Treatment and prevention of cancer using HER3 antigen-binding molecules

Abstract

Disclosed is a method for treating or preventing cancer in a subject, wherein the cancer comprises cells with a mutation that results in increased expression of a ligand for HER3, the method comprising administering to the subject a therapeutically or prophylactically effective amount of an antigen-binding molecule capable of binding to HER3.

Description

Technical Field

[0001] This application claims priority from GB1913079.8, filed on 11 September 2019, the content and elements of which are hereby incorporated by reference for all purposes.

[0002] Field of the Invention The present invention relates to the field of molecular biology, and more specifically to antibody technology and methods of medical treatment and prevention.

Background Art

[0003] Increased HER3 expression is associated with poor prognosis in multiple solid tumors, including breast, gastric, head and neck, pancreatic, ovarian, and lung cancers. HER3-mediated signaling results in detrimental outcomes for tumor progression, and upregulation of HER3 is associated with resistance to anti-HER2 and anti-EGFR therapies. Solid tumors refractory to anti-PD-1 therapy have been shown to express HER3 at higher levels compared to responders to anti-PD-1 therapy.

[0004] HER3-binding antibodies are described, for example, in Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1):39-48. LJM-716, an anti-HER3 antibody, binds to epitopes on subdomains II and IV of the HER3 extracellular domain and locks HER3 in an inactive conformation (Garner et al., Cancer Res (2013) 73:6024-6035). MM-121 (also known as seribantumab) has been shown to inhibit HER3-mediated signaling by blocking the binding of heregulin (HRG) to HER3 (Schoeberl et al., Sci. Signal. (2009) 2(77):ra31). Patritumab (also known as U-1287 and AMG-888) also blocks the binding of heregulin to HER3 (see, for example, Shimizu et al., Cancer Chemother Pharmacol. (2017), 79(3):489-495). RG7116 (also known as lumretuzumab and RO-5479599) recognizes an epitope within subdomain I of the HER3 extracellular domain (see, for example, Mirschberger et al., Cancer Research (2013) 73(16)5183-5194). KTN3379 binds to HER3 through interactions with amino acid residues within subdomain III (corresponding to positions below SEQ ID NO:1: Gly476, Pro477, Arg481, Gly452, Arg475, Ser450, Gly420, Ala451, Gly419, Arg421, Thr394, Leu423, Arg426, Gly427, Lys356, Leu358, Leu358, Lys356, Ala330, Lys329, and Gly337), as well as Met310, Glu311, and Pro328 of subdomain II (see Lee et al., Proc Natl Acad Sci USA. October 27, 2015, 112(43):13225).AV-203 (also known as CAN-017) has been shown to block the binding of NRG1 to HER3 and to promote the degradation of HER3 (see Meetze et al., Eur J Cancer 2012, 48:126). REGN1400 also inhibits the binding of the ligand to HER3 (see Zhang et al., Mol Cancer Ther (2014) 13:1345 - 1355). RG7597 (duligotumab) is a bispecific Fab (DAF) that can bind to both HER3 and EGFR and binds to subdomain III of HER3 (see Schaefer et al., Cancer Cell (2011) 20(4):472 - 486). MM-111 and MM-141 are bispecific antibodies with HER3-binding arms that inhibit the binding of the HRG ligand to HER3 (see McDonagh et al., Mol Cancer Ther (2012) 11:582 - 593 and Fitzgerald et al., Mol Cancer Ther (2014) 13:410 - 425).

Summary of the Invention

Means for Solving the Problems

[0005] The present invention provides an antigen-binding molecule capable of binding to HER3 according to any of the embodiments described herein for use in a method of treating or preventing cancer in a subject, wherein the cancer comprises cells characterized by the expression / overexpression of a HER3 ligand.

[0006] Also provided is the use of an antigen-binding molecule capable of binding to HER3 according to any of the embodiments described herein in the manufacture of a medicament for use in a method of treating or preventing cancer in a subject, wherein the cancer comprises cells characterized by the expression / overexpression of a HER3 ligand.

[0007] Also provided is a method of treating or preventing cancer in a subject, the cancer comprising cells characterized by the expression / overexpression of a HER3 ligand, the method comprising administering to the subject a therapeutically or prophylactically effective amount of an antigen-binding molecule capable of binding to HER3 according to any of the embodiments described herein.

[0008] In some embodiments, according to various aspects of the invention, the cancer comprises cells having a mutation that results in increased expression of a ligand for HER3.

[0009] More specifically, the invention provides an antigen-binding molecule capable of binding to HER3 for use in a method of treating or preventing cancer in a subject, the cancer comprising cells having a mutation that results in increased expression of a ligand for HER3, (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 43 HC-CDR2 having the amino acid sequence of SEQ ID NO: 46 HC-CDR3 having the amino acid sequence of SEQ ID NO: 51 incorporated into a heavy chain variable (VH) region, and (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 91 LC-CDR2 having the amino acid sequence of SEQ ID NO: 94 LC-CDR3 having the amino acid sequence of SEQ ID NO: 99 incorporated into a light chain variable (VL) region is provided.

[0010] Also provided is the use of an antigen-binding molecule capable of binding to HER3 in the manufacture of a medicament for use in a method of treating or preventing cancer in a subject, the cancer comprising cells having a mutation that results in increased expression of a ligand for HER3, the antigen-binding molecule being (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 43 HC-CDR2 having the amino acid sequence of SEQ ID NO: 46 HC-CDR3 having the amino acid sequence of SEQ ID NO: 51 a heavy chain variable (VH) region incorporating them, and (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 91 LC-CDR2 having the amino acid sequence of SEQ ID NO: 94 LC-CDR3 having the amino acid sequence of SEQ ID NO: 99 a light chain variable (VL) region incorporating them are also provided for use.

[0011] Also provided is a method of treating or preventing cancer in a subject, wherein the cancer comprises cells having a mutation that results in increased expression of a ligand for HER3, and the method comprises administering to the subject a therapeutically or prophylactically effective amount of an antigen-binding molecule that can bind to HER3, wherein the antigen-binding molecule (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 43 HC-CDR2 having the amino acid sequence of SEQ ID NO: 46 HC-CDR3 having the amino acid sequence of SEQ ID NO: 51 a heavy chain variable (VH) region incorporating them, and (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 91 LC-CDR2 having the amino acid sequence of SEQ ID NO: 94 LC-CDR3 having the amino acid sequence of SEQ ID NO: 99 a light chain variable (VL) region incorporating them is also provided.

[0012] In some embodiments according to various aspects of the invention, the ligand for HER3 comprises an amino acid sequence having at least 60% sequence identity to the EGF-like domain of NRG.

[0013] In some embodiments, the cancer comprises cells having an NRG gene fusion. In some embodiments, the NRG gene fusion is selected from CLU-NRG1, CD74-NRG1, DOC4-NRG1, SLC3A2-NRG1, RBPMS-NRG1, WRN-NRG1, SDC4-NRG1, RAB2IL1-NRG1, VAMP2-NRG1, KIF13B-NRG1, THAP7-NRG1, SMAD4-NRG1, MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1, DPYSL2-NRG1, ATP1B1-NRG1, CDH6-NRG1, APP-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1, CDK1-NRG1, BMPRIB-NRG1, TNFRSF10B-NRG1, MCPH1-NRG1 and SLC12A2-NRG2. In some embodiments, the NRG gene fusion is selected from CLU-NRG1, CD74-NRG1, SLC3A2-NRG1 or VAMP2-NRG1.

[0014] In some embodiments, the cancer is derived from the lung, breast, head, neck, kidney, ovary, pancreas, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue or nasopharynx.

[0015] In some embodiments, the cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, invasive breast cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, renal clear cell carcinoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, bladder cancer, urothelial bladder cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor and nasopharyngeal neuroendocrine tumor. In some embodiments, the cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma and lung squamous cell carcinoma.

[0016] In some embodiments, the antigen-binding molecule is (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48 a VH region incorporating them, and (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 a VL region incorporating them is included.

[0017] In some embodiments, the antigen-binding molecule is a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 36, and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 83 is included.

[0018] In some embodiments, the antigen-binding molecule is the following framework regions (FR): HC-FR1 having the amino acid sequence of SEQ ID NO: 53 HC-FR2 having the amino acid sequence of SEQ ID NO: 59 HC-FR3 having the amino acid sequence of SEQ ID NO: 66 HC-FR4 having the amino acid sequence of SEQ ID NO: 71 a VH region incorporating them is included.

[0019] In some embodiments, the antigen-binding molecule is the following framework regions (FR): LC-FR1 having the amino acid sequence of SEQ ID NO: 104 LC-FR2 having the amino acid sequence of SEQ ID NO: 110 LC-FR3 having the amino acid sequence of SEQ ID NO: 120 LC-FR4 having the amino acid sequence of SEQ ID NO: 125 and incorporates the VL region containing.

[0020] In some embodiments, the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 171.

[0021] In some embodiments, the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 177.

BEST MODE FOR CARRYING OUT THE INVENTION

[0022] Known anti-HER3 antibodies are broadly classified into two classes. Antibodies of the first class bind to domains I and / or III of HER3, thereby competitively inhibiting ligand binding to HER3. Seribantumab (MM-121) is a representative member of this class, and other members include patritumab (U3-1287 or AMG-888), lumretuzumab (RG-7116), AV-203, GSK2849330, and REGN1400. Antibodies of the second class lock HER3 into an inactive conformation through binding to the interface between domains II and IV, or between domains II and III. LJM-716, like KTN3379, is a representative example of this class.

[0023] The present invention relates to novel HER3-binding molecules having improved properties compared to known anti-HER3 antibodies.

[0024] The inventors contemplated the targeted generation of antigen-binding molecules that bind to specific regions of interest within the extracellular region of HER3. The HER3-binding molecules of the present invention have a combination of desired biophysical and / or functional properties compared to antigen-binding molecules disclosed in the prior art.

[0025] In an embodiment of the present invention, the antigen-binding molecule can bind to subdomain II (SEQ ID NO: 16) of the extracellular region of HER3 and inhibits the association of the bound HER3 molecule with its interaction partner.

[0026] In particular, the HER3-binding antigen-binding molecules described herein have been demonstrated to bind to an epitope of HER3 and result in (i) potent inhibition of the association of HER3 with its interaction partners (e.g., EGFR, HER2), and (ii) high-affinity binding to HER3 both in the presence and absence of the NRG ligand. This unique combination of properties results in potent inhibition of downstream signaling and exceptional anti-cancer activity against a broad range of cancers.

[0027] HER3 HER3 (also known as ERBB3 LCCS2, MDA-BF-1, for example) is a protein identified by UniProt P21860. Alternative splicing of the mRNA encoded by the human ERBB3 gene results in five different isoforms: isoform 1 (UniProt: P21860-1, v1; SEQ ID NO: 1); isoform 2 (UniProt: P21860-2; SEQ ID NO: 2), which contains a sequence different from SEQ ID NO: 1 at position 141 and lacks the amino acid sequence corresponding to positions 183 to 1342 of SEQ ID NO: 1; isoform 3 (UniProt: P21860-3; SEQ ID NO: 3), which contains the substitution C331F compared to SEQ ID NO: 1 and lacks the amino acid sequence corresponding to positions 332 to 1342 of SEQ ID NO: 1; isoform 4 (UniProt: P21860-4; SEQ ID NO: 4), which lacks the amino acid sequence corresponding to positions 1 to 59 of SEQ ID NO: 1; and isoform 5 (UniProt: P21860-5; SEQ ID NO: 5), which lacks the amino acid sequence corresponding to positions 1 to 643 of SEQ ID NO: 1.

[0028] Since the 19 amino acids at the N-terminus of SEQ ID NOs: 1 to 3 constitute a signal peptide, the mature forms of HER3 isoforms 1, 2, and 3 (i.e., after processing to remove the signal peptide) have the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively.

[0029] The structure and function of HER3 are described, for example, in Cho and Leahy Science (2002) 297(5585):1330 - 1333, Singer et al., Journal of Biological Chemistry (2001) 276, 44266 - 44274, Roskoski et al., Pharmacol.Res. (2014) 79:34 - 74, Bazley and Gullick Endocrine-Related Cancer (2005) S17 - S27, and Mujoo et al., Oncotarget (2014) 5(21):10222 - 10236, each of which is hereby incorporated by reference in its entirety. HER3 is a single-pass transmembrane ErbB receptor tyrosine kinase having an N-terminal extracellular region (SEQ ID NO: 9) that includes two leucine-rich subdomains (Domain I and III, shown in SEQ ID NOs: 15 and 17, respectively) and two cysteine-rich subdomains (Domain II and IV, shown in SEQ ID NOs: 16 and 18, respectively). Domain II includes a β-hairpin dimerization loop (SEQ ID NO: 19) that is involved in intermolecular interactions with other HER receptor molecules. The extracellular region is linked to the cytoplasmic region (SEQ ID NO: 11) via a transmembrane region (SEQ ID NO: 10). The cytoplasmic region includes a membrane-proximal segment (SEQ ID NO: 12), a protein kinase domain (SEQ ID NO: 13), and a C-terminal segment (SEQ ID NO: 14).

[0030] Signaling via HER3 involves receptor homodimerization (i.e., with other HER3 receptors) or heterodimerization (with other HER receptors, e.g., HER2), and as a consequence, autophosphorylation of the tyrosine protein kinase domain in the cytoplasmic region. Phosphorylated tyrosine residues recruit adapter / effector proteins (e.g., Grb2 and phospholipase Cγ (PLCγ)) that contain Src homology domain 2 (SH2) or phosphotyrosine-binding (PTB) domains.

[0031] Signaling via HER3 can be activated either ligand-dependently or ligand-independently. In the absence of ligand, HER3 receptor molecules are typically expressed as monomers on the cell surface in a conformation that prevents receptor dimerization, with the dimerization loop of subdomain II making an intramolecular contact with a pocket on subdomain IV. Binding of a HER3 ligand, such as neuregulin (NRG), e.g., NRG1 (also known as heregulin, HRG) or NRG2, to subdomains I and III of the extracellular region causes a conformational change, resulting in exposure of the dimerization loop of subdomain II, facilitating receptor dimerization and signaling. Some cancer-related mutations within HER3 can disrupt the interaction between subdomains II and IV required for formation of the inactive "closed" conformation, thereby causing constitutive presentation of the dimerization loop and activation of HER3-mediated signaling in the absence of ligand binding (see, e.g., Jaiswal et al., Cancer Cell (2013) 23(5):603-617).

[0032] As used herein, "HER3" refers to HER3 from any species, including isoforms, fragments, variants (including mutants), or homologs of HER3 from any species.

[0033] As used herein, a "fragment," "variant," or "homolog" of a protein may optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of a reference protein (e.g., a reference isoform). In some embodiments, fragments, variants, isoforms, and homologs of a reference protein may be characterized by their ability to perform the function performed by the reference protein.

[0034] A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that includes one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of a reference protein, but that retains a substantial degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein that is expressed by the same species as the reference protein (e.g., HER3 isoforms 1-5 are all isoforms of each other). A "homolog" generally refers to a variant of a reference protein that is produced by a different species compared to the species of the reference protein. For example, human HER3 isoform 1 (P21860-1, v1; SEQ ID NO: 1) and rhesus HER3 (UniProt: F7HEH3-1, v2; SEQ ID NO: 20) are homologs of each other. Homologs include orthologs.

[0035] A "fragment" of a reference protein can be of any length (by number of amino acids), but may optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment is derived), and may have a maximum length of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0036] The HER3 fragment may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids.

[0037] In some embodiments, HER3 is HER3 derived from a mammal (e.g., HER3 of a primate (e.g., rhesus monkey, cynomolgus monkey, non-human primate, or human), and / or a rodent (e.g., rat or mouse)). An isoform, fragment, variant, or homolog of HER3 may optionally have at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of a given species, e.g., immature or mature HER3 isoform, such as human HER3.

[0038] An isoform, fragment, variant, or homolog may optionally be a functional isoform, fragment, variant, or homolog having the functional characteristics / activities of a reference HER3 (e.g., human HER3 isoform 1), as determined by analysis, e.g., by an assay appropriate for the functional characteristics / activities. For example, an isoform, fragment, variant, or homolog of HER3 may exhibit an association with one or more of HER2, NRG1 (type I, II, III, IV, V, or VI), or NRG2 (α or β).

[0039] In some embodiments, HER3 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to one of SEQ ID NOs: 1-8.

[0040] In some embodiments, a fragment of HER3 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to one of SEQ ID NOs: 9-19, such as one of SEQ ID NOs: 9, 16, or 19.

[0041] Specific region of interest on the target molecule The antigen-binding molecules of the present invention are specifically designed to target specific regions of interest of HER3. In the two-step method, the HER3 region to be targeted was selected according to the analysis of predicted antigenicity, function, and safety. Then, a peptide corresponding to the target region was used as an immunogen to elicit specific monoclonal antibodies, and antibodies capable of binding to HER3 in the naive state were identified by subsequent screening. This approach provides precise control over antibody epitopes.

[0042] The antigen-binding molecules of the present invention can be defined by reference to the region of HER3 to which they bind. The antigen-binding molecules of the present invention can bind to specific regions of interest of HER3. In some embodiments, the antigen-binding molecule can bind to a linear epitope of HER3 consisting of a continuous sequence of amino acids (i.e., the primary sequence of amino acids). In some embodiments, the antigen-binding molecule can bind to a conformational epitope of HER3 consisting of a discontinuous sequence of amino acids within the amino acid sequence.

[0043] In some embodiments, the antigen-binding molecule of the present invention binds to HER3. In some embodiments, the antigen-binding molecule binds to the extracellular region of HER3 (e.g., the region shown in SEQ ID NO: 9). In some embodiments, the antigen-binding molecule binds to subdomain II of the extracellular region of HER3 (e.g., the region shown in SEQ ID NO: 16).

[0044] In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO: 229. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO: 229. In some embodiments, the antigen-binding molecule binds to the regions of HER3 shown in SEQ ID NOs: 230 and 231. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the regions of HER3 shown in SEQ ID NOs: 230 and 231. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO: 230. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO: 230. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO: 231. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO: 231.

[0045] In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 23. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 23. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 21. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 21. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 19. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 19. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 22. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 22.

[0046] In some embodiments, the antigen-binding molecule does not bind to the region of HER3 corresponding to positions 260-279 of SEQ ID NO: 1. In some embodiments, the antigen-binding molecule does not contact the amino acid residues of the region of HER3 corresponding to positions 260-279 of SEQ ID NO: 1. In some embodiments, the antigen-binding molecule does not bind to the region of HER3 set forth in SEQ ID NO: 23. In some embodiments, the antigen-binding molecule does not contact the amino acid residues of the region of HER3 set forth in SEQ ID NO: 23.

[0047] The region of the peptide / polypeptide to which the antibody binds can be determined by one of ordinary skill in the art using a variety of methods well known in the art, including X-ray co-crystallographic analysis of the antibody-antigen complex, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and protease-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is hereby incorporated by reference in its entirety.

[0048] In some embodiments, the antigen-binding molecule can bind to the same or overlapping HER3 region to which an antibody that includes the VH and VL sequences of one of the antibody clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6, 4-35-B2, or 4-35-B4, as described herein, binds. In some embodiments, the antigen-binding molecule can bind to the same or overlapping HER3 region to which an antibody that includes the VH and VL sequences of one of the antibody clones 10D1_c89, 10D1_c90, or 10D1_c91 binds. In some embodiments, the antigen-binding molecule can bind to the same or overlapping HER3 region to which an antibody that includes the VH and VL sequences of the antibody clone 10D1_c89 binds.

[0049] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length of about 2 to 50 amino acids within the region. "Polypeptide" refers to a polymeric chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

[0050] In some embodiments, the antigen-binding molecule of the present invention can bind to a polypeptide that comprises or consists of one of the amino acid sequences of SEQ ID NO: 1, 3, 4, 6, or 8.

[0051] In some embodiments, the antigen-binding molecule can bind to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antigen-binding molecule can bind to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0052] In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 229. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequences of SEQ ID NOs: 230 and 231. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 230. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 231. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 22.

[0053] In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of the amino acid sequence corresponding to positions 260-279 of SEQ ID NO: 1. In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 23.

[0054] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (e.g., Western blotting), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), or biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507).

[0055] In embodiments where the antigen-binding molecule can bind to a peptide / polypeptide comprising a reference amino acid sequence, the peptide / polypeptide can include one or more additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments, the peptide / polypeptide includes, at one or both ends of the reference amino acid sequence, for example, 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40, or 20-50 additional amino acids.

[0056] In some embodiments, in the context of the amino acid sequence of HER3, the additional amino acids provided at one or both ends (i.e., the N-terminus and C-terminus) of the reference sequence correspond to the positions at the ends of the reference sequence. By way of example, if an antigen-binding molecule can bind to a peptide comprising the sequence of SEQ ID NO: 23 and two additional amino acids at the C-terminus of SEQ ID NO: 23, the two additional amino acids can be threonine and lysine, corresponding to positions 278 and 279 of SEQ ID NO: 1.

[0057] In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide to which an antibody comprising the VH and VL sequences of one of the antibody clones described herein, 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6, 4-35-B2, or 4-35-B4, binds. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide to which an antibody comprising the VH and VL sequences of one of the antibody clones 10D1_c89, 10D1_c90, or 10D1_c91 binds. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide to which an antibody comprising the VH and VL sequences of the antibody clone 10D1_c89 binds.

[0058] Antigen-binding molecule The present invention provides an antigen-binding molecule capable of binding to HER3.

[0059] "Antigen-binding molecule" refers to a molecule capable of binding to a target antigen and includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies and multispecific antibodies (e.g., bispecific antibodies), as well as antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabody, triabody, scFv-Fc, minibody, single domain antibodies (e.g., VhH), etc.) insofar as they exhibit binding to the relevant target molecule.

[0060] The antigen-binding molecule of the present invention includes a portion capable of binding to a target antigen. In some embodiments, the portion capable of binding to a target antigen includes an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that can specifically bind to the target antigen. In some embodiments, the portion capable of binding to a target antigen includes or consists of an aptamer capable of binding to the target antigen, such as a nucleic acid aptamer (e.g., as reviewed in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). In some embodiments, the portion capable of binding to a target antigen includes or consists of an antigen-binding peptide / polypeptide, such as a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e., single domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody, or fibronectin (e.g., as reviewed in Reverdatto et al., Curr Top Med Chem., 2015;15(12):1082-1101, which is hereby incorporated by reference in its entirety (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48)).

[0061] The antigen-binding molecule of the present invention generally includes an antigen-binding domain that includes the VH and VL of an antibody that can specifically bind to a target antigen. As used herein, the antigen-binding domain formed by VH and VL may also be referred to as the Fv region.

[0062] The antigen-binding molecule may be an antigen-binding polypeptide, or an antigen-binding polypeptide complex, or may contain these. The antigen-binding molecule may also contain more than one polypeptide that together forms an antigen-binding domain. The polypeptides may be covalently associated or non-covalently associated. In some embodiments, the polypeptide forms part of a larger polypeptide that includes the polypeptide (e.g., in the case of an scFv that includes VH and VL, or in the case of an scFab that includes VH-CH1 and VL-CL).

[0063] The antigen-binding molecule may refer to a non-covalent or covalent complex of an IgG-like antigen-binding molecule that includes more than one polypeptide (e.g., 2, 3, 4, 6, or 8 polypeptides), such as two heavy-chain polypeptides and two light-chain polypeptides.

[0064] The antigen-binding molecule of the present invention can be designed and prepared using the sequence of a monoclonal antibody (mAb) that can bind to HER3. Antigen-binding regions of antibodies such as single-chain variable fragments (scFv), Fab, and F(ab’)2 fragments can also be used / provided. An “antigen-binding region” is any fragment of an antibody that can bind to the target to which a given antibody is specific.

[0065] Antibodies generally contain six complementarity-determining regions (CDRs) that are three in the heavy-chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light-chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody that binds to the target antigen.

