Treatment and prevention of cancer using HER3 antigen-binding molecules

Abstract

The present disclosure provides antigen-binding molecules that bind to HER3 for treating or preventing cancer, compositions comprising the molecules, and therapeutic and prophylactic methods using the molecules.

Description

[Technical Field]

[0001] This application claims priority from SG10202109667X, filed September 3, 2021, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to the field of molecular biology, and more particularly to antibody technology and methods of medical treatment and prevention. [Background technology]

[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 has deleterious consequences for tumor progression, HER3 upregulation is associated with resistance to anti-HER2 and anti-EGFR therapy, and solid tumors refractory to anti-PD-1 therapy have been shown to express higher levels of HER3 compared with those that respond 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. The anti-HER3 antibody, LJM-716, binds to an epitope on subdomains II and IV of the HER3 extracellular domain, locking 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 signal transduction 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 heregulin binding to HER3 (see, e.g., Shimizu et al., Cancer Chemother Pharmacol. (2017), 79(3):489-495). RG7116 (also known as lumletuzumab and RO-5479599) recognizes an epitope within subdomain I of the HER3 extracellular domain (see, e.g., Mirschberger et al., Cancer Research (2013) 73(16) 5183-5194). KTN3379 binds to HER3 through interactions with amino acid residues in subdomain III (corresponding to the following positions of 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) and Met310, Glu311, and Pro328 in subdomain II (see Lee et al., Proc Natl Acad Sci USA. 2015 Oct. 27, 112(43):13225).AV-203 (also known as CAN-017) has been shown to block the binding of NRG1 to HER3 and promote HER3 degradation (see Meetze et al., Eur J Cancer 2012, 48:126). REGN1400 also inhibits the binding of ligands to HER3 (see Zhang et al., Mol Cancer Ther (2014) 13:1345-1355). RG7597 (durigotuzumab) is a dual-acting 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 a HER3-binding arm that inhibit the binding of 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 problem]

[0005] This disclosure relates in part to compositions comprising antigen-binding molecules capable of binding to HER3. The compositions find use in the treatment of cancers and cancer cells that express HER3. Thus, in one aspect, the present disclosure provides a composition comprising an antigen-binding molecule capable of binding to HER3.

[0006] In some embodiments, the antigen-binding molecule is (i) The following CDR: 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 (ii) the following CDR: 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 Includes.

[0007] In some embodiments, the antigen-binding molecule is (i) The following CDR: 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 and a VH region incorporating (ii) the following CDR: 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 The VL region incorporating Includes.

[0008] In some embodiments, the composition comprises: (i) containing 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, and 0.001% to 0.1% (w / v) polysorbate-80, and having a pH of 4.0 to 7.0; or (ii) containing 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, and 0.001% to 0.1% (w / v) polysorbate-20, and having a pH of 4.0 to 7.0; or (iii) containing 2 mM to 200 mM histidine, 1 mM to 250 mM sodium chloride, and 0.001% to 0.1% (w / v) polysorbate-80, and having a pH of 4.0 to 7.0; or (iv) 2 mM to 200 mM histidine, 1 mM to 250 mM sodium chloride, and 0.001% to 0.1% (w / v) polysorbate-20, and having a pH of 4.0 to 7.0; or (v) 2 mM to 200 mM histidine, 1 mM to 250 mM arginine, and 0.001% to 0.1% (w / v) polysorbate-80, and having a pH of 4.0 to 7.0; or (vi) 2 mM to 200 mM histidine, 1 mM to 250 mM arginine, and 0.001% to 0.1% (w / v) polysorbate-20, and having a pH of 4.0 to 7.0; or (vii) 2 mM to 200 mM acetate, 1 mM to 250 mM sodium chloride, and 0.001% to 0.1% (w / v) polysorbate-20, and having a pH of 4.0 to 7.0.

[0009] In some embodiments, the composition comprises: (i) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, having a pH of 5.8; or (ii) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, having a pH of 5.1; or (iii) 20 mM histidine, 4% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or (iv) 20 mM histidine, 2% (w / v) sucrose; 0.02% (w / v) polysorbate-80, having a pH of 5.3; or (v) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, having a pH of 6.1; or (vi) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-20, and having a pH of 5.8; or (vii) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-20, and having a pH of 5.5; or (viii) 20 mM histidine, 150 mM sodium chloride; 0.02% (w / v) polysorbate-80, having a pH of 6.5; or (ix) 20 mM acetate, 150 mM sodium chloride; 0.05% (w / v) polysorbate-20, having a pH of 5.5.

[0010] In some embodiments, the composition comprises 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and has a pH of 5.8. In some embodiments, the composition contains at least 1.2 mg / mL of the antigen-binding molecule. In some embodiments, the composition contains up to 50 mg / mL of the antigen-binding molecule. In some embodiments, the composition contains between 1.2 mg / mL and 50 mg / mL of the antigen-binding molecule.

[0011] 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. Includes.

[0012] 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 VH region incorporating Includes.

[0013] 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 The VL region incorporating Includes.

[0014] In some embodiments, the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 171. In some embodiments, the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 177.

[0015] Also provided are compositions according to the present disclosure for use as pharmaceuticals. Also provided is a composition according to the present disclosure for use in a method of treating or preventing cancer in a subject.

[0016] Also provided is the use of a composition according to the present disclosure in the manufacture of a medicament for treating or preventing cancer in a subject. Also provided is a method of treating or preventing cancer in a subject, comprising administering a therapeutically or prophylactically effective amount of a composition according to the present disclosure.

[0017] In some embodiments, the cancer comprises cells that express a ligand for HER3, EGFR, HER2, HER4, NRG1, NRG2, and / or HER3. In some embodiments, the cancer comprises cells with a mutation that results in increased expression of a ligand for HER3. In some embodiments, the cancer comprises cells with an NRG gene fusion. In some embodiments, the NRG gene fusions include 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, P Selected from ARP8-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.

[0018] In some embodiments, the cancer originates from the lung, breast, head, neck, kidney, ovary, cervix, pancreas, stomach, liver, esophagus, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue, or nasopharynx.

[0019] 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, triple-negative breast cancer, 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, castration-resistant prostate cancer, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, RAS wild-type colorectal cancer, gastric cancer, hepatocellular carcinoma (HCC), esophageal cancer, bladder cancer, urothelial bladder cancer, cervical cancer, endometrial cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumors, and nasopharyngeal neuroendocrine tumors.

[0020] In some embodiments, the method includes detecting cancer cells in a subject that express HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3, and / or an NRG gene fusion. In some embodiments, the method includes obtaining the cells from the subject. In some embodiments, the cells are obtained from the subject. In some embodiments, the detecting is performed on an in vitro sample and / or is performed in vitro.

[0021] In some embodiments, if cancer cells expressing HER3, EGFR, HER2, HER4, NRG1, NRG2, ligands for HER3 and / or NRG gene fusions are detected, the subject is selected for treatment with the composition.

[0022] In some embodiments, the composition is administered in combination with one or more of HER2 targeted therapy, EGFR targeted therapy, and / or androgen receptor targeted therapy. In some embodiments, the composition is administered in combination with one or more of cetuximab, enzalutamide, and / or trastuzumab.

[0023] Also provided is an antigen-binding molecule capable of binding to HER3 for use in a method of treating or preventing cancer in a subject, comprising: (i) The following CDR: 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 (ii) the following CDR: 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 Also provided as part of this disclosure are antigen-binding molecules comprising:

[0024] Also provided is the use of an antigen-binding molecule capable of binding to HER3 in the manufacture of a medicament for treating or preventing cancer in a subject, comprising: (i) The following CDR: 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 (ii) the following CDR: 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 Also provided is an antigen-binding molecule comprising:

[0025] Also provided is a method for treating or preventing cancer in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of an antigen-binding molecule capable of binding to HER3, wherein the antigen-binding molecule: (i) The following CDR: 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 (ii) the following CDR: 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 A method is also provided, including:

[0026] In some embodiments, the antigen-binding molecule is (i) The following CDR: 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 and a VH region incorporating (ii) the following CDR: 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 The VL region incorporating Includes.

[0027] In some embodiments, the antigen-binding molecule comprises 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 The VH region comprises:

[0028] In some embodiments, the antigen-binding molecule comprises 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 The VL region incorporating Includes.

[0029] In some embodiments, the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 171. In some embodiments, the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 177.

[0030] In some embodiments, the cancer comprises cells that express a ligand for HER3, EGFR, HER2, HER4, NRG1, NRG2, and / or HER3. In some embodiments, the cancer comprises cells with a mutation that results in increased expression of a ligand for HER3. In some embodiments, the cancer comprises cells with an NRG gene fusion. In some embodiments, the NRG gene fusions include 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, P Selected from ARP8-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.

[0031] In some embodiments, the cancer originates from the lung, breast, head, neck, kidney, ovary, cervix, pancreas, stomach, liver, esophagus, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue, or nasopharynx.

[0032] 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, triple-negative breast cancer, 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, castration-resistant prostate cancer, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, RAS wild-type colorectal cancer, gastric cancer, hepatocellular carcinoma (HCC), esophageal cancer, bladder cancer, urothelial bladder cancer, cervical cancer, endometrial cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumors, and nasopharyngeal neuroendocrine tumors.

[0033] In some embodiments, the method includes detecting cancer cells in a subject that express HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3, and / or an NRG gene fusion. In some embodiments, the method includes obtaining the cells from the subject. In some embodiments, the cells are obtained from the subject. In some embodiments, the detecting step is performed on an in vitro sample and / or is performed in vitro. In some embodiments, if cancer cells that express HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3, and / or an NRG gene fusion are detected, the subject is selected for treatment with the antigen-binding molecule.

[0034] In some embodiments, the antigen-binding molecule is administered in combination with one or more of a HER2-targeted therapy, an EGFR-targeted therapy, and / or an androgen receptor-targeted therapy. In some embodiments, the antigen-binding molecule is administered in combination with one or more of cetuximab, enzalutamide, and / or trastuzumab.

[0035] In some embodiments, a composition or antigen-binding molecule according to the disclosure is administered once every 7 days, once every 14 days, once every 21 days, or once every 28 days. In some embodiments, a composition or antigen-binding molecule according to the disclosure is administered four times every 28 days, two times every 28 days, or three times every 21 days, e.g., over one or more 21-day or 28-day periods. In some embodiments, a composition or antigen-binding molecule according to the disclosure is administered over one, two, three, four, five, six, or more 21-day or 28-day periods.

[0036] In some embodiments, treatment according to the present disclosure comprises administering between 150 mg and 3000 mg of antigen-binding molecule per administration and / or over a 21-day or 28-day period.

[0037] In some embodiments, treatment according to the present disclosure comprises administering 1800-2500 mg of antigen-binding molecule per dose. In some embodiments, treatment according to the present disclosure provides a total of at least 600 mg, at least 900 mg, at least 1200 mg, at least 1500 mg, at least 1800 mg, at least 2100, at least 2400 mg, at least 2700 mg, at least 3000 mg, at least 3300 mg, at least 3600 mg, at least 3900 mg, at least 4200 mg, at least 4500 mg, at least 4800 mg, at least 5100 mg, at least 5400 mg, at least 5700 mg, at least 6000 mg, at least 6100 mg, at least 6200 mg, at least 6300 mg, at least 6400 mg, at least 6500 mg, at least 6600 mg, at least 6700 mg, at least 6800 mg, at least 6900 mg, at least 7000 mg, at least 7100 mg, at least 7200 mg, at least 7300 mg, at least 7400 mg, at least 7500 mg, at least 7600 mg, at least 7700 mg, at least 7800 mg, at least 7900 mg, at least 8000 mg, at least 8100 mg, at least 8200 mg, at least 8300 mg, at least 8400 mg, at least 8500 mg, at least 8600 mg, at least 8700 mg, at least 8800 mg, at least 8900 mg, at least 9000 mg, at least 9100 mg, at least 9200 mg, at least 9300 mg, at least 9400 mg, at least 9500 mg, at least 9600 mg, at least 97 mg, at least 6300 mg, at least 6600 mg, at least 6900 mg, at least 7200 mg, at least 7500 mg, at least 7800 mg, at least 8100 mg, at least 8400 mg, at least 8700 mg, at least 9000 mg, at least 9300 mg, at least 9600 mg, at least 9900 mg, at least 10200 mg, at least 10500 mg, at least 10800 mg, at least 11100 mg, at least 11400 mg, at least 11700 mg, or at least 12000 mg of the antigen-binding molecule.

[0038] In some embodiments, treatment according to the present disclosure comprises administering a total of about 3600 mg of antigen-binding molecules every 21 days, or about 4800 mg of antigen-binding molecules every 28 days. In some embodiments, treatment according to the present disclosure comprises administering a total of about 5400 mg of antigen-binding molecules every 21 days, or about 7200 mg of antigen-binding molecules every 28 days. In some embodiments, treatment according to the present disclosure comprises administering a total of about 6300 mg of antigen-binding molecules every 21 days, or about 8400 mg of antigen-binding molecules every 28 days. In some embodiments, treatment according to the present disclosure comprises administering a total of about 9000 mg of antigen-binding molecules every 21 days, or about 12000 mg of antigen-binding molecules every 28 days.

[0039] The antigen-binding molecule may be administered in multiple doses (e.g., once per week) to reach a total amount of antigen-binding molecule per 21 or 28 day period. In some embodiments, treatment according to the present disclosure comprises administering at least 150 mg, at least 300 mg, at least 600 mg, at least 900 mg, at least 1200 mg, at least 1500 mg, at least 1800 mg, at least 2100 mg, at least 2400, or at least 2800 mg of an antigen-binding molecule every 7 or 14 days (once per week or once per 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering 1500 mg to 3000 mg of an antigen-binding molecule every 7 or 14 days (once per week or once per 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering 1800 mg to 2500 mg of an antigen-binding molecule every 7 or 14 days (once per week or once per 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering about 1200 mg of an antigen-binding molecule every 7 or 14 days (once per week or once per 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering about 1500 mg of an antigen-binding molecule every 7 or 14 days (once per week or once per 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering about 1800 mg of an antigen-binding molecule every 7 or 14 days (once per week or once per 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering about 2100 mg of an antigen-binding molecule every 7 or 14 days (once per week or once per 2 weeks).

