antibodies or fragments thereof described herein, including but not limited to: specificity for human and/or cynomolgus HER3 Binding; Antibody binds HER3 in a phospho-HER assay and inhibits HER3 biological activity by inhibiting HER signaling activity.

The functional properties of altered antibodies can be assessed using standard assays available in the art and/or assays described herein, such as those shown in the Examples (eg, ELISA).

In certain embodiments of the methods of engineering antibodies of the invention, mutations may be introduced randomly or selectively along all or part of the antibody or fragment coding sequence, and the resulting modified HER3 antibodies may be screened for binding as described herein. activity and/or other functional properties. Mutation methods have been described in the art. For example, PCT Publication WO 02/092780 to Short describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication WO 03/074679 by Lazar et al. describes methods for optimizing physicochemical properties of antibodies using computational screening methods.

Characterization of the Antibodies of the Invention

Antibodies of the invention can be characterized by a variety of functional assays. For example, their ability to inhibit biological activity by inhibiting HER signaling in the phospho-HER assays described herein, their affinity for HER3 proteins (e.g., human and/or cynomolgus HER3), epitope binding, Their resistance to proteolysis was characterized by their ability to block signaling downstream of HER3. HER3-mediated signaling can be measured in a variety of ways. For example, it can be achieved by (i) measuring phospho-HER3, (ii) measuring phosphorylation of HER3 or other downstream signaling proteins (such as Akt), (iii) ligand blocking assays as described herein, (iv) heterologous two The HER signaling pathway is monitored by aggregate formation, (v) HER3-dependent gene expression signatures, (vi) receptor internalization, and (vii) HER3-driven cellular phenotypes such as proliferation.

The ability of an antibody to bind HER3 can be detected by directly labeling the antibody of interest, or the antibody can be unlabeled and binding can be detected indirectly using a variety of sandwich assay formats known in the art.

In some embodiments, the HER3 antibody blocks binding of the reference HER3 antibody to HER3 or competes with the reference HER3 antibody for binding to HER3. They may be fully human HER3 antibodies as described above. They can also be other mouse, chimeric or humanized HER3 antibodies that bind to the same epitope as the reference antibody. The ability to block binding of the reference antibody or to compete with the reference antibody for binding indicates that the tested HER3 antibody binds an epitope that is identical or similar to that defined by the reference antibody, or that binds an epitope sufficiently close to that bound by the reference HER3 antibody . Such antibodies are especially likely to share favorable properties identified for the reference antibody. The ability to block or compete with a reference antibody can be determined, for example, by a competition binding assay. Using a competition binding assay, the ability of the tested antibody to inhibit specific binding of a reference antibody to a common antigen (eg, HER3 polypeptide or protein) is examined. The test antibody competes with the reference antibody for specific binding to the antigen if the excess of the test antibody substantially inhibits the binding of the reference antibody. Substantial inhibition of a known test antibody typically reduces specific binding of the reference antibody by at least 10%, 25%, 50%, 75%, or 90%.

A number of known competition binding assays are available to assess the competitive binding of HER3 antibodies and reference HER3 antibodies to HER3. This includes, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., (1983) Methods in Enzymology 9:242-253); Solid phase direct biotin-avidin EIA (see Kirkland et al., (1986) J. Immunol. 137:3614-3619); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow & Lane, supra) ; Solid-phase direct-labeled RIA using 1-125 marker (see Morel et al., (1988) Molec. Immunol. 25:7-15); Solid-phase direct biotin-avidin EIA (Cheung et al., ( 1990) Virology 176:546-552); and directly labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test HER3-binding antibody or a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Test antibodies are usually present in excess. Due to steric hindrance, antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody binds and antibodies that bind to an adjacent epitope that is sufficiently close to the epitope that the reference antibody binds.

To determine whether selected HER3 monoclonal antibodies bind unique epitopes, individual antibodies can be biotinylated using commercially available reagents (eg, from Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using HER3 polypeptide-coated ELISA plates. Binding of biotinylated MAbs can be detected with a streptavidin-alkaline phosphatase probe. To determine the isotype of purified HER3-binding antibodies, an isotype ELISA can be performed. For example, wells of microtiter plates can be coated with 1 μg/ml anti-human IgG overnight at 4°C. After blocking with 1% BSA, plates were reacted with 1 μg/ml or less of monoclonal HER3 antibody or purified isotype control for 1-2 hours at ambient temperature. The wells can then be reacted with human IgGl or human IgM specific alkaline phosphatase-conjugated probes. The plate is then developed and analyzed so that the isotype of the purified antibody can be determined.

To demonstrate binding of monoclonal HER3 antibodies to living cells expressing HER3 polypeptide, flow cytometry can be used. Briefly, HER3-expressing cell lines (cultured under standard growth conditions) can be mixed with various concentrations of HER3-binding antibodies in PBS containing 0.1% BSA and 10% fetal bovine serum and incubated for 1 hour at 4°C . After washing, cells were reacted with fluorescein-labeled anti-human IgG antibody under the same conditions as for primary antibody staining. Samples can be analyzed by a FACScan instrument, using light scatter and side scatter properties to sort single cells. Alternative assays utilizing fluorescence microscopy may be used in addition to or instead of flow cytometry assays. Cells were stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of single cells, but may have reduced sensitivity depending on antigen density.

Antibodies of the invention or fragments thereof can be further tested for reactivity with HER3 polypeptides or antigen fragments by Western blotting. Briefly, purified HER3 polypeptides or fusion proteins, or cell extracts from cells expressing HER3, can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 10% fetal calf serum, and probed with the monoclonal antibodies to be tested. Binding of human IgG can be detected with anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

The potency and specificity of HER3 antibodies can be assessed in cell-based assays of ligand-induced heterodimer formation using a variety of readout formats. Activity can be assessed by one or more of the following:

(i) Inhibition of ligand-induced heterodimerization of HER2 with other EGF family members in target cell lines (eg, MCF-7 breast cancer cells). HER2 complexes can be immunoprecipitated from cell lysates with receptor-specific antibodies, and complexes can be analyzed for the absence of other EGF receptors and their biologically relevant ligands by probing with antibodies to other EGF receptors after electrophoresis/Western transfer /exist.

(ii) Inhibition of signaling pathways activated by ligand-activated heterodimers. Binding to HER3 appears to be key for other members of the EGF family of receptors to elicit maximal cellular responses following ligand binding. In the case of kinase-deficient HER3, HER2 presents a functional tyrosine kinase domain that enables signaling following growth factor ligand binding. Thus, cells co-expressing HER2 and HER3 can be treated with ligands such as heregulin in the absence and presence of inhibitors, and the effect on HER3 tyrosine phosphorylation monitored in a number of ways, including from treated HER3 was immunoprecipitated from cell lysates of the HER3 followed by Western blotting using an anti-phosphotyrosine antibody (see Agus supra for details). Alternatively, HER3 from the solubilized lysate can be detected by capturing onto the wells of a 96-well plate coated with anti-HER3 receptor antibody and using e.g. Waddleton et al., (2002) Anal. Biochem. 309:150-157 A europium-labeled anti-phosphotyrosine antibody was implemented to measure tyrosine phosphorylation levels to develop a high-throughput assay.

In a broader extension of this approach, effector molecules known to be activated downstream of activated receptor heterodimers, such as mitogen-activated protein kinase (MAPK) and Akt, can be obtained by immunization from processed lysates. Precipitation and blotting with antibodies that detect the activated form of these proteins, or direct analysis by analyzing the ability of these proteins to modify/activate specific substrates.

(iii) Inhibition of ligand-induced cell proliferation. Various cell lines are known to co-express combinations of ErbB receptors, such as many breast and prostate cancer cell lines. Assays can be performed in 24/48/96-well format with readout based on relative DNA synthesis (tritiated thymidine incorporation), increase in cell number (crystal violet staining), etc.

The potency and specificity of HER3 antibodies can be assessed in cell-based assays of ligand-independent homo- and heterodimer formation using a variety of readout formats. For example, HER2 overexpression triggers ligand-independent activation of the kinase domain due to spontaneous dimer formation. Overexpressed HER2 forms homo- or heterodimers with other HER molecules such as HER1, HER3, and HER4.

The ability of the antibody or fragment thereof to block in vivo growth of a tumor xenograft of a human tumor cell line whose tumorigenic phenotype is known to be at least in part dependent on ligand activation of HER3 heterodimer cell signaling, e.g. BxPC3 pancreatic cancer cells, etc. This ability can be assessed in immunocompromised mice alone or in combination with appropriate cytotoxic agents for the cell line in question. Examples of functional assays are also described in the Examples section below.

preventive and therapeutic use

The present invention provides methods of treating diseases or disorders associated with the HER3 signaling pathway by administering to an individual in need thereof an effective amount of an antibody or fragment thereof of the present invention. In particular embodiments, the invention provides for the treatment or prevention of cancer (e.g., breast, colorectal, lung, multiple myeloma, ovarian cancer) by administering to an individual in need thereof an effective amount of an antibody or fragment thereof of the invention. , liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myelogenous leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumor, schwannoma, head and neck cancer, bladder cancer, esophagus cancer, Barretts esophagus cancer, gelatinous glioma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, and endometriosis). In some embodiments, the invention provides methods of treating or preventing cancers associated with the HER3 signaling pathway by administering to an individual in need thereof an effective amount of an antibody of the invention.

In particular embodiments, the present invention provides methods for treating cancers associated with the HER3 signaling pathway, including but not limited to breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer , acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumor, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barretts esophagus cancer, glioblastoma, soft tissue clear cell Sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, and endometriosis.