[0066] The VH and VL regions each contain a framework region (FR) that provides a scaffold for each CDR, on either side of each CDR. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus - [HC-FR1] - [HC-CDR1] - [HC-FR2] - [HC-CDR2] - [HC-FR3] - [HC-CDR3] - [HC-FR4] - C-terminus, and the VL region has the following structure: N-terminus - [LC-FR1] - [LC-CDR1] - [LC-FR2] - [LC-CDR2] - [LC-FR3] - [LC-CDR3] - [LC-FR4] - C-terminus.

[0067] There are several different conventions for defining the CDRs and FRs of antibodies, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901 - 917 (1987), and Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671 - D674, such as VBASE2. The CDRs and FRs of the VH and VL regions of the antibody clones described herein are defined according to the IMGT V domain numbering rules described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55 - 77, in accordance with the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue): D413 - 22).

[0068] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule capable of binding to HER3. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule capable of binding to HER3. In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of an antigen-binding molecule capable of binding to HER3. That is, in some embodiments, the antigen-binding molecule comprises the VH and VL regions of an antigen-binding molecule capable of binding to HER3.

[0069] In some embodiments, the antigen-binding molecule comprises the VH / VL regions of a HER3-binding antibody clone described herein (i.e., anti-HER3 antibody clones 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10D1, 10A6, 4-35-B2, or 4-35-B4; e.g., 10D1_c89, 10D1_c90, or 10D1_c91; e.g., 10D1_c89), or the VH and VL regions derived therefrom.

[0070] In some embodiments, the antigen-binding molecule comprises a VH region according to one of the following (1)-(10).

[0071] (1) (derived from 10D1) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 43 HC-CDR2 having the amino acid sequence of SEQ ID NO: 46 HC-CDR3 having the amino acid sequence of SEQ ID NO: 51, or a VH region incorporating variants thereof in which one or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0072] (2) (10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c87, 10D1_c92, 10D1_c93) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 44 HC-CDR3 having the amino acid sequence of SEQ ID NO: 47, or a VH region incorporating these variants in which one or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0073] (3) (10D1_c85v1, 10D1_c85v2) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 47, or a VH region incorporating these variants in which one or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0074] (4) (10D1_c85o1) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 49, or a VH region incorporating these variants in which one or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0075] (5) (10D1_c85o2) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 50, or a VH region incorporating these variants in which one or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0076] (6) (10D1_c89, 10D1_c90) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48, or a VH region incorporating these variants in which one or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0077] (7) (10D1_c91) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 42 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48, or a VH region incorporating these variants in which one or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0078] (8) (10A6) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 158 HC-CDR2 having the amino acid sequence of SEQ ID NO: 159 HC-CDR3 having the amino acid sequence of SEQ ID NO: 160, A VH region incorporating these variants, in which one or more of one, two, or three amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0079] (9) (4-35-B2) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 128 HC-CDR2 having the amino acid sequence of SEQ ID NO: 129 HC-CDR3 having the amino acid sequence of SEQ ID NO: 130, A VH region incorporating these variants, in which one or more of one, two, or three amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0080] (10) (4-35-B4) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 144 HC-CDR2 having the amino acid sequence of SEQ ID NO: 145 HC-CDR3 having the amino acid sequence of SEQ ID NO: 146, A VH region incorporating these variants, in which one or more of one, two, or three amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0081] In some embodiments, the antigen-binding molecule comprises a VH region according to one of the following (11)-(24).

[0082] (11) (10D1) The following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO: 55 HC-FR2 having the amino acid sequence of SEQ ID NO: 58 HC-FR3 having the amino acid sequence of SEQ ID NO: 69 HC-FR4 having the amino acid sequence of SEQ ID NO: 73, A VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.

[0083] (12)(10D1_c75, 10D1_c92) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 52 HC-FR2 having the amino acid sequence of SEQ ID NO: 56 HC-FR3 having the amino acid sequence of SEQ ID NO: 61 HC-FR4 having the amino acid sequence of SEQ ID NO: 70, A VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.

[0084] (13)(10D1_c76, 10D1_c77, 10D1_c78v1) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 52 HC-FR2 having the amino acid sequence of SEQ ID NO: 56 HC-FR3 having the amino acid sequence of SEQ ID NO: 62 HC-FR4 having the amino acid sequence of SEQ ID NO: 70, A VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.

[0085] (14)(10D1_c78v2) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 52 HC-FR2 having the amino acid sequence of SEQ ID NO: 57 HC-FR3 having the amino acid sequence of SEQ ID NO: 62 HC-FR4 having the amino acid sequence of SEQ ID NO: 70, a VH region incorporating these variants in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0086] (15)(10D1_11B) The following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO: 224 HC-FR2 having the amino acid sequence of SEQ ID NO: 60 HC-FR3 having the amino acid sequence of SEQ ID NO: 63 HC-FR4 having the amino acid sequence of SEQ ID NO: 70, a VH region incorporating these variants in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0087] (16)(10D1_c85v1) The following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO: 52 HC-FR2 having the amino acid sequence of SEQ ID NO: 56 HC-FR3 having the amino acid sequence of SEQ ID NO: 64 HC-FR4 having the amino acid sequence of SEQ ID NO: 70, a VH region incorporating these variants in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0088] (17)(10D1_c85v2, 10D1_c85o1, 10D1_c85o2) The following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO: 52 HC-FR2 having the amino acid sequence of SEQ ID NO: 57 HC-FR3 having the amino acid sequence of SEQ ID NO: 64 HC-FR4 having the amino acid sequence of SEQ ID NO: 70, or a VH region incorporating variants thereof, in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are substituted with another amino acid.

[0089] (18) (10D1_c87, 10D1_c93) The following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO: 52 HC-FR2 having the amino acid sequence of SEQ ID NO: 56 HC-FR3 having the amino acid sequence of SEQ ID NO: 65 HC-FR4 having the amino acid sequence of SEQ ID NO: 70, or a VH region incorporating variants thereof, in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are substituted with another amino acid.

[0090] (19) (10D1_c89) The following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO: 53 HC-FR2 having the amino acid sequence of SEQ ID NO: 59 HC-FR3 having the amino acid sequence of SEQ ID NO: 66 HC-FR4 having the amino acid sequence of SEQ ID NO: 71, or a VH region incorporating variants thereof, in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are substituted with another amino acid.

[0091] (20) (10D1_c90) The following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO: 54 HC-FR2 having the amino acid sequence of SEQ ID NO: 59 HC-FR3 having the amino acid sequence of SEQ ID NO: 67 HC-FR4 having the amino acid sequence of SEQ ID NO: 71, or a VH region incorporating variants thereof, in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are substituted with another amino acid.

[0092] (21) (10D1_c91) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 53 HC-FR2 having the amino acid sequence of SEQ ID NO: 59 HC-FR3 having the amino acid sequence of SEQ ID NO: 68 HC-FR4 having the amino acid sequence of SEQ ID NO: 72, or a VH region incorporating variants thereof, in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are substituted with another amino acid.

[0093] (22) (10A6) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 161 HC-FR2 having the amino acid sequence of SEQ ID NO: 162 HC-FR3 having the amino acid sequence of SEQ ID NO: 163 HC-FR4 having the amino acid sequence of SEQ ID NO: 73, or a VH region incorporating variants thereof, in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are substituted with another amino acid.

[0094] (23) (4-35-B2) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 131 HC-FR2 having the amino acid sequence of SEQ ID NO: 132 HC-FR3 having the amino acid sequence of SEQ ID NO: 133 HC-FR4 having the amino acid sequence of SEQ ID NO: 134, or a VH region incorporating these variants in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced with another amino acid.

[0095] (24)(4 - 35 - B4) The following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO: 147 HC-FR2 having the amino acid sequence of SEQ ID NO: 148 HC-FR3 having the amino acid sequence of SEQ ID NO: 149 HC-FR4 having the amino acid sequence of SEQ ID NO: 73, or a VH region incorporating these variants in which one, two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced with another amino acid.

[0096] In some embodiments, the antigen-binding molecule comprises a VH region comprising a CDR according to one of (1)-(10) above and an FR according to one of (11)-(24) above.

[0097] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (25)-(41) below.

[0098] (25) A VH region comprising a CDR according to (1) and an FR according to (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), or (21).

[0099] (26) A VH region comprising a CDR according to (2) and an FR according to (11).

[0100] (27) A VH region comprising a CDR according to (2) and an FR according to (12).

[0101] The VH region containing a CDR according to (28)(2) and an FR according to (13).

[0102] (4) The VH region containing a CDR according to (29)(2) and an FR according to (14).

[0103] (5) The VH region containing a CDR according to (30)(2) and an FR according to (15).

[0104] (6) The VH region containing a CDR according to (31)(2) and an FR according to (18).

[0105] (7) The VH region containing a CDR according to (32)(3) and an FR according to (16).

[0106] (8) The VH region containing a CDR according to (33)(3) and an FR according to (17).

[0107] (9) The VH region containing a CDR according to (34)(4) and an FR according to (17).

[0108] (10) The VH region containing a CDR according to (35)(5) and an FR according to (17).

[0109] (11) The VH region containing a CDR according to (36)(6) and an FR according to (19).

[0110] (12) The VH region containing a CDR according to (37)(6) and an FR according to (20).

[0111] (13) The VH region containing a CDR according to (38)(7) and an FR according to (21).

[0112] (14) The VH region containing a CDR according to (39)(8) and an FR according to (22).

[0113] (15) The VH region containing a CDR according to (40)(9) and an FR according to (23).

[0114] (16) The VH region containing a CDR according to (41)(10) and an FR according to (24).

[0115] In some embodiments, the antigen-binding molecule comprises a VH region that follows one of the following (42)-(61).

[0116] (42) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0117] (43) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25.

[0118] (44) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26.

[0119] (45) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 27.

[0120] (46) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 28.

[0121] (47) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 29.

[0122] (48) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 30.

[0123] (49) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 31.

[0124] (50) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 32.

[0125] A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 33.

[0126] (52) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 34.

[0127] (53) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 35.

[0128] (54) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 36.

[0129] (55) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 37.

[0130] A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 38.

[0131] (57) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 39.

[0132] (58) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 40.

[0133] (59) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 127.

[0134] (60) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 143.

[0135] A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 157.

[0136] In some embodiments, the antigen-binding molecule comprises a VL region according to one of the following (62)-(71).

[0137] (62) (derived from 10D1) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 91 LC-CDR2 having the amino acid sequence of SEQ ID NO: 94 LC-CDR3 having the amino acid sequence of SEQ ID NO: 99; Or a VL region incorporating these variants in which one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0138] (63) (10D1, 10D1_c75, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c87, 10D1_c89, 10D1_c91, 10D1_c93) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95; Or a VL region incorporating these variants in which one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0139] (64) (10D1_c76) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 89 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95; or a VL region incorporating these variants in which one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0140] (65)(10D1_c77) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 90 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 96; or a VL region incorporating these variants in which one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0141] (66)(10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 93 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95; or a VL region incorporating these variants in which one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0142] (67)(10D1_c90) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 97; A VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0143] (68)(10D1_c92) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 98; A VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0144] (69)(10A6) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 165 LC-CDR2 having the amino acid sequence of SEQ ID NO: 166 LC-CDR3 having the amino acid sequence of SEQ ID NO: 167; A VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0145] (70)(4-35-B2) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 136 LC-CDR2 having the amino acid sequence of SEQ ID NO: 137 LC-CDR3 having the amino acid sequence of SEQ ID NO: 138; A VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0146] (71) The following CDRs of (4-35-B4): LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 152 LC-CDR3 having the amino acid sequence of SEQ ID NO: 153; Or a VL region incorporating these variants in which one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0147] In some embodiments, the antigen-binding molecule comprises a VL region according to one of the following (72)-(86).

[0148] (72) The following FRs of (10D1): LC-FR1 having the amino acid sequence of SEQ ID NO: 106 LC-FR2 having the amino acid sequence of SEQ ID NO: 113 LC-FR3 having the amino acid sequence of SEQ ID NO: 123 LC-FR4 having the amino acid sequence of SEQ ID NO: 126, Or a VL region incorporating these variants in which one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with another amino acid.

[0149] (73) The following FRs of (10D1_c75): LC-FR1 having the amino acid sequence of SEQ ID NO: 100 LC-FR2 having the amino acid sequence of SEQ ID NO: 107 LC-FR3 having the amino acid sequence of SEQ ID NO: 114 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, A VL region incorporating these variants, in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with another amino acid.

[0150] (74)(10D1_c76)The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 101 LC-FR2 having the amino acid sequence of SEQ ID NO: 108 LC-FR3 having the amino acid sequence of SEQ ID NO: 115 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, A VL region incorporating these variants, in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with another amino acid.

[0151] (75)(10D1_c77)The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 102 LC-FR2 having the amino acid sequence of SEQ ID NO: 108 LC-FR3 having the amino acid sequence of SEQ ID NO: 116 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, A VL region incorporating these variants, in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with another amino acid.

[0152] (76)(10D1_c78v1, 10D1_c78v2, 10D1_11B)The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 103 LC-FR2 having the amino acid sequence of SEQ ID NO: 108 LC-FR3 having the amino acid sequence of SEQ ID NO: 117 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, a VL region incorporating these variants in which one, two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

[0153] (77)(10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2) The following FRs: LC-FR1 having the amino acid sequence of SEQ ID NO: 103 LC-FR2 having the amino acid sequence of SEQ ID NO: 108 LC-FR3 having the amino acid sequence of SEQ ID NO: 118 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, a VL region incorporating these variants in which one, two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

[0154] (78)(10D1_c87) The following FRs: LC-FR1 having the amino acid sequence of SEQ ID NO: 103 LC-FR2 having the amino acid sequence of SEQ ID NO: 109 LC-FR3 having the amino acid sequence of SEQ ID NO: 119 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, a VL region incorporating these variants in which one, two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

[0155] (79)(10D1_c89) The following FRs: LC-FR1 having the amino acid sequence of SEQ ID NO: 104 LC-FR2 having the amino acid sequence of SEQ ID NO: 110 LC-FR3 having the amino acid sequence of SEQ ID NO: 120 LC-FR4 having the amino acid sequence of SEQ ID NO: 125, or a VL region incorporating these variants in which one, two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

[0156] (80)(10D1_c90) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 105 LC-FR2 having the amino acid sequence of SEQ ID NO: 110 LC-FR3 having the amino acid sequence of SEQ ID NO: 121 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, or a VL region incorporating these variants in which one, two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

[0157] (81)(10D1_c91) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 104 LC-FR2 having the amino acid sequence of SEQ ID NO: 111 LC-FR3 having the amino acid sequence of SEQ ID NO: 122 LC-FR4 having the amino acid sequence of SEQ ID NO: 125, or a VL region incorporating these variants in which one, two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

[0158] (82)(10D1_c92) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 100 LC-FR2 having the amino acid sequence of SEQ ID NO: 112 LC-FR3 having the amino acid sequence of SEQ ID NO: 114 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with another amino acid.

[0159] (83)(10D1_c93) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 103 LC-FR2 having the amino acid sequence of SEQ ID NO: 108 LC-FR3 having the amino acid sequence of SEQ ID NO: 119 LC-FR4 having the amino acid sequence of SEQ ID NO: 124, or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with another amino acid.

[0160] (84)(10A6) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 168 LC-FR2 having the amino acid sequence of SEQ ID NO: 169 LC-FR3 having the amino acid sequence of SEQ ID NO: 170 LC-FR4 having the amino acid sequence of SEQ ID NO: 142, or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with another amino acid.

[0161] (85)(4-35-B2) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 139 LC-FR2 having the amino acid sequence of SEQ ID NO: 140 LC-FR3 having the amino acid sequence of SEQ ID NO: 141 LC-FR4 having the amino acid sequence of SEQ ID NO: 142, or a VL region incorporating these variants in which one, two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

[0162] (86)(4-35-B4) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 154 LC-FR2 having the amino acid sequence of SEQ ID NO: 155 LC-FR3 having the amino acid sequence of SEQ ID NO: 156 LC-FR4 having the amino acid sequence of SEQ ID NO: 142, or a VL region incorporating these variants in which one, two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are substituted with another amino acid.

[0163] In some embodiments, the antigen-binding molecule comprises a VL region comprising a CDR according to one of (62)-(71) above and an FR according to one of (72)-(86) above.

[0164] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (87)-(102) below.

[0165] (87) A VL region comprising a CDR according to (62) and an FR according to (72), (73), (74), (75), (76), (77), (78), (79), (80), (81), (82), or (83).

[0166] (88) A VL region comprising a CDR according to (63) and an FR according to (72).

[0167] (89) A VL region comprising a CDR according to (63) and an FR according to (73).

[0168] The VL region including a CDR according to (90)(63) and an FR according to (76).

[0169] The VL region including a CDR according to (91)(63) and an FR according to (78).

[0170] The VL region including a CDR according to (92)(63) and an FR according to (79).

[0171] The VL region including a CDR according to (93)(63) and an FR according to (81).

[0172] The VL region including a CDR according to (94)(63) and an FR according to (83).

[0173] The VL region including a CDR according to (95)(64) and an FR according to (74).

[0174] The VL region including a CDR according to (96)(65) and an FR according to (75).

[0175] The VL region including a CDR according to (97)(66) and an FR according to (77).

[0176] The VL region including a CDR according to (98)(67) and an FR according to (80).

[0177] The VL region including a CDR according to (99)(68) and an FR according to (82).

[0178] The VL region including a CDR according to (100)(69) and an FR according to (84).

[0179] The VL region including a CDR according to (101)(70) and an FR according to (85).

[0180] The VL region including a CDR according to (102)(71) and an FR according to (86).

[0181] In some embodiments, the antigen-binding molecule comprises a VL region that follows one of the following (103)-(119).

[0182] (103) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 74.

[0183] (104) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 75.

[0184] (105) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 76.

[0185] (106) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 77.

[0186] (107) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 78.

[0187] (108) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 79.

[0188] (109) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 80.

[0189] (110) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 81.

[0190] (111) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 82.

[0191] A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 83.

[0192] (113) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 84.

[0193] (114) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 85.

[0194] (115) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 86.

[0195] (116) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 87.

[0196] A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 135.

[0197] (118) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 150.

[0198] (119) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 164.

[0199] In some embodiments, the antigen-binding molecule comprises a VH region according to any one of (1)-(61) above and a VL region according to any one of (62)-(119) above.

[0200] In embodiments according to the invention in which one or more amino acids are substituted with another amino acid, the substitution may be, for example, a conservative substitution according to the following table. In some embodiments, amino acids within the same block in the middle column are substituted. In some embodiments, amino acids in the same run in the rightmost column are substituted.

[0201] [Table 1] In some embodiments, the substitution may be functionally conservative. That is, in some embodiments, the substitution may not (or may substantially not) affect one or more functional properties (e.g., binding to a target) of the antigen-binding molecule comprising the substitution as compared to an equivalent unsubstituted molecule.

[0202] The VH and VL regions of the antigen-binding domain of an antibody together constitute the Fv region. In some embodiments, the antigen-binding molecule according to the invention comprises or consists of an Fv region that binds to HER3. In some embodiments, the VH and VL regions of the Fv are provided as a single polypeptide, i.e., a single-chain Fv (scFv), joined by a linker region.

[0203] In some embodiments, the antigen-binding molecule of the invention comprises one or more regions of the heavy-chain constant sequence of an immunoglobulin. In some embodiments, the heavy-chain constant sequence of the immunoglobulin is or is derived from the heavy-chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0204] In some embodiments, the heavy-chain constant sequence of the immunoglobulin is human immunoglobulin G1 constant (IGHG1; UniProt: P01857-1, v1; SEQ ID NO: 171). The 1st to 98th positions of SEQ ID NO: 171 form the CH1 region (SEQ ID NO: 172). The 99th to 110th positions of SEQ ID NO: 171 form the hinge region (SEQ ID NO: 173) between the CH1 and CH2 regions. The 111th to 223rd positions of SEQ ID NO: 171 form the CH2 region (SEQ ID NO: 174). The 224th to 330th positions of SEQ ID NO: 171 form the CH3 region (SEQ ID NO: 175).

[0205] The exemplified antigen-binding molecule can be prepared using pFUSE-CHIg-hG1, which contains substitution D356E, L358M (numbered according to EU numbering) within the CH3 region. The amino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is shown in SEQ ID NO: 176. It will be understood that the CH3 region can be further substituted according to the modification of the Fc region of the antigen-binding molecule described herein.

[0206] In some embodiments, the CH1 region comprises, or consists of, a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the sequence of SEQ ID NO: 172, or the amino acid sequence of SEQ ID NO: 172. In some embodiments, the hinge region between CH1-CH2 comprises, or consists of, a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the sequence of SEQ ID NO: 173, or the amino acid sequence of SEQ ID NO: 173. In some embodiments, the CH2 region comprises, or consists of, a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the sequence of SEQ ID NO: 174, or the amino acid sequence of SEQ ID NO: 174. In some embodiments, the CH3 region comprises, or consists of, a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the sequence of SEQ ID NO: 175 or 176, or the amino acid sequence of SEQ ID NO: 175 or 176.

[0207] In some embodiments, the antigen-binding molecule of the invention comprises one or more regions of the constant sequence of the light chain of an immunoglobulin. In some embodiments, the constant sequence of the light chain of the immunoglobulin is human immunoglobulin kappa constant (IGKC; Cκ; UniProt: P01834-1, v2; SEQ ID NO: 177). In some embodiments, the constant sequence of the light chain of the immunoglobulin is human immunoglobulin lambda constant (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In some embodiments, the CL region comprises or consists of a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the sequence of SEQ ID NO: 177 or the amino acid sequence of SEQ ID NO: 177.

[0208] The VL and constant light (CL) regions of the antigen-binding region of an antibody, and the VH region and constant heavy 1 (CH1) region together constitute the Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising VH, CH1, VL, and CL (e.g., Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising VL and CH (e.g., a VL-CH1 fusion polypeptide), i.e., in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of the Fab or CrossFab are provided as a single polypeptide joined by a linker region, i.e., a single-chain Fab (scFab) or a single-chain CrossFab (scCrossFab).

[0209] In some embodiments, the antigen-binding molecule of the invention comprises or consists of a Fab region that binds to HER3.

[0210] In some embodiments, the antigen-binding molecules described herein comprise or consist of an intact antibody that binds to HER3. As used herein, "intact antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different classes of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini, J Allergy Clin Immunol. (2010) 125(2 Suppl 2):S41-S52, which is incorporated herein by reference in its entirety.

[0211] The G class of immunoglobulins (i.e., IgG) is a glycoprotein of approximately 150 kDa that contains two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains a heavy chain constant region that follows VH and includes three constant domains (CH1, CH2, and CH3), and similarly, the light chain follows VL and includes CL. Depending on the heavy chain, the immunoglobulin can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be either kappa (κ) or lambda (λ).

[0212] In some embodiments, the antigen-binding molecules described herein comprise or consist of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that binds to HER3.

[0213] In some embodiments, the antigen-binding molecule of the invention is at least monovalent for HER3. The valence refers to the number of binding sites within the antigen-binding molecule for a given antigenic determinant. Thus, in some embodiments, the antigen-binding molecule comprises at least one binding site for HER3.

[0214] In some embodiments, the antigen-binding molecule comprises more than one binding site for HER3, e.g., two, three, or four binding sites. The binding sites may be the same or different. In some embodiments, the antigen-binding molecule is, e.g., bivalent, trivalent, or tetravalent with respect to HER3.

[0215] Aspects of the invention relate to multispecific antigen-binding molecules. "Multispecific" means that the antigen-binding molecule exhibits specific binding to more than one target. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains (i.e., at least two antigen-binding domains including, e.g., non-identical VH and VL).

[0216] In some embodiments, the antigen-binding molecule is at least bispecific as it binds to HER3 and another target (e.g., an antigen other than HER3). The term "bispecific" means that the antigen-binding molecule can specifically bind to at least two distinct antigenic determinants.

[0217] It will be understood that an antigen-binding molecule according to the invention (e.g., a multispecific antigen-binding molecule) may comprise antigen-binding molecules that can bind to the targets to which the antigen-binding molecule is specific. For example, an antigen-binding molecule that can bind to HER3 and an antigen other than HER3 may comprise (i) an antigen-binding molecule that can bind to HER3, and (ii) an antigen-binding molecule that can bind to an antigen other than HER3.