[0040] In some embodiments, administration of the antigen-binding molecule every 7 days (e.g., as described above) is carried out for at least 21 days or at least 28 days. In some embodiments, administration of the antigen-binding molecule every 7 days (e.g., as described above) is carried out for 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or more weeks, or for 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or more weeks. DETAILED DESCRIPTION OF THE INVENTION

[0041] 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; other members include patritumab (U3-1287 or AMG-888), lumletuzumab (RG-7116), AV-203, GSK2849330, and REGN1400. Antibodies of the second class lock HER3 in an inactive conformation via binding to the interface between domains II and IV or between domains II and III. LJM-716, as well as KTN3379, are representative examples of this class.

[0042] HER3 overexpression is frequently observed in multiple tumor types and is associated with poorer clinical outcomes. Enhanced HER3 expression is found in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and breast, gastric, ovarian, prostate, and bladder cancers. The impact of HER3 overexpression is greater in cancers that also overexpress HER2, such as breast, gastric, and ovarian cancers. In melanoma and pancreatic cancer, HER3 is the preferred heterodimer partner of EGFR. Upregulation of HER3 expression and activity is associated with resistance to multiple pathway inhibitors and poor prognosis.

[0043] The present invention relates to novel HER3 binding molecules that have improved properties compared to known anti-HER3 antibodies. The present inventors have contemplated the targeted generation of antigen-binding molecules that bind to specific regions of interest within the extracellular domain of HER3. The HER3-binding molecules of the present invention possess a combination of desirable biophysical and / or functional properties compared to antigen-binding molecules disclosed in the prior art.

[0044] In an embodiment of the invention, the antigen-binding molecule is capable of binding to subdomain II (SEQ ID NO: 16) of the extracellular region of HER3 and inhibits the association of the bound HER3 molecule with an interaction partner.

[0045] In particular, the HER3-binding antigen-binding molecules described herein are demonstrated to bind to an epitope on HER3, resulting in (i) potent inhibition of HER3 association with interacting partners (e.g., EGFR, HER2), and (ii) high-affinity binding to HER3 both in the presence and absence of NRG ligands. This unique combination of properties results in potent inhibition of downstream signaling and exceptional anti-cancer activity against a wide range of cancers.

[0046] HER3 HER3 (also known as, for example, ERBB3 LCCS2, MDA-BF-1) 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 that differs 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.

[0047] The N-terminal 19 amino acids of SEQ ID NOs: 1 to 3 constitute a signal peptide, and therefore the mature forms of HER3 isoforms 1, 2 and 3 (i.e., after processing to remove the signal peptide) have the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively.

[0048] 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 incorporated herein by reference in its entirety. HER3 is a single-pass transmembrane ErbB receptor tyrosine kinase with an N-terminal extracellular region (SEQ ID NO: 9) containing two leucine-rich subdomains (domains I and III, shown in SEQ ID NOs: 15 and 17, respectively) and two cysteine-rich subdomains (domains II and IV, shown in SEQ ID NOs: 16 and 18, respectively). Domain II contains a β-hairpin dimerization loop (SEQ ID NO: 19) that is involved in intermolecular interactions with other HER receptor molecules. The extracellular region is connected to the cytoplasmic region (SEQ ID NO: 11) via a transmembrane region (SEQ ID NO: 10). The cytoplasmic region contains 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).

[0049] Signaling through HER3 involves receptor homodimerization (i.e., with other HER3 receptors) or heterodimerization (with other HER receptors, e.g., HER2) and consequent autophosphorylation of the protein kinase domain on tyrosine in the cytoplasmic region. The phosphorylated tyrosine residue recruits adaptor / effector proteins containing src homology domain 2 (SH2) or phosphotyrosine-binding (PTB) domains, such as Grb2 and phospholipase Cγ (PLCγ).

[0050] The signal transduction through HER3 can be activated either in a ligand-dependent manner or in a ligand-independent manner.In the absence of ligand, HER3 receptor molecules are usually expressed as monomers on the cell surface, with a conformation that prevents receptor dimerization, in which the dimerization loop of subdomain II makes intramolecular contact with the pocket on subdomain IV.The binding of HER3 ligands, such as neuregulin (NRG), for example, NRG1 (also known as heregulin, HRG) or NRG2, to the subdomains I and III of the extracellular region causes a conformational change, resulting in the exposure of the dimerization loop of subdomain II, facilitating receptor dimerization and signal transduction. Some cancer-associated mutations in HER3 can disrupt the interaction between subdomains II and IV required for the formation of an inactive "closed" conformation, which can lead to 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).

[0051] As used herein, "HER3" refers to HER3 from any species, and includes isoforms, fragments, variants (including mutants), or homologs of HER3 from any species.

[0052] As used herein, a "fragment," "variant," or "homologue" 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 homologues of a reference protein may be characterized by their ability to perform a function performed by the reference protein.

[0053] A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of the reference protein, but that retains a significant 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 "homologue" 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 monkey HER3 (UniProt: F7HEH3-1, v2; SEQ ID NO: 20) are homologs of each other. Homologues include orthologs.

[0054] A "fragment" of a reference protein may 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 one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0055] A fragment of HER3 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 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.

[0056] In some embodiments, the HER3 is mammalian-derived HER3 (e.g., primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or rodent (e.g., rat or mouse) HER3). HER3 isoforms, fragments, variants, or homologs may optionally be characterized as having 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 an immature or mature HER3 isoform from a given species, e.g., human.

[0057] The isoform, fragment, variant, or homologue may optionally be a functional isoform, fragment, variant, or homologue that has the functional property / activity of a reference HER3 (e.g., human HER3 isoform 1), e.g., as determined by analysis with an assay appropriate for the functional property / activity. For example, an isoform, fragment, variant, or homologue of HER3 may exhibit association with one or more of HER2, NRG1 (types I, II, III, IV, V, or VI), or NRG2 (α or β).

[0058] 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.

[0059] In some embodiments, the 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, e.g., one of SEQ ID NOs: 9, 16, or 19.

[0060] A specific region of interest on the target molecule The antigen-binding molecules of the present invention were specifically designed to target specific regions of HER3. In a two-step approach, the HER3 region to be targeted was selected based on predicted antigenicity, function, and safety analyses. Then, peptides corresponding to the target region were used as immunogens to elicit specific monoclonal antibodies, thereby preparing antibodies specific to the target region of HER3. Subsequent screening identified antibodies capable of binding to HER3 in the naive state. This approach allows for precise control over the antibody epitope.

[0061] Antigen-binding molecules of the present invention can be defined by referring to the region of HER3 to which they bind. Antigen-binding molecules of the present invention can bind to a specific region of interest on HER3. In some embodiments, antigen-binding molecules can bind to a linear epitope of HER3 consisting of a continuous sequence of amino acids (i.e., a primary sequence of amino acids). In some embodiments, antigen-binding molecules can bind to a conformational epitope of HER3 consisting of a discontinuous sequence of amino acids within the amino acid sequence.

[0062] In some embodiments, the antigen-binding molecules of the present invention bind to HER3. In some embodiments, the antigen-binding molecules bind to the extracellular region of HER3 (e.g., the region set forth in SEQ ID NO: 9). In some embodiments, the antigen-binding molecules bind to subdomain II of the extracellular region of HER3 (e.g., the region set forth in SEQ ID NO: 16).

[0063] In some embodiments, the antigen-binding molecule binds to a 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 a region of HER3 set forth in SEQ ID NO: 229. In some embodiments, the antigen-binding molecule binds to a 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 a region of HER3 set forth in SEQ ID NO: 230 and 231. In some embodiments, the antigen-binding molecule binds to a 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 a region of HER3 set forth in SEQ ID NO: 230. In some embodiments, the antigen-binding molecule binds to a 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 a region of HER3 set forth in SEQ ID NO: 231.

[0064] 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.

[0065] 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 amino acid residues in 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 amino acid residues in the region of HER3 set forth in SEQ ID NO: 23.

[0066] The region of a peptide / polypeptide to which an antibody binds can be determined by those skilled in the art using a variety of methods well known in the art, including X-ray cocrystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, the entire contents of which are incorporated herein by reference.

[0067] In some embodiments, the antigen-binding molecule can bind to the same or overlapping region of HER3 as 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. In some embodiments, the antigen-binding molecule can bind to the same region of HER3 as, or an overlapping region of HER3, bound by, an antibody comprising the VH and VL sequences of one of the antibody clones 10D1_c89, 10D1_c90, or 10D1_c91. In some embodiments, the antigen-binding molecule can bind to the same region of HER3 as, or an overlapping region of HER3, bound by, an antibody comprising the VH and VL sequences of the antibody clone 10D1_c89.

[0068] As used herein, a "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length in the region of about 2 to 50 amino acids. A "polypeptide" is a polymeric chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

[0069] In some embodiments, an antigen-binding molecule of the invention is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of one of SEQ ID NOs: 1, 3, 4, 6, or 8.

[0070] In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0071] In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 229. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NOs: 230 and 231. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 230. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 231. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 22.

[0072] 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.

[0073] 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, immunoblot (e.g., Western blot), 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).

[0074] In embodiments in which the antigen-binding molecule can bind to a peptide / polypeptide comprising a reference amino acid sequence, the peptide / polypeptide may comprise one or more additional amino acids at one or both termini of the reference amino acid sequence. In some embodiments, the peptide / polypeptide comprises, for example, 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 50, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 20 to 30, 20 to 40, or 20 to 50 additional amino acids at one or both termini of the reference amino acid sequence.

[0075] 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 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, which correspond to positions 278 and 279 of SEQ ID NO:1.

[0076] In some embodiments, the antigen-binding molecule is capable of binding 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. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of one of the antibody clones 10D1_c89, 10D1_c90, or 10D1_c91. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of the antibody clone 10D1_c89.

[0077] antigen binding molecule The present invention provides antigen-binding molecules capable of binding to HER3. The term "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, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g., VhH), etc.), so long as they exhibit binding to the relevant target molecule.

[0078] The antigen-binding molecules of the present invention comprise a moiety capable of binding to a target antigen. In some embodiments, the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specifically binding to the target antigen. In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an aptamer, such as a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202), capable of binding to the target antigen. In some embodiments, the moiety capable of binding to a target antigen comprises 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 are incorporated by reference in their entireties (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (20113:38-48)).

[0079] The antigen-binding molecules of the present invention generally comprise an antigen-binding domain comprising VH and VL of an antibody capable of specifically binding to a target antigen. Herein, the antigen-binding domain formed by VH and VL may also be referred to as an Fv region.

[0080] An antigen-binding molecule may be or comprise an antigen-binding polypeptide or an antigen-binding polypeptide complex. An antigen-binding molecule may comprise more than one polypeptide that together form an antigen-binding domain. The polypeptides may be covalently or non-covalently associated. In some embodiments, the polypeptide forms part of a larger polypeptide that comprises the polypeptide (e.g., in the case of an scFv comprising a VH and a VL, or in the case of an scFab comprising a VH-CH1 and a VL-CL).

[0081] An antigen-binding molecule may refer to a non-covalent or covalent complex of IgG-like antigen-binding molecules that includes more than one polypeptide (e.g., two, three, four, six, or eight polypeptides), e.g., two heavy chain polypeptides and two light chain polypeptides.

[0082] The antigen-binding molecules 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 / derived. An "antigen-binding region" is any fragment of an antibody that can bind to the target for which the given antibody is specific.

[0083] Antibodies generally contain six complementarity-determining regions, or CDRs: 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. Together, the six CDRs define the antibody paratope, which is the portion of the antibody that binds to the target antigen.

[0084] The VH and VL regions comprise, on either side of each CDR, framework regions (FRs) that provide a scaffold for the CDRs. From N- to C-terminus, the VH region comprises 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 comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0085] 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 VBASE2, described in Retter et al., Nucl. Acids Res. (2005) 33(suppl 1):D671-D674. The CDRs and FRs of the VH and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue): D413-22) using the IMGT V-domain numbering convention described in Lefranc et al., Dev. Comp. Immunol. (2003) 27: 55-77.

[0086] 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 region and VL region of an antigen-binding molecule capable of binding to HER3.

[0087] In some embodiments, the antigen-binding molecule is selected from the group consisting of HER3-binding antibody clones 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_c85v3, 10D1_c85o1, 10D1_c85o2, 10D1_c85v4, 10D1_c85v5, 10D1_c85v6, 10D1_c85v7, 10D1_c85v8, 10D1_c85v9, 10D1_c85v1, 10D1_c85v2, 10D1_c85v1, 10D1_c85v2, 10D1_c85v2, 10D1_c85v3, 10D1_c85v4, 10D1_c85v5, 10D1_c85v6, 10D1_c85v7, 10D1_c85v8, 10D1_c85v9, 10D1_c85v1, 10D1_c85v2 ... and / or 4-35-B4; e.g., 10D1_c89, 10D1_c90, or 10D1_c91; e.g., 10D1_c89).

[0088] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (1) to (10) below. (1) (10D1 derived) The following CDR: 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 these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0089] (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, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0090] (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, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0091] (4)(10D1_c85o1) The following CDR: 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, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0092] (5)(10D1_c85o2) The following CDR: 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, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0093] (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, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0094] (7)(10D1_c91) The following CDR: 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, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0095] (8)(10A6) The following CDR: 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; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0096] (9)(4-35-B2) The following CDR: 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; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0097] (10)(4-35-B4) The following CDR: 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; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with another amino acid.

[0098] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (11) to (24) below. (11)(10D1) FR below: 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; 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.