Antibodies or fragments thereof of the present invention may also be used to treat or prevent other diseases associated with abnormal or defective HER3 signaling, including but not limited to respiratory diseases, osteoporosis, osteoarthritis, polycystic kidney disease, Diabetes, schizophrenia, vascular disease, heart disease, non-cancerous proliferative diseases, fibrosis, and neurodegenerative diseases such as Alzheimer's disease.

Drugs suitable for use in combination therapy with HER3 antibodies include standard therapeutic agents known in the art that are capable of modulating the ErbB signaling pathway. Suitable examples of standard therapeutic agents for HER2 include, but are not limited to, Herceptin and Lapatinib (Tykerb). Suitable examples of standard therapeutic agents for EGFR include, but are not limited to, Iressa, Tarceva, Erbitux and Vectibix mentioned above. Other agents that may be suitable for combination therapy with HER3 antibodies include, but are not limited to, modulating receptor tyrosine kinases, G protein-coupled receptors, growth/survival signaling pathways, nuclear hormone receptors, apoptotic pathways, cell cycle and Those drugs that cause angiogenesis.

diagnostic use

In one aspect, the invention includes methods for determining HER3 and/or nucleic acid expression and HER3 in the context of a biological sample (e.g., blood, serum, cells, tissue) or in an individual suffering from, or at risk of developing, cancer. Diagnostic assays of protein function.

Diagnostic assays, such as competition assays, rely on the ability of a labeled analog ("tracer") to compete with a test sample analyte for a limited number of binding sites on a common binding partner. The binding partner is generally insoluble either before or after competition, and tracer and analyte bound to the binding partner are then separated from unbound tracer and analyte. This separation is achieved by decantation (where the binding partner is not previously dissolved) or by centrifugation (where the binding partner is precipitated after the competition reaction). The amount of test sample analyte is inversely proportional to the amount of bound tracer, as determined by the amount of labeled substance. Dose response curves for known amounts of analyte are prepared and compared to test results to quantify the amount of analyte present in the test sample. When enzymes are used as detectable labels, these assays are referred to as ELISA systems. In this form of assay, competitive binding between the antibody and the HER3 antibody results in bound HER3, preferably a HER3 epitope of the invention, being a measure of antibody, most especially inhibitory antibody, in a serum sample .

A significant advantage of the assay is the direct measurement of inhibitory antibodies, ie those antibodies that interfere with HER3 binding, especially epitope binding. This assay, especially the ELISA test format, has considerable application in clinical settings and routine blood screening.

Another aspect of the invention provides methods for determining HER3 nucleic acid expression or HER3 activity in an individual, thereby selecting an appropriate therapeutic or prophylactic agent for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows selection of active agents (eg, drugs) for therapeutic or prophylactic treatment of an individual based on the individual's genotype (eg, examining an individual's genotype to determine the individual's ability to respond to a particular agent).

Another aspect of the invention relates to monitoring the effect of an active agent (eg, a drug) on HER3 expression or activity in a clinical trial.

pharmaceutical composition

To prepare a pharmaceutical or sterile composition comprising an antibody or fragment thereof, the antibody or fragment thereof is mixed with a pharmaceutically acceptable carrier or excipient. The composition may additionally contain a compound suitable for the treatment or prevention of cancer (breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myelogenous leukemia, chronic myelogenous leukemia, osteosarcoma, Squamous cell carcinoma, peripheral nerve sheath tumor, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma , Barretts esophagus cancer, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, and endometriosis).

Formulations of therapeutic and diagnostic agents can be prepared, for example, in the form of lyophilized powders, slurries, aqueous solutions, emulsions or suspensions by mixing with physiologically acceptable carriers, excipients or stabilizers (see e.g. , Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

The choice of administration regimen for a therapeutic agent will depend on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, the administration regimen maximizes the amount of therapeutic agent delivered to the patient at a level consistent with acceptable side effects. Thus, the amount of biological agent delivered depends in part on the particular entity and severity of the condition being treated. Guidance for selecting appropriate doses of antibodies, cytokines, and small molecules is available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis , Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al., (2003) New Engl.J.Med.348:601- 608; Milgrom et al., (1999) New Engl.J.Med.341:1966-1973; Slamon et al., (2001) New Engl.J.Med.344:783-792; Beniaminovitz et al., (2000) New Engl.J.Med.342:613-619; Ghosh et al., (2003) New Engl.J.Med.348:24-32; Lipsky et al., (2000) New Engl.J.Med.343:1594- 1602).

Appropriate dosages are determined by the clinician, for example, using parameters or factors known or suspected in the art to affect therapy or are expected to affect therapy. The dosage will generally be started at an amount slightly less than optimal and then increased by small increments until the desired or optimal effect relative to any negative side effects is achieved. Important diagnostic measures include those of symptoms such as inflammation or levels of inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied to obtain an amount of the active ingredient which is effective for a particular patient, composition and mode of administration to achieve the desired therapeutic response without being toxic to the patient . The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition utilized in the invention, or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound utilized, the therapeutic Other drugs, compounds and/or materials used in combination with the particular composition utilized, age, sex, weight, condition, general health and past medical history of the patient to be treated, and similar factors known in the medical arts.

Compositions comprising an antibody or fragment thereof of the invention may be provided by continuous infusion, or by dosing at intervals of, for example, 1 day, 1 week, or 1-7 times per week. Doses may be given intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally or by inhalation. A particular dosage regimen is one that involves the maximum dose or dose frequency that avoids significant adverse side effects. The total weekly dose may be at least 0.05 μg/kg body weight, at least 0.2 μg/kg, at least 0.5 μg/kg, at least 1 μg/kg, at least 10 μg/kg, at least 100 μg/kg, at least 0.2 mg/kg, at least 1.0 mg/kg kg, at least 2.0 mg/kg, at least 10 mg/kg, at least 25 mg/kg, or at least 50 mg/kg (see for example, Yang et al., (2003) New Engl. J. Med. 349:427-434; Herold et al., (2002) New Engl.J.Med.346:1692-1698; Liu et al., (1999) J.Neurol.Neurosurg.Psych.67:451-456; Portielji et al., (2003) Cancer Immunol.Immunother.52 :133-144). The ideal dosage for an antibody or fragment thereof is about the same as for an antibody or polypeptide, on a moles/kg body weight basis. Ideal plasma concentrations of antibodies or fragments thereof are on the order of moles/kg body weight. The dosage may be at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg , at least 95 μg, or at least 100 μg. Doses administered to an individual may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.

For the antibodies or fragments thereof of the present invention, the dosage administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight. The dosage can be in the range of 0.0001mg/kg to 20mg/kg, 0.0001mg/kg to 10mg/kg, 0.0001mg/kg to 5mg/kg, 0.0001 to 2mg/kg, 0.0001 to 1mg/kg, 0.0001mg/kg to 0.75mg/kg kg, 0.0001 mg/kg to 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or Between 0.01 and 0.10 mg/kg patient body weight.

Dosages of antibodies or fragments thereof of the invention can be calculated by multiplying the patient's body weight in kilograms (kg) by the dose to be administered in mg/kg. The dose of the antibody or fragment thereof of the invention may be 150 μg/kg or less, 125 μg/kg or less, 100 μg/kg or less, 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg or Lower, 80 μg/kg or lower, 75 μg/kg or lower, 70 μg/kg or lower, 65 μg/kg or lower, 60 μg/kg or lower, 55 μg/kg or lower, 50 μg/kg or lower Low, 45 μg/kg or less, 40 μg/kg or less, 35 μg/kg or less, 30 μg/kg or less, 25 μg/kg or less, 20 μg/kg or less, 15 μg/kg or less , 10 μg/kg or less, 5 μg/kg or less, 2.5 μg/kg or less, 2 μg/kg or less, 1.5 μg/kg or less, 1 μg/kg or less, 0.5 μg/kg or lower, or 0.5 μg/kg or lower patient body weight.

The unit dose of the antibody or fragment thereof of the present invention may be 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25mg to 20mg, 0.25 to 15mg, 0.25 to 12mg, 0.25 to 10mg, 0.25 to 8mg, 0.25mg to 7mg, 0.25mg to 5mg, 0.5mg to 2.5mg, 1mg to 20mg, 1mg to 15mg, 1mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

Doses of the antibodies or fragments thereof of the invention may achieve at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml in an individual , at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml , a serum titer of at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml. Alternatively, the dose of an antibody or fragment thereof of the invention may achieve at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, A serum titer of at least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml.

Doses of antibodies or fragments thereof of the invention may be repeated and may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or for at least 6 months.

Amounts effective in a particular patient may vary depending on factors such as the condition being treated, the general health of the patient, the method, route and dose of administration, and the severity of side effects (see, e.g., Maynard et al., (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

The route of administration can be by, for example, topical or dermal application, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional injection or infusion, or by sustained release system or implant (see, e.g., Sidman et al., (1983) Biopolymers 22:547-556; Langer et al., (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech. 12: 98-105; Epstein et al., (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang et al., (1980) Proc. Natl. 6,350,466 and 6,316,024). If necessary, the composition may further include a solubilizer and a local anesthetic (such as lidocaine) to relieve pain at the injection site. In addition, pulmonary administration can also be utilized, for example, by use of an inhaler or nebulizer, and formulations with aerosolizing agents. See, e.g., U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078, and PCT Publication Nos. Each of which is incorporated herein by reference in its entirety.

Compositions of the invention may also be administered via one or more routes of administration by one or more of a variety of methods known in the art. The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the desired result. Routes of administration selected for the antibodies or fragments thereof of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, eg, by injection or infusion. Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intrathecal, intraorbital, intracardiac, intradermal, Intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Alternatively, the compositions of the invention may be administered via non-parenteral routes, such as topical, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. In one embodiment, an antibody or fragment thereof of the invention is administered by infusion. In another embodiment, the multispecific epitope binding protein of the invention is administered subcutaneously.