[0218] Furthermore, it will be understood that an antigen-binding molecule according to the present invention (e.g., a multispecific antigen-binding molecule) may comprise an antigen-binding polypeptide, or an antigen-binding polypeptide complex, capable of binding to a target to which the antigen-binding molecule is specific. For example, an antigen-binding molecule according to the present invention may comprise, for example, (i) an antigen-binding polypeptide complex capable of binding to HER3, comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3), and (ii) an antigen-binding polypeptide complex capable of binding to an antigen other than HER3, comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3).

[0219] In some embodiments, the antigen-binding molecules that are components of larger antigen-binding molecules (e.g., multispecific antigen-binding molecules) may be referred to as, for example, "antigen-binding domains" or "antigen-binding regions" of the larger antigen-binding molecules.

[0220] In some embodiments, the antigen-binding molecule comprises an antigen-binding molecule capable of binding to HER3 and an antigen-binding molecule capable of binding to an antigen other than HER3. In some embodiments, the antigen other than HER3 is a surface molecule of an immune cell. In some embodiments, the antigen other than HER3 is a cancer cell antigen. In some embodiments, the antigen other than HER3 is a receptor molecule, e.g., a cell surface receptor. In some embodiments, the antigen other than HER3 is a cell signaling molecule, e.g., a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen other than HER3 is a growth factor or a hormone.

[0221] A cancer cell antigen is an antigen that is expressed or overexpressed by cancer cells. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The expression of a cancer cell antigen can be associated with cancer. A cancer cell antigen may be abnormally expressed by cancer cells (e.g., a cancer cell antigen can be expressed with abnormal localization), or may be expressed with an abnormal structure by cancer cells. A cancer cell antigen may be capable of inducing an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen to which the antigen-binding molecules described herein bind is presented on the outer surface of the cancer cell (i.e., extracellular). A cancer cell antigen can be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the onset, progression, or severity of symptoms of cancer. A cancer-associated antigen may be associated with the cause or pathology of cancer and may be abnormally expressed as a result of cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA level and / or protein level) by cancer cells as compared to the expression level by, for example, equivalent non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen is preferentially expressed by cancerous cells and may not be expressed by equivalent non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen can be the product of a mutated cancer gene or a mutated tumor suppressor gene. In some embodiments, a cancer-associated antigen can also be the product of an overexpressed intracellular protein, or a cancer antigen produced by an oncogenic virus, a cancer fetal antigen, or a cell surface glycolipid or glycoprotein.

[0222] In some embodiments, the antigen other than HER3 is an antigen expressed by cells of HER3-related cancer. HER3-related cancer can be a cancer that expresses HER3 (e.g., expresses HER3 protein on the cell surface), and such cancer can be referred to as a "HER3-positive" cancer. HER3-related cancers include cancers in which the expression of the HER3 gene / protein is a risk factor and / or is positively associated with the occurrence, onset, progression, or severity of symptoms, and / or metastasis of the cancer. HER3-related cancers include those cancers described in Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1):39-48, and Sithanandam and Anderson Cancer Gene Ther (2008) 15(7):413-448, which are hereby incorporated by reference in their entirety. In some embodiments, the HER3-related cancer can be lung cancer (e.g., NSCLC), melanoma, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, colon cancer, or oral cancer.

[0223] Surface molecules of immune cells can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof that is expressed on or at the cell surface of immune cells. In some embodiments, the portion of the surface molecule of an immune cell to which an antigen-binding molecule of the invention binds is on the outer surface of the immune cell (i.e., extracellular). Surface molecules of immune cells can be expressed on the cell surface of any immune cell. In some embodiments, the immune cell can be a cell derived from hematopoietic cells, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. Lymphocytes can be, for example, T cells, B cells, natural killer (NK) cells, NKT cells, or innate lymphoid cells (ILCs), or progenitor cells thereof (e.g., thymocytes or pre-B cells). In some embodiments, the surface molecule of an immune cell can be a costimulatory molecule (e.g., CD28, OX40, 4-1BB, ICOS, or CD27) or a ligand thereof. In some embodiments, the surface molecule of an immune cell can be a checkpoint molecule (e.g., PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, or BTLA) or a ligand thereof.

[0224] Multispecific antigen-binding molecules according to the present invention can be provided in any suitable format, such as the formats described in Brinkmann and Kontermann MAbs (2017) 9(2):182-212, which are incorporated herein by reference in their entirety. Suitable formats include the formats shown in Figure 2 of Brinkmann and Kontermann MAbs (2017) 9(2):182-212: antibody conjugates, such as IgG2, F(ab’)2, or CovX-Body; IgG or IgG-like molecules, such as IgG, chimeric IgG, common HC of κλ-body; CH1 / CL fusion proteins, such as scFv2-CH1 / CL, VHH2-CH1 / CL; bispecific antigen-binding molecules “of variable domains only”, such as tandem scFv (taFV), triple body, diabody (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAb, triple head, tandem dAb / VHH, tetravalent dAb.VHH; non-Ig fusion proteins, such as scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, minibody, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine 2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, such as scFv-Fc(kih), scFv-Fc(CH3 charge pair), scFv-Fc(EW-RVT), scFv-fc(HA-TF), scFv-Fc(SEEDbody), taFv-Fc(kih), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc(SEEDbody), DART-Fc, scFv-CH3(kih), TriFab; Fc fusions, such as diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions, such as dia-dibody, scDb-CH3; IgE / IgM CH2 fusions, such as scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, such as Fab-scFv (biobody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, such as DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine 2; asymmetric IgG or IgG-like molecules, such as IgG(kih), IgG(kih) common LC, ZW1IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-IgG(kih), Fab-scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pair + CH1 / CL charge pair, hinge / CH3 charge pair, SEED-body, Duo-body, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc. *; appended and Fc-modified IgGs, such as IgG(kih)-Fv, IgG HA-TF-Fv, IgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, halfDVD-Ig, DVI-Ig (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, such as Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; appended IgG-HC fusions, such as IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CαCβ)Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab) Fab-IgG(CαCβFab), Fab-IgG(CR3), Fab-hinge-IgG(CR3); appended IgG-LC fusions, such as IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; appended IgG-HC and LC fusions, such as DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, Zybody; Fc fusions, such as Fab-scFv-Fc, scFv4-Ig; F(ab’)2 fusions, such as F(ab’)2-scFv2; CH1 / CL fusion proteins, such as scFv2-CH1-hinge / CL; modified IgGs, such as DAF (two-in-one-IgG), DutaMab, Mab 2 ; and non-Ig fusions, such as DNL-Fab4-IgG.

[0225] One of ordinary skill in the art can design and prepare bispecific antigen-binding molecules. Methods for producing bispecific antigen-binding molecules include, for example, chemically cross-linking antigen-binding molecules or antibody fragments by means of a reducible disulfide bond or a non-reducible thioether bond, as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1 - 2.13.16, which is hereby incorporated by reference in its entirety. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically cross-link Fab fragments, for example, via the SH-groups in the hinge region, to create a disulfide-linked bispecific F(ab)2 heterodimer.

[0226] Other methods for producing bispecific antigen-binding molecules include fusing antibody-producing hybridomas, for example, with polyethylene glycol, to produce quadroma cells that can secrete bispecific antibodies, as described in D.M. and Bast, B.J. 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1 - 2.13.16.

[0227] The bispecific antigen-binding molecules according to the present invention can also be produced, for example, by recombinant expression from nucleic acid constructs encoding polypeptides for antigen-binding molecules as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz), or French, How to make bispecific antigen-binding molecules, Methods Mol. Med. 2000;40:333-339, the entire contents of both of which are incorporated herein by reference. For example, a DNA construct encoding light and heavy chain variable domains for two antigen-binding fragments (i.e., light and heavy chain variable domains for an antigen-binding fragment capable of binding to HER3, and light and heavy chain variable domains for an antigen-binding fragment capable of binding to another target protein) and containing a sequence encoding an appropriate linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques. Subsequently, the recombinant bispecific antibody is produced by expression of the construct (e.g., in vitro) in a suitable host cell (e.g., a mammalian host cell), and then the expressed recombinant bispecific antibody can optionally be purified.

[0228] Fc region In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region.

[0229] For the IgG, IgA, and IgD isotypes, the Fc region is composed of CH2 and CH3 regions derived from one polypeptide and CH2 and CH3 regions derived from another polypeptide. The CH2 and CH3 regions derived from the two polypeptides together form the Fc region. For the IgM and IgE isotypes, the Fc region contains three constant domains (CH2, CH3, and CH4), and the CH2-CH4 derived from two polypeptides together form the Fc region.

[0230] In a preferred embodiment according to various aspects of the present disclosure, the Fc region comprises two polypeptides, each polypeptide comprising a CH2 region and a CH3 region.

[0231] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising a modification that promotes the association of the Fc region in one or more of the CH2 and CH3 regions. The recombinant co-expression of the polypeptides that are components of the antigen-binding molecule, and subsequent association, results in several possible combinations. In recombinant production, it is advantageous to introduce a modification of the Fc region that promotes the association of the heavy-chain polypeptides of the desired combination in order to improve the yield of the polypeptides of the desired combination within the antigen-binding molecule. The modification can, for example, promote hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described, for example, in Ha et al., Front. Immunol (2016) 7:394, which is incorporated herein by reference in its entirety.

[0232] In some embodiments, the antigen-binding molecule of the present invention has the following format: KiH, KiH shown in Table 1 of Ha et al., Front. Immunol (2016) 7:394 S-S 、HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT S-S 、SEED, or A107, and comprises an Fc region comprising a pair of substitutions within the CH3 region of the Fc region.

[0233] In some embodiments, the Fc region comprises, for example, a "knob-into-hole" or "KiH" modification as described in, for example, US 7,695,936 and Carter, J Immunol Meth 248, 7-15 (2001). In such embodiments, one of the CH3 regions of the Fc region comprises a "knob" modification and the other CH3 region comprises a "hole" modification. The "knob" and "hole" modifications are positioned within their respective CH3 regions such that the "knob" can be positioned within the "hole" to promote (and inhibit homodimerization of) heterodimerization of the polypeptide and / or to stabilize the heterodimer. The knob is constructed by substituting an amino acid having a small side chain with an amino acid having a larger side chain (e.g., tyrosine or tryptophan). The hole is created by substituting an amino acid having a large side chain with an amino acid having a smaller side chain (e.g., alanine or threonine).

[0234] In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule of the invention comprises a substitution (the numbering of positions / substitutions in the Fc, CH2, and CH3 regions herein follows the EU numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) T366W, and the other CH3 region of the Fc region comprises a substitution Y407V. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises a substitution T366W, and the other CH3 region of the Fc region comprises substitutions T366S and L368A. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises a substitution T366W, and the other CH3 region of the Fc region comprises substitutions Y407V, T366S, and L368A.

[0235] In some embodiments, the Fc region comprises, for example, the "DD-KK" modification as described in WO2014 / 131694A1. In some embodiments, one of the CH3 regions comprises the substitutions K392D and K409D, and the other CH3 region of the Fc region comprises the substitutions E356K and D399K. The modifications promote electrostatic interactions between the CH3 regions.

[0236] In some embodiments, the antigen-binding molecule of the invention is modified as described by Labrijn et al., Proc Natl Acad Sci USA. (2013) 110(13):5145 - 50 and comprises an Fc region referred to as the "duobody" format. In some embodiments, one of the CH3 regions comprises the substitution K409R, and the other CH3 region of the Fc region comprises the substitution K405L.

[0237] In some embodiments, the antigen-binding molecule of the invention comprises an Fc region comprising the "EEE-RRR" modification as described by Strop et al., J Mol Biol. (2012) 420(3):204 - 19. In some embodiments, one of the CH3 regions comprises the substitutions D221E, P228E, and L368E, and the other CH3 region of the Fc region comprises the substitutions D221R, P228R, and K409R.

[0238] In some embodiments, the antigen-binding molecule comprises an Fc region comprising the "EW-RVT" modification as described by Choi et al., Mol Cancer Ther (2013) 12(12):2748 - 59. In some embodiments, one of the CH3 regions comprises the substitutions K360E and K409W, and the other CH3 region of the Fc region comprises the substitutions Q347R, D399V, and F405T.

[0239] In some embodiments, one of the CH3 regions comprises the substitution S354C and the other CH3 region of the Fc region comprises the substitution Y349C. The introduction of these cysteine residues results in the formation of a disulfide bridge between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001), J Immunol Methods, 248, 7-15).

[0240] In some embodiments, the Fc region comprises the "KiH" S-S modification. In some embodiments, one of the CH3 regions comprises the substitutions T366W and S354C and the other CH3 region of the Fc region comprises the substitutions T366S, L368A, Y407V, and Y349C.

[0241] In some embodiments, the antigen-binding molecule of the invention comprises an Fc region comprising the "SEED" modification in which the β-strand segment of human IgG1 CH3 and the β-strand segment of human IgA CH3 are exchanged, as described in Davis et al., Protein Eng Des Sel (2010) 23(4):195-202.

[0242] In some embodiments, one of the CH3 regions comprises the substitutions S364H and F405A and the other CH3 region of the Fc region comprises the substitutions Y349T and T394F (see, e.g., Moore et al., MAbs (2011) 3(6):546-57).

[0243] In some embodiments, one of the CH3 regions comprises the substitutions T350V, L351Y, F405A, and Y407V and the other CH3 region of the Fc region comprises the substitutions T350V, T366L, K392L, and T394W (see, e.g., Von Kreudenstein et al., MAbs (2013) 5(5):646-54).

[0244] In some embodiments, one of the CH3 regions contains the substitutions K360D, D399M, and Y407A, and the other CH3 region of the Fc region contains the substitutions E345R, Q347R, T366V, and K409V (see, e.g., Leaver-Fay et al., Structure (2016) 24(4):641-51).

[0245] In some embodiments, one of the CH3 regions contains the substitutions K370E and K409W, and the other CH3 region of the Fc region contains the substitutions E357N, D399V, and F405T (see, e.g., Choi et al., PLoS One (2015) 10(12):e0145349).

[0246] Fc-mediated functions include binding to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), degranulation from cells, production of cytokines and / or chemokines, and processing and presentation of antigens.

[0247] In the art, modifications to the Fc region of antibodies that affect Fc-mediated functions are known, such as those described, for example, in Wang et al., Protein Cell (2018) 9(1):63-73, which is incorporated herein by reference in its entirety. Exemplary Fc region modifications that are known to affect the effector functions of antibodies are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73.

[0248] The combination of substitutions F243L / R292P / Y300L / V305I / P396L, which is a combination of substitutions, has been described in Stavenhagen et al., Cancer Res. (2007) to increase binding to FcγRIIIa, thereby enhancing ADCC. The combination of substitutions S239D / I332E or S239D / I332E / A330L, which is a combination of substitutions, has been described in Lazar et al., Proc Natl Acad Sci USA. (2006) 103:4005-4010 to increase binding to FcγRIIIa, thereby increasing ADCC. The combination of substitutions S239D / I332E / A330L has also been described to decrease binding to FcγRIIb, thereby increasing ADCC. The combination of substitutions S298A / E333A / K334A, which is a combination of substitutions, has been described in Shields et al., J Biol Chem. (2001) 276:6591-6604 to increase binding to FcγRIIIa, thereby increasing ADCC. The combination of substitutions L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and the combination of substitutions D270E / K326D / A330M / K334E in the other heavy chain, which are combinations of substitutions, have been described in Mimoto et al., MAbs. (2013):5:229-236 to increase binding to FcγRIIIa, thereby increasing ADCC. The combination of substitutions G236A / S239D / I332E, which is a combination of substitutions, has been described in Richards et al., Mol Cancer Ther. (2008) 7:2517-2527 to increase binding to FcγRIIa and increase binding to FcγRIIIa, thereby increasing ADCP.

[0249] The combination of substitutions, K326W / E333S, is described in Idusogie et al., J Immunol. (2001) 166(4):2571-2575 to increase binding to C1q, thereby increasing CDC. The combination of substitutions, S267E / H268F / S324T, is described in Moore et al., MAbs. (2010) 2(2):181-189 to increase binding to C1q, thereby increasing CDC. The combination of substitutions described in Natsume et al., Cancer Res. (2008) 68(10):3863-3872 has been reported to increase binding to C1q, thereby increasing CDC. The combination of substitutions, E345R / E430G / S440Y, is described in Diebolder et al., Science (2014) 343(6176):1260-1263 to increase hexamerization, thereby increasing CDC.

[0250] The combination of substitutions, M252Y / S254T / T256E, is described in Dall’Acqua et al., J Immunol. (2002) 169:5171-5180 to increase binding to FcRn at pH 6.0, thereby extending the half-life of the antigen-binding molecule. The combination of substitutions, M428L / N434S, is described in Zalevsky et al., Nat Biotechnol. (2010) 28:157-159 to increase binding to FcRn at pH 6.0, thereby extending the half-life of the antigen-binding molecule.

[0251] As used herein, when the heavy chain constant region / Fc region / CH2-CH3 region / CH2 region / CH3 region is described as including a reference position / substitution "corresponding" position / substitution, equivalent positions / substitutions within the homologous heavy chain constant region / Fc region / CH2-CH3 region / CH2 region / CH3 region are contemplated.

[0252] When the Fc region is described as including a particular position / substitution, the position / substitution may be present in one or both of the polypeptide chains that together form the Fc region.

[0253] Unless otherwise specified, positions in this specification refer to positions in the amino acid sequence of the human immunoglobulin constant region numbered according to the EU numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. By way of example, substitutions L242C and K334C in human IgG1 correspond to an L>C substitution at position 125 and a K>C substitution at position 217 of the human IgG1 constant region numbered according to SEQ ID NO: 171.

[0254] The identical heavy chain constant region is a heavy chain constant region comprising an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the heavy chain constant region of human IgG1 (i.e., the amino acid sequence shown in SEQ ID NO: 171). The identical Fc region is an Fc region composed of a polypeptide comprising an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the CH2-CH3 region of human IgG1 (i.e., the amino acid sequences shown in SEQ ID NO: 174 and 175). The identical CH2 region is a CH2 region comprising an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the CH2 region of human IgG1 (i.e., the amino acid sequence shown in SEQ ID NO: 174). The identical CH3 region is a CH3 region comprising an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the CH3 region of human IgG1 (i.e., the amino acid sequence shown in SEQ ID NO: 175).

[0255] Positions corresponding to positions identified within human IgG1 can be identified by sequence alignment, which can be performed using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960).

[0256] In some embodiments, the antigen-binding molecule of the invention comprises an Fc region comprising a modification that increases Fc-mediated function. In some embodiments, the Fc region comprises a modification that increases ADCC. In some embodiments, the Fc region comprises a modification that increases ADCP. In some embodiments, the Fc region comprises a modification that increases CDC. An antigen-binding molecule comprising an Fc region comprising a modification that increases Fc-mediated function (e.g., ADCC, ADCP, CDC) induces an increase in the level of the associated effector function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

[0257] In some embodiments, the antigen-binding molecule of the invention comprises an Fc region comprising a modification that increases the affinity for one or more Fc receptors (e.g., FcγRIIa, FcγRIIIa). Modifications that increase the affinity for Fc receptors can increase Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the antigen-binding molecule of the invention comprises an Fc region comprising a modification that reduces the affinity for C1q, such a modification reducing complement-dependent cytotoxicity (CDC), which may be desirable. In some embodiments, the antigen-binding molecule of the invention comprises an Fc region comprising a modification that increases hexamer formation. Modifications to the Fc region that can increase the affinity for one or more Fc receptors, reduce the affinity for C1q, and / or increase hexamer formation are described, for example, in Saxena and Wu Front Immunol. (2016) 7:580, which is hereby incorporated by reference in its entirety. In some embodiments, the antigen-binding molecule of the invention comprises an Fc region comprising a CH2 / CH3 comprising one or more of the substitutions shown in Table 1 of Saxena and Wu Front Immunol. (2016) 7:580.

[0258] In some embodiments, the antigen-binding molecule of the invention comprises an Fc comprising a modification that increases binding to an Fc receptor. In some embodiments, the Fc region comprises a modification that increases binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification that increases binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification that increases binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification that increases binding to FcγRIIa. In some embodiments, the Fc region comprises a modification that increases binding to FcγRIIb. In some embodiments, the Fc region comprises a modification that increases binding to FcRn. In some embodiments, the Fc region comprises a modification that increases binding to a complement protein. In some embodiments, the Fc region comprises a modification that increases or reduces binding to C1q. In some embodiments, the Fc region comprises a modification that promotes hexamerization of the antigen-binding molecule. In some embodiments, the Fc region comprises a modification that extends the half-life of the antigen-binding molecule. In some embodiments, the Fc region comprises a modification that increases co-engagement.

[0259] As used herein, "Fcγ receptor" may be derived from any species and includes isoforms, fragments, variants (including mutants), or homologs derived from any species. Similarly, "FcγRI", "FcγRIIa", "FcγRIIb", "FcγRIIc", "FcγRIIIa", and "FcγRIIIb" each refer to FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb derived from any species and include isoforms, fragments, variants (including mutants), or homologs derived from any species. Humans have six different classes of Fcγ receptors (mouse orthologs shown in parentheses): FcγRI (mFcγRI), FcγRIIa (mFcγRIII), FcγRIIb (mFcγRIIb), FcγRIIc, FcγRIIIa (mFcγRIV), and FcγRIIIb. Variant Fcγ receptors include, for example, the 158V and 158F polymorphisms of human FcγRIIIa, and the 167H and 167R polymorphisms of human FcγRIIa.

[0260] In some embodiments, the antigen-binding molecule of the invention comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following: C at a position corresponding to position 242; C at a position corresponding to position 334; A at a position corresponding to position 236; D at a position corresponding to position 239; E at a position corresponding to position 332; L at a position corresponding to position 330; K at a position corresponding to position 345; and G at a position corresponding to position 430 (e.g., in a heavy chain constant region containing these, or in a further polypeptide containing a CH2-CH3 region containing these) in an Fc region.

[0261] In some embodiments, the antigen-binding molecule of the present invention comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following substitutions (or corresponding substitutions): L242C, K334C, G236A, S239D, I332E, A330L, E345K, and E430G (e.g., comprising these, a heavy chain constant region, or further comprising one polypeptide comprising a CH2-CH3 region) and comprises an Fc region.

[0262] In some embodiments, the antigen-binding molecule comprises an Fc region that comprises C at the position corresponding to position 242 (e.g., comprising these, a heavy chain constant region, a CH2-CH3 region, or further comprising one polypeptide comprising a CH2 region). In some embodiments, the Fc region comprises C at the position corresponding to position 334 (e.g., comprising these, a heavy chain constant region, a CH2-CH3 region, or further comprising one polypeptide comprising a CH2 region). In some embodiments, the Fc region comprises C at the position corresponding to position 242 and C at the position corresponding to position 334 (e.g., comprising these, a heavy chain constant region, a CH2-CH3 region, or further comprising one polypeptide comprising a CH2 region).

[0263] In some embodiments, the antigen-binding molecule comprises an Fc region that comprises A at the position corresponding to position 236 (e.g., comprising these, a heavy chain constant region, a CH2-CH3 region, or further comprising one polypeptide comprising a CH2 region). In some embodiments, the Fc region comprises D at the position corresponding to position 239 (e.g., comprising these, a heavy chain constant region, a CH2-CH3 region, or further comprising one polypeptide comprising a CH2 region). In some embodiments, the Fc region comprises A at the position corresponding to position 236 and D at the position corresponding to position 239 (e.g., comprising these, a heavy chain constant region, a CH2-CH3 region, or further comprising one polypeptide comprising a CH2 region).

[0264] In some embodiments, the antigen-binding molecule comprises an Fc region that includes an E at the position corresponding to position 332 (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide). In some embodiments, the Fc region includes an A at the position corresponding to position 236, a D at the position corresponding to position 239, and an E at the position corresponding to position 332 (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide).

[0265] In some embodiments, the antigen-binding molecule comprises an Fc region that includes an L at the position corresponding to position 330 (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide). In some embodiments, the Fc region includes an A at the position corresponding to position 236, a D at the position corresponding to position 239, an E at the position corresponding to position 332, and an L at the position corresponding to position 330 (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide).