[0099] (12)(10D1_c75, 10D1_c92) FR below: 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; 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.

[0100] (13)(10D1_c76, 10D1_c77, 10D1_c78v1) FR below: 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; 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.

[0101] (14)(10D1_c78v2) FR below: 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; 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.

[0102] (15)(10D1_11B) FR below: 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; 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.

[0103] (16)(10D1_c85v1) FR below: 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; 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.

[0104] (17)(10D1_c85v2, 10D1_c85o1, 10D1_c85o2) FR below: 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 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.

[0105] (18)(10D1_c87, 10D1_c93) FR below: 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 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.

[0106] (19)(10D1_c89) 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: 66 HC-FR4 having the amino acid sequence of SEQ ID NO: 71; 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.

[0107] (20)(10D1_c90) FR below: 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 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.

[0108] (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 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.

[0109] (22)(10A6) FR below: 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 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.

[0110] (23)(4-35-B2) FR below: 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.

[0111] (24)(4-35-B4) FR below: 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.

[0112] In some embodiments, the antigen-binding molecule comprises a VH region comprising a CDR according to one of (1) to (10) above and a FR according to one of (11) to (24) above.

[0113] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (25) to (41) below. (25) A VH region comprising a CDR according to (1) and a FR according to (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), or (21).

[0114] (26) A VH region comprising a CDR according to (2) and a FR according to (11). (27) A VH region comprising a CDR according to (2) and a FR according to (12). (28) A VH region comprising a CDR according to (2) and a FR according to (13).

[0115] (29) A VH region comprising a CDR according to (2) and a FR according to (14). (30) A VH region comprising a CDR according to (2) and a FR according to (15). (31) A VH region comprising a CDR according to (2) and a FR according to (18).

[0116] (32) A VH region comprising a CDR according to (3) and a FR according to (16). (33) A VH region comprising a CDR according to (3) and a FR according to (17). (34) A VH region comprising a CDR according to (4) and a FR according to (17).

[0117] (35) A VH region comprising a CDR according to (5) and a FR according to (17). (36) A VH region comprising a CDR according to (6) and a FR according to (19). (37) A VH region comprising a CDR according to (6) and a FR according to (20).

[0118] (38) A VH region comprising a CDR according to (7) and a FR according to (21). (39) A VH region comprising a CDR according to (8) and a FR according to (22). (40) A VH region comprising a CDR according to (9) and a FR according to (23).

[0119] (41) A VH region comprising a CDR according to (10) and a FR according to (24). In some embodiments, the antigen-binding molecule comprises a VH region according to one of (42) to (61) below.

[0120] (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.

[0121] (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.

[0122] (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.

[0123] (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.

[0124] (46) 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: 28.

[0125] (47) 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: 29.

[0126] (48) 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: 30.

[0127] (49) 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: 31.

[0128] (50) 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: 32.

[0129] (51) 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: 33.

[0130] (52) 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: 34.

[0131] (53) 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: 35.

[0132] (54) 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: 36.

[0133] (55) 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: 37.

[0134] (56) 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: 38.

[0135] (57) 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: 39.

[0136] (58) 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: 40.

[0137] (59) 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: 127.

[0138] (60) 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: 143.

[0139] (61) 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.

[0140] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (62) to (71) below. (62) (derived from 10D1) CDR: 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, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0141] (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, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0142] (64)(10D1_c76)The following CDR: 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, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0143] (65)(10D1_c77)The following CDR: 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, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0144] (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, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0145] (67)(10D1_c90)The following CDR: 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; or 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 replaced with another amino acid.

[0146] (68)(10D1_c92)The following CDR: 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; or 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 replaced with another amino acid.

[0147] (69)(10A6) The following CDR: 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; or 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 replaced with another amino acid.

[0148] (70)(4-35-B2) The following CDR: 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; or 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 replaced with another amino acid.

[0149] (71)(4-35-B4) The following CDR: 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, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0150] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (72) to (86) below. (72)(10D1) FR below: 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, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0151] (73)(10D1_c75) FR below: 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; 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 replaced with another amino acid.

[0152] (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; 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 replaced with another amino acid.

[0153] (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; 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 replaced with another amino acid.

[0154] (76)(10D1_c78v1, 10D1_c78v2, 10D1_11B) FR below: 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; 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 replaced with another amino acid.

[0155] (77)(10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2) FR below: 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; 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 replaced with another amino acid.

[0156] (78)(10D1_c87) 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: 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; 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 replaced with another amino acid.

[0157] (79)(10D1_c89) 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: 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 replaced with another amino acid.

[0158] (80)(10D1_c90) FR below: 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 replaced with another amino acid.

[0159] (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 replaced with another amino acid.

[0160] (82)(10D1_c92) FR below: 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 replaced with another amino acid.

[0161] (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 replaced with another amino acid.

[0162] (84)(10A6) FR below: 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 replaced with another amino acid.

[0163] (85)(4-35-B2) FR below: 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 replaced with another amino acid.

[0164] (86)(4-35-B4) FR below: 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 replaced with another amino acid.

[0165] In some embodiments, the antigen-binding molecule comprises a VL region comprising a CDR according to one of (62) to (71) above and a FR according to one of (72) to (86) above.

[0166] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (87) to (102) below. (87) A VL region comprising a CDR according to (62) and a FR according to (72), (73), (74), (75), (76), (77), (78), (79), (80), (81), (82), or (83).

[0167] (88) A VL region comprising CDRs according to (63) and FRs according to (72). (89) A VL region comprising CDRs according to (63) and FRs according to (73). (90) A VL region comprising CDRs according to (63) and FRs according to (76).

[0168] (91) A VL region comprising CDRs according to (63) and FRs according to (78). (92) A VL region comprising CDRs according to (63) and FRs according to (79). (93) A VL region comprising CDRs according to (63) and FRs according to (81).

[0169] (94) A VL region comprising CDRs according to (63) and FRs according to (83). (95) A VL region comprising CDRs according to (64) and FRs according to (74). (96) A VL region comprising CDRs according to (65) and FRs according to (75).

[0170] (97) A VL region comprising CDRs according to (66) and FRs according to (77). (98) A VL region comprising CDRs according to (67) and FRs according to (80). (99) A VL region comprising CDRs according to (68) and FRs according to (82).

[0171] (100) A VL region comprising CDRs according to (69) and FRs according to (84). (101) A VL region comprising CDRs according to (70) and FRs according to (85). (102) A VL region comprising CDRs according to (71) and FRs according to (86).

[0172] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (103) to (119) below. (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.

[0173] (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.

[0174] (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.

[0175] (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.

[0176] (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.

[0177] (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.

[0178] (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.

[0179] (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.

[0180] (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.

[0181] (112) 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: 83.

[0182] (113) 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: 84.

[0183] (114) 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: 85.

[0184] (115) 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: 86.

[0185] (116) 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: 87.

[0186] (117) 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: 135.

[0187] (118) 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: 150.

[0188] (119) 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: 164.

[0189] In some embodiments, the antigen-binding molecule comprises a VH region according to any one of (1) to (61) above and a VL region according to any one of (62) to (119) above.

[0190] In embodiments according to the invention in which one or more amino acids are substituted with another amino acid, the substitution may be a conservative substitution, for example, according to the following table: In some embodiments, amino acids in the same block in the middle column are substituted; In some embodiments, amino acids in the same stretch in the rightmost column are substituted.

[0191] [Table 1]

[0192] In some embodiments, substitutions may be functionally conservative, i.e., in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule comprising the substitution compared to a comparable unsubstituted molecule.

[0193] The VH and VL regions of the antigen-binding region of an antibody together constitute an Fv region. In some embodiments, an antigen-binding molecule according to the present 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.

[0194] In some embodiments, the antigen-binding molecules of the present invention comprise one or more regions of an immunoglobulin heavy chain constant sequence, which is or is derived from an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM heavy chain constant sequence.

[0195] In some embodiments, the immunoglobulin heavy chain constant sequence is human immunoglobulin G1 constant (IGHG1; UniProt: P01857-1, v1; SEQ ID NO: 171). Positions 1 to 98 of SEQ ID NO: 171 form the CH1 region (SEQ ID NO: 172). Positions 99 to 110 of SEQ ID NO: 171 form the hinge region between the CH1 and CH2 regions (SEQ ID NO: 173). Positions 111 to 223 of SEQ ID NO: 171 form the CH2 region (SEQ ID NO: 174). Positions 224 to 330 of SEQ ID NO: 171 form the CH3 region (SEQ ID NO: 175).

[0196] An exemplary antigen-binding molecule can be prepared using pFUSE-CHIg-hG1, which contains the substitutions D356E, L358M (positions numbered according to EU numbering) within the CH3 region. The amino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is set forth in SEQ ID NO: 176. It will be understood that the CH3 region can be further substituted in accordance with the modifications to the Fc region of the antigen-binding molecule described herein.

[0197] In some embodiments, the CH1 region comprises or consists of the sequence of SEQ ID NO: 172, or 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 amino acid sequence of SEQ ID NO: 172. In some embodiments, the hinge region between CH1 and CH2 comprises or consists of the sequence of SEQ ID NO: 173, or 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 amino acid sequence of SEQ ID NO: 173. In some embodiments, the CH2 region comprises or consists of the sequence of SEQ ID NO: 174, or 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 amino acid sequence of SEQ ID NO: 174. In some embodiments, the CH3 region comprises or consists of the sequence of SEQ ID NO: 175 or 176, or 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 amino acid sequence of SEQ ID NO: 175 or 176.

[0198] In some embodiments, the antigen-binding molecules of the present invention comprise one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; Cκ; UniProt: P01834-1, v2; SEQ ID NO: 177). In some embodiments, the immunoglobulin light chain constant sequence 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 the sequence of SEQ ID NO: 177 or 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 amino acid sequence of SEQ ID NO: 177.

[0199] The VL and light chain constant (CL) regions, and the VH region and heavy chain constant 1 (CH1) region of the antigen-binding region of an antibody together constitute a Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising a VH, CH1, VL, and CL (e.g., CK or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising a VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising a VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising a 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 a Fab or CrossFab are provided as a single polypeptide joined by a linker region, i.e., a single-chain Fab (scFab) or single-chain CrossFab (scCrossFab).

[0200] In some embodiments, the antigen-binding molecules of the invention comprise or consist of a Fab region that binds to HER3. In some embodiments, the antigen-binding molecules described herein comprise or consist of a whole antibody that binds to HER3. As used herein, "whole antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different types of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini, J Allergy Clin Immunol. (2010) 125(202):S41-S52, which is incorporated herein by reference in its entirety.

[0201] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa that contain two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains a VH followed by a heavy chain constant region containing three constant domains (CH1, CH2, and CH3); similarly, the light chain contains a VL followed by a CL. Depending on the heavy chain, immunoglobulins 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 kappa (κ) or lambda (λ).

[0202] In some embodiments, the antigen-binding molecules described herein comprise or consist of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that binds to HER3.

[0203] In some embodiments, the antigen-binding molecules of the present invention bind to HER3 at least monovalently. Valency refers to the number of binding sites within an antigen-binding molecule for a given antigenic determinant. Thus, in some embodiments, the antigen-binding molecule comprises at least one binding site for HER3.

[0204] 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, for example, bivalent, trivalent, or tetravalent for HER3.

[0205] Aspects of the present invention relate to multispecific antigen-binding molecules. "Multispecific" means that an 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 comprising, for example, non-identical VH and VL).

[0206] 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.

[0207] It will be understood that antigen-binding molecules (e.g., multispecific antigen-binding molecules) according to the present invention may include antigen-binding molecules capable of binding to a target for which the antigen-binding molecule is specific. For example, antigen-binding molecules capable of binding to HER3 and antigens other than HER3 may include (i) antigen-binding molecules capable of binding to HER3 and (ii) antigen-binding molecules capable of binding to antigens other than HER3.

[0208] It will also be understood that antigen-binding molecules (e.g., multispecific antigen-binding molecules) according to the present invention may comprise antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to a target for which the antigen-binding molecule is specific. For example, antigen-binding molecules according to the present invention may include, for example, (i) antigen-binding polypeptide complexes 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) antigen-binding polypeptide complexes 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).

[0209] In some embodiments, an antigen-binding molecule that is a component of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to as, for example, an "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0210] 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, for example, a cell surface receptor. In some embodiments, the antigen other than HER3 is a cell signaling molecule, for example, a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen other than HER3 is a growth factor or hormone.

[0211] A cancer cell antigen is an antigen expressed or overexpressed by cancer cells. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Expression of a cancer cell antigen can be associated with cancer. A cancer cell antigen can be aberrantly expressed by cancer cells (e.g., the cancer cell antigen can be expressed with abnormal localization) or with abnormal structure by cancer cells. A cancer cell antigen can be capable of eliciting an immune response. In some embodiments, the antigen is expressed on the cell surface of a cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (i.e., is 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 can be associated with the cause or pathology of cancer, or can be aberrantly expressed as a consequence 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, e.g., compared to expression levels by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be preferentially expressed by cancerous cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, a cancer-associated antigen may be the product of an overexpressed intracellular protein, or may be a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.

[0212] In some embodiments, the antigen other than HER3 is an antigen expressed by cells of a HER3-associated cancer. A HER3-associated cancer can be a cancer that expresses HER3 (e.g., expresses HER3 protein on the cell surface); such cancers can be referred to as "HER3-positive" cancers. HER3-associated cancers include cancers in which expression of the HER3 gene / protein is a risk factor and / or is positively associated with cancer onset, development, progression, or symptom severity, and / or metastasis. HER3-associated cancers include those 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, all of which are incorporated herein by reference in their entireties. In some embodiments, a HER3-associated cancer can be lung cancer (e.g., NSCLC), melanoma, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, colon cancer, or oral cancer.