If the antibody or fragment thereof of the invention is administered in a controlled or sustained release system, pumps can be used to achieve the controlled or sustained release (see Langer, supra; Sefton, (1987) CRCCrit. Ref Biomed. Eng. 14:20; Buchwald et al., (1980), Surgery 88:507; Saudek et al., (1989) N. Engl. J. Med. 321:574). Controlled or sustained release of the therapeutics of the invention can be achieved using polymeric materials (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds), CRC Press., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds), Wiley, New York (1984); Ranger and Peppas, (1983) J.Macromol.Sci.Rev.Macromol.Chem.23:61; see also Levy et al., (1985 ) Science 228:190; During et al., (1989) Ann.Neurol.25:351; Howard et al., (1989) J.Neurosurg.71:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly-2-hydroxyethyl methacrylate, polymethyl methacrylate, polyacrylic acid, ethylene-vinyl acetate copolymer, polymethacrylic acid, polyethylene glycol Lactide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), polyvinyl alcohol, polyacrylamide, polyethylene glycol, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, stable on storage, sterile and biodegradable. Controlled-release or sustained-release systems can be placed in close proximity to prophylactic or therapeutic targets, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, Vol. 2, No. 115- 138 pages (1984)).

Controlled release systems are discussed in the review by Langer, (1990), Science 249:1527-1533. Sustained release formulations comprising one or more antibodies or fragments thereof of the invention can be produced by any technique known to those of skill in the art. See, e.g., U.S. Patent No. 4,526,938; PCT Publication WO 91/05548; PCT Publication WO 96/20698; Ning et al., (1996), Radiotherapy & Oncology 39:179-189; Song et al., (1995) PDA Journal of Pharmaceutical Science & Technology 50 :372-397; Cleek et al., (1997) Pro.Int'l.Symp.Control.Rel.Bioact.Mater.24:853-854; and Lam et al., (1997) Proc.Int'l.Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.

If the antibodies or fragments thereof of the invention are administered topically, they may be formulated as ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, emulsions, or as present Other forms known to those skilled in the art. See, eg, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, a viscous to semi-solid or solid form comprising a carrier or excipient(s) compatible with topical application and in some cases having a dynamic viscosity greater than water is generally used . Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, ointments, and the like, which, if desired, are sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, emollients, etc.) Wetting agents, buffers or salts) to affect various properties such as osmotic pressure. Other suitable topical dosage forms include sprayable aerosols, wherein the active ingredient (in some cases in combination with a solid or liquid inert carrier) is packaged in a in the mix or in a plastic squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms, if desired. Examples of such additional ingredients are well known in the art.

If the composition comprising the antibody or fragment thereof is administered intranasally, it may be formulated as an aerosol, spray, mist or drops. In particular, prophylactic or therapeutic agents for use in accordance with the present invention may be conveniently delivered in the form of an aerosol spray in the form of a pressurized pack or nebulizer, using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, methane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (eg, consisting of gelatin) for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Methods of co-administration or treatment with a second therapeutic agent (e.g., cytokines, steroids, chemotherapeutics, antibiotics, or radiation) are known in the art (see, e.g., Hardman et al., (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (ed.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of a therapeutic agent can reduce symptoms by at least 10%, at least 20%, at least about 30%, at least 40%, or at least 50%.

Additional therapies (e.g., prophylactic or therapeutic agents) that may be administered in combination with an antibody or fragment thereof of the invention may be less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart from an antibody or fragment thereof of the invention hours, about 1 to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours apart to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, About 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, Administration is 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. Two or more therapies can be administered within the same patient visit.

Antibodies or fragments thereof of the invention and other therapies can be administered cyclically. Cycling therapy involves administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally followed by a third therapy ( For example, a prophylactic or therapeutic agent) over a period of time, etc., and this sequential administration is repeated, i.e., cycled to reduce resistance to one of the therapies, to avoid or reduce side effects of one of the therapies, and/or to increase the efficacy of the therapy.

In certain embodiments, antibodies or fragments thereof of the invention may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. See, eg, US Patent Nos. 4,522,811, 5,374,548 and 5,399,331 for methods of preparing liposomes. Liposomes may contain one or more moieties that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, eg, Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, e.g., US Pat. et al., (1995) FEBS Lett.357:140; Owais et al., (1995) Antimicrob.Agents Chemother.39:180); Surfactant protein A receptor (Briscoe et al., (1995) Am.J.Physiol .1233:134); p 120 (Schreier et al., (1994) J.Biol.Chem.269:9090); see also K.Keinanen; M.L.Laukkanen (1994) FEBS Lett.346:123; J.J.Killion; I.J.Fidler (1994) Immunomethods 4:273.

The invention provides regimens for administering a pharmaceutical composition comprising an antibody or fragment thereof of the invention, alone or in combination with other therapies, to an individual in need thereof. The regimens (eg, prophylactic or therapeutic agents) of the combination therapy of the invention can be administered to an individual simultaneously or sequentially. The regimens (eg, prophylactic or therapeutic agents) of the combination therapy of the invention may also be administered in cycles. Cycling therapy involves administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration, ie, cycling to reduce Developing resistance to one of the therapies (eg, drugs), avoiding or reducing side effects of one of the therapies (eg, drugs), and/or increasing the efficacy of the therapy.

The therapies (eg, prophylactic or therapeutic agents) of the combination therapy of the invention can be administered to an individual concurrently. The term "simultaneously" is not limited to administering a therapy (e.g., a prophylactic or therapeutic agent) at the exact same time, but refers to administering a pharmaceutical composition comprising an antibody or fragment thereof of the invention to an individual sequentially and at intervals such that Antibodies of the invention can be used in conjunction with other therapies to provide enhanced benefits over administering them in other ways. For example, the treatments can be administered to the subject sequentially in any order, at the same time or at different points in time; however, if not administered at the same time, they should be administered in close enough time to provide the desired therapeutic or prophylactic effect. Each therapy may be administered to the subject separately, in any suitable form and by any suitable route. In various embodiments, in less than 15 minutes, less than 30 minutes, less than 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours apart hours to about 4 hours, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart , about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart to administer the therapy (e.g., prophylactic or therapeutic agent). In other embodiments, two or more therapies (eg, prophylactic or therapeutic agents) are administered within the same patient visit.

The prophylactic or therapeutic agents of the combination therapy can be administered to an individual in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapy may be administered to an individual simultaneously in separate pharmaceutical compositions. Prophylactic or therapeutic agents can be administered to an individual by the same or different routes of administration.

Having now fully described the invention, it is further illustrated by the following examples and claims, which are illustrative and are not intended to be further limiting.

Example

Embodiment 1: method, material and antibody screening

(i) cell lines

SK-Br-3, BT-474 and MCF-7 cell lines were purchased from ATCC and routinely cultured in media supplemented with 10% fetal bovine serum (FBS).

(ii) Production of recombinant human, cynomolgus, mouse and rat HER3 vectors

The murine HER3 extracellular domain was PCR amplified from mouse brain cDNA (Clontech) and sequence verified by comparison with Refseq NM_010153. Rat HER3ECD was reverse transcribed from Rat-2 cell mRNA and the sequence was verified by comparison with NM_017218. Cynomolgus HER3 cDNA templates were generated using RNA from various cynomolgus monkey tissues (Zyagen Laboratories), and the RT-PCR products were cloned into (Invitrogen), followed by double-strand sequencing. Human HER3 was derived from a human fetal brain cDNA library (Source) and the sequence was verified by comparison with NM_001982.

To generate tagged recombinant proteins, human, mouse, rat and cynomolgus HER3 were PCR amplified with Pwo Taq polymerase (Roche Diagnostics). The amplified PCR product was gel purified and cloned into the pDonR201 (Invitrogen) Gateway entry vector, which had been previously modified to include an in-frame N-terminal CD33 leader sequence and a C-terminal TAG, eg, FLAG TAG. TAG allows the purification of monomeric proteins by anti-TAG monoclonal antibodies. The target gene is flanked by AttB1 and AttB2, allowing the use of Cloning technology (Invitrogen) to recombine into a proprietary destination vector suitable for Gateway (eg, pcDNA3.1). A Gateway LR reaction was used to perform a recombination reaction with a proprietary destination vector containing the CMV promoter to generate the TAG expression vector, although any commercially available vector could also be used.

Additional recombinant HER3 proteins were generated that fused the HER3 ECD at the C-terminal Factor X cleavage site and upstream of the human IgG hinge and Fc domain to generate an Fc-tagged protein. To this end, multiple HER3 ECDs were PCR amplified and cloned into a vector (eg, pcDNA3.1) modified to contain an in-frame C-terminal fusion of Factor X site-hinge-hFc. The resulting open reading frame was flanked by AttB1 and AttB2 sites for further use Cloning by recombinant cloning technology (Invitrogen). The LR Gateway reaction was used to transfer HER3-Fc into the expression construct of interest containing the CMV promoter. HER3 point mutation expression constructs were generated using standard site-directed mutagenesis protocols and the resulting vector sequences were verified.