[0266] In some embodiments, the antigen-binding molecule comprises an Fc region that includes a K at the position corresponding to position 345 (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH3 region, and further including one polypeptide). In some embodiments, the Fc region includes a G at the position corresponding to position 430 (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH3 region, and further including one polypeptide). In some embodiments, the Fc region includes a K at the position corresponding to position 345 and a G at the position corresponding to position 430 (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide).

[0267] In some embodiments, the antigen-binding molecule comprises an Fc region comprising C at the position corresponding to position 242, C at the position corresponding to position 334, A at the position corresponding to position 236, and D at the position corresponding to position 239 (e.g., comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing these, and further comprising one polypeptide).

[0268] In some embodiments, the antigen-binding molecule comprises an Fc region comprising C at the position corresponding to position 242, C at the position corresponding to position 334, A at the position corresponding to position 236, D at the position corresponding to position 239, and E at the position corresponding to position 332 (e.g., comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing these, and further comprising one polypeptide).

[0269] In some embodiments, the antigen-binding molecule comprises an Fc region comprising C at the position corresponding to position 242, C at the position corresponding to position 334, A at the position corresponding to position 236, D at the position corresponding to position 239, E at the position corresponding to position 332, and L at the position corresponding to position 330 (e.g., comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing these, and further comprising one polypeptide).

[0270] In some embodiments, the antigen-binding molecule comprises an Fc region comprising C at the position corresponding to position 242, C at the position corresponding to position 334, K at the position corresponding to position 345, and G at the position corresponding to position 430 (e.g., comprising a heavy chain constant region or a CH2-CH3 region containing these, and further comprising one polypeptide).

[0271] In some embodiments, the antigen-binding molecule comprises an Fc region that includes a substituted L242C (or equivalent substitution) (e.g., including these, including a heavy chain constant region, CH2-CH3 region, or CH2 region, and further including one polypeptide). In some embodiments, the Fc region includes a substituted K334C (or equivalent substitution) (e.g., including these, including a heavy chain constant region, CH2-CH3 region, or CH2 region, and further including one polypeptide). In some embodiments, the Fc region includes a substituted L242C (or equivalent substitution) and a substituted K334C (or equivalent substitution) (e.g., including these, including a heavy chain constant region, CH2-CH3 region, or CH2 region, and further including one polypeptide).

[0272] In some embodiments, the antigen-binding molecule comprises an Fc region that includes a substituted G236A (or equivalent substitution) (e.g., including these, including a heavy chain constant region, CH2-CH3 region, or CH2 region, and further including one polypeptide). In some embodiments, the Fc region includes a substituted S239D (or equivalent substitution) (e.g., including these, including a heavy chain constant region, CH2-CH3 region, or CH2 region, and further including one polypeptide). In some embodiments, the Fc region includes a substituted G236A (or equivalent substitution) and a substituted S239D (or equivalent substitution) (e.g., including these, including a heavy chain constant region, CH2-CH3 region, or CH2 region, and further including one polypeptide).

[0273] In some embodiments, the antigen-binding molecule comprises an Fc region that includes a substituted I332E (or equivalent substitution) (e.g., including these, including a heavy chain constant region, CH2-CH3 region, or CH2 region, and further including one polypeptide). In some embodiments, the Fc region includes a substituted G236A (or equivalent substitution), a substituted S239D (or equivalent substitution), and a substituted I332E (or equivalent substitution) (e.g., including these, including a heavy chain constant region, CH2-CH3 region, or CH2 region, and further including one polypeptide).

[0274] In some embodiments, the antigen-binding molecule comprises an Fc region that includes the substitution A330L (or an equivalent substitution) (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide). In some embodiments, the Fc region includes the substitutions G236A (or an equivalent substitution), S239D (or an equivalent substitution), I332E (or an equivalent substitution), and A330L (or an equivalent substitution) (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide).

[0275] In some embodiments, the antigen-binding molecule comprises an Fc region that includes the substitution E345K (or an equivalent substitution) (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH3 region, and further including one polypeptide). In some embodiments, the Fc region includes the substitution E430G (or an equivalent substitution) (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH3 region, and further including one polypeptide). In some embodiments, the Fc region includes the substitutions E345K (or an equivalent substitution) and E430G (or an equivalent substitution) (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide).

[0276] In some embodiments, the antigen-binding molecule comprises an Fc region that includes the substitutions L242C (or an equivalent substitution), K334C (or an equivalent substitution), G236A (or an equivalent substitution), and S239D (or an equivalent substitution) (e.g., including these, a heavy chain constant region, a CH2-CH3 region, or a CH2 region, and further including one polypeptide).

[0277] In some embodiments, the antigen-binding molecule comprises an Fc region that includes a substituted L242C (or equivalent substitution), a substituted K334C (or equivalent substitution), a substituted G236A (or equivalent substitution), a substituted S239D (or equivalent substitution), and a substituted I332E (or equivalent substitution) (e.g., a further polypeptide including these, including a heavy chain constant region, a CH2-CH3 region, or a CH2 region).

[0278] In some embodiments, the antigen-binding molecule comprises an Fc region that includes a substituted L242C (or equivalent substitution), a substituted K334C (or equivalent substitution), a substituted G236A (or equivalent substitution), a substituted S239D (or equivalent substitution), a substituted I332E (or equivalent substitution), and a substituted A330L (or equivalent substitution) (e.g., a further polypeptide including these, including a heavy chain constant region, a CH2-CH3 region, or a CH2 region).

[0279] In some embodiments, the antigen-binding molecule comprises an Fc region that includes a substituted L242C (or equivalent substitution), a substituted K334C (or equivalent substitution), a substituted E345K (or equivalent substitution), and a substituted E430G (or equivalent substitution) (e.g., a further polypeptide including these, including a heavy chain constant region or a CH2-CH3 region).

[0280] In some embodiments, the antigen-binding molecule comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) of the following: L at the position corresponding to position 243, P at the position corresponding to position 292, L at the position corresponding to position 300, I at the position corresponding to position 305, and L at the position corresponding to position 396; D at the position corresponding to position 239, and E at the position corresponding to position 332; D at the position corresponding to position 239, E at the position corresponding to position 332, and L at the position corresponding to position 330; A at the position corresponding to position 298, A at the position corresponding to position 333, and A at the position corresponding to position 334; Y at the position corresponding to position 234, Q at the position corresponding to position 235, W at the position corresponding to position 236, M at the position corresponding to position 239, D at the position corresponding to position 268, E at the position corresponding to position 270, and A at the position corresponding to position 298; E at the position corresponding to position 270, D at the position corresponding to position 326, M at the position corresponding to position 330, and E at the position corresponding to position 334; A at the position corresponding to position 236, D at the position corresponding to position 239, and E at the position corresponding to position 332; W at the position corresponding to position 326, and S at the position corresponding to position 333; E at the position corresponding to position 267, F at the position corresponding to position 268, and T at the position corresponding to position 324; R at the position corresponding to position 345, G at the position corresponding to position 430, and Y at the position corresponding to position 440; Y at the position corresponding to position 252, T at the position corresponding to position 254, and E at the position corresponding to position 256; and L at the position corresponding to position 428, and S at the position corresponding to position 434 (e.g., including these, including the heavy chain constant region, or including one further polypeptide including the CH2-CH3 region) and comprises an Fc region.

[0281] In some embodiments, the antigen-binding molecule comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following combinations of substitutions (or corresponding substitutions): F243L / R292P / Y300L / V305I / P396L; S239D / I332E; S239D / I332E / A330L; S298A / E333A / K334A; L234Y / L235Q / G236W / S239M / H268D / D270E / S298A; D270E / K326D / A330M / K334E; G236A / S239D / I332E; K326W / E333S; S267E / H268F / S324T; E345R / E430G / S440Y; M252Y / S254T / T256E; and M428L / N434S (e.g., a heavy chain constant region comprising these, or a further polypeptide comprising a CH2-CH3 region comprising these).

[0282] Polypeptide The invention also provides a polypeptide component of the antigen-binding molecule. The polypeptide may be provided in isolated form or in substantially purified form.

[0283] The antigen-binding molecule of the invention can be or can comprise a complex of polypeptides.

[0284] It will be understood that where a polypeptide comprises more than one domain or region herein, it is preferred that the multiple domains / regions are present within the same polypeptide chain. That is, a polypeptide comprising more than one domain or region is a fusion polypeptide comprising the domains / regions.

[0285] In some embodiments, a polypeptide according to the invention comprises or consists of a VH as described herein. In some embodiments, a polypeptide according to the invention comprises or consists of a VL as described herein.

[0286] In some embodiments, the polypeptide further comprises the heavy chain constant region (CH) of one or more antibodies. In some embodiments, the polypeptide further comprises the light chain constant region (CL) of one or more antibodies. In some embodiments, the polypeptide comprises the CH1, CH2 region, and / or CH3 region of an immunoglobulin (Ig).

[0287] In some embodiments, the polypeptide comprises one or more regions of the heavy chain constant sequence of an immunoglobulin. In some embodiments, the polypeptide comprises the CH1 region described herein. In some embodiments, the polypeptide comprises the CH1-CH2 hinge region described herein. In some embodiments, the polypeptide comprises the CH2 region described herein. In some embodiments, the polypeptide comprises the CH3 region described herein. In some embodiments, the polypeptide comprises the CH2-CH3 region described herein.

[0288] In some embodiments, the polypeptide comprises a CH3 region comprising any one of the following amino acid substitutions / combinations of amino acid substitutions (e.g., shown in Table 1 of Ha et al., Front. Immunol (2016) 7:394, incorporated herein by reference above): T366W; T366S, L368A, and Y407V; T366W and S354C; T366S, L368A, Y407V, and Y349C; S364H and F405A; Y349T and T394F; T350V, L351Y, F405A, and Y407V; T350V, T366L, K392L, and T394W; K360D, D399M, and Y407A; E345R, Q347R, T366V, and K409V; K409D and K392D; D399K and E356K; K360E and K409W; Q347R, D399V, and F405T; K360E, K409W, and Y349C; Q347R, D399V, F405T, and S354C; K370E and K409W; and E357N, D399V, and F405T.

[0289] In some embodiments, the CH2 and / or CH3 regions of the polypeptide comprise one or more amino acid substitutions to promote the association of the polypeptide with another polypeptide comprising the CH2 and / or CH3 region of the polypeptide.

[0290] In some embodiments, the polypeptide comprises one or more regions of the constant sequence of the light chain of an immunoglobulin. In some embodiments, the polypeptide comprises the CL region described herein.

[0291] In some embodiments, the polypeptide according to the invention, from the N-terminus to the C-terminus, is as follows: (i) VH (ii) VL (iii) VH-CH1 (iv) VL-CL (v) VL-CH1 (vi) VH-CL (vii) VH-CH1-CH2-CH3 (viii) VL-CL-CH2-CH3 (ix) VL-CH1-CH2-CH3 (x) VH-CL-CH2-CH3 and comprises a structure according to one of the following:

[0292] Also provided by the present invention is an antigen-binding molecule composed of the polypeptide of the present invention. In some embodiments, the antigen-binding molecule of the present invention is a combination of the following polypeptides: (A) VH + VL (B) VH-CH1 + VL-CL (C) VL-CH1 + VH-CL (D) VH-CH1-CH2-CH3 + VL-CL (E) VH-CL-CH2-CH3 + VL-CH1 (F) VL-CH1-CH2-CH3 + VH-CL (G) VL-CL-CH2-CH3 + VH-CH1 (H) VH-CH1-CH2-CH3 + VL-CL-CH2-CH3 (I) VH-CL-CH2-CH3 + VL-CH1-CH2-CH3 includes one of the following.

[0293] In some embodiments, the antigen-binding molecule comprises more than one of the polypeptides of the combinations shown in (A) - (I) above. By way of example, referring to (D) above, in some embodiments, the antigen-binding molecule comprises two polypeptides comprising the structure VH-CH1-CH2-CH3 and two polypeptides comprising the structure VL-CL.

[0294] In some embodiments, the antigen-binding molecule of the invention is a combination of the following polypeptides: (J) VH(anti-HER3) + VL(anti-HER3) (K) VH(anti-HER3)-CH1 + VL(anti-HER3)-CL (L) VL(anti-HER3)-CH1 + VH(anti-HER3)-CL (M) VH(anti-HER3)-CH1-CH2-CH3 + VL(anti-HER3)-CL (N) VH(anti-HER3)-CL-CH2-CH3 + VL(anti-HER3)-CH1 (O) VL(anti-HER3)-CH1-CH2-CH3 + VH(anti-HER3)-CL (P) VL(anti-HER3)-CL-CH2-CH3 + VH(anti-HER3)-CH1 (Q) VH(anti-HER3)-CH1-CH2-CH3 + VL(anti-HER3)-CL-CH2-CH3 (R) VH(anti-HER3)-CL-CH2-CH3 + VL(anti-HER3)-CH1-CH2-CH3 includes one of the following, where "VH(anti-HER3)" refers to the VH of an antigen-binding molecule capable of binding to HER3 as defined in one of (1) - (61) described herein; "VL(anti-HER3)" refers to the VL of an antigen-binding molecule capable of binding to HER3 as defined in one of (62) - (119) described herein.

[0295] In some embodiments, the polypeptide comprises, or consists of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to one of the amino acid sequences of SEQ ID NOs: 187-223.

[0296] Linker and additional sequences In some embodiments, the antigen-binding molecule and polypeptide of the invention comprise a hinge region. In some embodiments, the hinge region is provided between the CH1 region and the CH2 region. In some embodiments, the hinge region is provided between the CL region and the CH2 region. In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 173.

[0297] In some embodiments, the antigen-binding molecule and polypeptide of the invention comprise one or more linker sequences between amino acid sequences. The linker sequence can be provided at one or both ends of one or more of the VH, VL, CH1-CH2 hinge region, CH2 region, and CH3 region of the antigen-binding molecule / polypeptide.

[0298] Those skilled in the art are aware that linker sequences are well known and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369, which is incorporated herein by reference in its entirety. In some embodiments, the linker sequence can be a flexible linker sequence. A flexible linker sequence allows for relative movement of the amino acid sequences linked by the linker sequence. Those skilled in the art are aware that flexible linkers are well known and some have been identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369. Flexible linker sequences often contain a high proportion of glycine residues and / or serine residues.

[0299] In some embodiments, the linker sequence contains at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, or 1-10 amino acids.

[0300] The antigen-binding molecules and polypeptides of the present invention may further contain additional amino acids or amino acid sequences. For example, the antigen-binding molecules and polypeptides may contain amino acid sequences that facilitate the expression, folding, transport, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecule / polypeptide may optionally contain a sequence encoding His (e.g., 6×His), Myc, GST, MBP, FLAG, HA, E, or a biotin tag at the N-terminus or C-terminus of the antigen-binding molecule / polypeptide. In some embodiments, the antigen-binding molecule / polypeptide contains a detectable moiety, such as a fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzyme label.

[0301] The antigen-binding molecules and polypeptides of the present invention may further comprise a signal peptide (also known as a leader sequence or signal sequence). A signal peptide typically consists of a sequence of 5 to 30 hydrophobic amino acids that forms a single alpha helix. Proteins that are secreted and expressed on the cell surface often contain a signal peptide.

[0302] The signal peptide can be present at the N-terminus of the antigen-binding molecule / polypeptide and can be present within the newly synthesized antigen-binding molecule / polypeptide. The signal peptide results in efficient transport and secretion of the antigen-binding molecule / polypeptide. Since the signal peptide is often removed by cleavage, it is not included in the mature antigen-binding molecule / polypeptide secreted from the cell expressing the antigen-binding molecule / polypeptide.

[0303] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0304] In some embodiments, the signal peptide of the antigen-binding molecule / polypeptide of the present invention comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of the amino acid sequences of SEQ ID NOs: 178-186.

[0305] Labels and conjugates In some embodiments, the antigen-binding molecule of the invention further comprises a detectable moiety.

[0306] In some embodiments, the antigen-binding molecule comprises a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radioactive label, a chemical, nucleic acid, or enzyme label. The antigen-binding molecule may be labeled covalently or non-covalently with the detectable moiety.

[0307] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), chelating agents of rare earths such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radioactive labels include iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 , bromine 77 , technetium 99m , indium 111 , indium 113m , gallium 67 , gallium 68 , ruthenium 95 , ruthenium 97 , ruthenium 103 , ruthenium 105 , mercury 207 , mercury 203 , rhenium 99m , rhenium 101 , rhenium 105 , scandium 47 , tellurium 121m , tellurium 122m , tellurium 125m , thulium 165 , thulium 167 , thulium 168 , copper 67 , fluorine 18 , yttrium 90 , palladium100 , bismuth 217 , and antimony 211 and other radioisotopes. The luminescent labels include radioluminescent labels, chemiluminescent labels (e.g., acridinium esters, luminol, isoluminol), and bioluminescent labels. The immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands such as biotin, avidin, streptavidin, or digoxigenin. The nucleic acid labels include aptamers. The enzyme labels include, for example, peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, and luciferase.

[0308] In some embodiments, the antigen-binding molecule of the present invention is conjugated to a chemical moiety. The chemical moiety can be a moiety for providing a therapeutic effect. Antibody-drug conjugates are reviewed, for example, in Parslow et al., Biomedicines. September 2016; 4(3):14. In some embodiments, the chemical moiety can be a drug moiety (e.g., a cytotoxic agent). In some embodiments, the drug moiety can be a chemotherapeutic agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD.

[0309] Specific exemplary embodiments of the antigen-binding molecule In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 187, and (ii) Two polypeptides that contain, or consist of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 188 contain, or consist of.

[0310] In some embodiments, the antigen-binding molecule is (i) Two polypeptides that contain, or consist of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 189, and (ii) Two polypeptides that contain, or consist of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 190 contain, or consist of.

[0311] In some embodiments, the antigen-binding molecule is (i) Two polypeptides that contain, or consist of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 191, and (ii) Two polypeptides that contain, or consist of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 192 contain, or consist of.

[0312] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 193, and (ii) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 195 and comprises, or consists of, the same.

[0313] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 194, and (ii) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 195 and comprises, or consists of, the same.

[0314] In some embodiments, the antigen-binding molecule is (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 196, or consisting of such an amino acid sequence, two polypeptides, and (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 195, or consisting of such an amino acid sequence, two polypeptides comprising or consisting of.

[0315] In some embodiments, the antigen-binding molecule is (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 197, or consisting of such an amino acid sequence, two polypeptides, and (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 199, or consisting of such an amino acid sequence, two polypeptides comprising or consisting of.

[0316] In some embodiments, the antigen-binding molecule is (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 198, or consisting of such an amino acid sequence, two polypeptides, and (ii) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 199, or consisting of such an amino acid sequence, and two polypeptides comprising or consisting of.

[0317] In some embodiments, the antigen-binding molecule is (i) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 200, or consisting of such an amino acid sequence, and two polypeptides, and (ii) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 201, or consisting of such an amino acid sequence, and two polypeptides comprising or consisting of.

[0318] In some embodiments, the antigen-binding molecule is (i) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 202, or consisting of such an amino acid sequence, and two polypeptides, and (ii) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 203, or consisting of such an amino acid sequence, and two polypeptides comprising or consisting of.

[0319] In some embodiments, the antigen-binding molecule (i) comprises, or consists of, two polypeptides having an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 204, and (ii) comprises, or consists of, two polypeptides having an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 205 and comprises, or consists of, the same.

[0320] In some embodiments, the antigen-binding molecule (i) comprises, or consists of, two polypeptides having an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 206, and (ii) comprises, or consists of, two polypeptides having an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 207 and comprises, or consists of, the same.

[0321] In some embodiments, the antigen-binding molecule (i) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 208, two polypeptides comprising or consisting of such an amino acid sequence, and (ii) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 209, two polypeptides comprising or consisting of such an amino acid sequence comprising or consisting of.

[0322] In some embodiments, the antigen-binding molecule is (i) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 210, two polypeptides comprising or consisting of such an amino acid sequence, and (ii) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 211, two polypeptides comprising or consisting of such an amino acid sequence comprising or consisting of.

[0323] In some embodiments, the antigen-binding molecule is (i) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 212, two polypeptides comprising or consisting of such an amino acid sequence, and (ii) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 213, or consisting of such an amino acid sequence, two polypeptides comprising or consisting of.

[0324] In some embodiments, the antigen-binding molecule is (i) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 214, or consisting of such an amino acid sequence, two polypeptides, and (ii) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 215, or consisting of such an amino acid sequence, two polypeptides comprising or consisting of.

[0325] In some embodiments, the antigen-binding molecule is (i) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 216, or consisting of such an amino acid sequence, two polypeptides, and (ii) An amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 217, or consisting of such an amino acid sequence, two polypeptides comprising or consisting of.

[0326] In some embodiments, the antigen-binding molecule (i) comprises or consists of two polypeptides having one of at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 218, and (ii) two polypeptides comprising or consisting of an amino acid sequence having one of at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 219 and comprises or consists of the same.

[0327] In some embodiments, the antigen-binding molecule (i) comprises or consists of two polypeptides having one of at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 220, and (ii) two polypeptides comprising or consisting of an amino acid sequence having one of at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 221 and comprises or consists of the same.

[0328] In some embodiments, the antigen-binding molecule (i) Two polypeptides comprising or consisting of an amino acid sequence having at least one of 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 222, and (ii) Two polypeptides comprising or consisting of an amino acid sequence having at least one of 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 223 comprising or consisting of.

[0329] In some embodiments, the antigen-binding molecule is (i) Two polypeptides comprising or consisting of an amino acid sequence having at least one of 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 225, and (ii) Two polypeptides comprising or consisting of an amino acid sequence having at least one of 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 207 comprising or consisting of.

[0330] In some embodiments, the antigen-binding molecule is (i) Two polypeptides comprising or consisting of an amino acid sequence having at least one of 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 226, and (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 207 comprising, or consisting of.

[0331] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 227, and (ii) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 217 comprising, or consisting of.

[0332] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 228, and (ii) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 217 comprising, or consisting of.

[0333] Functional characteristics of the antigen-binding molecule The antigen-binding molecules described herein can be characterized by referring to certain functional characteristics. In some embodiments, the antigen-binding molecules described herein have the following characteristics: Binding to HER3 (e.g., human, mouse, rat, or cynomolgus HER3); Not binding to EGFR and / or HER2; Binding to HER3-expressing cells; Binding to subdomain II of the extracellular region of HER3; Binding to HER3 when HER3 is in the open and closed conformations; Binding to HER3 independently of NRG; Not competing with MM-121 and / or LJM-716 for binding to HER3; Not competing with M-05-74 and / or M-08-11 for binding to HER3; Inhibiting the interaction between HER3 and its interaction partners (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet); Inhibiting HER3-mediated signaling; Inhibiting the proliferation of HER3-expressing cells (e.g., in response to stimulation by NRG); Inhibiting PI3K / AKT / mTOR and / or MAPK signaling (e.g., in response to stimulation by NRG) by HER3-expressing cells; Binding to activating Fcγ receptors (e.g., FcγRIIIa); Increased binding to activating Fcγ receptors; Increased binding to activating Fcγ receptors compared to equivalent antigen-binding molecules having an Fc region composed of CH2-CH3 with the amino acid sequences of SEQ ID NOs: 174-175; Reduction in binding to inhibitory Fcγ receptors, compared to an equivalent antigen-binding molecule, having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175; Increase in binding to activating Fcγ receptors, compared to an equivalent antigen-binding molecule, having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175, that exceeds binding to inhibitory Fcγ receptors; Increase or decrease in binding to complement proteins (e.g., C1q), compared to an equivalent antigen-binding molecule, having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175; Increase in hexamerization, compared to an equivalent antigen-binding molecule, having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175; Increase in ADCC activity, compared to an equivalent antigen-binding molecule, having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175; Increase in ADCP activity, compared to an equivalent antigen-binding molecule, having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175; Increase or decrease in CDC activity, compared to an equivalent antigen-binding molecule, having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175; Similar or increased thermal stability, compared to an equivalent antigen-binding molecule, having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175; Increasing the killing of HER3-expressing cells; Reducing the number / ratio of HER3-expressing cells; And Inhibiting cancer onset and / or progression in vivo May have one or more of the above.