[0213] The immune cell surface molecule can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed on or at the cell surface of the immune cell. In some embodiments, the portion of the immune cell surface molecule bound by the antigen-binding molecule of the present invention is on the external surface of the immune cell (i.e., extracellular). The immune cell surface molecule can be expressed on the cell surface of any immune cell. In some embodiments, the immune cell can be a hematopoietic cell-derived cell, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte can be, for example, a T cell, a B cell, a natural killer (NK) cell, a NKT cell, or an innate lymphoid cell (ILC), or a precursor cell thereof (e.g., a thymocyte or a pre-B cell). In some embodiments, the immune cell surface molecule 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.

[0214] Multispecific antigen-binding molecules according to the present invention may be provided in any suitable format, such as those described in Brinkmann and Kontermann MAbs (2017) 9(2):182-212, which is incorporated herein by reference in its entirety. Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann MAbs (2017) 9(2):182-212: antibody conjugates, e.g., IgG2, F(ab')2, or CovX-Body; IgG or IgG-like molecules, e.g., common HC of IgG, chimeric IgG, κλ-body; CH1 / CL fusion proteins, e.g., scFv2-CH1 / CL, VHH2-CH1 / CL; "variable domain-only" bispecific antigen-binding molecules, e.g., 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, e.g., scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibodies, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g., scFv-Fc(kih), scFv-Fc(CH3 charge pair), scFv-Fc(EW-RVT), scFv-fc(HA-TF), scFv-Fc(SEEDbody), taFv-Fc(kih), scF v-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, e.g., di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions, e.g., dia-diabody, scDb-CH3; IgE / IgM CH2 fusions, e.g., scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, e.g., Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, e.g., DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g., 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, Duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc. *appended and Fc-modified IgG, e.g., 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, e.g., Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; appended IgG-HC fusions, e.g., IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CαCβ)Fab, scFv-HC-IgG, tandem Fa b-IgG (orthogonal Fab), Fab-IgG (CαCβFab), Fab-IgG (CR3), Fab-hinge-IgG (CR3); appended IgG-LC fusions, e.g., IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; appended IgG-HC and LC fusions, e.g., DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, Zybody; Fc fusions, e.g., Fab-scFv-Fc, scFv4-Ig; F(ab')2 fusions, e.g., F(ab')2-scFv2; CH1 / CL fusion proteins, e.g., scFv2-CH1-hinge / CL; modified IgGs, e.g., DAF (two-in-one-IgG), DutaMab, Mab 2 as well as non-Ig fusions, eg, DNL-Fab4-IgG.

[0215] Those skilled in the art can design and prepare bispecific antigen-binding molecules. Methods for producing bispecific antigen-binding molecules include chemically crosslinking antigen-binding molecules or antibody fragments, for example, via a reducible disulfide bond or a non-reducible thioether bond, as described, for example, 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, the entire contents of which are incorporated herein by reference. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink Fab fragments, for example, via SH groups in the hinge region, to create disulfide-linked bispecific F(ab)2 heterodimers.

[0216] Other methods for producing bispecific antigen-binding molecules include fusing antibody-producing hybridomas, for example, with polyethylene glycol, to produce quadroma cells capable of secreting bispecific antibodies, as described, for example, in DM and Bast, BJ 2001. Production of Bispecific Antigen-binding Molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

[0217] Bispecific antigen-binding molecules according to the present invention can also be produced by recombinant expression from nucleic acid constructs encoding polypeptides for the antigen-binding molecules, as described, for example, 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 sequences encoding appropriate linkers or dimerization domains between the antigen-binding fragments, can be prepared by molecular cloning techniques. The recombinant bispecific antibody is then produced by expression (e.g., in vitro) of the construct in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibody can then optionally be purified.

[0218] Fc area In some embodiments, the antigen-binding molecules of the invention comprise an Fc region. In IgG, IgA, and IgD isotypes, the Fc region is composed of a CH2 and CH3 region derived from one polypeptide and a CH2 and CH3 region derived from another polypeptide. The CH2 and CH3 regions derived from the two polypeptides together form the Fc region. In IgM and IgE isotypes, the Fc region contains three constant domains (CH2, CH3, and CH4), and the CH2 to CH4 regions derived from the two polypeptides together form the Fc region.

[0219] In preferred embodiments according to various aspects of the present disclosure, the Fc region comprises two polypeptides, each polypeptide comprising a CH2 region and a CH3 region. In some embodiments, the antigen-binding molecules of the present invention comprise an Fc region containing modifications in one or more of the CH2 and CH3 regions that promote Fc region assembly. Recombinant coexpression of polypeptides that constitute the antigen-binding molecule and subsequent assembly results in several possible combinations. In recombinant production, to improve the yield of the desired combination of polypeptides within the antigen-binding molecule, it is advantageous to introduce modifications to the Fc region that promote the assembly of the desired combination of heavy chain polypeptides. Modifications 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, the entire contents of which are incorporated herein by reference.

[0220] In some embodiments, the antigen-binding molecules of the invention are in the following formats: KiH, KiH, as 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.

[0221] In some embodiments, the Fc region comprises, for example, a "knob-into-hole" or "KiH" modification, as described, for example, in U.S. Pat. No. 7,695,936 and Carter, J. Immunol. Meth. 248, 7-15 (2001). In such embodiments, one CH3 region of the Fc region comprises a "knob" modification and the other CH3 region comprises a "hole" modification. The "knob" and "hole" modifications are placed within the respective CH3 regions such that the "knob" can be placed within the "hole" to promote heterodimerization (and inhibit homodimerization) of the polypeptide and / or stabilize the heterodimer. Knobs are constructed by substituting amino acids with larger side chains (e.g., tyrosine or tryptophan) for amino acids with larger side chains (e.g., alanine or threonine). Holes are created by substituting amino acids with larger side chains (e.g., alanine or threonine) for amino acids with smaller side chains (e.g., alanine or threonine).

[0222] In some embodiments, one CH3 region of the Fc region of an antigen-binding molecule of the invention comprises the substitution T366W (the numbering of positions / substitutions of the Fc, CH2, and CH3 regions herein is 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), and the other CH3 region of the Fc region comprises the substitution Y407V. In some embodiments, one CH3 region of the Fc region of an antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions T366S and L368A. In some embodiments, one CH3 region of the Fc region of an antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions Y407V, T366S, and L368A.

[0223] In some embodiments, the Fc region comprises a "DD-KK" modification, e.g., as described in WO2014 / 131694A1. In some embodiments, one of the CH3 regions comprises substitutions K392D and K409D, and the other CH3 region of the Fc region comprises substitutions E356K and D399K. The modifications promote electrostatic interactions between the CH3 regions.

[0224] In some embodiments, the antigen-binding molecules of the invention comprise Fc regions modified as described in Labrijn et al., Proc Natl Acad Sci USA. (2013) 110(13):5145-50, referred to as a "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.

[0225] In some embodiments, antigen-binding molecules of the invention comprise an Fc region comprising the "EEE-RRR" modification described in Strop et al., J Mol Biol. (2012) 420(3):204-19. In some embodiments, one of the CH3 regions comprises substitutions D221E, P228E, and L368E, and the other CH3 region of the Fc region comprises substitutions D221R, P228R, and K409R.

[0226] In some embodiments, the antigen-binding molecule comprises an Fc region comprising the "EW-RVT" modification described in Choi et al., Mol Cancer Ther (2013) 12(12):2748-59. In some embodiments, one of the CH3 regions comprises substitutions K360E and K409W, and the other CH3 region of the Fc region comprises substitutions Q347R, D399V, and F405T.

[0227] In some embodiments, one of the CH3 regions contains the substitution S354C and the other CH3 region of the Fc region contains the substitution Y349C. The introduction of these cysteine ​​residues results in the formation of disulfide bridges between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001), J Immunol Methods, 248, 7-15).

[0228] In some embodiments, the Fc region is S-S " modification. In some embodiments, one of the CH3 regions comprises substitutions T366W and S354C, and the other CH3 region of the Fc region comprises substitutions T366S, L368A, Y407V, and Y349C.

[0229] In some embodiments, the antigen-binding molecules of the invention comprise an Fc region comprising the "SEED" modification, in which the β-strand segment of human IgG1 CH3 is exchanged for the β-strand segment of human IgA CH3, as described by Davis et al., Protein Eng Des Sel (2010) 23(4):195-202.

[0230] In some embodiments, one of the CH3 regions contains substitutions S364H and F405A, and the other CH3 region of the Fc region contains substitutions Y349T and T394F (see, e.g., Moore et al., MAbs (2011) 3(6):546-57).

[0231] 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).

[0232] In some embodiments, one of the CH3 regions comprises substitutions K360D, D399M, and Y407A, and the other CH3 region of the Fc region comprises substitutions E345R, Q347R, T366V, and K409V (see, e.g., Leaver-Fay et al., Structure (2016) 24(4):641-51).

[0233] In some embodiments, one of the CH3 regions contains substitutions K370E and K409W, and the other CH3 region of the Fc region contains substitutions E357N, D399V, and F405T (see, e.g., Choi et al., PLoS One (2015) 10(12):e0145349).

[0234] Fc-mediated functions include binding to Fc receptors, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cellular degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0235] Modifications to the Fc region of antibodies that affect Fc-mediated function are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73, which is incorporated herein by reference in its entirety. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73.

[0236] The substitution combination F243L / R292P / Y300L / V305I / P396L has been described in Stavenhagen et al., Cancer Res. (2007) to increase binding to FcγRIIIa, thereby enhancing ADCC. The substitution combination S239D / I332E or S239D / I332E / A330L has been described in Lazar et al., Proc Natl Acad Sci USA. (2006) 103:4005-4010 to increase binding to FcγRIIIa, thereby enhancing ADCC. The substitution combination S239D / I332E / A330L has also been described to decrease binding to FcγRIIb, thereby enhancing ADCC. The substitution combination S298A / E333A / K334A has been described in Shields et al., J. Biol. Chem. (2001) 276:6591-6604 to increase binding to FcγRIIIa, thereby increasing ADCC. The substitution combination L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the other heavy chain have been described in Mimoto et al., MAbs. (2013):5:229-236 to increase binding to FcγRIIIa, thereby increasing ADCC. The substitution combination G236A / S239D / I332E 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.

[0237] The substitution combination K326W / E333S has been described in Idusogie et al., J Immunol. (2001) 166(4):2571-5 as increasing binding to C1q, thereby increasing CDC. The substitution combination S267E / H268F / S324T has been described in Moore et al., MAbs. (2010) 2(2):181-9 as increasing binding to C1q, thereby increasing CDC. The substitution combination described in Natsume et al., Cancer Res. (2008) 68(10):3863-72 has been reported to increase binding to C1q, thereby increasing CDC. The substitution combination E345R / E430G / S440Y has been described in Diebolder et al., Science (2014) 343(6176):1260-3 to increase hexamerization and thereby increase CDC.

[0238] The substitution combination M252Y / S254T / T256E has been described in Dall'Acqua et al., J Immunol. (2002) 169:5171-5180 as increasing FcRn binding at pH 6.0, thereby extending the half-life of antigen-binding molecules. The substitution combination M428L / N434S has been described in Zalevsky et al., Nat Biotechnol. (2010) 28:157-159 as increasing FcRn binding at pH 6.0, thereby extending the half-life of antigen-binding molecules.

[0239] When a heavy chain constant region / Fc region / CH2-CH3 region / CH2 region / CH3 region is described herein as containing a position / substitution "corresponding to" a referenced position / substitution, the equivalent position / substitution in a homologous heavy chain constant region / Fc region / CH2-CH3 region / CH2 region / CH3 region is assumed.

[0240] When an Fc region is described as comprising a particular position / substitution, the position / substitution may be present in one or both of the polypeptide chains that together form the Fc region. Unless otherwise specified, positions herein refer to positions in the amino acid sequence of human immunoglobulin constant regions, numbered according to the EU numbering system, as set forth 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, the substitutions L242C and K334C in human IgG1 correspond to the L>C substitution at position 125 and the K>C substitution at position 217 of the human IgG1 constant region, numbered according to SEQ ID NO: 171.

[0241] A homologous 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 set forth in SEQ ID NO: 171). A homologous 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 sequence set forth in SEQ ID NOs: 174 and 175). A homologous 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 set forth in SEQ ID NO: 174). A homologous 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 set forth in SEQ ID NO: 175).

[0242] Positions corresponding to those identified in 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).

[0243] In some embodiments, antigen-binding molecules of the present invention comprise an Fc region comprising a modification that increases an 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. Antigen-binding molecules comprising an Fc region comprising a modification that increases an Fc-mediated function (e.g., ADCC, ADCP, CDC) induce increased levels of the associated effector function compared to an antigen-binding molecule comprising a corresponding unmodified Fc region.

[0244] In some embodiments, antigen-binding molecules of the present invention comprise an Fc region comprising a modification that increases affinity for one or more Fc receptors (e.g., FcγRIIa, FcγRIIIa). Modifications that increase affinity for an Fc receptor may increase Fc-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, antigen-binding molecules of the present invention comprise an Fc region comprising a modification that reduces affinity for C1q; such modifications reduce complement-dependent cytotoxicity (CDC), which may be desirable. In some embodiments, antigen-binding molecules of the present invention comprise an Fc region comprising a modification that increases hexamer formation. Modifications to an Fc region that can increase affinity for one or more Fc receptors, reduce affinity for C1q, and / or increase hexamer formation are described, for example, in Saxena and Wu Front Immunol. (2016) 7:580, which is incorporated herein by reference in its entirety. In some embodiments, the antigen-binding molecules of the invention comprise an Fc region comprising a CH2 / CH3 that comprises one or more of the substitutions set out in Table 1 of Saxena and Wu Front Immunol. (2016) 7:580.

[0245] In some embodiments, the antigen-binding molecules of the present invention comprise an Fc region 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 decreases 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.