Table 3: Generation of HER3 expression vectors. HER3 amino acid numbering is based on NP_001973 (human), NP_034283 (mouse) and NP_058914 (rat).

name describe hHER3 CD33-[Human HER3, residues 20-640]-TAG murine HER3 CD33-[mouse HER3, residues 20-643]-TAG rat HER3 CD33-[rat HER3, residues 20-643]-TAG cynomolgus monkey HER3 CD33-[cynomolgus HER3, residues 20-643]-TAG HER3D1-2 CD33-[Human HER3, residues 20-329]-TAG HER3D2 CD33-[Human HER3, residues 185-329]-TAG HER3D3-4 CD33-[Human HER3, residues 330-643]-TAG HER3D3 CD33-[Human HER3, residues 330-495]-TAG HER3D4 CD33-[Human HER3, residues 496-643]-TAG

humanHER3-Fc [Human HER3, residues 1-643]-Fc mouseHER3-Fc [mouse HER3, residues 1-643]-Fc Cynomolgus monkey HER3-Fc [cynomolgus HER3, residues 1-643]-Fc Rat HER3-Fc [rat HER3, residues 1-643]-Fc HER3D2-Fc [Human HER3 residues 207-329]-Fc

(iii) Expression of recombinant HER3 protein

Desired HER3 recombinant proteins are expressed in HEK293-derived cell lines previously adapted to suspension culture and grown in Novartis proprietary serum-free medium. Small-scale expression validation was performed in a lipofection-based transient 6-well plate transfection assay. Large-scale protein production was performed by transient transfection and performed at a 10-20 L scale in a Wave ^™ bioreactor system (Wave Biotech). DNA polyethylenimine (Polysciences) was used as a plasmid carrier at a ratio of 1:3 (w:w). Cell culture supernatants were harvested 7–10 days after transfection and concentrated by tangential flow filtration and diafiltration prior to purification.

(iv) Purification of labeled protein

Purify recombinant tagged HER3 protein (e.g., TAG-HER3) by collecting cell culture supernatant and concentrating 10-fold by tangential flow filtration with a 10 kDa cut-off filter (Fresenius). Prepare an anti-TAG column by coupling anti-TAG monoclonal antibody to CNBr-activated Sepharose 4B at a final ratio of 10 mg antibody/mL resin. Apply the concentrated supernatant to a 35 ml anti-Tag column at a flow rate of 1-2 mL/min. After a baseline wash with PBS, bound material was eluted with 100 mM glycine (pH 2.7), neutralized and sterile filtered. Protein concentration was determined by measuring the absorbance at 280 nm and converting with a theoretical coefficient of 0.66 AU/mg. The purified protein was finally characterized by SDS-PAGE, N-terminal sequencing and LC-MS.

(v) Fc Tag purification

The concentrated cell culture supernatant was applied to a 50 ml protein A Sepharose Fast Flow column at a flow rate of 1 ml/min. After a baseline wash with PBS, the column was washed with 10 column volumes of 10 mM NaH ₂ PO ₄ /30% (v/v) isopropanol pH 7.3, followed by 5 column volumes of PBS. Finally, bound material was eluted with 50 mM citrate/140 mM NaCl (pH 2.7), neutralized and sterile filtered.

(vi) HuCAL or panning

To select antibodies that recognize human HER3, various panning strategies were utilized. MorphoSys HuCAL, a commercially available phage display library or The library serves as a source of antibody variant proteins to generate therapeutic antibodies against human HER3 protein by selecting clones with high binding affinity. Phagemid libraries are based on The concept of (Knappik et al., (2000) J MolBiol 296:57-86), and using technology to display Fabs on the surface of phage (WO 01/05950 by Lohning).

To isolate anti-HER3 antibodies, standard panning strategies and RapMAT panning strategies were performed using solid-phase, solution, whole-cell, and differential whole-cell panning methods.

(vii) Solid phase panning

To identify anti-HER3 antibodies, various solid-phase panning strategies were performed with different recombinant HER3 proteins. For each round of solid phase panning, Maxisorp plates (Nunc) were coated with HER3 protein. Tagged proteins were captured with plates previously coated with anti-Fc (goat or mouse anti-human IgG, Jackson Immuno Research), anti-Tag antibody or by passive adsorption. Coated plates were washed with PBS and blocked. Wash the coated plate 2 times with PBS, then add HuCAL or For phage-antibody, incubate for 2 hours at room temperature on a shaker. Bound phage were eluted, added to E. coli TG-1 and incubated for phage infection. Infected bacteria were subsequently isolated and plated on agar plates. Colonies were scraped off the plate, and phage were rescued and amplified. Each HER3 panning strategy consists of rounds of panning and contains unique antigens, antigen concentrations, and wash stringencies.

(viii) Solution phase panning

Each round of solution phase panning was performed with various biotinylated recombinant HER3 proteins in the presence or absence of neuregulin 1-β1 (R&D Systems). Proteins were biotinylated using the EZ-Link Sulfo-NHS-LC Biotinylation Kit (Pierce) according to the manufacturer's instructions. 800 μl of streptavidin-linked magnetic beads (Dynabeads, Dynal) were washed once with PBS and blocked overnight with Chemiblocker (Chemicon). Incubate HuCAL in reaction tubes or Phage-antibody and appropriate biotinylated HER3. Streptavidin magnetic beads were added for 20 minutes and the beads were collected with a magnetic particle separator (Dynal). Bound phage were eluted from Dynabeads by adding DTT-containing buffer to each tube and added to E. coli TG-1. Phage infection was performed in the same manner as described for solid phase panning. Each HER3 panning strategy consists of rounds of panning and contains unique antigens, antigen concentrations, and wash stringencies. To isolate antibodies targeting specific epitopes, competitive panning was performed. In these panning strategies, HER3 was incubated and pre-blocked with a reference antibody before addition of HuCAL or Phage-antibody. As an alternative strategy, a reference antibody was used to specifically elute phage-antibodies complexed with HER3.

(ix) Cell-based panning

For cell panning, HuCAL or Phage-antibody was incubated with approximately ¹⁰⁷ cells for 2 hours at room temperature on a rotator, followed by centrifugation. The cell pellet is separated and the phage are eluted from the cells. The supernatant was collected and added to the E. coli TG-1 culture, followed by the procedure described above. Two cell-based strategies were utilized to identify anti-HER3 antibodies:

a) Whole cell panning: In this strategy, various whole cell lines are used as antigens.

b) Differential whole-cell panning: In this strategy, the antigen consists sequentially of cells and recombinant HER3 protein. Cell-based panning was performed as described above, while a solid-phase panning protocol was utilized when recombinant proteins were used as antigens. Washes were performed with PBS (2-3X) and PBST (2-3X).

(x) RapMAT ^TM library generation and panning

To increase antibody binding affinity while maintaining library diversity, both solution and solid-phase panning second-round outputs were fed into the RapMAT ^™ process, while whole-cell and differential whole-cell panning strategies were third-round outputs into this process (Prassler et al., (2009) Immunotherapy; 1:571-583). By subcloning the Fab-encoding insert of phage selected by panning into a display vector 25_bla_LHC to generate the RapMAT ^™ library, and by further digestion with specific restriction enzymes to generate the H-CDR2RapMAT ^™ library or the L-CDR3RapMAT ^™ library. Inserts were replaced with H-CDR2 or L-CDR3 with the TRIM maturation cassette (Virnekas et al., (1994) Nucleic Acids Research 22:5600-5607) depending on library composition. Library sizes are expected to be in the range of ^8x106 - ^1x108 clones. RapMAT antibody-phage were generated and subjected to two additional rounds of solution, solid-phase or cell-based panning using the protocol described previously.

This broad panning strategy involving iterative refinement of library design was developed specifically to divert the screen away from pure ligand-competing antibodies by including ligand-blocking antibodies directly in the panning. Second, the FAB-to-IgG conversion process was modified to maximize the recovery of candidate clones and ensure that all selective binders were analyzed in functional assays. From 44 initial pannings, approximately 28 families of specific Her3 binding antibodies were generated, with only three antibody families showing the desired property of blocking both ligand-dependent and independent signal transduction. Family A of isolated domains 1-2 and 2 that bind Her3. Family B that binds domains 3-4 in isolation but not only domain 4 and family C that binds domain 3.

Example 2: Transient expression of anti-HER3 IgG

Suspension-adapted HEK293-6E cells were cultured in BioWave20. Cells were transiently transfected with the relevant sterile DNA: PEI-MIX and further cultured. After transfection, cells were removed by tangential flow filtration using Fresenius filters. The cell-free material was concentrated by tangential flow filtration with a cut-off filter (Fresenius) and the concentrate was sterile filtered through a stericup filter. Store the sterile supernatant at 4°C.

Example 3: Purification of anti-HER3 IgG

in the cooling cabinet Purification of IgG was performed on a 100explorer Air chromatography system using an XK16/20 column (both GE Healthcare) with 25 mL of self-packing MabSelect SuRe resin. All flow rates were 3.5 mL/min with a pressure limit of 5 bar, except for sample loading. Equilibrate the column with 3 column volumes of PBS and load the filtered fermentation supernatant. The column was washed with PBS. IgG was eluted with a pH gradient starting from citrate/NaCl (pH 4.5) and decreasing linearly to citrate/NaCl (pH 2.5), followed by a constant step of the same pH 2.5 buffer. Fractions containing IgG were pooled, immediately neutralized, and sterile filtered (Millipore Steriflip, 0.22um). The _OD280 was measured and the protein concentration was calculated based on the sequence data. Pools were tested for aggregation (SEC-MALS) and purity (SDS-PAGE and MS), respectively.

Example 4: Expression and purification of HuCAL-Fab antibody in Escherichia coli

in shake flask cultures with YT medium supplemented with chloramphenicol Expression of encoded Fab fragments in TG-1 cells. Shake the culture until the OD600nm reaches 0.5. Expression was induced by adding IPTG (isopropyl-β-D-thiogalactoside). Cells were disrupted with lysozyme. His6-tagged Fab fragments were isolated by IMAC (Bio-Rad). The buffer was exchanged to 1x Dulbecco's PBS (pH 7.2) using a PD10 column. Samples were sterile filtered. Protein concentration was determined by UV spectrophotometry. Sample purity was analyzed on denaturing, reducing 15% SDS-PAGE. The homogeneity of the native Fab preparations was determined by size exclusion chromatography (HP-SEC) using calibration standards.