[0334] The antigen-binding molecules described herein preferably exhibit specific binding to HER3. As used herein, "specific binding" refers to binding that is selective for an antigen and distinguishable from non-specific binding to non-target antigens. An antigen-binding molecule that specifically binds to a target molecule preferably binds to the target with greater affinity and / or for a longer duration than it binds to other non-target molecules.

[0335] The ability of a given polypeptide to specifically bind to a given molecule can be determined by assays according to methods known in the art, such as ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507), flow cytometry, or a radio-labeled antigen-binding assay (RIA), a method by an enzyme immunoassay. By such assays, binding to a given molecule can be measured and quantified. In some embodiments, the binding can be a response detected in a given assay.

[0336] In some embodiments, the degree of binding of the antigen-binding molecule to non-target molecules is less than about 10% of the binding of the antibody to the target molecule, as measured, for example, by ELISA, SPR, biolayer interferometry, or by RIA. Alternatively, the binding specificity is such that the antigen-binding molecule binds with a dissociation constant (K D with at least 0.1 more digits (i.e., 0.1×10 n , where n is an integer representing the number of digits) than the dissociation constant (K D ) for binding to the non-target molecule, which can optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0337] In some embodiments, the antigen-binding molecule exhibits binding to human HER3, mouse HER3, rat HER3, and / or cynomolgus macaque (Macaca fascicularis) HER3. That is, in some embodiments, the antigen-binding molecule is cross-reactive with human HER3, mouse HER3, rat HER3, and / or cynomolgus macaque HER3. In some embodiments, the antigen-binding molecule of the present invention exhibits cross-reactivity with HER3 of non-human primates. Cross-reactivity with HER3 in a model species enables in vivo investigation of efficacy in an orthologous model without relying on a surrogate molecule.

[0338] In some embodiments, the antigen-binding molecule binds to human HER3, mouse HER3, rat HER3, and / or cynomolgus macaque HER3 and does not bind to HER2 and / or EGFR (e.g., human HER2 and / or human EGFR).

[0339] In some embodiments, the antigen-binding molecule does not exhibit specific binding to EGFR (e.g., human EGFR). In some embodiments, the antigen-binding molecule does not exhibit specific binding to HER2 (e.g., human HER2). In some embodiments, the antigen-binding molecule does not exhibit specific binding to members of the EGFR family of proteins other than HER3 (i.e., does not cross-react with them). In some embodiments, the antigen-binding molecule does not exhibit specific binding to EGFR, HER2, and / or HER4.

[0340] In some embodiments, the antigen-binding molecule of the present invention has a K of 10 μM or less, preferably ≤5 μM, ≤2 μM, ≤1 μM, ≤500 nM, ≤400 nM, ≤300 nM, ≤200 nM, ≤100 nM, ≤95 nM, ≤90 nM, ≤85 nM, ≤80 nM, ≤75 nM, ≤70 nM, ≤65 nM, ≤60 nM, ≤55 nM, ≤50 nM, ≤45 nM, ≤40 nM, ≤35 nM, ≤30 nM, ≤25 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤90 pM, ≤80 pM, ≤70 pM, ≤60 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM, ≤9 pM, ≤8 pM, ≤7 pM, ≤6 pM, ≤5 pM, ≤4 pM, ≤3 pM, ≤2 pM, ≤1 pM, and binds to HER3 (e.g., human HER3). D and binds to HER3 (e.g., human HER3).

[0341] The antigen-binding molecule of the present invention can bind to a specific target region of HER3. The antigen-binding region of the antigen-binding molecule according to the present invention can bind to a linear epitope of HER3 consisting of a continuous sequence of amino acids (i.e., the primary sequence of amino acids). In some embodiments, the antigen-binding molecule can bind to a conformational epitope of HER3 consisting of a discontinuous sequence of amino acids in the amino acid sequence.

[0342] In some embodiments, the antigen-binding molecule of the present invention binds to HER3. In some embodiments, the antigen-binding molecule binds to the extracellular region of HER3 (e.g., the region shown in SEQ ID NO: 9). In some embodiments, the antigen-binding molecule binds to subdomain II of the extracellular region of HER3 (e.g., the region shown in SEQ ID NO: 16).

[0343] In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 229. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 229. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NOs: 230 and 231. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NOs: 230 and 231. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 230. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 230. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 231. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 231. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 23. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 23. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 21. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 21. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 19. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 19. In some embodiments, the antigen-binding molecule binds to the region of HER3 set forth in SEQ ID NO: 22. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 set forth in SEQ ID NO: 22.

[0344] In some embodiments, the antigen-binding molecule of the present invention can bind to a polypeptide comprising or consisting of one of the amino acid sequences of SEQ ID NO: 1, 3, 4, 6, or 8. In some embodiments, the antigen-binding molecule can bind to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antigen-binding molecule can bind to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 229. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequences of SEQ ID NO: 230 and 231. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 230. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 231. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 22.

[0345] In some embodiments, the antigen-binding molecule does not bind to the region of HER3 corresponding to positions 260 to 279 of SEQ ID NO: 1. In some embodiments, the antigen-binding molecule does not contact the amino acid residues in the region of HER3 corresponding to positions 260 to 279 of SEQ ID NO: 1. In some embodiments, the antigen-binding molecule does not bind to the region of HER3 shown in SEQ ID NO: 23. In some embodiments, the antigen-binding molecule does not contact the amino acid residues in the region of HER3 shown in SEQ ID NO: 23. In some embodiments, the antigen-binding molecule is unable to bind to a peptide consisting of the amino acid sequence corresponding to positions 260 to 279 of SEQ ID NO: 1. In some embodiments, the antigen-binding molecule is unable to bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 23.

[0346] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length of about 2 to 50 amino acids within a region. "Polypeptide" refers to a polymeric chain of two or more peptides. Polypeptides typically have a length of more than about 50 amino acids.

[0347] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (e.g., Western blotting), immunoprecipitation, surface plasmon resonance, and biolayer interferometry.

[0348] Binding of a ligand to HER3 results in activation of downstream pathways as a result of promoting a conformational change that enables HER3 to homodimerize or heterodimerize. HER3 exhibits "closed" and "open" conformations. The closed conformation means that HER3 is in a tethered conformation and is not available for homodimerization or heterodimerization of the receptor. The open conformation means that HER3 is in an extended conformation and is available for homodimerization or heterodimerization of the receptor.

[0349] In some embodiments, when HER3 is in an open conformation, the antigen-binding molecule can bind to HER3. In some embodiments, when HER3 is in a closed conformation, the antigen-binding molecule can bind to HER3. In some embodiments, when HER3 is in an open and / or closed conformation, the antigen-binding molecule can bind to HER3. In some embodiments, when HER3 is in an open and / or closed conformation, the antigen-binding molecule can bind to the extracellular domain of HER3. In some embodiments, when HER3 is in an open and / or closed conformation, the antigen-binding molecule can bind to the dimerization arm of HER3. Binding to the dimerization arm allows the antigen-binding molecule to prevent the interaction of HER3 with, for example, an interaction partner of HER3 as described herein.

[0350] In some embodiments, the antigen-binding molecule can bind to HER3 in the presence and / or absence of a ligand for HER3. In some embodiments, the antigen-binding molecule can bind to HER3 independent of a ligand for HER3. In some embodiments, the ligand is NRG, NRG-1, and / or NRG-2. HER3 is activated by the binding of a ligand to its extracellular domain, which promotes a conformational change that allows HER3 to homodimerize or heterodimerize. Binding of the antigen-binding molecule to HER3 allows the antigen-binding molecule to inhibit the action of HER3, independent of the binding of the ligand, in either the conformational state in the absence of the ligand or in the presence of the ligand. In some embodiments, the antigen-binding molecule does not compete with the ligand that binds to HER3. In some embodiments, the antigen-binding molecule does not bind to HER3 at the ligand-binding site.

[0351] In some embodiments, the antigen-binding molecule binds HER3 equally well in the presence or absence of a ligand for HER3 (i.e., regardless of whether HER3 is provided in a ligand-bound or unbound form).

[0352] In some embodiments, the antigen-binding molecule binds HER3 in the presence of a ligand for HER3 with an affinity similar to the affinity for binding of the antigen-binding molecule to HER3 in the absence of the ligand for HER3. Examples 8.10 and FIGS. 78A and 78B of the present disclosure demonstrate that 10D1F binds human HER3 with sub-picomolar affinity when HER3 is provided both in the NRG1-bound form and in the absence of NRG1.

[0353] As used herein, a reference binding affinity and a "similar" binding affinity, determined under equivalent conditions, mean a binding affinity within 50% of the reference binding affinity, such as within 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the reference binding affinity.

[0354] In some embodiments, the antigen-binding molecule has a K D for binding to HER3 in the absence of the ligand that is within 50% of the K D of the antigen-binding molecule for binding to HER3, such as within 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the K

[0355] In some embodiments, the antigen-binding molecule has a K onwithin 50% of, for example, 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of K on binds to HER3 in the presence of a ligand (e.g., NRG1 or NRG2) for HER3.

[0356] In some embodiments, the antigen-binding molecule has a K that is within 50% of the K of the antigen-binding molecule for binding to HER3 in the absence of ligand (determined under equivalent conditions), for example, within 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of K off within 50% of, for example, 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of K off binds to HER3 in the presence of a ligand (e.g., NRG1 or NRG2) for HER3.

[0357] In some embodiments, the antigen-binding molecule can bind to the same or overlapping regions of HER3 to which an antibody comprising the VH and VL sequences of one of the clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6, 4-35-B2, or 4-35-B4 binds. In some embodiments, the antigen-binding molecule can bind to the same or overlapping regions of HER3 to which an antibody comprising the VH and VL sequences of one of the clones 10D1_c89, 10D1_c90, or 10D1_c91 binds. In some embodiments, the antigen-binding molecule can bind to the same or overlapping regions of HER3 to which an antibody comprising the VH and VL sequences of the clone 10D1_c89 binds.

[0358] The region of the peptide / polypeptide to which the antibody binds can be determined by one of ordinary skill in the art using a variety of methods well known in the art, including X-ray co-crystallography of the antibody-antigen complex, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and protease-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is hereby incorporated by reference in its entirety. Such methods can also be used to determine whether an antigen-binding molecule can bind to proteins in different conformations.

[0359] In some embodiments, the antigen-binding molecule of the invention does not bind to HER3 in the same or overlapping regions of HER3 as an antibody comprising the VH and VL sequences of the anti-HER3 antibody clones MM-121 (described, for example, in Schoeberl et al., Sci. Signal. (2009) 2(77):ra31) and / or LJM-716 (described, for example, in Garner et al., Cancer Res (2013) 73:6024-6035). In some embodiments, the antigen-binding molecule of the invention does not exhibit competition for binding to HER3 with an antibody comprising the VH and VL sequences of the anti-HER3 antibody clones MM-121 and / or LJM-716, as determined, for example, by SPR analysis.

[0360] In some embodiments, the antigen-binding molecule of the invention binds to HER3 within a region accessible to the antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when HER3 is expressed on the cell surface (i.e., within or on the cell membrane). In some embodiments, the antigen-binding molecule can bind to HER3 expressed on the cell surface of a cell expressing HER3. In some embodiments, the antigen-binding molecule can bind to HER3-expressing cells (e.g., HER3+ cells, e.g., HER3+ cancer cells).

[0361] The ability of an antigen-binding molecule to bind to a given cell type can be analyzed, for example, by contacting the cells with the antigen-binding molecule and detecting the antigen-binding molecule bound to the cells, after a washing step to remove unbound antigen-binding molecules. The ability of an antigen-binding molecule to bind to immune cell surface molecule-expressing cells and / or cancer cell antigen-expressing cells can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0362] The antigen-binding molecule of the present invention can be an antagonist of HER3. In some embodiments, the antigen-binding molecule can inhibit a function or process (e.g., interaction, signal transduction, or other activity) mediated by HER3 and / or a binding partner of HER3 (e.g., HER3 (i.e., in the case of homodimerization), HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet). As used herein, "inhibit" refers to a reduction, decrease, or diminution as compared to control conditions.

[0363] In some embodiments, the antigen-binding molecule of the present invention can inhibit the interaction between HER3 and an interaction partner of HER3. The interaction partner of HER3 can be expressed by the same cell as HER3. The interaction partner or HER3 can be expressed on the cell surface (i.e., within or on the cell membrane). In some embodiments, the interaction partner of HER3 can be a member of the EGFR family of proteins, such as HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet. In some embodiments, the interaction partner of HER3 can be IGF1R and / or cMet. The interaction between HER3 and an interaction partner of HER3 can result in the formation of a polypeptide complex. The interaction between HER3 and an interaction partner of HER3 that forms a polypeptide complex can be referred to as multimerization. When multimerization is between polypeptide monomers, it can be referred to as dimerization.

[0364] In some embodiments, the antigen-binding molecule can inhibit the interaction between HER3 monomers. In some embodiments, the antigen-binding molecule can inhibit the interaction between HER3 and HER2. In some embodiments, the antigen-binding molecule can inhibit the interaction between HER3 and EGFR. In some embodiments, the antigen-binding molecule can inhibit the interaction between HER3 and HER4. In some embodiments, the antigen-binding molecule can inhibit the interaction between HER3 and HGFR. In some embodiments, the antigen-binding molecule can inhibit the interaction between HER3 and IGF1R. In some embodiments, the antigen-binding molecule can inhibit the interaction between HER3 and cMet.

[0365] Inhibition of the interaction can be achieved by binding of the antigen-binding molecule to the region of HER3 required for the interaction of HER3 with its interaction partner (e.g., the dimerization loop of HER3 shown in SEQ ID NO: 19). In some embodiments, the antigen-binding molecule contacts one or more residues of HER3 necessary for the interaction of HER3 with its interaction partner, and in this way, the antigen-binding molecule renders the region unavailable, thereby inhibiting the interaction. In some embodiments, the antigen-binding molecule binds to HER3 in a manner that inhibits / prevents the interaction of HER3 with its interaction partner. In some embodiments, the antigen-binding molecule inhibits / prevents access of the HER3 interaction partner to the region of HER3 required for the interaction of HER3 with its interaction partner, which can be achieved, for example, by steric hindrance of access of the HER3 interaction partner to the region of HER3 required for the interaction of HER3 with its interaction partner, even if the antigen-binding molecule does not contact the region of HER3 required for the interaction of HER3 with its interaction partner.

[0366] In some embodiments, the antigen-binding molecule can inhibit the homodimerization of the HER3 monomer. In some embodiments, the antigen-binding molecule can inhibit the dimerization of HER3 and HER2. In some embodiments, the antigen-binding molecule can inhibit the dimerization of HER3 and EGFR. In some embodiments, the antigen-binding molecule can inhibit the dimerization of HER3 and HER4. In some embodiments, the antigen-binding molecule can inhibit the dimerization of HER3 and HGFR. In some embodiments, the antigen-binding molecule can inhibit the dimerization of HER3 and IGF1R. In some embodiments, the antigen-binding molecule can inhibit the dimerization of HER3 and cMet.

[0367] The ability of the antigen-binding molecule to inhibit the interaction between two factors can be determined, for example, by analyzing the interaction in the presence of the antibody / fragment or after incubation with one or both of the interaction partners of the antibody / fragment. Assays for determining whether a given antigen-binding molecule can inhibit the interaction between two interaction partners include competitive ELISA assays and SPR-based analysis. In some embodiments, the antigen-binding molecule is a competitive inhibitor of the interaction between HER3 and an interaction partner of HER3.

[0368] In some embodiments, the antigen-binding molecule of the present invention inhibits the interaction between HER3 and an interaction partner of HER3 (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet) in a suitable assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the level of the interaction between HER3 and an interaction partner of HER3 in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0369] The ability of an antigen-binding molecule to inhibit the interaction between interaction partners can also be determined by analyzing the functional consequences downstream of such an interaction. For example, the functional consequences downstream of the interaction between HER3 and its interaction partner include PI3K / AKT / mTOR and / or MAPK signaling. For example, the ability of an antigen-binding molecule to inhibit the interaction between HER3 and its interaction partner can be determined by analyzing PI3K / AKT / mTOR and / or MAPK signaling after treatment with NRG in the presence of the antigen-binding molecule. PI3K / AKT / mTOR and / or MAPK signaling can be detected and quantified, for example, using antibodies capable of detecting phosphorylated members of the signaling pathway.

[0370] The ability of an antigen-binding molecule to inhibit the interaction between HER3 and its interaction partner can also be determined by analyzing the proliferation of cells expressing HER3 after treatment with NRG in the presence of the antigen-binding molecule. Cell proliferation can be determined, for example, by detecting changes in cell number over time, or 3 by in vitro analysis of the incorporation of H-thymidine, or, for example, by a CFSE dilution assay as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564, which is incorporated herein by reference in its entirety.

[0371] In some embodiments, the antigen-binding molecule of the invention can inhibit the proliferation of cells carrying a mutation at V600 of BRAF, such as cells containing the V600E or V600K mutation of BRAF (see Example 10).

[0372] In some embodiments, the antigen-binding molecule inhibits HER3-mediated signaling. HER3-mediated signaling can be analyzed, for example, using an assay for a correlative factor of HER3-mediated signaling, such as cell proliferation, and / or phosphorylation of one or more signaling molecules in the PI3K / AKT / mTOR, and / or MAPK signaling pathways.

[0373] In some embodiments, the antigen-binding molecule of the invention can inhibit PI3K / AKT / mTOR, and / or MAPK signaling by HER3-expressing cells. The level of PI3K / AKT / mTOR, and / or MAPK signaling can be analyzed, for example, by detecting phosphorylation of one or more of the components of the PI3K / AKT / mTOR, and / or MAPK pathway and quantifying this level after stimulation with NRG (see Example 4.3).

[0374] In some embodiments, the antigen-binding molecule of the invention can inhibit the proliferation of HER3-expressing cells, for example, in response to stimulation with NRG. In some embodiments, the antigen-binding molecule of the invention inhibits the proliferation of HER3-expressing cells in a suitable assay to less than 1-fold, such as ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold of the level of proliferation of HER3-expressing cells in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0375] In some embodiments, the antigen-binding molecule of the invention inhibits PI3K / AKT / mTOR and / or MAPK signaling by HER3-expressing cells in a suitable assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the level of signaling by HER3-expressing cells in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0376] HER3-mediated signaling can be examined in vitro, for example, as described in Example 8.9, or in vivo, for example, as described in Example 11.

[0377] ADCC activity can be analyzed, for example, according to the method described in Yamashita et al., Scientific Reports (2016) 6:19772, which is incorporated herein by reference in its entirety, or, for example, according to the method described in Jedema et al., Blood (2004) 103:2677-82, which is incorporated herein by reference in its entirety. 51 It can be analyzed by a Cr release assay. ADCC activity can also be analyzed using the Pierce LDH cytotoxicity assay kit according to the manufacturer's instructions (described in Example 5 herein).

[0378] ADCP can be analyzed, for example, according to the method described in Kamen et al., J Immunol (2017) 198(1 Supplement) 157.17, which is incorporated herein by reference in its entirety.

[0379] The ability to induce CDC can be analyzed, for example, using a C1q binding assay as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466 (incorporated herein by reference in its entirety).

[0380] The thermal stability of an antigen-binding molecule can be analyzed by methods well known to those skilled in the art, including differential scanning fluorimetry and differential scanning calorimetry (DSC), as described in He et al., J Pharm Sci. (2010), incorporated herein by reference in its entirety. Thermal stability can be reflected in terms of the melting temperature (T m m), unfolding temperature, or degradation temperature (e.g., represented in °C or °F).

[0381] In some embodiments, an antigen-binding molecule comprising an Fc region described herein binds to an activating Fcγ receptor (e.g., hFcγRIIa (e.g., hFcγRIIa167H, hFcγRIIa167R), hFcγRIIIa (e.g., hFcγRIIIa158V, hFcγRIIIa158F), mFcγRIV, mFcγRIII) with a binding affinity greater than 1-fold, e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or greater than 20-fold, that of an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs: 174-175. In some embodiments, the K D for an antigen-binding molecule comprising an Fc region described herein with respect to binding to an activating Fcγ receptor is less than 1-fold, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06-fold, or less than 0.05-fold, that of an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs: 174-175 for the activating Fcγ receptor. D

[0382] ​In some embodiments, the antigen-binding molecule comprising the Fc region described herein has a K of 1000 nM or less, preferably ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, or ≤1 nM. D and binds to activating Fcγ receptors (e.g., hFcγRIIa (e.g., hFcγRIIa167H, hFcγRIIa167R), hFcγRIIIa (e.g., hFcγRIIIa158V, hFcγRIIIa158F), mFcγRIV, mFcγRIII).

[0383] In some embodiments, the antigen-binding molecule comprising the Fc region described herein binds to FcRn (e.g., hFcRn, mFcRn) with a binding affinity that is more than 1-fold, e.g., more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or more than 20-fold, the binding affinity of an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175 for FcRn. In some embodiments, the K of the antigen-binding molecule comprising the Fc region described herein for binding to FcRn D is less than 1-fold, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06-fold, or less than 0.05-fold, the K of an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175 for FcRn. D

[0384] In some embodiments, the antigen-binding molecule comprising the Fc region described herein has a K of 1000 nM or less, preferably ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, or ≤1 nM. Dand binds to FcRn (e.g., hFcRn, mFcRn).

[0385] In some embodiments, the antigen-binding molecule comprising the Fc region described herein binds to an inhibitory Fcγ receptor (e.g., hFcγRIIb, mFcγRIIb) with a binding affinity less than 1-fold, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2-fold, or less than 0.1-fold, of the binding affinity of an equivalent antigen-binding molecule having an Fc region consisting of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175 for the inhibitory Fcγ receptor. In some embodiments, the K D for the antigen-binding molecule comprising the Fc region described herein for binding to the inhibitory Fcγ receptor is D more than 1-fold, e.g., more than 2, 3, 4, 5, 6, 7, 8, 9-fold, or more than 10-fold, of the K

[0386] for an equivalent antigen-binding molecule having an Fc region consisting of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175 for the inhibitory Fcγ receptor. In some embodiments, the antigen-binding molecule comprising the Fc region described herein binds to an inhibitory Fcγ receptor (e.g., hFcγRIIb, mFcγRIIb) with a K D of 1 nM or more, preferably, one of ≧5 nM, ≧10 nM, ≧50 nM, ≧100 nM, ≧500 nM, ≧1000 nM, ≧2000 nM, ≧3000 nM, ≧4000 nM, or ≧5000 nM.

[0387] In some embodiments, the selectivity of binding of the antigen-binding molecule comprising the Fc region described herein to an activating Fcγ receptor (e.g., hFcγRIIa) compared to an inhibitory Fcγ receptor (e.g., hFcγRIIb) is more than 1-fold, e.g., more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or more than 20-fold, of the selectivity of binding exhibited by an equivalent antigen-binding molecule having an Fc region consisting of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175.

[0388] In some embodiments, the antigen-binding molecule comprising the Fc region described herein exhibits ADCC that is more than 1-fold, e.g., more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or more than 20-fold the ADCC exhibited by an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175.

[0389] In some embodiments, in an assay of ADCC activity, the EC50 (ng / ml) determined for the antigen-binding molecule comprising the Fc region described herein is less than 1-fold, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2-fold, or less than 0.1-fold the EC50 (ng / ml) determined for an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs: 174-175.

[0390] In some embodiments, in an assay of ADCC activity, the EC50 (ng / ml) for the antigen-binding molecule comprising the Fc region described herein is one of 500 ng / ml or less, preferably, ≤400 ng / ml, ≤300 ng / ml, ≤200 ng / ml, ≤100 ng / ml, ≤90 ng / ml, ≤80 ng / ml, ≤70 ng / ml, ≤60 ng / ml, ≤50 ng / ml, ≤40 ng / ml, ≤30 ng / ml, ≤20 ng / ml, or ≤10 ng / ml.