[0246] 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" refer to FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb, respectively, derived from any species and include isoforms, fragments, variants (including mutants), or homologs derived from any species. Humans have six distinct classes of Fcγ receptors (murine orthologs are 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 in human FcγRIIIa and the 167H and 167R polymorphisms in human FcγRIIa.

[0247] In some embodiments, the antigen-binding molecules of the invention comprise an Fc region (e.g., a heavy chain constant region comprising, or further comprising, a polypeptide comprising a CH2-CH3 region) that comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following: C at a position corresponding to 242; C at a position corresponding to 334; A at a position corresponding to 236; D at a position corresponding to 239; E at a position corresponding to 332; L at a position corresponding to 330; K at a position corresponding to 345; and G at a position corresponding to 430.

[0248] In some embodiments, antigen-binding molecules of the invention comprise an Fc region (e.g., a heavy chain constant region, or a further polypeptide comprising CH2-CH3 regions) that 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.

[0249] In some embodiments, the antigen-binding molecule comprises an Fc region that comprises a C at a position corresponding to 242 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising these). In some embodiments, the Fc region comprises a C at a position corresponding to 334 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising these). In some embodiments, the Fc region comprises a C at a position corresponding to 242 and a C at a position corresponding to 334 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising these).

[0250] In some embodiments, the antigen-binding molecule comprises an Fc region that comprises an A at a position corresponding to 236 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising these). In some embodiments, the Fc region comprises a D at a position corresponding to 239 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising these). In some embodiments, the Fc region comprises an A at a position corresponding to 236 and a D at a position corresponding to 239 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising these).

[0251] In some embodiments, the antigen-binding molecule comprises an Fc region that comprises an E at a position corresponding to 332 (e.g., a heavy chain constant region, a CH2-CH3 region, or a CH2 region, including one further polypeptide). In some embodiments, the Fc region comprises an A at a position corresponding to 236, a D at a position corresponding to 239, and an E at a position corresponding to 332 (e.g., a heavy chain constant region, a CH2-CH3 region, or a CH2 region, including one further polypeptide).

[0252] In some embodiments, the antigen-binding molecule comprises an Fc region that comprises an L at a position corresponding to 330 (e.g., a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising one or more polypeptides). In some embodiments, the Fc region comprises an A at a position corresponding to 236, a D at a position corresponding to 239, an E at a position corresponding to 332, and an L at a position corresponding to 330 (e.g., a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising one or more polypeptides).

[0253] In some embodiments, the antigen-binding molecule comprises an Fc region that comprises a K at a position corresponding to 345 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH3 region comprising these). In some embodiments, the Fc region comprises a G at a position corresponding to 430 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH3 region comprising these). In some embodiments, the Fc region comprises a K at a position corresponding to 345 and a G at a position corresponding to 430 (e.g., comprises one further polypeptide that comprises a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising these).

[0254] In some embodiments, the antigen-binding molecule comprises an Fc region comprising (e.g., a heavy chain constant region, a CH2-CH3 region, or a further polypeptide comprising a CH2 region) a C at a position corresponding to 242, a C at a position corresponding to 334, an A at a position corresponding to 236, and a D at a position corresponding to 239.

[0255] In some embodiments, the antigen-binding molecule comprises an Fc region comprising (e.g., a heavy chain constant region, a CH2-CH3 region, or a further polypeptide comprising a CH2 region) a C at a position corresponding to 242, a C at a position corresponding to 334, an A at a position corresponding to 236, a D at a position corresponding to 239, and an E at a position corresponding to 332.

[0256] In some embodiments, the antigen-binding molecule comprises an Fc region comprising (e.g., a heavy chain constant region, a CH2-CH3 region, or a further polypeptide comprising a CH2 region) a C at a position corresponding to 242, a C at a position corresponding to 334, an A at a position corresponding to 236, a D at a position corresponding to 239, an E at a position corresponding to 332, and an L at a position corresponding to 330.

[0257] In some embodiments, the antigen-binding molecule comprises an Fc region comprising (e.g., a heavy chain constant region comprising, or a further polypeptide comprising, a CH2-CH3 region) a C at a position corresponding to 242, a C at a position corresponding to 334, a K at a position corresponding to 345, and a G at a position corresponding to 430.

[0258] In some embodiments, the antigen-binding molecule comprises an Fc region comprising substitution L242C (or an equivalent substitution) (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising these). In some embodiments, the Fc region comprises substitution K334C (or an equivalent substitution) (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising these). In some embodiments, the Fc region comprises substitution L242C (or an equivalent substitution) and substitution K334C (or an equivalent substitution) (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising these).

[0259] In some embodiments, the antigen-binding molecule comprises an Fc region (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising the substitution G236A (or an equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising the substitution S239D (or an equivalent substitution)). In some embodiments, the Fc region comprises (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising the substitution G236A (or an equivalent substitution) and substitution S239D (or an equivalent substitution) (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising the substitution).

[0260] In some embodiments, the antigen-binding molecule comprises an Fc region (e.g., comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising the substitution I332E (or an equivalent substitution)). In some embodiments, the Fc region comprises (e.g., comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising the substitution G236A (or an equivalent substitution), the substitution S239D (or an equivalent substitution), and the substitution I332E (or an equivalent substitution) (e.g., comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising the substitution G236A (or an equivalent substitution), the substitution S239D (or an equivalent substitution), and the substitution I332E (or an equivalent substitution).

[0261] In some embodiments, the antigen-binding molecule comprises an Fc region (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising the substitution A330L (or an equivalent substitution). In some embodiments, the Fc region comprises (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising the substitution G236A (or an equivalent substitution), substitution S239D (or an equivalent substitution), substitution I332E (or an equivalent substitution), and substitution A330L (or an equivalent substitution).

[0262] In some embodiments, the antigen-binding molecule comprises an Fc region comprising the substitution E345K (or an equivalent substitution) (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH3 region comprising the same). In some embodiments, the Fc region comprises the substitution E430G (or an equivalent substitution) (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH3 region comprising the same). In some embodiments, the Fc region comprises the substitution E345K (or an equivalent substitution) and the substitution E430G (or an equivalent substitution) (e.g., comprising one further polypeptide comprising a heavy chain constant region, CH2-CH3 region, or CH2 region comprising the same).

[0263] In some embodiments, the antigen-binding molecule comprises an Fc region comprising (e.g., a heavy chain constant region, a CH2-CH3 region, or a further polypeptide comprising a CH2 region) the substitution L242C (or an equivalent substitution), the substitution K334C (or an equivalent substitution), the substitution G236A (or an equivalent substitution), and the substitution S239D (or an equivalent substitution).

[0264] In some embodiments, the antigen-binding molecule comprises an Fc region comprising (e.g., a heavy chain constant region, a CH2-CH3 region, or a further polypeptide comprising a CH2 region) substitution L242C (or an equivalent substitution), substitution K334C (or an equivalent substitution), substitution G236A (or an equivalent substitution), substitution S239D (or an equivalent substitution), and substitution I332E (or an equivalent substitution).

[0265] In some embodiments, the antigen-binding molecule comprises an Fc region comprising (e.g., a heavy chain constant region, a CH2-CH3 region, or a further polypeptide comprising) the substitution L242C (or an equivalent substitution), the substitution K334C (or an equivalent substitution), the substitution G236A (or an equivalent substitution), the substitution S239D (or an equivalent substitution), the substitution I332E (or an equivalent substitution), and the substitution A330L (or an equivalent substitution).

[0266] In some embodiments, the antigen-binding molecule comprises an Fc region comprising (e.g., a heavy chain constant region, or a further polypeptide comprising CH2-CH3 regions) the substitution L242C (or an equivalent substitution), the substitution K334C (or an equivalent substitution), the substitution E345K (or an equivalent substitution), and the substitution E430G (or an equivalent substitution).

[0267] In some embodiments, the antigen-binding molecule contains one of the following amino acids: L at position corresponding to 243, P at position corresponding to 292, L at position corresponding to 300, I at position corresponding to 305, and L at position corresponding to 396; D at position corresponding to 239 and E at position corresponding to 332; D at position corresponding to 239, E at position corresponding to 332, and L at position corresponding to 330; A at position corresponding to 298, A at position corresponding to 333, and A at position corresponding to 334; Y at position corresponding to 234, Q at position corresponding to 235, W at position corresponding to 236, M at position corresponding to 239, D at position corresponding to 268, E at position corresponding to 270, and A at position corresponding to 298; E at position corresponding to 270, D at position corresponding to 326, M at position corresponding to 330, and and E at positions corresponding to 334; A at position corresponding to 236, D at position corresponding to 239, and E at position corresponding to 332; W at position corresponding to position 326 and S at position corresponding to 333; E at position corresponding to position 267, F at position corresponding to position 268, and T at position corresponding to position 324; R at position corresponding to position 345, G at position corresponding to position 430, and Y at position corresponding to position 440; Y at position corresponding to position 252, T at position corresponding to position 254, and E at position corresponding to position 256; and L at position corresponding to position 428, and S at position corresponding to position 434 (e.g., a heavy chain constant region comprising these, or further comprising one polypeptide comprising a CH2-CH3 region).

[0268] In some embodiments, the antigen-binding molecule comprises 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 and M428L / N434S).

[0269] Polypeptides The present invention also provides polypeptide components of antigen-binding molecules. The polypeptides may be provided in isolated or substantially purified form.

[0270] The antigen-binding molecules of the present invention may be or may comprise a complex of polypeptides. Where a polypeptide comprises more than one domain or region herein, it will be understood that the multiple domains / regions are preferably present within the same polypeptide chain, i.e., a polypeptide comprising more than one domain or region is a fusion polypeptide comprising the domains / regions.

[0271] In some embodiments, a polypeptide in accordance with the invention comprises or consists of a VH described herein. In some embodiments, a polypeptide in accordance with the invention comprises or consists of a VL described herein.

[0272] In some embodiments, the polypeptide further comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide further comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises an immunoglobulin (Ig) CH1, CH2, and / or CH3 region.

[0273] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region described herein. In some embodiments, the polypeptide comprises a CH1-CH2 hinge region described herein. In some embodiments, the polypeptide comprises a CH2 region described herein. In some embodiments, the polypeptide comprises a CH3 region described herein. In some embodiments, the polypeptide comprises a CH2-CH3 region described herein.

[0274] In some embodiments, the polypeptides have the following amino acid substitutions / combinations of amino acid substitutions (e.g., as shown in Table 1 of Ha et al., Front. Immunol (2016) 7:394, incorporated herein by reference): 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, T366 L, 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 a CH3 region comprising any one of E357N, D399V, and F405T.

[0275] In some embodiments, the CH2 and / or CH3 region of the polypeptide comprises one or more amino acid substitutions to promote association of the polypeptide with another polypeptide that comprises a CH2 and / or CH3 region.

[0276] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence, hi some embodiments, the polypeptide comprises a CL region described herein.

[0277] In some embodiments, the polypeptides in accordance with the invention comprise, from N-terminus to C-terminus: (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 It contains a structure that conforms to one of the following:

[0278] The present invention also provides antigen-binding molecules composed of the polypeptides of the present invention. In some embodiments, the antigen-binding molecules of the present invention comprise 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 Contains one of the following:

[0279] In some embodiments, an antigen-binding molecule comprises more than one of the combinations of polypeptides set forth in (A)-(I) above. By way of example, and referring to (D) above, in some embodiments, an antigen-binding molecule comprises two polypeptides comprising the structure VH-CH1-CH2-CH3 and two polypeptides comprising the structure VL-CL.

[0280] In some embodiments, the antigen-binding molecules of the invention comprise 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 wherein "VH(anti-HER3)" refers to the VH of an antigen-binding molecule capable of binding to HER3 as described herein, for example, as defined in one of (1) to (61) above; and "VL(anti-HER3)" refers to the VL of an antigen-binding molecule capable of binding to HER3 as described herein, for example, as defined in one of (62) to (119) above.

[0281] 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 the amino acid sequence of one of SEQ ID NOs: 187-223.

[0282] Linkers and further sequences In some embodiments, the antigen-binding molecules and polypeptides of the present 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.

[0283] In some embodiments, the antigen-binding molecules and polypeptides of the present invention comprise one or more linker sequences between amino acid sequences, which may 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.

[0284] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those skilled in the art, and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0285] In some embodiments, the linker sequence comprises 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 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 10 amino acids.

[0286] The antigen-binding molecules and polypeptides of the present invention may further comprise additional amino acids or amino acid sequences. For example, the antigen-binding molecules and polypeptides may comprise amino acid sequences that facilitate the expression, folding, transport, processing, purification, or detection of the antigen-binding molecules / polypeptides. For example, the antigen-binding molecules / polypeptides may optionally comprise a sequence encoding His (e.g., 6xHis), Myc, GST, MBP, FLAG, HA, E, or biotin tag at the N- or C-terminus of the antigen-binding molecules / polypeptides. In some embodiments, the antigen-binding molecules / polypeptides comprise a detectable moiety, such as a fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzyme label.

[0287] 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 usually 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.

[0288] A signal peptide can be present at the N-terminus of an antigen-binding molecule / polypeptide or can be present in a newly synthesized antigen-binding molecule / polypeptide. The signal peptide ensures efficient transport and secretion of the antigen-binding molecule / polypeptide. The signal peptide is often removed by cleavage and is therefore not included in the mature antigen-binding molecule / polypeptide secreted from cells expressing the antigen-binding molecule / polypeptide.

[0289] 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, for example, SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0290] In some embodiments, the signal peptide of an 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 the amino acid sequence of one of SEQ ID NOs: 178-186.

[0291] Labels and conjugates In some embodiments, the antigen-binding molecules of the invention further comprise a detectable moiety. 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 label, a nucleic acid label, or an enzymatic label. The antigen-binding molecule may be covalently or non-covalently labeled with the detectable moiety.

[0292] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), rare earth chelators 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 ,palladium 100 , bismuth 217, and antimony 211 Examples of suitable labels include radioisotopes such as fluorophore, fluorophore ...