Example 5: Measurement of HER3 antibody affinity (K _D ) by solution equilibrium titration (SET)

Affinity determinations were performed in solution essentially as described previously (Friguet et al., (1985) J Immunol Methods 77:305-19). To improve the sensitivity and accuracy of the SET method, it was changed from the classical ELISA to an ECL-based technique (Haenel et al., (2005) Anal biochem 339:182-84).

Affinity determinations were performed by SET with previously described unlabeled HER3-Tag (human, rat, mouse or cynomolgus monkey).

Data were evaluated using XLfit software (ID Business Solutions) using custom fit models. For the _KD determination of each IgG, the following model (modified from Piehler et al. (Piehler et al., (1997) J Immunol Methods 201 :189-206)) was used.

$\mathrm{<span\; class="notranslate">\; the\; y</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; 2</span>}\mathrm{<span\; class="notranslate">\; B</span>}}_{\mathrm{<span\; class="notranslate">\; max</span>}}}{<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; IgG</span>}<span\; class="notranslate">\; ]</span>}<span\; class="notranslate">\; (</span>\frac{<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; IgG</span>}<span\; class="notranslate">\; ]</span>}{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>\frac{{<span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; +</span><span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; IgG</span>}<span\; class="notranslate">\; ]</span><span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; K</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}}}{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>\sqrt{\frac{{<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; +</span><span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; kgG</span>}<span\; class="notranslate">\; ]</span><span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; K</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}{\mathrm{<span\; class="notranslate">\; 4</span>}}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; IgG</span>}<span\; class="notranslate">\; ]</span>}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}{\mathrm{<span\; class="notranslate">\; 2</span>}<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; IgG</span>}<span\; class="notranslate">\; ]</span>}<span\; class="notranslate">\; )</span>$

[IgG]: Applied total IgG concentration

X: Applied total soluble antigen concentration (binding site)

B _max : maximum signal of IgG without antigen

K _D : Affinity.

Example 6: Determination of Antibody Cell Binding by FACS

Binding of antibodies to endogenous human antigens expressed on human cancer cells was assessed by FACS. To determine antibody EC ₅₀ values, SK-Br-3 cells were harvested with accutase and diluted to 1×10 ⁶ cells/mL in FACS buffer (PBS/3% FBS/0.2% NaN3). 1×10 ⁵ cells/well were added to each well of a 96-well plate (Nunc) and centrifuged at 210 g for 5 minutes at 4° C., then the supernatant was removed. Serial dilutions of the test antibody (diluted in 1:4 dilution steps with FACS buffer) were added to the pelleted cells and incubated on ice for 1 h. Wash and pellet cells 3 times with 100 μL FACS buffer. PE-conjugated goat anti-human IgG (Jackson ImmunoResearch) diluted 1/200 in FACS buffer was added to the cells and incubated on ice for 1 hour. Three additional wash steps with 100 μL of FACS buffer were performed, followed by a centrifugation step at 210 g for 5 min at 4°C. Finally, cells were resuspended in 200 μL of FACS buffer, and fluorescence was measured with a FACSArray (BD Biosciences). The amount of anti-HER3 antibody bound to the cell surface was assessed by measuring the mean channel fluorescence.

Example 7: HER3 Domain Binding

96-well Maxisorp plates (Nunc) were coated overnight with 200 ng of the appropriate recombinant human proteins (HER3-tagged, D1-2-tagged, D2-tagged, D3-4-tagged, D4-tagged and tagged irrelevant control protein). All wells were then washed with PBS/0.1% Tween-20, blocked with PBS/1%BSA/0.1% Tween-20, and washed with PBS/0.1% Tween-20. Add anti-HER3 antibody to relevant wells to a final concentration of 10 μg/mL and incubate at room temperature. Plates were washed with PBS/0.1% Tween-20, then the appropriate peroxidase-linked detection antibody diluted 1/10000 in PBS/1% BSA/0.1% Tween-20 was added. The detection antibodies used were goat anti-mouse (Pierce, 31432), rabbit anti-goat (Pierce, 31402) and goat anti-human (Pierce, 31412). Plates were incubated for 1 hour at room temperature and then washed with PBS/0.1% Tween-20. 100 μl of TMB (3,3',5,5' tetramethylbenzidine) substrate solution (BioFx) was added to all wells, and then the reaction was stopped with 50 μl of 2.5% H ₂ SO ₄ . The degree of binding of HER3 antibody to each recombinant protein was determined by measuring _OD450 with a SpectraMax microplate reader (Molecular Devices). When appropriate, dose response curves were analyzed with Graphpad Prism.

Example 8: Determination of X-ray Crystal Structure of Human HER3/MOR12604 Fab Complex

This example presents the Crystal structure of HER3 bound to the Fab fragment of MOR12604 determined at resolution. The tagged human HER3 extracellular domain was further purified on a HiLoad 26/60 Superdex 200 PrepGrade column (GE Healthcare) equilibrated in PBS (pH 7.3). MOR12604 Fab was expressed in E. coli and purified as previously described. HER3/MOR12604-Fab complexes were prepared by mixing excess MOR12604Fab with labeled HER3 at a molar ratio of 2:1 (concentration estimated by LCUV method) and Superdex 200 equilibrated in 25 mM Tris (pH 7.5), 150 mM NaCl 10 /300 column (GE Healthcare). Peak fractions were analyzed by SDS-PAGE and LCMS. Fractions containing approximately equimolar ratios of both HER3 and Fab were pooled and concentrated to 10 mg/ml. HER3/MOR12604 crystals were grown at 293K by sitting drop vapor diffusion from droplets containing 150 nl HER3/MOR12604 complex and 150 nl pool solution (200 mM dipotassium phosphate and 20% PEG 3350). Crystals were transferred to 200 mM dipotassium phosphate, 25% PEG 3350 and 15% glycerol and snap frozen in liquid nitrogen.

Data were collected at beamline 17-ID at the Advanced Photon Source (Argonne National Laboratory). The data of the HER3/MOR12604 Fab complex were processed and adjusted with autoPROC (Global Phasing, LTD) in the space group P2 ₁ 2 ₁ 2 ₁ to Unit cell size a=56.15, b=174.71, Statistically good. The structure of HER3/MOR12604 Fab was solved by molecular replacement with Phaser (McCoy et al., (2007) J. Appl. Cryst. 40:658-674), using the published HER3 ECD structure 1mb6 and the proprietary Fab as search models. In COOT (Emsley & Cowtan (2004) Acta Cryst.60:2126-2132), the final model of each asymmetric unit containing 1 molecule of HER3/MOR12604Fab complex was established, and the R and R _free values were calculated by BUSTER (Global Phasing, LTD). The refinements are 19.9% and 23.3%, and the root mean square deviations of bond length and bond angle are and 1.19°. In PyMOL ( LLC) containing any atom in the MOR12604 Fab HER3 residues within atoms are listed in Tables 5 and 6.

Example 9: Phospho-HER3 in vitro cell assay

MCF-7 cells were routinely maintained in DMEM/F12, 15mM HEPES, L-glutamine, 10% FBS, BT474 cells were routinely maintained in DMEM, 10% FBS, McCoy's5a, 10% FBS, 1.5mM L- SK-Br-3 cells were routinely maintained in glutamine. Near confluent cells were trypsinized, washed with PBS, and diluted to 5 x ¹⁰⁵ cells/mL. 100 μL of the cell suspension was then added to each well of a 96-well flat bottom plate (Nunc), resulting in a final density of ^5x104 cells/well. The MCF7 cells were allowed to attach for about 3 hours, and then the medium was changed to starvation medium containing 0.5% FBS. All plates were then incubated overnight at 37°C before being treated with appropriate concentrations of HER3 antibodies for 80 minutes at 37°C. MCF7 cells were treated with 50ng/mL NRG1 for the last 20 minutes to stimulate HER3 and AKT phosphorylation, while BT474/SK-Br-3 cells did not need additional stimulation. All medium was slowly aspirated and cells were washed with ice-cold PBS containing 1 mM _CaCl2 and 0.5 mM _MgCl2 (Gibco). By adding 50 μL ice-cold lysis buffer (20mM Tris (pH8.0)/137mM NaCl/10% glycerol/2mM EDTA/1%NP-40/1mM sodium orthovanadate/1xPhospho-Stop/1x Complete mini protease inhibitor ( Roche)/0.1mM PMSF) to lyse the cells and incubate on ice for 30 minutes with shaking. Lysates were then collected and centrifuged at 1800g for 15 minutes at 4°C to remove cell debris.

HER3 capture plates were prepared using carbon plates (Mesoscale Discovery) that were coated with 20 μL of 4 μg/mL MAB3481 capture antibody (R&D Systems) diluted in PBS overnight at 4 °C, followed by 1x 3% bovine serum albumin. Tris buffer (Mesoscale Discovery)/0.1% Tween-20 blocking. HER3 was captured by adding an appropriate amount of lysate and incubating the plate with shaking for 1 hour at room temperature, after which the lysate was aspirated and the wells were washed with 1x Tris buffer (Mesoscale Discovery)/0.1% Tween-20. Phosphorylated HER3 was detected with an anti-pY1197 antibody (Cell Signaling) prepared at 1:8000 in 3% milk powder/1x Tris/0.1% Tween-20 by incubation at room temperature for 1 hour. Wash wells 4 times with 1x Tris/0.1% Tween-20 and detect phosphorylated proteins by incubating with S-Tag-labeled goat anti-rabbit Ab (#R32AB) diluted in 3% blocking buffer for 1 hour at room temperature . Aspirate each well, wash 4 times with 1x Tris/0.1% Tween-20, then add 20 μL of reading buffer T (Mesoscale Discovery) containing surfactant, and quantify the signal with Mesoscale Sector Imager.