[0391] In some embodiments, the antigen-binding molecule comprising the Fc region described herein has a melting temperature, unfolding temperature, or degradation temperature that is ≧0.75-fold and ≦1.25-fold, e.g., ≧0.8-fold and ≦1.2-fold, ≧0.85-fold and ≦1.15-fold, ≧0.9-fold and ≦1.1-fold, ≧0.91-fold and ≦1.09-fold, ≧0.92-fold and ≦1.08-fold, ≧0.93-fold and ≦1.07-fold, ≧0.94-fold and ≦1.06-fold, ≧0.95-fold and ≦1.05-fold, ≧0.96-fold and ≦1.04-fold, ≧0.97-fold and ≦1.03-fold, ≧0.98-fold and ≦1.02-fold, or ≧0.99-fold and ≦1.01-fold of the melting temperature, unfolding temperature, or degradation temperature of an equivalent antigen-binding molecule having an Fc region composed of the amino acid sequences of SEQ ID NOs: 174-175, and may have a melting temperature, unfolding temperature, or degradation temperature.

[0392] In some embodiments, the antigen-binding molecule of the present invention can increase the killing of HER3-expressing cells. The killing of HER3-expressing cells can be increased via the effector function of the antigen-binding molecule. In embodiments where the antigen-binding molecule comprises an Fc region, the antigen-binding molecule can increase the killing of HER3-expressing cells via one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0393] An antigen-binding molecule that can increase the killing of HER3-expressing cells can be identified by observing an increase in the level of killing of HER3-expressing cells in the presence of the antigen-binding molecule (or after incubation of HER3-expressing cells with the antigen-binding molecule) compared to the level of cell killing detected in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule) in a suitable assay. Assays for CDC, ADCC, and ADCP are well known to those of skill in the art. The level of killing of HER3-expressing cells can also be determined by measuring the number / ratio of surviving and / or non-surviving HER3-expressing cells after exposure to different treatment conditions.

[0394] In some embodiments, the antigen-binding molecule of the present invention increases the killing of HER3-expressing cells (e.g., HER3-expressing cancer cells) by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold compared to the level of killing observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0395] In some embodiments, the antigen-binding molecule of the present invention reduces the number of HER3-expressing cells (e.g., HER3-expressing cancer cells) in an equivalent assay to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the number of HER3-expressing cells (e.g., HER3-expressing cancer cells) detected after incubation in the absence of the antigen-binding molecule (or after incubation in the presence of a suitable control antigen-binding molecule).

[0396] In some embodiments, the antigen-binding molecule of the present invention inhibits cancer onset and / or progression in vivo.

[0397] In some embodiments, the antigen-binding molecule causes an increase in the killing of cancer cells, for example, by effector immune cells. In some embodiments, the antigen-binding molecule causes a reduction in the number of cancer cells in vivo compared to appropriate control conditions. In some embodiments, the antigen-binding molecule inhibits tumor growth as determined, for example, by measuring tumor size / volume over time.

[0398] The antigen-binding molecules of the present invention can be analyzed for their ability to inhibit cancer onset and / or progression in a suitable in vivo model, such as a xenograft model derived from a cell line. The xenograft model derived from a cell line can be derived from HER3-expressing cancer cells. In some embodiments, the model is a model derived from N87 cells, a model derived from SNU16 cells, a model derived from FaDu cells, a model derived from OvCAR8 cells, a model derived from HCC95 cells, a model derived from A549 cells, a model derived from ACHN cells, or a model derived from HT29 cells.

[0399] The cancer can be a HER3-related cancer as described herein (i.e., a cancer in which the expression of the HER3 gene / protein is a risk factor and / or is positively associated with cancer development, onset, progression, or the severity of symptoms, and / or metastasis). The cancer can contain HER3-expressing cells. In some embodiments, the cancer contains HER3+ tumors.

[0400] In some embodiments, administration of the antigen-binding molecules according to the present invention can cause one or more of inhibition of cancer onset / progression, delay / prevention of cancer development, reduction / delay / prevention of tumor growth, reduction / delay / prevention of metastasis, reduction in the severity of cancer symptoms, reduction in the number of cancer cells, reduction in tumor size / volume, and / or, for example, extension of survival time (e.g., progression-free survival time) as determined in a suitable xenograft model derived from HER3-expressing cancer cell lines.

[0401] In some embodiments, the antigen-binding molecules of the present invention can inhibit tumor growth in a xenograft model derived from HER3-expressing cancer cell lines to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the tumor growth observed in the absence of treatment with the antigen-binding molecule (or after treatment with a suitable negative control antigen-binding molecule).

[0402] Chimeric antigen receptor (CAR) The present invention also provides a chimeric antigen receptor (CAR) comprising the antigen-binding molecule or polypeptide of the present invention.

[0403] A CAR is a recombinant receptor that provides both antigen-binding and T cell activation functions. The structure and engineering of CARs are reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1), which is hereby incorporated by reference in its entirety. A CAR comprises an antigen-binding region linked to a transmembrane anchor region and a signaling region. An optional hinge region can provide a separation between the antigen-binding region and the transmembrane anchor region and can act as a flexible linker.

[0404] The CAR of the present invention comprises an antigen-binding region that comprises the antigen-binding molecule of the present invention or consists of the antigen-binding molecule of the present invention, or that comprises a polypeptide according to the present invention or consists of a polypeptide according to the present invention.

[0405] The transmembrane anchor region provides anchoring of the CAR to the cell membrane of a cell expressing the CAR, bringing the antigen-binding region of the CAR into the extracellular lumen and the signaling region into the interior of the cell, between the antigen-binding region and the signaling region of the CAR. In some embodiments, the CAR comprises an amino acid sequence of a transmembrane region for one of CD3-ζ, CD4, CD8, or CD28, or consists of an amino acid sequence of a transmembrane region for one of CD3-ζ, CD4, CD8, or CD28, or comprises an amino acid sequence derived from an amino acid sequence of a transmembrane region for one of CD3-ζ, CD4, CD8, or CD28, or consists of an amino acid sequence derived from an amino acid sequence of a transmembrane region for one of CD3-ζ, CD4, CD8, or CD28. As used herein, a region "derived from" a reference amino acid sequence comprises an amino acid sequence having one of at least 60%, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference sequence.

[0406] The signaling domain of the CAR enables the activation of T cells. The signaling domain of the CAR can include the amino acid sequence of the intracellular domain of CD3-ζ, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. In CARs, signaling domains containing the sequences of other ITAM-containing proteins, such as FcγRI, have also been utilized (Haynes et al., 2001 J Immunol 166(1):182-187). The signaling domain of the CAR can also include a co-stimulatory sequence derived from the signaling domain of a co-stimulatory molecule so as to promote the activation of CAR-expressing T cells upon binding to the target protein. Suitable co-stimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, the CAR is engineered to provide co-stimulation of different intracellular signaling pathways. For example, signaling associated with CD28 co-stimulation preferentially activates the phosphatidylinositol 3 kinase (PI3K) pathway, whereas 4-1BB-mediated signaling occurs via the TNF receptor-associated factor (TRAF) adapter protein. Thus, the signaling domain of the CAR can, in some cases, contain co-stimulatory sequences derived from the signaling domains of more than one co-stimulatory molecule. In some embodiments, the CAR of the invention comprises, consists of, or consists essentially of an amino acid sequence of the intracellular domain of one or more of CD28, OX40, 4-1BB, ICOS, and CD27, or comprises, consists of, or consists essentially of an amino acid sequence derived from one or more co-stimulatory sequences thereof.

[0407] An optional hinge region can provide a separation between the antigen-binding domain and the transmembrane domain and can act as a flexible linker. The hinge region can be derived from IgG1. In some embodiments, the CAR of the invention comprises, consists of, or consists essentially of an amino acid sequence of the hinge region of IgG1, or comprises, consists of, or consists essentially of an amino acid sequence derived from the hinge region thereof.

[0408] Also provided are cells comprising a CAR according to the present invention. The CAR according to the present invention can be used to generate CAR-expressing immune cells, such as CAR-T cells or CAR-NK cells. Manipulation of the CAR into immune cells can be carried out during in vitro culture.

[0409] The antigen-binding region of the CAR of the present invention can be provided in any suitable format, such as scFv, scFab, etc.

[0410] Nucleic Acids and Vectors The present invention provides one nucleic acid or a plurality of nucleic acids encoding an antigen-binding molecule, polypeptide, or CAR according to the present invention.

[0411] In some embodiments, the nucleic acid is purified or isolated, for example, from other nucleic acids or naturally occurring biological materials. In some embodiments, the nucleic acid comprises and / or consists of DNA and / or RNA.

[0412] The present invention also provides one vector or a plurality of vectors comprising one nucleic acid or a plurality of nucleic acids according to the present invention.

[0413] The nucleotide sequence can be contained within a vector, such as an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle for transferring an exogenous nucleic acid into a cell. The vector can be a vector for expressing a nucleic acid in a cell. Such a vector can include a promoter sequence operably linked to a nucleotide sequence encoding the sequence to be expressed. The vector can also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from the vector according to the present invention.

[0414] The term "operably linked" can include situations where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked so as to place the expression of the nucleic acid sequence under the influence or control of the regulatory array (thereby forming an expression cassette). Thus, if a regulatory sequence is capable of effecting transcription of a nucleic acid sequence, the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.

[0415] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma-retroviral vectors (e.g., vectors derived from murine leukemia virus (MLV)), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia virus vectors, and herpes virus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0416] In some embodiments, the vector can be a eukaryotic vector, e.g., a vector that contains elements necessary for protein expression from a vector within a eukaryotic cell. In some embodiments, the vector can be a mammalian vector, e.g., that contains a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0417] The polypeptides that are components of the antigen-binding molecules according to the invention can be encoded by different nucleic acids among a plurality of nucleic acids, or by different vectors among a plurality of vectors.

[0418] Cells comprising / expressing antigen-binding molecules and polypeptides The invention also provides cells that comprise or express an antigen-binding molecule, polypeptide, or CAR according to the invention. Also provided are cells that comprise or express one nucleic acid, a plurality of nucleic acids, one vector, or a plurality of vectors according to the invention.

[0419] The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human), or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse, or other rodent (including any animal within the order Rodentia), cat, dog, pig, sheep, goat, cattle (cow, e.g., dairy cow, or any animal within the order Bovidae), horse (including any animal within the order Equidae), donkey, and non-human primate).

[0420] The invention also provides a method for producing a cell comprising a nucleic acid or vector according to the invention, the method comprising introducing into the cell one nucleic acid, a plurality of nucleic acids, one vector, or a plurality of vectors according to the invention. In some embodiments, the step of introducing an isolated nucleic acid or vector according to the invention into the cell comprises transformation, transfection, electroporation, or transduction (e.g., transduction by a retrovirus).

[0421] The invention also provides a method for producing a cell expressing / comprising an antigen-binding molecule, polypeptide, or CAR according to the invention, the method comprising introducing into the cell one nucleic acid, a plurality of nucleic acids, one vector, or a plurality of vectors according to the invention. In some embodiments, the method further comprises culturing the cell under conditions suitable for expression of the nucleic acid or vector by the cell. In some embodiments, the method is performed in vitro.

[0422] The invention also provides a cell obtainable or obtained by a method according to the invention.

[0423] Production of antigen-binding molecules and polypeptides The antigen-binding molecules and polypeptides according to the invention can be prepared according to methods for producing polypeptides known to those skilled in the art.

[0424] The polypeptide can be prepared by chemical synthesis, for example, by liquid phase or solid phase synthesis. For example, the peptide / polypeptide can be synthesized using, for example, the methods described in Chandrudu et al., Molecules (2013), 18:4373 - 4388, which is incorporated herein by reference in its entirety.

[0425] Alternatively, the antigen - binding molecule and the polypeptide can also be produced by recombinant expression. In the art, molecular biological techniques suitable for the recombinant production of polypeptides, such as those shown in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Press, 2012, as well as Nat Methods. (2008); 5(2):135 - 146 (both of which are incorporated herein by reference in their entirety), are well - known. Methods for the recombinant production of antigen - binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4:217, as well as Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451 - 3461 (both of which are incorporated herein by reference in their entirety).

[0426] In some cases, the antigen - binding molecule of the present invention is composed of more than one polypeptide chain. In such cases, the production of the antigen - binding molecule can include the transcription and translation of more than one polypeptide to form the antigen - binding molecule and the subsequent association of the polypeptide chains.

[0427] For recombinant production according to the present invention, any cell suitable for the expression of a polypeptide can be used. The cell may be a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell such as an archaeal or bacterial cell. In some embodiments, the bacterium can be a gram-negative bacterium such as a bacterium of the family Enterobacteraceae, for example, Escherichia coli. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, for example, a CHO, HEK (e.g., HEK293), HeLa, or COS cell. In some embodiments, the cell is a CHO cell that transiently or stably expresses the polypeptide.

[0428] In some cases, since some prokaryotic cells cannot perform the same folding or post-translational modifications as eukaryotic cells, the cell is not a prokaryotic cell. In addition, within eukaryotic cells, extremely high expression levels are possible, and the protein can be easily purified from eukaryotic cells using appropriate tags. Specific plasmids that enhance the secretion of the protein into the medium can also be utilized.

[0429] In some embodiments, the polypeptide can be prepared by cell-free protein synthesis (CFPS) according to, for example, using the system described in Zemella et al., Chembiochem (2015) 16(17):2420 - 2431, which is incorporated herein by reference in its entirety.

[0430] Production can involve culturing or fermenting eukaryotic cells modified to express the polypeptide of interest. The culturing or fermenting can be carried out in a bioreactor with an appropriate supply of nutrients, air / oxygen, and / or growth factors. The secreted protein can be recovered by separating the culture medium / fermentation broth from the cells, extracting the protein-containing material, and isolating the individual proteins to isolate the secreted polypeptide. Culture, fermentation, and separation techniques are well known to those skilled in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition; incorporated herein by reference above).

[0431] The bioreactor includes one or more reaction vessels in which cells can be cultured. The culturing within the bioreactor can be carried out continuously by a continuous inflow of reactants into the reactor and a continuous outflow of the cultured cells from the reactor. Alternatively, the culturing can be carried out in batch. The bioreactor monitors and controls environmental conditions such as pH, oxygen, the flow rates of inflow into and outflow from the reaction vessel, and agitation within the reaction vessel so as to provide optimal conditions for the cells being cultured.

[0432] After culturing the cells expressing the antigen-binding molecule / polypeptide, the polypeptide of interest can be isolated. Any suitable method known in the art for separating proteins from cells can be used. It may be necessary to separate the cells from the nutrient medium to isolate the polypeptide. If the polypeptide is secreted from the cells, the cells can be separated from the culture medium containing the secreted polypeptide of interest by centrifugation. If the polypeptide of interest accumulates intracellularly, the isolation of the protein can include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with lysis buffer, and disruption of the cells by, for example, sonication, rapid freeze-thaw, or osmotic lysis.

[0433] Next, it may be desirable to isolate the polypeptide of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common technique for separating protein components from the supernatant or culture medium is precipitation. Proteins with different solubilities precipitate with different concentrations of a precipitating agent such as ammonium sulfate. For example, at low concentrations of the precipitating agent, water-soluble proteins are extracted. Thus, by adding increasing different concentrations of the precipitating agent, proteins with different solubilities can be discriminated. Subsequently, dialysis can be used to remove ammonium sulfate from the separated proteins.

[0434] In the art, other methods for discriminating different proteins, such as ion exchange chromatography and size exclusion chromatography, are known. These can be used as alternatives to precipitation or can be carried out subsequent to precipitation.

[0435] Once the polypeptide of interest has been isolated from the culture, it may be desirable or necessary to concentrate the polypeptide. In the art, numerous methods for concentrating proteins, such as ultrafiltration or lyophilization, are known.

[0436] Composition The invention also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein.

[0437] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The compositions may be formulated for local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intracorneal, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal routes of administration, which may include injection or infusion.

[0438] Suitable formulations may contain antigen-binding molecules in a sterile medium or an isotonic medium. The medicaments and pharmaceutical compositions can be formulated in a fluid, including in a gel form. The fluid formulations can be formulated for administration by injection or infusion (e.g., via a catheter) into a selected region in a human or animal body.

[0439] In some embodiments, the composition is formulated for injection or infusion, for example, into a blood vessel or a tumor.

[0440] Also provided according to the invention described herein is a method for producing a pharmaceutically useful composition, such a production method comprising one or more steps selected from the steps of producing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell as described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell as described herein; and / or mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0441] For example, a further aspect of the invention described herein is a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease / condition (e.g., cancer), the method comprising formulating a medicament or pharmaceutical composition by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0442] Therapeutic and prophylactic applications The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells, and compositions described herein are used in methods of treatment and prevention.

[0443] The present invention provides an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein for use in a method of medical treatment or prevention. Also provided is the use of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition. Also provided is a method of treating or preventing a disease or condition, the method comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein.

[0444] The method can be effective in reducing the onset or progression of a disease / condition, alleviating the symptoms of a disease / condition, or reducing the pathology of a disease / condition. The method can be effective in preventing the progression of a disease / condition, e.g., preventing the worsening of a disease / condition or slowing the rate of onset of a disease / condition. In some embodiments, the method can result in the improvement of a disease / condition, e.g., reducing the symptoms of a disease / condition or reducing other correlating factors of the severity / activity of a disease / condition. In some embodiments, the method can prevent the onset of a late stage (e.g., chronic or metastatic) of a disease / condition.

[0445] It will be understood that the articles of the present invention can be used for the treatment or prevention of any disease / condition that derives a therapeutic or preventive benefit from a reduction in the number and / or activity of cells expressing HER3. For example, the disease / condition is a disease / condition in which cells expressing HER3 are pathologically involved, for example, an increase in the number / ratio of cells expressing HER3 is positively associated with the occurrence, onset, or progression of the disease / condition, and / or the severity of one or more symptoms of the disease / condition, or an increase in the number / ratio of cells expressing HER3 is a risk factor for the occurrence, onset, or progression of the disease / condition.

[0446] In some embodiments, the disease / condition treated / prevented according to the present invention is a disease / condition characterized by an increase in the number / ratio / activity of cells expressing HER3, for example, compared to the number / ratio / activity of cells expressing HER3 in the absence of the disease / condition.

[0447] In some embodiments, the disease / condition treated / prevented is cancer.

[0448] Cancer can be any unwanted cell proliferation (or any disease manifested by itself by unwanted cell proliferation), neoplasm, or tumor. Cancer can be benign or malignant, primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. Cancer can be, for example, cancer of the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, jaw, mediastinum, mesentery, myometrium, nasopharynx, reticulum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, and / or cancer of tissues / cells derived from white blood cells.

[0449] The tumor to be treated can be a nervous system or non-nervous system tumor. Nervous system tumors, such as gliomas, medulloblastomas, meningiomas, neurofibromas, ependymomas, schwannomas, neurofibrosarcomas, astrocytomas, and oligodendrogliomas, can be derived from the central or peripheral nervous system. Non-nervous system cancers / tumors may be derived from any other non-nervous tissue, and examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, squamous cell carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[0450] HER3, as well as its association with cancer and its role in cancer, are reviewed, for example, in Karachaliou et al., BioDrugs. (2017) 31(1):63-73, and Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1):39-48, both of which are incorporated herein by reference in their entirety.

[0451] In some embodiments, the cancer is selected from cancers that include HER3-expressing cells, solid tumors, breast cancer, breast carcinoma, ductal carcinoma of the breast, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, head and neck squamous cell carcinoma (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lung cancer, ovarian cancer, ovarian carcinoma, serous ovarian adenocarcinoma, kidney cancer, renal cell carcinoma, clear cell renal carcinoma, renal cell adenocarcinoma, papillary renal cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, endometrial carcinoma of the uterus, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, urothelial carcinoma of the bladder, prostate cancer, prostatic adenocarcinoma, sarcoma, and thymoma.

[0452] In some embodiments, the cancer treated according to the present invention is selected from HER3-expressing cancer, gastric cancer (e.g., gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), breast cancer, ovarian cancer (e.g., ovarian carcinoma), lung cancer (e.g., NSCLC, lung adenocarcinoma, squamous lung cell carcinoma), melanoma, prostate cancer, oral cancer (e.g., oropharyngeal cancer), kidney cancer (e.g., renal cell carcinoma), and / or colorectal cancer (e.g., colorectal carcinoma), esophageal cancer, pancreatic cancer, solid cancer, and / or liquid cancer.

[0453] Treatment / prevention can be targeted at one or more of delaying / preventing the occurrence / progression of cancer symptoms, reducing the severity of cancer symptoms, reducing the survival period / growth / invasion / metastasis of cancer cells, reducing the number of cancer cells, and / or prolonging the survival period of the subject.

[0454] In some embodiments, the cancer to be treated / prevented comprises cells that express an EGFR family member (e.g., HER3, EGFR, HER2, or HER4), and / or cells that express a ligand for an EGFR family member. In some embodiments, the cancer to be treated / prevented is a cancer that is positive for an EGFR family member. In some embodiments, the cancer overexpresses an EGFR family member, and / or a ligand for an EGFR family member. Overexpression can be determined by detecting a level of expression that exceeds the level of expression by equivalent non-cancerous cells / non-tumor tissue.

[0455] Expression can be determined by any suitable means. Expression can be gene expression or protein expression. Gene expression can be determined, for example, by quantitative real-time PCR (qRT-PCR), for example, by detection of mRNA encoding HER3. For example, protein expression can be determined, for example, by antibody-based methods, for example, Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[0456] In some embodiments, the cancer to be treated / prevented comprises cells that express HER3. In some embodiments, the cancer to be treated / prevented is a cancer that is positive for HER3. In some embodiments, the cancer overexpresses HER3. Overexpression of HER3 can be determined by detecting the expression level of HER3 that exceeds the level of expression by equivalent non-cancerous cells / non-tumor tissues.

[0457] In some embodiments, a patient can be selected for the treatment described herein based on, for example, the detection of cancer that expresses HER3 or overexpresses HER3 in a sample obtained from the subject.

[0458] In some embodiments, the cancer to be treated / prevented comprises cells that express a ligand for HER3 (e.g., NRG1 and / or NRG2). In some embodiments, the cancer to be treated / prevented comprises cells that express an expression level of NRG1 and / or NRG2 that exceeds the level of expression by equivalent non-cancerous cells / non-tumor tissues.

[0459] The HER3-binding antigen-binding molecules described herein have been demonstrated to bind to HER3 with extremely high affinity when HER3 is bound to NRG (i.e., when HER3 is presented in the "open" conformation), and also when HER3 is not bound to NRG (i.e., when HER3 is presented in the "closed" conformation).

[0460] Accordingly, the antigen-binding molecules of the invention are particularly useful for the treatment / prevention of cancers characterized by HER3 ligand expression / overexpression, e.g., cancers / tumors that comprise cells that express / overexpress a ligand for HER3.

[0461] In some embodiments, the cancer to be treated according to the present invention includes cells carrying a genetic variant (e.g., a mutation) that causes increased (gene and / or protein) expression of a ligand for HER as compared to equivalent cells carrying a reference allele that does not contain the genetic variant (e.g., a non-mutated, or “wild-type” allele). The genetic variant may be, or may include, an insertion, deletion, substitution, or more large-scale translocation / rearrangement of the nucleotide sequence relative to the reference allele.

[0462] A mutation that “results in” increased expression of a ligand for HER3 may be known to, or predicted to, or associated with, increased gene / protein expression of a ligand for HER3. A mutation that results in increased expression of a ligand for HER3 may be referred to as an “activating” mutation.

[0463] A mutation that causes increased expression of a ligand for HER3 may result in gene or protein expression of a ligand for HER3 that is not expressed by, and / or not encoded by, the genomic nucleic acid of equivalent cells that do not carry the mutation. That is, since the ligand for HER3 may be a neoantigen that results from the mutation, “increased expression” may not be from expression. By way of example, cells containing a CD47-NRG1 gene fusion exhibit increased expression of the CD47-NRG1 fusion polypeptide encoded by the gene fusion as compared to cells lacking the CD47-NRG1 gene fusion.

[0464] A mutation that causes increased expression of a ligand for HER3 may result in increased gene or protein expression of a ligand for HER3 that is expressed by, and / or encoded by, the genomic nucleic acid of equivalent cells that do not contain the mutation. By way of example, cells may contain a mutation that results in an increase in the level of transcription of the nucleic acid encoding NRG1 as compared to the level of transcription of the nucleic acid encoding NRG1 by equivalent cells that do not contain the mutation.