[0293] In some embodiments, the antigen-binding molecules of the present invention are 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. 2016 September;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.

[0294] Certain exemplary embodiments of antigen-binding molecules 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 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: 188. It comprises or consists of:

[0295] 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: 189; 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: 190. It comprises or consists of:

[0296] 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: 191; 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: 192. It comprises or consists of:

[0297] 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. It comprises or consists of:

[0298] 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. It comprises or consists of:

[0299] 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: 196; 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. It comprises or consists of:

[0300] 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: 197; 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: 199. It comprises or consists of:

[0301] 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: 198; 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: 199. It comprises or consists of:

[0302] 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: 200; 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: 201. It comprises or consists of:

[0303] 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: 202; 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: 203. It comprises or consists of:

[0304] 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: 204; 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: 205. It comprises or consists of:

[0305] 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: 206; 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: 207. It comprises or consists of:

[0306] 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: 208; 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: 209. It comprises or consists of:

[0307] 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: 210; 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: 211. It comprises or consists of:

[0308] 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: 212; 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: 213. It comprises or consists of:

[0309] 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: 214; 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: 215. It comprises or consists of:

[0310] 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: 216; 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. It comprises or consists of:

[0311] 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: 218; 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: 219. It comprises or consists 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: 220; 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: 221. It comprises or consists of:

[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: 222; 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: 223. It comprises or consists of:

[0314] 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: 225; 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: 207. It comprises or consists of:

[0315] 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: 226; 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: 207. It comprises or consists of:

[0316] 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. It comprises or consists of:

[0317] 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. It comprises or consists of:

[0318] In some embodiments, the antigen-binding molecule is produced by the cell line deposited on May 7, 2021 as ATCC Patent Deposit No. PTA-127062, e.g., as described in GB2108449.6, which is incorporated herein by reference in its entirety. Functional properties of antigen-binding molecules The antigen-binding molecules described herein can be characterized by reference to certain functional properties. In some embodiments, the antigen-binding molecules described herein may have one or more of the following properties: binding to HER3 (e.g., human, mouse, rat, or cynomolgus macaque HER3); lack of binding to EGFR and / or HER2; binding to cells expressing HER3; binding to subdomain II of the extracellular region of HER3; binding to HER3 when HER3 is in the open and closed conformation; Binding to HER3 independently of NRG; does not compete with MM-121 and / or LJM-716 for binding to HER3; does not compete with M-05-74 and / or M-08-11 for binding to HER3; inhibiting the interaction of HER3 with its interacting partners (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet); inhibiting HER3-mediated signaling; inhibiting proliferation of cells expressing HER3 (e.g., in response to stimulation with NRG); inhibiting PI3K / AKT / mTOR and / or MAPK signaling by cells expressing HER3 (e.g., in response to stimulation with NRG); inhibiting HER3 and / or AKT phosphorylation in the presence and / or absence of NRG1; binding to activating Fcγ receptors (e.g., FcγRIIIa); increased binding to activating Fcγ receptors; increased binding to activating Fcγ receptors compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs: 174-175; reduced binding to inhibitory Fcγ receptors compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs: 174-175; increased binding to activating Fcγ receptors over inhibitory Fcγ receptors compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs: 174-175; increased or decreased 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 sequence of SEQ ID NOs: 174-175; increased hexamerization compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs: 174-175; increased 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; increased 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; Increased or decreased CDC activity compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence 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 sequence of SEQ ID NOs: 174-175; increasing the killing of cells expressing HER3; Reducing the number / proportion of cells expressing HER3; inhibiting tumor cell proliferation (e.g., to a greater extent compared to MM-121 and / or LJM-716); and Inhibiting cancer initiation and / or progression in vivo.

[0319] 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 can be distinguished 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 longer duration than it binds to other non-target molecules.

[0320] The ability of a given polypeptide to specifically bind to a given molecule can be determined by analysis 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 radiolabeled antigen binding assay (RIA), enzyme-linked immunosorbent assay. Such analysis can measure and quantitate binding to a given molecule. In some embodiments, binding can be a response detected in a given assay.

[0321] In some embodiments, the extent of binding of the antigen-binding molecule to the non-target molecule 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 RIA. Alternatively, binding specificity can be measured by the K D Therefore, it is necessary to calculate the number of digits to at least 0.1 (i.e., 0.1 × 10 n , where n is an integer representing the number of digits) D), which may optionally be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0322] In some embodiments, the antigen-binding molecules exhibit binding to human HER3, mouse HER3, rat HER3, and / or cynomolgus monkey (Macaca fascicularis) HER3. That is, in some embodiments, the antigen-binding molecules are cross-reactive with human HER3, mouse HER3, rat HER3, and / or cynomolgus monkey HER3. In some embodiments, the antigen-binding molecules of the present invention exhibit cross-reactivity with HER3 of non-human primates. Cross-reactivity to HER3 in model species allows for in vivo investigation of efficacy in syngeneic models without relying on surrogate molecules.

[0323] In some embodiments, the antigen-binding molecule binds to human HER3, mouse HER3, rat HER3, and / or cynomolgus HER3, but does not bind to HER2 and / or EGFR (eg, human HER2 and / or human EGFR).

[0324] 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 (i.e., does not cross-react with) members of the EGFR family of proteins other than HER3. In some embodiments, the antigen-binding molecule does not exhibit specific binding to EGFR, HER2, and / or HER4.

[0325] In some embodiments, the antigen-binding molecules of the invention have a cytotoxicity 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, ≦ K of one of the following: 8nM, ≦7nM, ≦6nM, ≦5nM, ≦4nM, ≦3nM, ≦2nM, ≦1nM, ≦900pM, ≦800pM, ≦700pM, ≦600pM, ≦500pM, ≦400pM, ≦300pM, ≦200pM, ≦100pM, ≦90pM, ≦80pM, ≦70pM, ≦60pM, ≦50pM, ≦40pM, ≦30pM, ≦20pM, ≦10pM, ≦9pM, ≦8pM, ≦7pM, ≦6pM, ≦5pM, ≦4pM, ≦3pM, ≦2pM, ≦1pM D and binds to HER3 (e.g., human HER3).

[0326] The antigen-binding molecules of the present invention can bind to specific target regions of HER3. The antigen-binding region of an 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., a 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.

[0327] In some embodiments, the antigen-binding molecules of the present invention bind to HER3. In some embodiments, the antigen-binding molecules bind to the extracellular region of HER3 (e.g., the region set forth in SEQ ID NO: 9). In some embodiments, the antigen-binding molecules bind to subdomain II of the extracellular region of HER3 (e.g., the region set forth in SEQ ID NO: 16).

[0328] 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 NO: 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 in 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 in 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 in 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 in the region of HER3 set forth in SEQ ID NO: 22.

[0329] In some embodiments, antigen-binding molecules of the present invention are capable of binding to a polypeptide comprising or consisting of the amino acid sequence of one of SEQ ID NOs: 1, 3, 4, 6, or 8. In some embodiments, antigen-binding molecules are capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, antigen-binding molecules are capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 16. In some embodiments, antigen-binding molecules are capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 229. In some embodiments, antigen-binding molecules are capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NOs: 230 and 231. In some embodiments, antigen-binding molecules are capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 230. In some embodiments, antigen-binding molecules are capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 231. In some embodiments, antigen-binding molecules are capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 22.

[0330] 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 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 set forth in SEQ ID NO: 23. In some embodiments, the antigen-binding molecule does not contact amino acid residues in the region of HER3 set forth 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.

[0331] As used herein, a "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length in the region of about 2 to 50 amino acids. A "polypeptide" is a polymeric chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

[0332] 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 ELISA, immunoblot (e.g., Western blot), immunoprecipitation, surface plasmon resonance, and biolayer interferometry analysis.

[0333] Binding of a ligand to HER3 promotes a conformational change that allows HER3 to homo- or heterodimerize, resulting in activation of downstream pathways. HER3 exhibits "closed" and "open" conformations. The closed conformation means that HER3 is in a tethered conformation and is unavailable for receptor homo- or heterodimerization. The open conformation means that HER3 is in an extended conformation and is available for receptor homo- or heterodimerization.

[0334] In some embodiments, the antigen-binding molecule can bind to HER3 when HER3 is in an open conformation. In some embodiments, the antigen-binding molecule can bind to HER3 when HER3 is in a closed conformation. In some embodiments, the antigen-binding molecule can bind to HER3 when HER3 is in an open and / or closed conformation. In some embodiments, the antigen-binding molecule can bind to the extracellular domain of HER3 when HER3 is in an open and / or closed conformation. In some embodiments, the antigen-binding molecule can bind to the dimerization arm of HER3 when HER3 is in an open and / or closed conformation. Binding to the dimerization arm allows the antigen-binding molecule to block the interaction of HER3 with, for example, an interaction partner of HER3 described herein.

[0335] 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 binding of a ligand to its extracellular domain, which promotes a conformational change that allows HER3 to homo- or heterodimerize. Binding of the antigen-binding molecule to HER3 allows the antigen-binding molecule to inhibit the action of HER3 independent of ligand binding, both in the absence and presence of a ligand. In some embodiments, the antigen-binding molecule does not compete with a ligand for binding to HER3. In some embodiments, the antigen-binding molecule does not bind to HER3 at the ligand-binding site.

[0336] In some embodiments, the antigen-binding molecule binds to HER3 sufficiently similarly 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).

[0337] In some embodiments, the antigen-binding molecule binds to 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 a ligand for HER3. Example 8.10 and Figures 78A and 78B of the present disclosure demonstrate that 10D1F binds to human HER3 with sub-picomolar affinity when HER3 is provided both in an NRG1-bound form and in the absence of NRG1.

[0338] As used herein, a binding affinity that is "similar" to a reference binding affinity means a binding affinity that is within 50%, e.g., within one of 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, determined under equivalent conditions.

[0339] In some embodiments, the antigen-binding molecule is characterized by the K of the antigen-binding molecule for binding to HER3 in the absence of a ligand (determined under equivalent conditions). D within 50% of the K, e.g., within one of 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% D and binds to HER3 in the presence of a ligand for HER3 (e.g., NRG1 or NRG2).

[0340] In some embodiments, the antigen-binding molecule is characterized by the K of the antigen-binding molecule for binding to HER3 in the absence of a ligand (determined under equivalent conditions). onwithin 50% of the K, e.g., within one of 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% on and binds to HER3 in the presence of a ligand for HER3 (e.g., NRG1 or NRG2).

[0341] In some embodiments, the antigen-binding molecule is characterized by the K of the antigen-binding molecule for binding to HER3 in the absence of a ligand (determined under equivalent conditions). off within 50% of the K, e.g., within one of 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% off and binds to HER3 in the presence of a ligand for HER3 (e.g., NRG1 or NRG2).

[0342] In some embodiments, the antigen-binding molecule can bind to the same or overlapping region of HER3 as an antibody comprising the VH and VL sequences of one of the following 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. In some embodiments, the antigen-binding molecule can bind to the same region of HER3 as, or an overlapping region of HER3 as, an antibody comprising the VH and VL sequences of one of clones 10D1_c89, 10D1_c90, or 10D1_c91 binds. In some embodiments, the antigen-binding molecule can bind to the same region of HER3 as, or an overlapping region of HER3 as, an antibody comprising the VH and VL sequences of clone 10D1_c89 binds.

[0343] The region of a peptide / polypeptide to which an antibody binds can be determined by those skilled in the art using a variety of methods well known in the art, including X-ray cocrystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, the entire contents of which are incorporated herein by reference. Such methods can also be used to determine whether an antigen-binding molecule can bind to proteins in different conformations.

[0344] In some embodiments, the antigen-binding molecules of the invention do not bind to HER3 at the same or overlapping regions of HER3 as antibodies comprising the VH and VL sequences of the anti-HER3 antibody clones MM-121 (e.g., as described in Schoeberl et al., Sci. Signal. (2009) 2(77):ra31) and / or LJM-716 (e.g., as described in Garner et al., Cancer Res (2013) 73:6024-6035). In some embodiments, the antigen-binding molecules of the invention do not compete for binding to HER3 with antibodies 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.

[0345] In some embodiments, the antigen-binding molecules of the present invention bind to HER3 within a region accessible to antigen-binding molecules (i.e., extracellular antigen-binding molecules) when HER3 is expressed on the cell surface (i.e., within or on the cell membrane). In some embodiments, the antigen-binding molecules can bind to HER3 expressed on the cell surface of HER3-expressing cells. In some embodiments, the antigen-binding molecules can bind to HER3-expressing cells (e.g., HER3+ cells, e.g., HER3+ cancer cells).

[0346] 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.

[0347] The antigen-binding molecules of the present invention may be antagonists of HER3. In some embodiments, the antigen-binding molecules can inhibit a function or process (e.g., an 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, "inhibition" refers to a decrease, reduction, or decrease compared to a control condition.

[0348] In some embodiments, the antigen-binding molecules of the present invention can inhibit the interaction between HER3 and its interaction partner. The HER3 interaction partner may be expressed by the same cell as HER3. The interaction partner or HER3 may be expressed on the cell surface (i.e., in or on the cell membrane). In some embodiments, the HER3 interaction partner may be a member of the EGFR family of proteins, such as HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet. In some embodiments, the HER3 interaction partner may be IGF1R and / or cMet. The interaction between HER3 and its interaction partner may result in the formation of a polypeptide complex. The interaction between HER3 and its interaction partner to form a polypeptide complex may be referred to as multimerization. When multimerization is between polypeptide monomers, it may be referred to as dimerization.

[0349] 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.