Example 10: Phospho-Akt (S473) in vitro cell assay

Near-confluent MCF7, SK-Br-3 and BT-474 cells cultured in complete medium were harvested with accutase (PAA Laboratories) and resuspended in appropriate medium at a final concentration of ^5x105 cells/mL. 100 μL of cell suspension was then added to each well of a 96-well flat bottom plate (Nunc) to yield a final density of ^5x104 cells/well. The MCF7 cells were allowed to attach for about 3 hours, and then the medium was changed to starvation medium containing 0.5% FBS. All plates were then incubated overnight at 37°C before being treated with appropriate concentrations of HER3 antibodies for 80 minutes at 37°C. MCF7 cells were treated with 50ng/mL NRG1 for the last 20 minutes to stimulate HER3 and AKT phosphorylation, while SK-Br-3 cells did not need additional stimulation. All medium was slowly aspirated and cells were washed with ice-cold PBS containing 1 mM _CaCl2 and 0.5 mM _MgCl2 (Gibco). By adding 50 μL of ice-cold lysis buffer (20 mM Tris (pH8.0)/137 mM NaCl/10% glycerol/2 mM EDTA/1% NP-40/1 mM sodium orthovanadate/aprotinin (10 μg/mL)/leupeptin Peptide (10 μg/mL)) to lyse the cells and incubate on ice for 30 minutes with shaking. Lysates were then collected and centrifuged at 1800g for 15 minutes at 4°C to remove cell debris. 20 [mu]L of lysate was added to a multispot 384-well phospho-Akt carbon plate (Mesoscale Discovery) that had been previously blocked with 3% BSA/1x Tris/0.1% Tween-20. The plate was incubated with shaking at room temperature for 2 hours, then the lysate was aspirated and the wells were washed 4 times with 1× Tris buffer (Mesoscale Discovery)/0.1% Tween-20. Phosphorylated Akt was detected with 20 μL of SULFO-TAG anti-phospho-Akt (S473) antibody (Mesoscale Discovery) diluted 50-fold in 1% BSA/1×Tris/0.1% Tween-20 by incubation at room temperature for 2 hours with shaking. The wells were washed 4 times with 1xTris/0.1% Tween-20, then 20 μL of surfactant-containing reading buffer T (Mesoscale Discovery) was added, and the signal was quantified with a Mesoscale Sector Imager.

Example 11: Cell Line Proliferation Assay

SK-Br-3 cells were routinely cultured in modified McCoy's 5A medium supplemented with 10% fetal bovine serum, and BT-474 cells were cultured in DMEM supplemented with 10% FBS. Near-confluent cells were trypsinized, washed with PBS, diluted to 5 x ¹⁰⁴ cells/mL with growth medium, and seeded at a density of 5000 cells/well into 96-well clear bottom black plates (Costar 3904). Cells were incubated overnight at 37°C, and then an appropriate concentration of HER3 antibody was added (usually a final concentration of 10 or 1 μg/mL). Plates were returned to the incubator and cell viability was assessed with CellTiter-Glo (Promega) after 6 days. Add 100 μL of CellTiter-Glo solution to each well and incubate for 10 minutes at room temperature with gentle shaking. Luminescence was measured with a SpectraMax microplate reader (Molecular Devices). The degree of growth inhibition obtained with each antibody was calculated by comparing the luminescence value obtained with each HER3 antibody to that obtained with a standard isotype control antibody.

For proliferation assays, MCF-7 cells were routinely cultured in DMEM/F12 (1 :1 ) containing 4 mM L-glutamine/15 mM HEPES/10% FBS. Trypsinize near-confluent cells, wash with PBS, and dilute to 1 x ¹⁰⁵ cells with DMEM/F12 (1:1) containing 4 mM L-glutamine/15 mM HEPES/10 μg/mL human transferrin/0.2% BSA /mL. Cells were seeded into 96-well clear bottom black plates (Costar) at a density of 5000 cells/well. An appropriate concentration of HER3 antibody is then added (usually a final concentration of 10 or 1 μg/mL). Cell growth was also stimulated by adding 10 ng/mL NRG1-β1EGF domain (R&D Systems) to appropriate wells. Plates were returned to the incubator and cell viability was assessed with CellTiter-Glo (Promega) after 6 days. The extent of growth inhibition obtained with each antibody was calculated by subtracting the background (neuregulin-free) luminescence value and comparing the value obtained with each anti-HER3 antibody to that obtained with a standard isotype control antibody.

Example 12: In Vivo BxPC3 Potency Study

BxPC3 cells were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum without antibiotics until implantation.

Female athymic nu/nu Balb/C mice (Harlan Laboratories) were implanted subcutaneously with ^10x106 cells in a mixture of 50% phosphate buffered saline and 50% Matrigel. The total injection volume containing suspended cells was 200 µL. Animals were entered into efficacy studies once tumors reached a size of approximately ^200mm3 . In general, a total of 10 animals per group were included in the study. Animals were excluded from the study if they displayed unusual tumor growth characteristics prior to study entry.

Animals were dosed intravenously by lateral tail vein injection. Animals were on a regimen of 20 mg/kg twice weekly for the duration of the study. Tumor volumes and T/C values were calculated as detailed for the BT-474 study.

Example 13: In Vivo BT474 Potency Study

BT-474 cells were cultured in DMEM containing 10% heat-inactivated fetal calf serum without antibiotics until implantation.

One day prior to cell inoculation, female athymic nu/nu Balb/C mice (Harlan Laboratories) were subcutaneously implanted with sustained-release 17[beta]-estradiol particles (Innovative Research of America) to maintain serum estrogen levels. One day after 17β-estradiol particle implantation, 5× ¹⁰ cells were orthotopically injected into the fourth mammary fat pad in a suspension containing 50% phenol red-free matrigel in Hank’s balanced salt solution ( BD Biosciences). The total injection volume containing suspended cells was 200 µL. Twenty days after cell implantation, animals with tumor volumes of approximately 200 ^mm3 were entered into the efficacy study. In general, a total of 10 animals per group were entered into the efficacy study.

For single agent studies, animals were dosed intravenously with control IgG or MOR12606 or MOR13655 via lateral tail vein injection. Animals were administered a dosing regimen of 20 mg/kg twice weekly during the study period. For the combination study, both MOR10703 and MOR12606 were dosed to animals twice weekly at 20 mg/kg. Tumor volumes were measured twice weekly by diameter determination during the study. Percent treatment/control (T/C) values were calculated using the following formula:

%T/C＝100×ΔT/ΔC if ΔT>0

in:

T = mean tumor volume in the drug treatment group on the last day of the study;

ΔT = mean tumor volume of the drug treatment group on the last day of the study - mean tumor volume of the drug treatment group on the first day of administration;

C = mean tumor volume in the control group on the last day of the study; and

ΔC = average tumor volume of the control group on the last day of treatment - average tumor volume of the control group on the first day of administration.

Body weight was measured twice a week, and the dosage was adjusted according to body weight. % change in body weight was calculated as (BW _current - BW _initial )/(BW _initial ) x 100. Data are expressed as percent body weight change from the first day of treatment.

All data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using delta tumor volume and body weight. Between-group comparisons were performed using one-way ANOVA with post hoc Tukey. For all statistical evaluations, the significance level was set at p<0.05. Significance compared to the vehicle control group is reported.

Results and discussion

Collectively, these results show a class of antibodies that bind amino acid residues within domains 3 or 4. Binding of these antibodies inhibits both ligand-dependent and ligand-independent signaling.

(i) Affinity determination

Antibody affinity was determined by solution equilibrium titration (SET) as described above. The results are summarized in Table 3 and example titration curves for MOR12615 and MOR12604 are included in Figure 1 . The data revealed that a number of antibodies that tightly bound human HER3 were identified.

Table 3: _KD values of anti-HER3 IgG determined by solution equilibrium titration (SET). Hu (human), Cy (cynomolgus monkey), Mu (mouse) and ra (rat), nd (not determined).

(ii) Determination of EC ₅₀ of SK-Br-3 cells

The ability of the identified antibodies to bind to HER3-expressing cells was determined by calculating the _EC50 values of the identified antibodies binding to the HER2-amplified cell line SK-Br-3 (see FIG. 2, Table 4).

Table 4: FACS _EC50 values of anti-HER3 IgG on cells.

MOR# SK-Br-3FACS _EC50 (pM) 14537 179 14538 279 14533 42 14534 28

(iii) HER3 domain binding

The ability of a subset of anti-HER3 antibodies to bind various extracellular domains of human HER3 was characterized in an ELISA assay. To this end, the extracellular domain of HER3 was divided into its 4 constitutive domains and various combinations of these domains were cloned, expressed and purified as individual proteins as described above. Using this strategy, the following domains were successfully produced as soluble proteins: domains 1 and 2 (D1-2), domain 2 (D2), domains 3 and 4 (D3-4), and domain 4 (D4). The integrity of each isolated domain was previously confirmed using a series of in-house generated antibodies as positive controls.

As shown in Figure 3, successful binding of MOR12615 and MOR12604 to the HER3 extracellular domain and isolated D3-4 protein was observed. No binding to D1-2 or D2 proteins was observed. This binding data suggests that these antibodies recognize epitopes predominantly contained within domains 3 or 4. Interestingly, MOR12604 can bind isolated D3 protein, suggesting that its epitope can be further refined to residues within domain 3. Since MOR12615 and MOR12604 are representative members of two different families of anti-HER3 antibodies, according to their hCDR3 sequences, antibodies MOR12514, MOR12515, MOR12516, MOR12615, MOR12920, MOR12921, MOR12922, MOR13654, MOR13660, MOR13661, MOR13662, MOR13663, MOR13664, MOR13665, MOR13666, MOR13667, MOR13668, MOR13669, MOR13670, MOR14537 and MOR14538 can be classified as D3-4 binders. Antibodies MOR12603, MOR12604, MOR12605, MOR12606, MOR14533 and MOR14534 can be classified as D3 binders.