[0465] In some embodiments, a mutation that causes increased expression of the ligand for HER3 can cause an increase in the gene expression of the ligand for HER3 as compared to equivalent cells without the mutation. In some embodiments, a mutation that causes increased expression of the ligand for HER3 can cause an increase in the protein expression of the ligand for HER3 as compared to equivalent cells without the mutation.

[0466] In some embodiments, a mutation that causes increased expression of the ligand for HER3 can cause an increase in the level of the ligand for HER3 on or at the cell surface of cells containing the mutation as compared to equivalent cells without the mutation. In some embodiments, a mutation that causes increased expression of the ligand for HER3 can cause an increase in the level of secretion of the ligand for HER3 from cells containing the mutation as compared to equivalent cells without the mutation.

[0467] Cells having increased expression of the ligand for HER3 can be described as “overexpressing” the ligand for HER3 or having “upregulated expression” of the ligand for HER3 as compared to the level of expression of the ligand for HER3 by reference cells (e.g., as a result of a mutation). For example, a cancer comprising cells carrying a mutation that results in increased expression of the ligand for HER3 can be described as a cancer comprising cells that exhibit overexpression / upregulated expression of the ligand for HER3 as compared to equivalent cells lacking the mutation. In some embodiments, the reference cells lacking the mutation can be non-cancerous cells (e.g., of an equivalent cell type) or cancerous cells (e.g., of an equivalent cancer type).

[0468] As used herein, the "ligand for HER3" is generally intended to refer to a molecule that can bind to HER3 via the ligand-binding region of HER3 formed by domains I and III of HER3. In some embodiments, the ligand for HER3 binds to HER3 through interaction with domains I and / or III of HER3. Exemplary ligands for HER3 include neuregulins such as NRG1 and NRG2, which bind to HER3 through the interaction of their EGF-like domains with the ligand-binding region of HER3.

[0469] The HER3 ligand preferably binds to the HER3 receptor and / or the receptor complex containing HER3, and can induce signal transduction through them. As is apparent from the present disclosure, the receptor complex containing HER3 may further include interaction partners for HER3 described herein, such as HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet).

[0470] In some embodiments, the ligand for HER3 can bind to the HER3 receptor / receptor complex expressed by cells other than the cells having increased expression of the HER3 ligand. For example, in some embodiments, the ligand for HER3 can bind to HER3-expressing cancer cells.

[0471] In some embodiments, the ligand for HER3 can bind to the HER3 receptor / receptor complex expressed by cells having increased expression of the HER3 ligand.

[0472] In some embodiments, the cancer to be treated includes (i) cells expressing HER3, and (ii) cells expressing a ligand for HER3 (e.g., as a result of a mutation that results in increased expression of the ligand for HER3, e.g., having increased expression of the ligand for HER3).

[0473] In some embodiments, the cancer to be treated comprises cells that (i) express HER3 and (ii) also express a ligand for HER3 (e.g., as a result of a mutation that results in increased expression of the ligand for HER3, e.g., having increased expression of the ligand for HER3).

[0474] In some embodiments, the ligand for HER3 comprises, or consists of, the amino acid sequence of the HER3-binding region of the ligand for HER3, or an amino acid sequence derived from the HER3-binding region of the ligand for HER3. The amino acid sequence derived from the HER3-binding region of the ligand for HER3 can have at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the amino acid sequence from which it is derived.

[0475] In some embodiments, the ligand for HER3 comprises an EGF-like domain that can bind to HER3, or a HER3-binding fragment thereof. In some embodiments, the HER3-binding EGF-like domain / fragment is an EGF family member (e.g., heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen, betacellulin (BTC), NRG1, NRG2, NRG3, or NRG4), or is derived from these.

[0476] EGF family members contain one or more repeats of the conserved amino acid sequence shown in SEQ ID NO: 240, which results in three structural loops required for high affinity binding to their cognate receptors and contains six cysteine residues that form three intramolecular disulfide bonds (see Harris et al., Experimental Cell Research (2003) 284(1):2-13). In some embodiments, the ligand for HER3 comprises one or more copies of an amino acid sequence that is identical to the consensus sequence shown in SEQ ID NO: 240.

[0477] Exemplary ligands for HER3 include neuregulin (NRG). Neuregulin includes NRG1, NRG2, NRG3, and NRG4. The amino acid sequence of human NRG1 (alpha isoform) is shown in SEQ ID NO: 232. The alpha isoform and several other isoforms of human NRG1 (including the alpha 1a isoform (see UnitProt: Q02297-2), the alpha 2b isoform (see UnitProt: Q02297-3), and the alpha 3 isoform (see UnitProt: Q02297-4)) include the EGF-like domain shown in SEQ ID NO: 233, and through this EGF-like domain, they bind to HER3. The amino acid sequence of human NRG2 (isoform 1) is shown in SEQ ID NO: 234. Isoform 1 and several other isoforms of human NRG2 (including isoform 3 (see UniProt: O14511-3), isoform 5 (see UniProt: O14511-5), isoform 6 (see UniProt: O14511-6), isoform DON-1B (see UniProt: O14511-7), and isoform DON-1R (see UniProt: O14511-8)) include the EGF-like domain shown in SEQ ID NO: 235, and through this EGF-like domain, they bind to HER3. The amino acid sequence of human NRG3 is shown in SEQ ID NO: 236, and the EGF-like domain of human NRG3 is shown in SEQ ID NO: 237. The amino acid sequence of human NRG4 is shown in SEQ ID NO: 238, and the EGF-like domain of human NRG3 is shown in SEQ ID NO: 239. In some embodiments, NRG is selected from NRG1, NRG2, NRG3, and NRG4. In some embodiments, NRG is selected from NRG1 and NRG2.

[0478] In some embodiments, the EGF-like domain / fragment comprises, or consists of, an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG (NRG1, NRG2, NRG3, or NRG4). In some embodiments, the EGF-like domain / fragment comprises, or consists of, an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to one of SEQ ID NO: 233, 235, 237, or 239.

[0479] In some embodiments, the ligand for HER3 is NRG (e.g., NRG1, NRG2, NRG3, or NRG4, e.g., NRG1, or NRG2), or comprises an amino acid sequence derived from the amino acid sequence of NRG (i.e., comprises an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the amino acid sequence of NRG).

[0480] In some embodiments, the ligand for HER3 comprises, or consists of, an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the HER3-binding region of a ligand for HER3 (e.g., NRG, e.g., NRG1, NRG2, NRG3, or NRG4, e.g., NRG1, or NRG2). In some embodiments, the ligand for HER3 comprises, or consists of, an amino acid sequence having at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG (e.g., NRG1, NRG2, NRG3, or NRG4, e.g., NRG1, or NRG2).

[0481] In some embodiments, the ligand for HER3 is not an EGFR family protein (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, cMet).

[0482] In some embodiments, the mutation that results in increased expression of the ligand for HER3 is an NRG gene fusion. In some embodiments, the ligand for HER3 is the product of an NRG gene fusion (i.e., the polypeptide encoded by the NRG gene fusion). In some embodiments, the cancer comprises cells having the NRG gene fusion.

[0483] As used herein, an “NRG gene fusion” refers to a genetic variant encoding a polypeptide comprising (i) the amino acid sequence of an NRG protein (e.g., NRG1, NRG2, NRG3, or NRG4, e.g., NRG1, or NRG2), and (ii) the amino acid sequence of a protein other than an NRG protein.

[0484] It will be understood that the NRG gene fusion preferably encodes an HER3 ligand as described herein. In some embodiments, the NRG gene fusion encodes a polypeptide comprising the HER3-binding region of the NRG protein. In some embodiments, the NRG gene fusion encodes a polypeptide comprising the EGF-like domain of the NRG protein, or an amino acid sequence that binds to HER3 and has at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of the NRG protein.

[0485] In some embodiments, the NRG gene fusion encodes a fusion polypeptide comprising a transmembrane domain. In some embodiments, the NRG gene fusion encodes a fusion polypeptide comprising a transmembrane domain of a protein other than the NRG protein.

[0486] In some embodiments, the NRG gene fusion is an NRG1 gene fusion. In some embodiments, the NRG1 gene fusion encodes a polypeptide comprising the EGF-like domain of NRG1, or an amino acid sequence that binds to HER3 and has at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG1.

[0487] NRG1 gene fusions are described, for example, in WO2018 / 182422 A1, WO2019 / 051155 A1, Dhanasekaran et al., Nat Commun. (2014) 5:5893, Drilon et al., Cancer Discov. (2018) 8(6):686-695, Nagasaka et al., Journal of Thoracic Oncology (2019) 14(8):1354-1359, and Jonna et al., Clin Cancer Res. (2019) 25(16):4966-4972, all of which are hereby incorporated by reference in their entirety. The diversity of NRG1 gene fusions can be attributed, in particular, to NRG1 located on chromosome 8, which is susceptible to genomic translocation events (Adelaide et al., Genes Chromosomes Cancer. (2003) 37(4):333-45).

[0488] In some embodiments, the NRG1 gene fusion is selected from CLU-NRG1, CD74-NRG1, DOC4-NRG1, SLC3A2-NRG1, RBPMS-NRG1, WRN-NRG1, SDC4-NRG1, RAB2IL1-NRG1, VAMP2-NRG1, KIF13B-NRG1, THAP7-NRG1, SMAD4-NRG1, MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1, DPYSL2-NRG1, ATP1B1-NRG1, CDH6-NRG1, APP-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1, CDK1-NRG1, BMPRIB-NRG1, TNFRSF10B-NRG1, and MCPH1-NRG1. In some embodiments, the NRG1 gene fusion is CLU-NRG1. The CD74-NRG1 gene fusion is described, for example, in Fernandez-Cuesta et al., Cancer Discov. (2014) 4:415-22, and Nakaoku et al., Clin Cancer Res (2014) 20:3087-93. The DOC4-NRG1 gene fusion is described, for example, in Liu et al., Oncogene. (1999) 18(50):7110-4 and Wang et al., Oncogene. (1999) 18(41):5718-21. The SLC3A2-NRG1 gene fusion is described, for example, in Nakaoku et al., Clin Cancer Res (2014) 20:3087-93, Shin et al., Oncotarget (2016) 7:69450-65, and Shin et al., Mol Cancer Ther. (2018) 17(9):2024-2033. The RBPMS-NRG1, WRN-NRG1, RAB2IL1-NRG1, and SDC4-NRG1 gene fusions are described, for example, in Dhanasekaran et al., Nat Commun. (2014) 5:5893. The VAMP2-NRG1 gene fusion is described, for example, in Jung et al., J Thorac Oncol. (2015) 10(7):1107-11, and Shim et al., J Thorac Oncol. (2015) 10(8):1156-62.The KIF13B-NRG1 gene fusion is described, for example, in Xia et al., Int J Surg Pathol. (2017) 25(3):238-240. The SMAD4-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1, and THAP7-NRG1 gene fusions are described, for example, in Drilon et al., Cancer Discov. (2018) 8(6):686-695. The MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1, and DPYSL2-NRG1 gene fusions are described, for example, in Jonna et al., Clin Cancer Res. (2019) 25(16):4966-4972. The ATP1B1-NRG1 gene fusion is described, for example, in Drilon et al., Cancer Discov. (2018) 8(6):686-695, and Jones et al., Annals of Oncology (2017) 28:3092-3097. The CLU-NRG1 gene fusion is described, for example, in Drilon et al., Cancer Discov. (2018) 8(6):686-695, and Nagasaka et al., Journal of Thoracic Oncology (2019) 14(8):1354-1359.

[0489] In some embodiments, the NRG gene fusion is an NRG2 gene fusion. In some embodiments, the NRG2 gene fusion encodes a polypeptide comprising the EGF-like domain of NRG2, or an amino acid sequence that binds to HER3 and has at least 60% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG2.

[0490] NRG2 gene fusions include, for example, SLC12A2-NRG2 described in WO2015 / 093557 A1, and ZNF208-NRG2 described in Dupain et al., Mol Ther. (2019) 27(1):200-218.

[0491] Cancer comprising cells having a mutation that results in increased expression of a ligand for HER3 (e.g., comprising cells having an NRG gene fusion, e.g., an NRG1 gene fusion, or an NRG2 gene fusion) can be any cancer described herein. In some embodiments, such cancer can be cancer of tissue / cells derived from the lung, breast, head, neck, kidney, ovary, pancreas, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue or nasopharynx.

[0492] In some embodiments, cancer comprising cells having a mutation that results in increased expression of a ligand for HER3 (e.g., comprising cells having an NRG gene fusion, e.g., an NRG1 gene fusion, or an NRG2 gene fusion) is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, invasive breast cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, renal clear cell carcinoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, bladder cancer, urothelial bladder cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor and nasopharyngeal neuroendocrine tumor.

[0493] In certain embodiments, the cancer treated according to the present invention is lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, or lung squamous cell carcinoma) comprising cells having an NRG1 gene fusion.

[0494] It will be understood that in the embodiments herein, cancer comprising cells having certain characteristics can be or can include a tumor comprising cells having these characteristics.

[0495] As is common in the art, cancers / tumors containing cells with certain characteristics may herein simply be referred to as cancers / tumors having these characteristics. By way of example, a cancer / tumor containing cells having an NRG1 gene fusion may simply be referred to as a "cancer / tumor containing an NRG1 gene fusion", or an "NRG1 gene fusion cancer / tumor".

[0496] Administration of the articles of the invention is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is an amount sufficient to demonstrate a therapeutic or prophylactic benefit to the subject. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease / condition, and on the particular article being administered. Prescription of treatment, e.g., decisions regarding dosage, etc., is within the responsibility of a general practitioner and other physicians, and typically takes into account the disease / disorder being treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols referred to above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0497] Administration may be made alone, or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated. The antigen-binding molecules or compositions, and therapeutic agents described herein may be administered simultaneously or sequentially.

[0498] In some embodiments, the method includes additional therapeutic or prophylactic interventions, for example, for the treatment / prevention of cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination, and / or hormone therapy. In some embodiments, the therapeutic or prophylactic intervention includes leukapheresis. In some embodiments, the therapeutic or prophylactic intervention includes stem cell transplantation.

[0499] In some embodiments, the antigen-binding molecule is administered in combination with an agent capable of inhibiting signal transduction mediated by an EGFR family member.

[0500] Accordingly, the present invention provides a composition comprising an article according to the present invention (e.g., an antigen-binding molecule according to the present invention) and another agent capable of inhibiting signal transduction mediated by an EGFR family member (e.g., EGFR, HER2, HER3, or HER4). Also provided is the use of such a composition in the medical treatment and prevention of the diseases / conditions described herein.

[0501] Also provided is a method for treating / preventing a disease / condition described herein, the method comprising administering an article of the present invention according to the present invention (e.g., an antigen-binding molecule according to the present invention) and another agent capable of inhibiting signal transduction mediated by an EGFR family member.

[0502] In the art, agents capable of inhibiting signal transduction mediated by an EGFR family member are known and include, for example, small molecule inhibitors (e.g., tyrosine kinase inhibitors), monoclonal antibodies (and antigen-binding fragments thereof), peptide / polypeptide inhibitors (e.g., decoy ligands / receptors or peptide aptamers), and nucleic acids (e.g., antisense nucleic acids, splice-switching nucleic acids, or nucleic acid aptamers). Inhibitors of signal transduction mediated by an EGFR family member include agents that inhibit signal transduction through a direct effect on an interaction partner that is an EGFR family member and thus also and / or inhibit downstream factors involved in signal transduction mediated by an EGFR family member.

[0503] In some embodiments, an antagonist of signal transduction mediated by an EGFR family member inhibits signal transduction mediated by one or more of EGFR, HER2, HER4, and HER3. Inhibitors of signal transduction mediated by EGFR family members are described, for example, in Yamaoka et al., Int. J. Mol. Sci. (2018), 19, 3491, which is incorporated herein by reference in its entirety. In some embodiments, the antagonist is a pan-ErbB inhibitor. In some embodiments, the antagonist is an inhibitor of signal transduction mediated by EGFR (e.g., cetuximab, panitumumab, gefitinib, erlotinib, lapatinib, afatinib, brigatinib, icotinib, osimertinib, zalutumumab, vandetanib, necitumumab, nimotuzumab, dacomitinib, durvalumab, or matuzumab). In some embodiments, the antagonist is an inhibitor of signal transduction mediated by HER2 (e.g., trastuzumab, pertuzumab, lapatinib, neratinib, afatinib, dacomitinib, MM-111, MCLA-128, or margetuximab). In some embodiments, the antagonist is an inhibitor of signal transduction mediated by HER3 (e.g., seribantumab, lumretuzumab, elgemtumab, KTN3379, AV-203, GSK2849330, REGN1400, MP-RM-1, EV20, durvalumab, MM-111, istiratumab, MCLA-128, patritumab, EZN-3920, RB200, or U3-1402). In some embodiments, the antagonist is an inhibitor of signal transduction mediated by HER4 (e.g., lapatinib, ibrutinib, afatinib, dacomitinib, or neratinib).

[0504] In some embodiments, antagonists of signal transduction mediated by EGFR family members inhibit effectors downstream of signal transduction by EGFR family members. Effectors downstream of signal transduction by EGFR family members include, for example, PI3K, AKT, KRAS, BRAF, MEK / ERK, and mTOR. In some embodiments, antagonists of signal transduction mediated by EGFR family members are inhibitors of the MAPK / ERK pathway. In some embodiments, antagonists of signal transduction mediated by EGFR family members are inhibitors of the PI3K / ATK / mTOR pathway. In some embodiments, the antagonist is a PI3K inhibitor (e.g., pictilisib, buparlisib, idelalisib, copanlisib, or duvelisib). In some embodiments, the antagonist is an AKT inhibitor (e.g., MK-2206, AZD5363, ipatasertib, VQD-002, perifosine, or miltefosine). In some embodiments, the antagonist is a BRAF inhibitor (e.g., vemurafenib, dabrafenib, SB590885, XL281, RAF265, encorafenib, GDC-0879, PLX-4720, sorafenib, or LGX818). In some embodiments, the antagonist is a MEK / ERK inhibitor (e.g., trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, PD035901, or TAK-733). In some embodiments, the antagonist is an mTOR inhibitor (e.g., rapamycin, deforolimus, temsirolimus, everolimus, ridaforolimus, or sapitinib).

[0505] In some embodiments, the cancer being treated according to an aspect of the invention (including monotherapy or combination therapy) is a cancer that is resistant to treatment with an antagonist of signal transduction mediated by an EGFR family member (e.g., EGFR, HER2, HER4, and / or HER3), such as the antagonists described in the three preceding paragraphs. In some embodiments, the subject being treated has a cancer that is resistant to treatment with an antagonist of signal transduction mediated by an EGFR family member. In some embodiments, the subject being treated has a cancer that has developed resistance to treatment with an antagonist of signal transduction mediated by an EGFR family member. In some embodiments, the subject being treated has a cancer that was once responsive to treatment with an antagonist of signal transduction mediated by an EGFR family member but is now resistant to treatment with the antagonist. In some embodiments, the subject being treated has a cancer that has recurred and / or progressed after treatment with an antagonist of signal transduction mediated by an EGFR family member. In some embodiments, the subject being treated has a cancer that initially responded to treatment with an antagonist of signal transduction mediated by an EGFR family member but then progressed on said treatment.

[0506] In some embodiments, it may be determined (i.e., diagnosed) that the subject being treated according to the invention has a cancer comprising cells having a mutation that causes increased expression of a ligand for HER3 (e.g., as described herein). In some embodiments, the method of the invention may include determining whether the subject has a cancer comprising cells having a mutation that causes increased expression of a ligand for HER3. In some embodiments, the method includes analyzing a nucleic acid from the cancer cells. In some embodiments, the method includes detecting a mutation that causes increased expression of a ligand for HER3.

[0507] One skilled in the art can readily identify the cancers and subjects described herein. Such cancers and subjects can be identified, for example, by monitoring over time the onset / progression of the cancer (and / or this correlating factor), for example, during treatment with an antagonist of signal transduction mediated by an EGFR family member. In some embodiments, such identification of the subject / cancer can include, for example, analysis of a sample (e.g., a biopsy) in vitro. In some embodiments, it can be determined that the cancer includes cells having mutations associated with reduced sensitivity and / or resistance to treatment with the antagonist. In some embodiments, it can be determined that the cancer includes cells with upregulated expression of an EGFR family member.

[0508] In certain embodiments, the cancer being treated is a cancer that is resistant to treatment with an antagonist of signal transduction mediated by EGFR and / or HER2. In some embodiments, the subject being treated has a cancer that is resistant to treatment with an antagonist of signal transduction mediated by EGFR and / or HER2. In some embodiments, the subject being treated has a cancer that has developed resistance to treatment with an antagonist of signal transduction mediated by EGFR and / or HER2. In some embodiments, the subject being treated has a cancer that was once responsive to treatment with an antagonist of signal transduction mediated by EGFR and / or HER2 but is now resistant to treatment with the antagonist. In some embodiments, the subject being treated has a cancer that has recurred and / or progressed after treatment with an antagonist of signal transduction mediated by EGFR and / or HER2. In some embodiments, the subject being treated has a cancer that initially responded to treatment with an antagonist of signal transduction mediated by EGFR and / or HER2 but then progressed with said treatment.

[0509] In certain embodiments, the cancer being treated comprises a mutation that confers resistance to treatment with BRAF inhibitors. In some embodiments, the mutation is a mutation at V600 of BRAF. In some embodiments, the mutation is BRAF V600E or V600K.

[0510] In certain embodiments, the cancer being treated comprises a mutation (e.g., a mutation at V600 of BRAF) that confers resistance to treatment with BRAF inhibitors, and the treatment comprises administration of vemurafenib or dabrafenib.

[0511] In some embodiments, the antigen-binding molecule is administered in combination with an agent that can inhibit signal transduction mediated by an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is, for example, PD-1, CTLA-4, LAG-3, VISTA, TIM-3, TIGIT, or BTLA. In some embodiments, the antigen-binding molecule is administered in combination with an agent that can promote signal transduction mediated by a co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is, for example, CD28, CD80, CD40L, CD86, OX40, 4-1BB, CD27, or ICOS.

[0512] Accordingly, the present invention provides a composition comprising an article according to the present invention (e.g., an antigen-binding molecule according to the present invention) and an agent that can inhibit signal transduction mediated by an immune checkpoint molecule. Also provided is a composition comprising an article of the present invention and an agent that can promote signal transduction mediated by a co-stimulatory receptor. Also provided is the use of such a composition in a method for the medical treatment and prevention of the diseases / conditions described herein.

[0513] Also provided are methods for treating / preventing a disease / condition described herein, the methods comprising administering an article of the invention (e.g., an antigen-binding molecule according to the invention) of the invention and an agent capable of inhibiting signal transduction mediated by an immune checkpoint molecule. Also provided are methods for treating / preventing a disease / condition described herein, the methods comprising administering an article of the invention (e.g., an antigen-binding molecule according to the invention) of the invention and an agent capable of promoting signal transduction mediated by a costimulatory receptor.

[0514] In the art, agents capable of inhibiting signal transduction mediated by immune checkpoint molecules are known and include, for example, antibodies that bind to immune checkpoint molecules or their ligands and are capable of inhibiting signal transduction mediated by immune checkpoint molecules. Other agents capable of inhibiting signal transduction mediated by immune checkpoint molecules include agents that can reduce the expression of the gene / protein of an immune checkpoint molecule or a ligand for an immune checkpoint molecule (e.g., by inhibiting transcription of the gene encoding the immune checkpoint molecule / ligand, inhibiting post-transcriptional processing of the RNA encoding the immune checkpoint molecule / ligand, reducing the stability of the RNA encoding the immune checkpoint molecule / ligand, promoting degradation of the RNA encoding the immune checkpoint molecule / ligand, inhibiting post-translational processing of the immune checkpoint molecule / ligand, reducing the stability of the immune checkpoint molecule / ligand, or promoting degradation of the immune checkpoint molecule / ligand), and small molecule inhibitors.