[0350] Inhibition of interaction can be achieved by binding of an antigen-binding molecule to a region of HER3 required for interaction between HER3 and its interaction partner (e.g., the dimerization loop of HER3 set forth in SEQ ID NO: 19). In some embodiments, the antigen-binding molecule contacts one or more residues of HER3 required for interaction between HER3 and its interaction partner, thus making the region unavailable to the antigen-binding molecule, thereby inhibiting the interaction. In some embodiments, the antigen-binding molecule binds to HER3 in a manner that inhibits / blocks the interaction between HER3 and its interaction partner. In some embodiments, the antigen-binding molecule inhibits / blocks access of an interaction partner of HER3 to a region of HER3 required for interaction between HER3 and its interaction partner, which can be achieved even if the antigen-binding molecule does not contact a region of HER3 required for interaction between HER3 and its interaction partner, for example, by steric blocking of access of an interaction partner of HER3 to a region of HER3 required for interaction between HER3 and its interaction partner.

[0351] In some embodiments, the antigen-binding molecule can inhibit the homodimerization of HER3 monomers. 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.

[0352] The ability of an antigen-binding molecule to inhibit the interaction between two factors can be determined, for example, by analyzing the interaction in the presence of an antibody / fragment, or after incubating one or both of the interaction partners with 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 analysis. In some embodiments, the antigen-binding molecule is a competitive inhibitor of the interaction between HER3 and its interaction partner.

[0353] In some embodiments, an antigen-binding molecule of the invention can inhibit the interaction of HER3 with an interaction partner of HER3 (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet) in a suitable assay by 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 interaction of HER3 with the interaction partner of HER3 in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0354] The ability of an antigen-binding molecule to inhibit the interaction between interaction partners can also be determined by analyzing the downstream functional consequences of such interaction. For example, downstream functional consequences 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 an antibody that can detect phosphorylated members of the signaling pathway.

[0355] The ability of an antigen-binding molecule to inhibit the interaction of HER3 with an interacting partner of HER3 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 a change in cell number over time, or 3 H-thymidine incorporation or by the CFSE dilution assay, for example, as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564, which is incorporated herein by reference in its entirety.

[0356] In some embodiments, the antigen binding molecules of the invention can inhibit the proliferation of cells harboring a mutation to V600 of BRAF, for example, cells containing the V600E or V600K mutation of BRAF (see Example 10).

[0357] In some embodiments, the antigen-binding molecule inhibits HER3-mediated signaling. HER3-mediated signaling can be analyzed, for example, using assays for correlates 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.

[0358] In some embodiments, the antigen-binding molecules 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 and quantifying the phosphorylation of one or more components of the PI3K / AKT / mTOR and / or MAPK pathways after stimulation with NRG (see Example 4.3).

[0359] In some embodiments, antigen-binding molecules of the invention can inhibit the proliferation of HER3-expressing cells, for example, in response to stimulation with NRG. In some embodiments, antigen-binding molecules of the invention can inhibit the proliferation of HER3-expressing cells in a suitable assay by 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 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).

[0360] In some embodiments, antigen-binding molecules of the invention are capable of inhibiting PI3K / AKT / mTOR and / or MAPK signaling by HER3-expressing cells in a suitable assay by 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, the level of signaling by the HER3-expressing cells in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0361] 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.

[0362] ADCC activity can be analyzed, for example, according to the method described in Yamashita et al., Scientific Reports (2016) 6:19772 (incorporated herein by reference in its entirety), or, for example, according to the method described in Jedema et al., Blood (2004) 103:2677-82 (incorporated herein by reference in its entirety). 51 ADCC activity can also be analyzed using the Pierce LDH Cytotoxicity Assay Kit according to the manufacturer's instructions (described in Example 5 herein).

[0363] ADCPs can be analyzed, for example, according to the methods described in Kamen et al., J Immunol (2017) 198(1 Supplement) 157.17, which is incorporated herein by reference in its entirety.

[0364] The ability to induce CDC can be analyzed using, for example, a C1q binding assay, as described, for example, in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, which is incorporated herein by reference in its entirety.

[0365] 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), for example, as described in He et al., J Pharm Sci. (2010), which is incorporated herein by reference in its entirety. Thermal stability can be measured by measuring the melting temperature (T m ), unfolding temperature, or decomposition temperature (expressed, for example, in °C or F°).

[0366] 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 that is greater than 1-fold, e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or greater than 20-fold greater than the binding affinity for an activating Fcγ receptor by 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 of an antigen-binding molecule comprising an Fc region described herein for binding to an activating Fcγ receptor is D is 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 to 175 for an activating Fcγ receptor. D of the total amount of the α-tocopherol-containing compound, for example, less than 1 times, for example, 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 times, or less than 0.05 times.

[0367] In some embodiments, an antigen-binding molecule comprising an Fc region described herein has a K of 1000 nM or less, preferably one of: <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).

[0368] In some embodiments, an antigen-binding molecule comprising an Fc region described herein binds to FcRn (e.g., hFcRn, mFcRn) with a binding affinity that is 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 of an antigen-binding molecule comprising an Fc region described herein for binding to FcRn is D is 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 to 175 for FcRn. D of the total amount of the α-tocopherol-containing compound, for example, less than 1 times, for example, 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 times, or less than 0.05 times.

[0369] In some embodiments, an antigen-binding molecule comprising an Fc region described herein has a K of 1000 nM or less, preferably one of: <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).

[0370] In some embodiments, an antigen-binding molecule comprising an Fc region described herein binds to an inhibitory Fcγ receptor (e.g., hFcγRIIb, mFcγRIIb) with a binding affinity that 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, of the binding affinity to an inhibitory Fcγ receptor 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 is 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 to 175 for an inhibitory Fcγ receptor. D of more than 1-fold, e.g., more than 2, 3, 4, 5, 6, 7, 8, 9-fold, or more than 10-fold.

[0371] In some embodiments, an antigen-binding molecule comprising an Fc region described herein binds to an inhibitory Fcγ receptor (e.g., hFcγRIIb, mFcγRIIb) with a K 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. D Combine with.

[0372] In some embodiments, the binding selectivity for an activating Fcγ receptor (e.g., hFcγRIIa) relative to an inhibitory Fcγ receptor (e.g., hFcγRIIb) of an antigen-binding molecule comprising an Fc region described herein is more than 1-fold, e.g., more than 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, or more than 20-fold the binding selectivity exhibited by an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs: 174-175.

[0373] In some embodiments, antigen-binding molecules comprising an Fc region described herein exhibit ADCC that is greater than 1-fold, e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15-fold, or greater than 20-fold greater than that exhibited by an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs: 174-175.

[0374] In some embodiments, in an ADCC activity assay, the EC50 (ng / ml) determined for an antigen-binding molecule comprising an Fc region described herein is less than 1-fold, for example, 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 sequence of SEQ ID NOs: 174-175.

[0375] In some embodiments, in an assay for ADCC activity, the EC50 (ng / ml) for an antigen-binding molecule comprising an Fc region described herein is 500 ng / ml or less, preferably one of <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.

[0376] In some embodiments, an antigen-binding molecule comprising an Fc region described herein has a melting temperature, unfolding temperature, or decomposition temperature that is ≥ 0.75 and ≤ 1.25 times, for example, ≥ 0.8 and ≤ 1.2 times, ≥ 0.85 and ≤ 1.15 times, ≥ 0.9 and ≤ 1.1 times, ≥ 0.9 ... The polymer may have a melting temperature, unfolding temperature, or decomposition temperature that is 0.91x and ≦1.09x, ≧0.92x and ≦1.08x, ≧0.93x and ≦1.07x, ≧0.94x and ≦1.06x, ≧0.95x and ≦1.05x, ≧0.96x and ≦1.04x, ≧0.97x and ≦1.03x, ≧0.98x and ≦1.02x, or ≧0.99x and ≦1.01x.

[0377] In some embodiments, the antigen-binding molecules of the present invention can enhance the killing of HER3-expressing cells. The killing of HER3-expressing cells can be enhanced through the effector function of the antigen-binding molecule. In embodiments in which the antigen-binding molecule comprises an Fc region, the antigen-binding molecule can enhance the killing of HER3-expressing cells through one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0378] Antigen-binding molecules that can increase the killing of HER3-expressing cells can be identified by observing an increased 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 an appropriate assay. CDC, ADCC, and ADCP assays are well known to those skilled 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.

[0379] In some embodiments, antigen-binding molecules of the invention can increase 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, the level of killing observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0380] In some embodiments, an antigen-binding molecule of the invention can reduce the number of HER3-expressing cells (e.g., HER3-expressing cancer cells) in a comparable assay by 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, 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).

[0381] In some embodiments, the antigen-binding molecules of the invention inhibit the onset and / or progression of cancer in vivo. In some embodiments, the antigen-binding molecule causes increased killing of cancer cells, e.g., by effector immune cells. In some embodiments, the antigen-binding molecule causes a reduction in the number of cancer cells in vivo, e.g., compared to appropriate control conditions. In some embodiments, the antigen-binding molecule inhibits tumor growth, e.g., as determined by measuring tumor size / volume over time.

[0382] The antigen-binding molecules of the present invention can be analyzed for their ability to inhibit the onset and / or progression of cancer in suitable in vivo models, such as cell line-derived xenograft models. The cell line-derived xenograft models can be derived from HER3-expressing cancer cells. In some embodiments, the models are N87 cell-derived models, SNU16 cell-derived models, FaDu cell-derived models, OvCAR8 cell-derived models, HCC95 cell-derived models, A549 cell-derived models, ACHN cell-derived models, or HT29 cell-derived models.

[0383] The cancer may be a HER3-associated cancer (i.e., a cancer in which expression of the HER3 gene / protein is a risk factor and / or positively correlated with cancer occurrence, development, progression, or symptom severity, and / or metastasis), as described herein. The cancer may comprise HER3-expressing cells. In some embodiments, the cancer comprises a HER3+ tumor.

[0384] In some embodiments, administration of an antigen-binding molecule according to the invention may result in one or more of: inhibiting cancer onset / progression; delaying cancer onset / preventing cancer onset; reducing tumor growth / delaying tumor growth / preventing tumor growth; reducing metastasis / delaying metastasis / preventing metastasis; reducing the severity of cancer symptoms; reducing cancer cell number; reducing tumor size / volume; and / or increasing survival (e.g., progression-free survival) as determined, for example, in a suitable HER3-expressing cancer cell line-derived xenograft model.

[0385] In some embodiments, antigen-binding molecules of the invention are capable of inhibiting tumor growth in a xenograft model derived from a HER3-expressing cancer cell line by 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 tumor growth observed in the absence of treatment with the antigen-binding molecule (or after treatment with an appropriate negative control antigen-binding molecule).

[0386] In some embodiments, treatment of a subject with an antigen-binding molecule or other article (e.g., composition, nucleic acid, etc.) disclosed herein, e.g., when the antigen-binding molecule / article is administered to a subject at the dosages described herein and / or according to the dosing regimens described herein, may be associated with one or more of the following outcomes: · Absence of adverse events (AEs); · Absence of serious adverse events (SAEs); · The frequency of adverse events (AEs) is minimal or reduced, e.g., compared with other anti-HER3 therapeutic antibodies; · The frequency of serious adverse events (SAEs) is minimal or reduced, e.g., compared with other anti-HER3 therapeutic antibodies; · Absence of dose-limiting toxicities (DLTs); · The frequency of dose-limiting toxicities (DLTs) is minimal or reduced compared to other anti-HER3 therapeutic antibodies, such as elgemtumab and AV-203; - e.g., circulating tumor markers (e.g., reduction in cell-free (cf) DNA altered allele fraction / tumor fraction, ctDNA, soluble HER3 and / or soluble NRG1); · Reduction of pHER3, pERK, p-70S6K, Ki67 and cleaved caspase 3 after treatment, e.g., compared to the same subject before treatment; · Absence or minimal or reduced frequency of infusion-related reactions, e.g., cytokine release syndrome; · Absence or minimal or reduced incidence of gastrointestinal toxicity compared to other anti-HER3 therapeutic antibodies, such as elgemtumab or AV-203; - absence, minimal or reduced incidence of hypokalemia and / or hypomagnesemia, e.g., compared to other anti-HER3 therapeutic antibodies; -Rash or dermatitis is absent or is minimal or reduced in frequency, e.g., compared with other anti-HER3 therapeutic antibodies; Absence or minimal or reduced frequency of hematological changes, e.g., compared with other anti-HER3 therapeutic antibodies; absence, minimal or reduced incidence of cardiac toxicity, e.g., compared with other anti-HER3 therapeutic antibodies; and / or Absence, minimal frequency or reduced albumin elevation compared to other anti-HER3 therapeutic antibodies, such as GSK2849330.

[0387] An adverse event (AE) is any untoward, unwanted, or unplanned medical occurrence in a patient administered an investigational medicinal product (IMP), comparator, or approved drug. An AE may be a sign, symptom, disease, and / or laboratory or physiological observation that may or may not be related to the IMP or comparator. AEs include, but are not limited to, those listed below.

[0388] A clinically significant worsening of a pre-existing condition. An example of this would be a condition that has completely resolved and may become abnormal again. AEs caused by an excessive dose of IMP, whether accidental or intentional.

[0389] AEs caused by lack of efficacy of an IMP, for example, when an investigator suspects that a drug batch is ineffective, or when an investigator suspects that an IMP is contributing to disease progression.

[0390] Serious adverse events (SAEs) are those that occur regardless of dose, causality, or predictability. Any AE resulting in death; Any life-threatening AE, i.e., an event when the patient was at substantial risk of death at the time of the adverse event or with continued use of a device or other medicinal product that could cause the patient's death; Any AE that requires inpatient hospitalization or prolongs an existing inpatient hospitalization (some inpatient hospitalizations, e.g., hospitalizations that were planned before the patient entered the trial; overnight stays for planned procedures, such as blood transfusions, are exempt from reporting as SAEs); ·Any AE that causes persistent or significant incapacity or disability; ·Any AE that is a congenital malformation or birth defect; Any other AE that is a medically significant event, i.e., any event that may endanger the patient or require intervention to prevent one of the outcomes listed above. is.