(iv) Crystal structure of HER3/MOR12604

Analysis of the MOR12604 Fab fragment binding to the extracellular domain of HER3 High resolution X-ray crystal structures to further define the HER3 epitopes recognized by related antibodies of this family (see Figure 4). Superposition of the MOR12604/HER3 crystal structure with the published HER3 crystal structure shows that MOR12604-bound HER3 is all in a tethered (inactive) conformation (see Figure 4B). This conformation is characterized by a prominent interaction interface between domains 2 and 4 mediated by the β-hairpin dimerization loop in domain 2. The observed conformation of HER3 is similar to that previously described by Cho et al. (Cho & Leahy, (2002), Science 297:1330-1333), who published the crystal structure of the HER3 extracellular domain in the absence of neuregulin. Since neuregulin can activate HER3, it is speculated that the bound conformation of HER3 is inactive. Related EGFR family members HER4 (Bouyain et al., (2005) Proc. Natl. Acad. Sci. USA, 102:15024-15029) and HER1 (Ferguson et al., (2003) Molec. Cell 11:507) have been crystallized -517), a similar bound conformation was also observed.

The spatial relationship between domains 1 to 4 of HER3 in the inactive (bound) state is significantly different from that in the extended (activated) state. This finding is based on the crystal structures of related EGFR family members HER2 and ligand-bound HER1 (Cho et al., (2003) Nature 421:756-760; Ogiso et al., (2002) Cell 110:775-787; Garrett et al., (2002) Cell 110:763-773), both are in the stretched (activated) state. In the extended state, the β-hairpin dimerization loop of domain 2 is released from its inhibitory interaction with domain 4 and is thus free to interact with its dimerization partner protein. Thus, the β-hairpin dimerization loop of domain 2 has an important function in maintaining the tethered (inactive) state and mediating dimerization of the EGF receptor in the extended state, leading to activation of the intracellular kinase domain.

In the crystal structure, the electron densities of MOR12604 Fab, HER3 domain 3, HER3 domain 4 and part of domain 2 (residues 261-278) including the β-hairpin dimerization loop are all well defined. Weak or no electron density was observed for HER3 residues 20-260 and 279-303 localized in domains 1 and 2, suggesting that HER3 retains some degree of flexibility when it binds to MOR12604. This finding is consistent with comparisons of other crystal structures of HER3 alone and bound to various Fab fragments, which showed slight differences in relative domain positioning within the bound state.

The crystal structure also revealed that the HER3 epitope recognized by MOR12604 is a non-linear epitope comprising residues from domain 3 (see Figure 4, Tables 5 and 6). The HER3 epitopes recognized by this highly related antibody family can thus be defined as:

Domain 3: Residues 335-342, 362-376, 398, 400, 424-428, 431, 433-434 and 455.

The MOR12604 binding surface can be further subdivided into two surfaces marked as solid and dashed circles in Figure 4D:

Surface A: residues 362-376;

Surface B: Residues 335-342, 398, 400, 424-428, 431, 433-434 and 455.

Surface A or Surface B contribute approximately the same surface area to the overall HER3/12604 interface (Surface surface ).

Interestingly, binding of MOR12604 to domain 3 results in a dramatic conformational change in the loop defined by HER3 residues 371–377. This conformation of this loop is different from other published HER3 structures, thus suggesting that it is induced by MOR12604 binding. The MOR12604 epitope determined from the crystal structure is consistent with our ELISA domain binding data in which MOR12604 was determined to bind the isolated D3 domain. Furthermore, comparison of the MOR12604 epitope with the EGFR residues contacted by TGFα showed a high degree of overlap (Garrett et al., (2002) Cell 110:763-773). Since neuregulin is thought to interact with HER3 in a manner similar to TGFα/EGFR, it is likely that MOR12604 will prevent ligand binding and thereby block neuregulin-induced HER3 activation.

Binding of MOR12604 within domain 3 may indicate that MOR12604 may act by any one (or combination) of the following mechanisms:

- Block HER3 residues required for ligand binding;

- HER3 is prevented from adopting an active conformation due to steric hindrance between the antibody and the HER3 domain;

- prevent HER3 from adopting an active conformation by reducing the degree of flexibility in the HER3 hinge region (e.g. domain 3);

- Induces a conformational change in loop 371-377 of domain 3 that prevents HER3 from transitioning to an open conformation;

- destabilizes HER3, making it susceptible to degradation;

- Acts as a partial agonist to accelerate the downregulation of HER3;

- Inhibition of dimerization with binding partners;

- Binding of one molecule of HER3 via each arm of MOR12604 allows the antibody to generate non-native HER3 dimers that are susceptible to proteolytic degradation or unable to dimerize with other receptor tyrosine kinases.

Table 5: Interaction between MOR12604 Fab heavy chain and human HER3. The numbering of Fab VH residues is based on its linear amino acid sequence (SEQ ID NO: 1). The numbering of HER3 residues is based on NP_001973. The HER3 residues shown have at least one atom in the MOR12604 Fab atoms within.

Table 6: Interaction between MOR12604 Fab light chain and human HER3. The numbering of Fab VL residues is based on its linear amino acid sequence (SEQ ID NO: 1). The numbering of HER3 residues is based on NP_001973. The HER3 residues shown have at least one atom in the MOR12604 Fab atoms within.

(v) Inhibition of cell signaling

To determine the effect of anti-HER3 antibodies on ligand-dependent HER3 activity, MCF7 cells were incubated with IgG and then stimulated with neuregulin. Example inhibition curves are shown in Figure 5 and summarized in Table 7. The effect of anti-HER3 antibodies on HER2-mediated activation of HER3 was also investigated with the HER2-amplified cell lines SK-Br-3 and BT474 (Figure 6, Figure 7 and Table 7).

Table 7: pHER3 IC ₅₀ and inhibition degree values of anti-HER3 IgG in MCF7, BT474 and SK-Br-3 cells

To determine whether inhibition of HER3 activity affects downstream cell signaling, Akt phosphorylation was measured in NRG-stimulated MCF7 cells and HER2-amplified SK-Br-3/BT474 cells following treatment with anti-HER3 antibodies (see Figure 5, Figure 6 and Table 8).

Table 8: pAkt(S ⁴⁷³ ) IC ₅₀ and inhibition degree values of anti-HER3 IgG in SK-Br-3, BT-474 and MCF7 cells

总之，MOR12514、MOR12515、MOR12516、MOR12615、MOR12920、MOR12921、MOR12922、MOR13654、MOR13655、MOR13656、MOR13657、MOR13658、MOR13659、MOR13660、MOR13661、MOR13662、MOR13663、MOR13664、MOR13665、MOR13666、MOR13667、MOR13668、MOR13669、MOR13670、 MOR14537, MOR14538, MOR12603, MOR12604, MOR12605, MOR12606, MOR14533, and MOR14534 are each capable of inhibiting cellular HER3 activity and downstream signaling in a ligand-dependent and ligand-independent manner.

(vi) Inhibition of proliferation

由于MOR12514、MOR12515、MOR12516、MOR12615、MOR12920、MOR12921、MOR12922、MOR13654、MOR13655、MOR13656、MOR13657、MOR13658、MOR13659、MOR13660、MOR13661、MOR13662、MOR13663、MOR13664、MOR13665、MOR13666、MOR13667、MOR13668、MOR13669、MOR13670、MOR14537 , MOR14538, MOR12603, MOR12604, MOR12605, MOR12606, MOR14533, and MOR14534 were able to inhibit HER3 activity, and subsets thereof were tested for their ability to block ligand-dependent and independent cell growth in vitro (example data are shown in Figure 8, Figure 9, in Figure 10 and summarized in Table 9). Anti-HER3 antibodies tested were all potent inhibitors of cell proliferation, confirming that antibodies that bind D3 or D3-4 are able to suppress HER3-driven phenotypes.

Table 9: Proliferation inhibition after treatment of SK-Br-3, BT-474 and MCF7 cells with anti-HER3 IgG

(vii) In vivo inhibition of tumor growth

To determine the in vivo activity of the anti-HER3 antibodies, the antitumor activity of MOR12606 and MOR13655 was tested in both BxPC3 and BT-474 tumor models. Repeated dosing of the BxPC3 model with MOR12606 or MOR13655 produced 25% regression and 5% T/C, respectively (Fig. 11A).

Single drug treatment of the HER2-driven BT474 model with MOR12606 or MOR13655 resulted in 53% or 46% T/C, respectively (Fig. 1 IB).

We also examined the effect of combining two HER3-targeting antibodies that bind two different non-overlapping epitopes. Repeated dosing of the BT474 model with the combination of MOR10703 and MOR12606 produced a 7% T/C (Figure 12).