[0515] In the art, agents that can promote signal transduction mediated by co-stimulatory receptors are known, for example, including agonist antibodies that can bind to co-stimulatory receptors and induce or increase signal transduction mediated by co-stimulatory receptors. Other agents that can promote signal transduction mediated by co-stimulatory receptors include agents that can increase the expression of co-stimulatory receptors or ligands for co-stimulatory receptors (e.g., by promoting transcription of genes encoding co-stimulatory receptors / ligands, promoting post-transcriptional processing of RNAs encoding co-stimulatory receptors / ligands, increasing the stability of RNAs encoding co-stimulatory receptors / ligands, inhibiting the degradation of RNAs encoding co-stimulatory receptors / ligands, promoting post-translational processing of co-stimulatory receptors / ligands, increasing the stability of co-stimulatory receptors / ligands, or inhibiting the degradation of co-stimulatory receptors / ligands), and small molecule agonists.

[0516] In certain embodiments, the antigen-binding molecule of the invention is administered in combination with an agent that can inhibit signal transduction mediated by PD-1. An agent that can inhibit signal transduction mediated by PD-1 can be an agent that targets PD-1 or PD-L1. An agent that can inhibit signal transduction mediated by PD-1 can be, for example, an antibody that can bind to PD-1 or PD-L1 and inhibit PD-1-mediated signal transduction.

[0517] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent that can inhibit signal transduction mediated by CTLA-4. The agent that can inhibit signal transduction mediated by CTLA-4 can be an agent that targets CTLA-4, or an agent that targets a ligand for CTLA-4, such as CD80 or CD86. In some embodiments, the agent that can inhibit signal transduction mediated by CTLA-4 can be, for example, an antibody that binds to CTLA-4, CD80, or CD86 and can inhibit CTLA-4-mediated signal transduction.

[0518] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent that can inhibit signal transduction mediated by LAG-3. The agent that can inhibit signal transduction mediated by LAG-3 can be an agent that targets LAG-3, or an agent that targets a ligand for LAG-3, such as MHC class II. In some embodiments, the agent that can inhibit signal transduction mediated by LAG-3 can be, for example, an antibody that binds to LAG-3 or MHC class II and can inhibit LAG-3-mediated signal transduction.

[0519] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent that can inhibit signal transduction mediated by VISTA. The agent that can inhibit signal transduction mediated by VISTA can be an agent that targets VISTA, or an agent that targets a ligand for VISTA, such as VSIG-3 or VSIG-8. In some embodiments, the agent that can inhibit signal transduction mediated by VISTA can be, for example, an antibody that binds to VISTA, VSIG-3, or VSIG-8 and can inhibit VISTA-mediated signal transduction.

[0520] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signal transduction mediated by TIM-3. An agent capable of inhibiting signal transduction mediated by TIM-3 can be an agent that targets TIM-3 or an agent that targets a ligand for TIM-3, such as galectin 9. In some embodiments, an agent capable of inhibiting signal transduction mediated by TIM-3 can be, for example, an antibody that binds to TIM-3 or galectin 9 and can inhibit TIM-3-mediated signal transduction.

[0521] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signal transduction mediated by TIGIT. An agent capable of inhibiting signal transduction mediated by TIGIT can be an agent that targets TIGIT or an agent that targets a ligand for TIGIT, such as CD113, CD112, or CD155. In some embodiments, an agent capable of inhibiting signal transduction mediated by TIGIT can be, for example, an antibody that binds to TIGIT, CD113, CD112, or CD155 and can inhibit TIGIT-mediated signal transduction.

[0522] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signal transduction mediated by BTLA. An agent capable of inhibiting signal transduction mediated by BTLA can be an agent that targets BTLA or an agent that targets a ligand for BTLA, such as HVEM. In some embodiments, an agent capable of inhibiting signal transduction mediated by BTLA can be, for example, an antibody that binds to BTLA or HVEM and can inhibit BTLA-mediated signal transduction.

[0523] In some embodiments, methods of using a combination of an antigen-binding molecule of the invention and an agent capable of inhibiting signal transduction mediated by an immune checkpoint molecule (e.g., PD-1) result in an improved treatment effect as compared to the effects observed when either agent is used as monotherapy. In some embodiments, a combination of an antigen-binding molecule of the invention and an agent capable of inhibiting signal transduction mediated by an immune checkpoint molecule (e.g., PD-1) results in a synergistic (i.e., greater than additive) treatment effect.

[0524] Co-administration refers to the combined administration of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition and a therapeutic agent, e.g., administration as a pharmaceutical composition (combination preparation) containing both agents, or administration via the same route of administration, optionally, immediately following one another, e.g., administration to the same artery, vein, or other blood vessel. Sequential administration refers to the separate administration of the other agent after a given time interval following the administration of one of the antigen-binding molecule / composition or therapeutic agent. It is not required that the two agents be administered by the same route, although in some embodiments this applies. The time interval can be any time interval.

[0525] Chemotherapy and radiotherapy each refer to the treatment of cancer with a drug or ionizing radiation (e.g., radiotherapy using X-rays or γ-rays). The drug can be a chemical entity, e.g., a small molecule pharmaceutical, an antibiotic, a DNA intercalator, a protein inhibitor (e.g., a kinase inhibitor), or a biological agent, e.g., an antibody, antibody fragment, aptamer, nucleic acid (e.g., DNA, RNA), peptide, polypeptide, or protein. The drug can be formulated as a pharmaceutical composition or a medicament. The formulation can include one or more drugs (e.g., one or more active agents) in combination with one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0526] The treatment may involve the administration of more than one drug. The drugs may be administered alone, or in combination with other treatments, simultaneously or sequentially, depending on the condition being treated. For example, chemotherapy may be a combination therapy involving the administration of two drugs, one or more of which may be intended to treat cancer.

[0527] Chemotherapy may be administered by one or more routes of administration, such as parenterally, intravenously, orally, subcutaneously, intradermally, or intratumorally.

[0528] Chemotherapy may be administered according to a treatment regimen. A treatment regimen may be a predetermined schedule, plan, scheme, or schedule for the administration of chemotherapy, which may be created by a physician or healthcare provider and may be adjusted to suit the patient requiring the treatment. The treatment regimen may indicate one or more of the type of chemotherapy to be administered to the patient; the dosage of each drug or the dose of radiation; the time intervals between administrations; the length of each treatment; the number and nature of any drug-free days, if any. In combination therapy, a single treatment regimen may be presented indicating how each drug should be administered.

[0529] Chemotherapeutic drugs include abemaciclib, abiraterone acetate, abitrexate (methotrexate), Abraxane (albumin-stabilized nanoparticle formulation of paclitaxel), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, adriamycin (doxorubicin hydrochloride), afatinib dimaleate, afinitol (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Amboclorin (chlorambucil), amifostine, aminolevulinic acid, anastrozole, aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), arsenic trioxide, Arzerra (ofatumumab), asparaginase from Erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axicabtagene ciloleucel, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Beleodaq (belinostat), belinostat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I-131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-malate),Cabozantinib-S-malate, CAF, Calquence (acalabrutinib), Campath (alemtuzumab), Camptosar (irinotecan hydrochloride), Capecitabine, CAPOX, Carac (topical fluorouracil), Carboplatin, Carboplatin-TAXOL, Carfilzomib, Carmubris (carmustine), Carmustine, Carmustine implant agent, Casodex (bicalutamide), CEM, Ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, CEV, Chlorambucil, Chlorambucil-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (cyclophosphamide), Clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cobimetinib, Cometriq (cabozantinib-S-malate), Copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), Cytarabine, Cytarabine liposome, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (decitabine), Dactinomycin, Daratumumab, Darzalex (daratumumab), Dasatinib, Daunorubicin hydrochloride, Daunorubicin hydrochloride and cytarabine liposome, Decitabine, Defibrotide sodium, Defitelio (defibrotide sodium), Degarelix, Denileukin diftitox, Denosumab, DepoCyt (cytarabine liposome), Dexamethasone, Dexrazoxane hydrochloride, Dinutuximab, Docetaxel, Doxil (doxorubicin hydrochloride liposome), Doxorubicin hydrochloride, Doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), Durvalumab, Efudex (topical fluorouracil), Elitek (rasburicase), Ellence (epirubicin hydrochloride), Elotuzumab, Eloxatin (oxaliplatin),eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinaze (asparaginase from Erwinia chrysanthemi), Ethyol (amifostine), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposome), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (topical fluorouracil), Fareston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (topical fluorouracil), fluorouracil injection, topical fluorouracil, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI - bevacizumab, FOLFIRI - cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant quadrivalent HPV vaccine), Gardasil9 (recombinant nonavalent HPV vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine - cisplatin, gemcitabine - oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), bivalent HPV vaccine, recombinant nonavalent HPV vaccine, recombinant quadrivalent HPV vaccineRecombinant Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, Ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), IL-2 (aldesleukin), imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlygic (talimogene laherparepvec), Inlyta (axitinib), inotuzumab ozogamicin, interferon alpha-2b, recombinant, interleukin-2 (aldesleukin), Intron A (recombinant interferon alpha-2b), iodine I-131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposome, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), Lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate)Lupron Depot (Leuprolide Acetate), Lupron Pediatric Depot (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Albumin-Stabilized Nanoparticle Formulation of Paclitaxel), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olalatumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Albumin-Stabilized Nanoparticle Formulation of Paclitaxel, PAD, Palbociclib,Palifermin, Paroxetine Hydrochloride, Paroxetine Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim. Peg Interferon Alpha-2b, PEG-Intron (Pe, g interferon alpha-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), pre-leucovorin, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portrazza (nesitumumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenge (sipuleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), [not described], radium 223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and human hyaluronidase), rituximab, rituximab and human hyaluronidase, lorlatinib hydrochloride, romidepsin, romiplostim, Rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), rucaparib camsylate, luxolitinib phosphate, Rydapt (midostaurin), sclerosing agent pleural aerosol (talc), siltuximab, sipuleucel-T, somatostatin depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORDV, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peg Interferon Alpha-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Topical Fluorouracil), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I-131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Bemrafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, VincasarIt may be selected from PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, bismodib, Vistogard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (axicabtagene ciloleucel), Yondelis (trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0530] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with one or more of trastuzumab, cetuximab, cisplatin, 5-FU, or capecitabine. In some embodiments, the antigen-binding molecule of the present invention is administered in combination with trastuzumab and cisplatin, and 5-FU or capecitabine.

[0531] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with cetuximab. In particular, for the treatment of head and neck cancer (e.g., head and neck squamous cell carcinoma), administration in combination with cetuximab is contemplated.

[0532] Multiple administrations of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition may be provided. One or more administrations, or each administration, may be accompanied by the simultaneous or sequential administration of another therapeutic agent.

[0533] Multiple administrations may be selected to be at predetermined time intervals, which may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, administrations may be given every 7, 14, 21, or 28 days (±3, 2, or 1 day).

[0534] Detection methods The present invention also provides the articles of the present invention for use in methods of detecting, localizing, or imaging HER3, or cells expressing HER3.

[0535] The antigen-binding molecules described herein may be used in methods involving antigen-binding molecules to HER3. Such methods may involve the detection of a binding complex between the antigen-binding molecule and HER3.

[0536] Thus, provided are methods comprising contacting a sample suspected of containing or containing HER3, and detecting the formation of a complex between the antigen-binding molecule and HER3. Also provided are methods comprising contacting a sample suspected of containing or containing cells expressing HER3, and detecting the formation of a complex between the antigen-binding molecule and the cells expressing HER3.

[0537] In the art, suitable method formats are well known, including sandwich assays, such as immunoassays like ELISA. The method may involve labeling an antigen-binding molecule, or a target, or both, with a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label, a radioactive label, a chemical, nucleic acid, or enzyme label as described herein. Detection techniques are well known to those skilled in the art and can be selected to correspond to the labeling agent.

[0538] This type of method can provide the basis for a method for diagnostic and / or prognostic assessment of a disease or condition, such as cancer. Such methods can be performed on in vitro patient samples or after processing of patient samples. Once the sample is collected, since the patient is not required to be present during the performance of the in vitro method, the method can be one that is not performed on or within a human or animal body. In some embodiments, the method is performed in vivo.

[0539] Detection in a sample can be used for the purpose of diagnosing a disease / condition (e.g., cancer), a predisposition to a disease / condition, or for prognosticating (predicting) a disease / condition, such as those described herein. The diagnosis or prognosis can relate to an existing (already diagnosed) disease / condition.

[0540] Such methods can involve, for example, detecting or quantifying HER3 or cells expressing HER3 in a patient sample. If the method includes quantifying a relevant factor, the method can further include comparing the determined amount to a standard or reference value as part of a diagnostic or prognostic assessment. Other diagnostic / prognostic tests can be used in conjunction with the diagnostic / prognostic tests described herein to enhance the accuracy of the diagnosis or prognosis or to confirm the results obtained by using the tests described herein.

[0541] The sample can be taken from any tissue or body fluid. The sample can include a large amount of blood; a large amount of serum derived from an individual's blood, which can include the fluid portion of the blood obtained after removing fibrin clots and blood cells; a tissue sample or biopsy; pleural effusion; cerebrospinal fluid (CSF); or can contain or be derived from cells isolated from said individual. In some embodiments, the sample can be obtained from or be derived from one or more tissues affected by the disease / condition (e.g., one or more tissues in which the symptoms of the disease manifest or are involved in the pathogenesis of the disease / condition).

[0542] The present invention also provides a method for selecting / stratifying a subject for treatment with a HER3-targeted agent. In some embodiments, the subject is selected for treatment / prevention according to the present invention or is identified as a subject likely to benefit from such treatment / prevention based on the detection / quantification of HER3 or cells expressing HER3 in a sample obtained from an individual, for example.

[0543] Subject The subject according to the aspects of the present invention described herein can be any animal or human. The subject is preferably a mammal, more preferably a human. The subject can be a non-human mammal, but more preferably is a human. The subject can be male or female. The subject can be a patient. The subject may be diagnosed with a disease or condition (e.g., cancer) that requires treatment, may be suspected of having such a disease / condition, or may be at risk of developing / suffering from such a disease / condition.

[0544] In embodiments according to the present invention, the subject is preferably a human subject. In some embodiments, the subject to be treated according to the therapeutic or prophylactic methods of the present invention herein is a subject having cancer or at risk of developing cancer. In embodiments according to the present invention, the subject can be selected for treatment according to a method based on the characterization of certain markers of such a disease / condition.

[0545] Kit In some aspects of the invention described herein, a member kit is provided. In some embodiments, the kit can have at least one container having a predetermined quantity of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein.

[0546] In some embodiments, the kit can include materials for producing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein.

[0547] The kit can be provided together with instructions for administration to a patient for treating a designated disease / condition of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition.

[0548] In some embodiments, the kit can further include at least one container having a predetermined quantity of another therapeutic agent (e.g., an anti-infective agent or a chemotherapeutic agent). In such embodiments, the kit can also include a second pharmaceutical or pharmaceutical composition such that the two pharmaceuticals or pharmaceutical compositions can be administered simultaneously or separately such that they provide a combination treatment for a particular disease or condition. The therapeutic agent can also be formulated to be suitable for injection or infusion into a tumor or the blood.

[0549] Sequence identity As used herein, "sequence identity" refers to the percentage of nucleotide / amino acid residues in a subject sequence that are identical to the nucleotide / amino acid residues in a reference sequence after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps if necessary. Multiple sequence alignments in pairs for the purpose of determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved by various means known to those skilled in the art using commercially available computer software such as Clustal Omega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al., 2000, J. Mol. Biol. (2000), 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4), 772-780). When using such software, it is preferred to use the default parameters, for example, for gap penalties and extension penalties.

[0550]

Table 2-1

[0551]

Table 2-2

[0552]

Table 2-3

[0553]

Table 2-4

[0554]

Table 2-5

[0555]

Table 2-6

[0556]

Table 2-7

[0557]

Table 2-8

[0558]

Table 2-9

[0559]

Table 2-10

[0560]

Table 2-11

[0561]

Table 2-12

[0562]

Table 2-13

[0563]

Table 2-14

[0564]

Table 2-15

[0565]

Table 2-16

[0566] 2. The antigen-binding molecule according to item 1, which inhibits the interaction between HER3 and the interaction partner of HER3.

[0567] 3. The antigen-binding molecule according to item 1 or item 2, which is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0568] 4. The antigen-binding molecule according to any one of items 1 to 3, which is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 229.

[0569] 5. The antigen-binding molecule according to any one of items 1 to 4, which is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 229.

[0570] 6. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 43 HC-CDR2 having the amino acid sequence of SEQ ID NO: 46 HC-CDR3 having the amino acid sequence of SEQ ID NO: 51 A heavy chain variable (VH) region incorporating them, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 91 LC-CDR2 having the amino acid sequence of SEQ ID NO: 94 The LC-CDR3 having the amino acid sequence of SEQ ID NO: 99 The light chain variable (VL) region incorporating the same The antigen-binding molecule according to any one of Items 1 to 5, comprising the same

[0571] 7. (i) The following CDRs: The HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 The HC-CDR2 having the amino acid sequence of SEQ ID NO: 44 The HC-CDR3 having the amino acid sequence of SEQ ID NO: 47 The heavy chain variable (VH) region incorporating the same, and (ii) The following CDRs: The LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 The LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 The LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 The light chain variable (VL) region incorporating the same The antigen-binding molecule according to any one of Items 1 to 6, comprising the same

[0572] 8. (i) The following CDRs: The HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 The HC-CDR2 having the amino acid sequence of SEQ ID NO: 44 The HC-CDR3 having the amino acid sequence of SEQ ID NO: 47 The heavy chain variable (VH) region incorporating the same, and (ii) The following CDRs: The LC-CDR1 having the amino acid sequence of SEQ ID NO: 89 The LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 The LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 The light chain variable (VL) region incorporating the same The antigen-binding molecule according to any one of Items 1 to 6, comprising the same

[0573] 9. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 44 HC-CDR3 having the amino acid sequence of SEQ ID NO: 47 A heavy chain variable (VH) region incorporating the same, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 90 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 96 A light chain variable (VL) region incorporating the same An antigen-binding molecule according to any one of items 1 to 6, comprising the same.

[0574] 10. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 44 HC-CDR3 having the amino acid sequence of SEQ ID NO: 47 A heavy chain variable (VH) region incorporating the same, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 98 A light chain variable (VL) region incorporating the same An antigen-binding molecule according to any one of items 1 to 6, comprising the same.

[0575] 11. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 47 A heavy chain variable (VH) region incorporating the same, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 93 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 A light chain variable (VL) region incorporating the above An antigen-binding molecule according to any one of items 1 to 6, comprising the above.

[0576] 12. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 49 A heavy chain variable (VH) region incorporating the above, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 93 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 A light chain variable (VL) region incorporating the above An antigen-binding molecule according to any one of items 1 to 6, comprising the above.

[0577] 13. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 50 A heavy chain variable (VH) region incorporating the above, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 93 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 A light chain variable (VL) region incorporating the above An antigen-binding molecule according to any one of items 1 to 6, comprising the above.

[0578] 14. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48 a heavy chain variable (VH) region incorporating them, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 a light chain variable (VL) region incorporating them An antigen-binding molecule according to any one of items 1 to 6.

[0579] 15. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48 a heavy chain variable (VH) region incorporating them, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 97 a light chain variable (VL) region incorporating them An antigen-binding molecule according to any one of items 1 to 6.

[0580] 16. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 42 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48 a heavy chain variable (VH) region incorporating them, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 A light chain variable (VL) region incorporating them An antigen-binding molecule according to any one of items 1 to 6, comprising the same.

[0581] 17. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 158 HC-CDR2 having the amino acid sequence of SEQ ID NO: 159 HC-CDR3 having the amino acid sequence of SEQ ID NO: 160 A heavy chain variable (VH) region incorporating them, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 165 LC-CDR2 having the amino acid sequence of SEQ ID NO: 166 LC-CDR3 having the amino acid sequence of SEQ ID NO: 167 A light chain variable (VL) region incorporating them An antigen-binding molecule according to any one of items 1 to 5, comprising the same.

[0582] 18. An antigen-binding molecule according to any one of items 1 to 4, which can bind to a polypeptide comprising the amino acid sequence of SEQ ID NO: 22.

[0583] 19. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 128 HC-CDR2 having the amino acid sequence of SEQ ID NO: 129 HC-CDR3 having the amino acid sequence of SEQ ID NO: 130 A heavy chain variable (VH) region incorporating them, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 136 LC-CDR2 having the amino acid sequence of SEQ ID NO: 137 LC-CDR3 having the amino acid sequence of SEQ ID NO: 138 and a light chain variable (VL) region incorporating the same An antigen-binding molecule according to any one of items 1 to 4 or item 18, comprising the same

[0584] 20. (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 144 HC-CDR2 having the amino acid sequence of SEQ ID NO: 145 HC-CDR3 having the amino acid sequence of SEQ ID NO: 146 and a heavy chain variable (VH) region incorporating the same, and (ii) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 152 LC-CDR3 having the amino acid sequence of SEQ ID NO: 153 and a light chain variable (VL) region incorporating the same An antigen-binding molecule according to any one of items 1 to 4 or item 18, comprising the same

[0585] 21. (i) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 24, and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 74, or (ii) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 25, and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 75, or (iii) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 26, and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 76, or (iv) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 27, and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 77, or (v) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 28, and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 78, or (vi) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 29, and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 78, or (vii) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 30, and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 78, or (viii) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 31, and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 79, or (ix) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequenc...

Claims

1. A medicament for treating or preventing cancer in a subject, the medicament comprising, as an active ingredient, an antigen-binding molecule capable of binding to HER3, wherein the cancer comprises cells having an NRG gene fusion, and the antigen-binding molecule (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 41 HC-CDR2 having the amino acid sequence of SEQ ID NO: 45 HC-CDR3 having the amino acid sequence of SEQ ID NO: 48 A heavy chain variable (VH) region incorporating the above, and (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 88 LC-CDR2 having the amino acid sequence of SEQ ID NO: 92 LC-CDR3 having the amino acid sequence of SEQ ID NO: 95 A light chain variable (VL) region incorporating the above The medicament as described above.

2. The medicament according to claim 1, wherein the NRG gene fusion is selected from CLU-NRG1, CD74-NRG1, DOC4-NRG1, SLC3A2-NRG1, RBPM-S-NRG1, WRN-NRG1, SDC4-NRG1, RAB2IL1-NRG1, VAMP2-NRG1, KIF13B-NRG1, THAP7-NRG1, SMAD4-NRG1, MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1, DPYSL2-NRG1, ATP1B1-NRG1, CDH6-NRG1, APP-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1, CDK1-NRG1, BMPRIB-NRG1, TNFRSF10B-NRG1, MCPH1-NRG1 and SLC12A2-NRG2.

3. The medicament according to claim 1 or claim 2, wherein the NRG gene fusion is selected from CLU-NRG1, CD74-NRG1, SLC3A2-NRG1 or VAMP2-NRG1.

4. The medicament according to any one of claims 1 to 3, wherein the cancer is derived from the lung, breast, head, neck, kidney, ovary, pancreas, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue or nasopharynx.

5. The medicament according to any one of claims 1 to 4, wherein the cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, invasive breast cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, renal clear cell carcinoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, bladder cancer, urothelial bladder cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor, and nasopharyngeal neuroendocrine tumor.

6. The medicament according to any one of claims 1 to 5, wherein the cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, and lung squamous cell carcinoma.

7. The antigen-binding molecule is a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 36, and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 83 The medicament according to any one of claims 1 to 6.

8. The antigen-binding molecule is the following framework regions (FRs): HC-FR1 having the amino acid sequence of SEQ ID NO: 53 HC-FR2 having the amino acid sequence of SEQ ID NO: 59 HC-FR3 having the amino acid sequence of SEQ ID NO: 66 HC-FR4 having the amino acid sequence of SEQ ID NO: 71 and a VH region incorporating them The medicament according to any one of claims 1 to 7.

9. The antigen-binding molecule is the following framework regions (FRs): LC-FR1 having the amino acid sequence of SEQ ID NO: 104 LC-FR2 having the amino acid sequence of SEQ ID NO: 110 LC-FR3 having the amino acid sequence of SEQ ID NO: 120 LC-FR4 having the amino acid sequence of SEQ ID NO: 125 and a VL region incorporating them The medicament according to any one of claims 1 to 8.

10. The medicament according to any one of claims 1 to 9, wherein the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:

171.

11. The medicament according to any one of claims 1 to 10, wherein the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 177.

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