Introduce references

All references cited herein (including patents, patent applications, treatises, textbooks, etc.) and references cited therein (to the extent not listed) are hereby incorporated by reference in their entirety.

equivalent

We believe the foregoing description will be sufficient to enable one skilled in the art to practice the invention. The above description and examples detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It should be understood, however, that the invention, no matter how exhaustively described above, can be practiced in various ways and that the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims (47)

1. An isolated antibody or fragment thereof that recognizes a non-linear epitope of the HER3 receptor, wherein the non-linear epitope comprises amino acid residues within domain 3 of the HER3 receptor, wherein the antibody or fragment thereof binds to at least one selected from the group consisting of The binding surface of the amino acid residues of Surface B, and wherein the antibody or fragment thereof blocks both ligand-dependent and ligand-independent signal transduction. 2. The isolated antibody or fragment thereof of claim 1, wherein binding surface B comprises at least one amino acid residue selected from the group consisting of amino acid residues 335-342, 398, 400, 424-428, 431, 433-434 and 455. 3. The isolated antibody or fragment of claim 1, wherein the antibody or fragment thereof is further bound to a binding surface A. 4. The isolated antibody or fragment thereof of claim 3, wherein binding surface A comprises at least one amino acid residue selected from amino acid residues 362-376. 5. An isolated antibody or fragment thereof that recognizes a non-linear epitope of the HER3 receptor, wherein the non-linear epitope comprises amino acid residues within domain 3 of the HER3 receptor, wherein the antibody or fragment thereof binds to at least one selected from the group consisting of The amino acid residues of surface A and at least one binding surface selected from the amino acid residues of surface B, and wherein the antibody or fragment thereof blocks both ligand-dependent and ligand-independent signal transduction. 6. The isolated antibody or fragment thereof of claim 5, wherein the antibody or fragment thereof blocks HER3 ligand binding on the HER3 receptor. 7. The isolated antibody or fragment thereof of claim 6, wherein the HER3 ligand is selected from the group consisting of neuregulin 1 (NRG), neuregulin 2, beta cellulose, heparin-binding epidermal growth factor and epiregulin. 8. The isolated antibody or fragment thereof of claim 5, wherein the antibody or fragment thereof has any one of the following characteristics: binding to an inactive state of the HER3 receptor; Prevents HER3 from adopting an active conformation by steric hindrance; prevents HER3 from adopting an active conformation by reducing the degree of flexibility in domain 3; induces a conformational change in domain 3 residues 371-377 that prevents HER3 from adopting an active conformation; destabilizes HER3 so that it Susceptible to degradation; accelerated downregulation of HER3 on the cell surface; and generation of non-native HER3 dimers that are susceptible to proteolytic degradation or unable to dimerize with other receptor tyrosine kinases. 9. The isolated antibody or fragment thereof of claim 5, wherein binding surface A comprises amino acid residues 362-376. 10. The isolated antibody or fragment thereof of claim 5, wherein binding surface B comprises amino acid residues 335-342, 398, 400, 424-428, 431, 433-434 and 455. 11. The isolated antibody of claim 5, wherein the non-linear epitope comprises amino acid residues 335-342, 362-376, 398, 400, 424-428, 431, 433-434, and 455 (within domain 3), or its subset. 12. The isolated antibody of claim 5, wherein the VH of the antibody or fragment thereof binds at least one of the following HER3 residues: Ile365, Thr366, Asn369, Gly370, Asp371 , Pro372, Trp373, His374, Lys375, Gln400 and Lys434. 13. The isolated antibody of claim 5, wherein the VL of the antibody or fragment thereof binds at least one of the following HER3 residues: Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376 , Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455. 14. The isolated antibody or fragment thereof of claim 5, wherein binding of the antibody or fragment thereof to the HER3 receptor in the absence of a HER3 ligand reduces uncoordination of the HER2-HER3 protein complex in cells expressing HER2 and HER3 body-dependent formation. 15. The isolated antibody or fragment thereof of claim 5, wherein the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a HER3 ligand-independent phosphorylation assay. 16. The isolated antibody or fragment thereof of claim 15, wherein the HER3 ligand-independent phosphorylation assay uses HER2-amplified cells, wherein the HER2-amplified cells are SK-Br-3 cells and BT-474. 17. The isolated antibody or fragment thereof of claim 5, wherein binding of the antibody or fragment thereof to the HER3 receptor in the presence of a HER3 ligand reduces ligand dependence of the HER2-HER3 protein complex in cells expressing HER2 and HER3 sexual formation. 18. The isolated antibody or fragment thereof of claim 5, wherein the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a HER3 ligand-dependent phosphorylation assay. 19. The isolated antibody or fragment thereof of claim 18, wherein the HER3 ligand-dependent phosphorylation assay uses MCF7 cells stimulated in the presence of neuregulin (NRG). 20. The isolated antibody or fragment thereof of claim 5, wherein the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and synthetic antibodies. 21. An isolated antibody or fragment thereof that recognizes an epitope of the HER3 receptor, wherein the epitope comprises amino acid residues within domains 3-4 of the HER3 receptor, and wherein the antibody or fragment thereof blocks ligand-dependent and independent Ligand-dependent signal transduction of both. 22. The isolated antibody of claim 21, wherein the epitope comprises at least one amino acid residue selected from amino acid residues 329-498 (domain 3) of SEQ ID NO:1 and at least one amino acid residue selected from SEQ ID NO:1 Amino acid residues from bases 499-642 (domain 4). 23. The isolated antibody of claim 21, wherein the epitope comprising amino acid residues within domains 3-4 is selected from the group consisting of a linear epitope, a non-linear epitope and a conformational epitope. 24. The isolated antibody of claim 21, wherein the binding of the antibody or fragment thereof to the HER3 receptor in the absence of a HER3 ligand reduces ligand-independence of the HER2-HER3 protein complex in cells expressing HER2 and HER3 form. 25. The isolated antibody or fragment thereof of claim 21, wherein the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a HER3 ligand-independent phosphorylation assay. 26. The isolated antibody or fragment thereof of claim 25, wherein the HER3 ligand-independent phosphorylation assay uses HER2-amplified cells, wherein the HER2-amplified cells are SK-Br-3 cells and BT-474. 27. The isolated antibody or fragment thereof of claim 21, wherein binding of the antibody or fragment thereof to the HER3 receptor in the presence of a HER3 ligand reduces ligand dependence of the HER2-HER3 protein complex in cells expressing HER2 and HER3 sexual formation. 28. The isolated antibody or fragment thereof of claim 21, wherein the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a HER3 ligand-dependent phosphorylation assay. 29. The isolated antibody or fragment thereof of claim 28, wherein the HER3 ligand-dependent phosphorylation assay uses MCF7 cells stimulated in the presence of neuregulin (NRG). 30. An isolated antibody or fragment thereof against the HER3 receptor having at least 1×10 ⁷ M ⁻¹ , 10 ⁸ M ⁻¹ , 10 ⁹ M ⁻¹ , 10 ¹⁰ M ⁻¹ , 10 ¹¹ M ⁻¹ , 10 ¹² Dissociation (K _D ) of M ⁻¹ , 10 ¹³ M ⁻¹ , wherein the antibody or fragment thereof blocks both ligand-dependent and ligand-independent signal transduction. 31. The isolated antibody or fragment thereof of claim 30, wherein the antibody or fragment thereof inhibits phosphorylation of HER3 as measured by an in vitro phosphorylation assay selected from phospho-HER3 and phospho-Akt. 32. An isolated antibody or fragment thereof that binds the same non-linear epitope within domain 3 of HER3 as the antibody described in Table 1. 33. An isolated antibody or fragment thereof that binds to the same amino acid residues within domains 3-4 of HER3 as the antibody described in Table 2. 34. An antibody fragment binding to HER3 selected from Fab, F(ab ₂ )', F(ab) ₂ ', scFv, VHH, VH, VL, dAb, wherein the antibody fragment blocks ligand-dependent and ligand-independent dependent signal transduction both. 35. A pharmaceutical composition comprising an antibody or fragment thereof, and a pharmaceutically acceptable carrier. 36. The pharmaceutical composition of claim 35, further comprising an additional therapeutic agent. 37. The pharmaceutical composition of claim 36, wherein the additional therapeutic agent is selected from the group consisting of HER1 inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, mTOR inhibitors, and PI3 kinase inhibitors. 38. The pharmaceutical composition of claim 37, wherein the additional therapeutic agent is selected from Matuzumab (EMD72000), Cetuximab, Panitumumab, mAb 806, Nimotuzumab, Gefitinib, CI-1033 (PD183805), lapatinib (GW-572016), lapatinib ditosylate, Erlotinib hydrochloride (OSI-774), PKI-166 and HER1 inhibitors; selected from Pertuzumab, Trastuzumab, MM-111, Neratinib, Lapatinib, or Lapatinib Ditosylate HER2 inhibitors; selected from MM-121, MM-111, IB4C3, 2DID12 (U3PharmaAG), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech), MOR10703 (Novartis) and small molecules that inhibit HER3 HER3 inhibitors; and HER4 inhibitors. 39. The pharmaceutical composition of claim 37, wherein the additional therapeutic agent is a HER3 inhibitor, wherein the HER3 inhibitor is MOR10703. 40. The pharmaceutical composition of claim 37, wherein the additional therapeutic agent is selected from temsirolimus ridaforolimus/rapamycin, AP23573, MK8669, everolimus mTOR inhibitors. 41. The pharmaceutical composition of claim 37, wherein the additional therapeutic agent is a PI3 kinase inhibitor selected from GDC 0941, BEZ235, BKM120 and BYL719. 42. A method of treating cancer comprising selecting an individual with a HER3 expressing cancer, administering to said individual an effective amount of a composition comprising an antibody or fragment thereof disclosed in Table 1 or Table 2. 43. The method of claim 42, wherein the individual is human and the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, acute myelogenous leukemia, chronic myelogenous leukemia, osteosarcoma, Squamous cell carcinoma, peripheral nerve sheath tumor, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barretts esophagus cancer, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer , melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, and endometriosis. 44. The method of claim 43, wherein the cancer is breast cancer. 45. The antibody or fragment thereof of claims 1-34 for use as a medicament. 46. The antibody or fragment thereof of claims 1-34 for use in the treatment of cancer mediated by a HER3 ligand-dependent or ligand-independent signal transduction pathway. 47. Use of the antibody or fragment thereof of claims 1-34 for the preparation of cancers mediated by HER3 ligand-dependent signal transduction pathways or ligand-independent signal transduction pathways, said cancer being selected from Breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumor, nerve sheath tumor, head and neck cancer, bladder cancer, esophageal cancer, Barretts esophagus cancer, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia ( BPH), gynecomastia, and endometriosis.