[0391] In some embodiments, response to treatment according to the present disclosure can be characterized by reference to tumor / lesion response. In some embodiments, tumor / lesion response is assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, e.g., RECIST 1.1 criteria, as described in Eisenhauer et al., Eur J Cancer. 2009 Jan;45(2):228-47, which is incorporated herein by reference in its entirety. In some embodiments, tumor / lesion response is assessed according to Prostate Cancer Working Group 3 (PCWG3) criteria, as described in Scher et al., J Clin Oncol. 2016 Apr 20;34(12):1402-18, which is incorporated herein by reference in its entirety.

[0392] In some embodiments, treatment of a subject with an antigen-binding molecule or article described herein, e.g., when the antigen-binding molecule / article is administered to a subject at the dosages described herein and / or according to the dosing regimens described herein, may be associated with one or more of the following outcomes, e.g., at 12 and / or 24 months from the start of treatment (assessed according to RECIST 1.1 or PCWG3 criteria, as appropriate; see Example 16.10 for details and methods of assessment): Antitumor response; Complete response (CR). CR refers to the complete macroscopic disappearance of all target and / or non-target tumors. CR may also include normalization of tumor marker levels; an increased likelihood of complete response (CR), for example, compared to the likelihood of CR in the same subject not treated with the antigen binding molecule, compared to subjects not receiving the antigen binding molecule, or compared to subjects treated with a different anti-HER3 antigen binding molecule; an increase in the proportion of subjects experiencing a complete response (CR), e.g., compared to the proportion of subjects experiencing a CR who are not treated with the antigen binding molecule or who are treated with a different anti-HER3 antigen binding molecule; · Overall survival (OS), defined as the time from treatment initiation to death due to any cause; an increased likelihood of overall survival (OS), e.g., compared to the likelihood of OS in the same subject not treated with the antigen binding molecule, a subject not receiving the antigen binding molecule, or a subject treated with a different anti-HER3 antigen binding molecule; an increase in the proportion of subjects demonstrating overall survival (OS), e.g., compared to the proportion of subjects demonstrating OS who are not treated with the antigen binding molecule or who are treated with a different anti-HER3 antigen binding molecule; Progression-free survival (PFS). PFS refers to the time from the start of treatment to disease progression or death.

[0393] an increased likelihood of progression-free survival (PFS), e.g., compared to the likelihood of PFS in the same subject not treated with the antigen binding molecule, a subject not receiving the antigen binding molecule, or a subject treated with a different anti-HER3 antigen binding molecule; an increase in the proportion of subjects demonstrating progression-free survival (PFS), e.g., compared to the proportion of subjects exhibiting PFS who are not treated with the antigen binding molecule or who are treated with a different anti-HER3 antigen binding molecule; Partial response (PR). PR refers to a reduction of at least 30% in the sum of all target tumor diameters compared to the baseline sum diameter calculated before treatment; an increased likelihood of partial response (PR), e.g., compared to the likelihood of PR in the same subject not treated with the antigen binding molecule, a subject not receiving the antigen binding molecule, or a subject treated with a different anti-HER3 antigen binding molecule; an increase in the proportion of subjects demonstrating a partial response (PR), e.g., compared to the proportion of subjects showing a PR not treated with the antigen binding molecule or treated with a different anti-HER3 antigen binding molecule; · Mixed response (MR). MR refers to one or more tumor lesions that meet the criteria for PR and other tumor lesions that meet the criteria for progressive disease (an increase in the sum of all tumor diameters of at least 20% from the smallest tumor size and / or the appearance of new tumor lesions); an increased likelihood of mixed reaction (MR), e.g., compared to the likelihood of MR in the same subject not treated with the antigen-binding molecule, a subject not receiving the antigen-binding molecule, or a subject treated with a different anti-HER3 antigen-binding molecule; an increase in the proportion of subjects demonstrating a mixed response (MR), e.g., compared to the proportion of subjects exhibiting MR who are not treated with the antigen-binding molecule or who are treated with a different anti-HER3 antigen-binding molecule; Stable disease (SD). SD refers to the absence of partial response or progressive disease compared with the tumor burden at the start of treatment.

[0394] an increased likelihood of stable disease (SD), e.g., compared to the likelihood of SD in the same subject not treated with the antigen binding molecule, a subject not receiving the antigen binding molecule, or a subject treated with a different anti-HER3 antigen binding molecule; an increase in the proportion of subjects demonstrating stable disease (SD), e.g., compared to the proportion of subjects showing SD who are not treated with the antigen binding molecule or who are treated with a different anti-HER3 antigen binding molecule; · Overall response (OR), where OR is defined as achieving a complete response (CR) or partial response (PR); · Overall response rate (ORR). ORR is defined as the proportion of patients who achieve a complete response (CR) or partial response (PR); an increase in ORR, e.g., compared to the proportion of subjects exhibiting OR who are not treated with the antigen-binding molecule or who are treated with a different anti-HER3 antigen-binding molecule; · Reduction of HER3 phosphorylation in tumor tissue; · Reduced downstream pathway activation in tumor tissue, evidenced by downstream marker changes, for example, pERK and p-70SK6; · Reduction of tumor fraction in serial cfDNA samples; · Reduced tumor cell proliferation evidenced by altered Ki67 expression; Increased tumor cell death evidenced by changes in cleaved caspase 3; Demonstration of antitumor activity as evidenced through determination of overall response rate, where overall response rate is defined as the proportion of patients achieving a complete or partial response based on Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 or Prostate Working Group 3 (PCWG3) criteria, as applicable in the selected tumor type.

[0395] Tumor response can be assessed using appropriate imaging techniques according to the tumor and its location, such as CT scan, MRI scan, and FDG-PET. Suitable techniques are well known to those skilled in the art and are described in Eisenhauer et al., supra, and / or in Example 16.10 herein.

[0396] The subject may be a subject as defined herein, e.g., a subject as defined herein that has or is determined to have cancer or a solid tumor, e.g., a cancer or solid tumor according to the present disclosure.

[0397] Chimeric antigen receptor (CAR) The present invention also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule or polypeptide of the present invention.

[0398] CAR is a recombinant receptor that provides both antigen binding and T cell activation function.The structure and operation of CAR are generally described in, for example, Dotti et al., Immunol Rev (2014) 257 (1), which is incorporated herein by reference in its entirety.CAR comprises an antigen binding region that is connected to a cell membrane anchor region and a signal transduction region.Optionally, hinge region can separate the antigen binding region and the cell membrane anchor region, and can act as a flexible linker.

[0399] The CAR of the present invention comprises an antigen-binding region that comprises or consists of an antigen-binding molecule of the present invention or that comprises or consists of a polypeptide according to the present invention. The cell membrane anchor region is provided between the antigen-binding region and the signaling region of the CAR, and allows for anchoring of the CAR to the cell membrane of a cell expressing the CAR, with the antigen-binding region in the extracellular space and the signaling region inside the cell. In some embodiments, the CAR comprises a cell membrane anchor region that comprises, consists of, or is derived from the amino acid sequence of the transmembrane region of one of CD3-zeta, CD4, CD8, or CD28. As used herein, a region "derived from" a reference amino acid sequence includes an amino acid sequence that has at least 60%, e.g., 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.

[0400] The signaling region of a CAR enables T cell activation. The signaling region of a CAR may contain the amino acid sequence of the intracellular domain of CD3-zeta, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. CARs have also utilized signaling regions containing sequences from other ITAM-containing proteins, such as FcγRI (Haynes et al., 2001 J Immunol 166(1):182-187). The signaling region of a CAR may also contain a costimulatory sequence derived from the signaling region of a costimulatory molecule to promote activation of CAR-expressing T cells upon binding to a target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, CARs are engineered to provide costimulation of different intracellular signaling pathways. For example, signaling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1BB-mediated signaling is mediated by the TNF receptor-associated factor (TRAF) adaptor protein. Thus, the signaling region of a CAR may contain costimulatory sequences derived from the signaling region of more than one costimulatory molecule. In some embodiments, the CAR of the present invention comprises one or more costimulatory sequences that comprise, consist of, or are derived from the amino acid sequence of one or more intracellular domains of CD28, OX40, 4-1BB, ICOS, and CD27.

[0401] The optional hinge region can separate 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 present invention comprises a hinge region that comprises, consists of, or is derived from the amino acid sequence of the hinge region of IgG1.

[0402] 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. The engineering of CARs into immune cells can be performed during in vitro culture.

[0403] The antigen-binding region of the CAR of the present invention may be provided in any suitable format, such as, for example, scFv, scFab, etc. Nucleic acids and vectors The invention provides a nucleic acid or nucleic acids encoding an antigen-binding molecule, polypeptide, or CAR according to the invention.

[0404] In some embodiments, the nucleic acid is purified or isolated, e.g., from other nucleic acids or naturally occurring biological material. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[0405] The present invention also provides a vector or vectors comprising a nucleic acid or nucleic acids according to the invention. The nucleotide sequence may be contained within a vector, for example, an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle for transferring exogenous nucleic acids into cells. The vector may be a vector for expressing a nucleic acid within a cell. Such a vector may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present invention.

[0406] The term "operably linked" can include the situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked to place expression of the nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of effecting transcription of the nucleic acid sequence. The resulting transcription product can then be translated into a desired peptide / polypeptide.

[0407] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gammaretroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0408] In some embodiments, the vector may be a eukaryotic vector, e.g., a vector containing elements necessary for expression of a protein from a vector in a eukaryotic cell, hi some embodiments, the vector may be a mammalian vector, e.g., containing a cytomegalovirus (CMV) or SV40 promoter to drive expression of the protein.

[0409] The polypeptides that are components of the antigen-binding molecules according to the present invention can be encoded by different nucleic acids among the plurality of nucleic acids or different vectors among the plurality of vectors. Antigen-binding molecules and cells containing / expressing polypeptides The present invention also provides cells comprising or expressing an antigen-binding molecule, polypeptide, or CAR according to the invention. Also provided are cells comprising or expressing a nucleic acid, multiple nucleic acids, single vector, or multiple vectors according to the invention.

[0410] The cell can be a eukaryotic cell, e.g., 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 in the order Murine), cat, dog, pig, sheep, goat, cattle (including bovine, e.g., dairy cow, or any animal in the order Bovidae), horse (including any animal in the order Equine), donkey, and non-human primate).

[0411] The present invention also provides methods for producing cells containing nucleic acids or vectors in accordance with the invention, comprising introducing into a cell a nucleic acid, multiple nucleic acids, vector, or multiple vectors in accordance with the invention. In some embodiments, introducing into a cell an isolated nucleic acid or vector in accordance with the invention comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0412] The present invention also provides a method for producing a cell that expresses / contains an antigen-binding molecule, polypeptide, or CAR according to the present invention, comprising introducing into the cell a nucleic acid, multiple nucleic acids, single vector, or multiple vectors according to the present 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.

[0413] The present invention also provides a cell obtained or obtainable by a method according to the invention. Production of antigen-binding molecules and polypeptides Antigen-binding molecules and polypeptides according to the present invention can be prepared according to methods for producing polypeptides known to those skilled in the art.

[0414] Polypeptides can be prepared by chemical synthesis, e.g., liquid phase or solid phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described in, for example, Chandrudu et al., Molecules (2013), 18:4373-4388, which is incorporated herein by reference in its entirety.

[0415] Alternatively, antigen-binding molecules and polypeptides can also be produced by recombinant expression.Molecular biology techniques suitable for the recombinant production of polypeptides are well known in the art, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Press, 2012, and Nat Methods.(2008);5(2):135-146 (both of which are incorporated herein by reference in their entirety).Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol.(2013);4:217, and Kunert and Reinhart, Appl Microbiol Biotechnol.(2016)100:3451-3461 (both of which are incorporated herein by reference in their entirety).

[0416] In some cases, an antigen-binding molecule of the present invention is composed of more than one polypeptide chain. In such cases, production of the antigen-binding molecule may involve transcription and translation of more than one polypeptide and subsequent assembly of the polypeptide chains to form the antigen-binding molecule.

[0417] For recombinant production according to the present invention, any cell suitable for expressing 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 may be a gram-negative bacterium such as a bacterium of the Enterobacteraceae family, e.g., 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, e.g., a CHO, HEK (e.g., HEK293), HeLa, or COS cell. In some embodiments, the cell is a CHO cell that transiently or stably expresses a polypeptide.

[0418] In some cases, the cells are not prokaryotic because some prokaryotic cells are unable to fold or post-translationally modify proteins in the same way as eukaryotic cells. In addition, extremely high expression levels are possible in eukaryotic cells, and proteins can be easily purified from eukaryotic cells using appropriate tags. Specific plasmids that enhance the secretion of proteins into the medium can also be used.

[0419] In some embodiments, polypeptides may be prepared by cell-free protein synthesis (CFPS), for example, according to the use of the system described in Zemella et al., Chembiochem (2015) 16(17):2420-2431, which is incorporated herein by reference in its entirety.

[0420] Production may involve the culture or fermentation of eukaryotic cells modified to express the polypeptide of interest. The culture or fermentation may be carried out in a bioreactor with an appropriate supply of nutrients, air / oxygen, and / or growth factors. The secreted protein may be recovered by fractionating the culture medium / fermentation broth from the cells, extracting the protein content, separating the individual proteins, and isolating the secreted polypeptide. Culture, fermentation, and separation techniques are well known to those of skill in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.; incorporated herein by reference).

[0421] A bioreactor contains one or more reaction vessels in which cells can be cultured. Cultivation in a bioreactor can be carried out continuously by continuously inflowing reactants into the reactor and continuously effluent of cultured cells from the reactor. Alternatively, cultivation can be carried out in batch mode. Bioreactors monitor and control environmental conditions such as pH, oxygen, flow rates into and out of the reactor, and agitation within the reactor to provide optimal conditions for the cultured cells.

[0422] After culturing cells that express antigen-binding molecules / polypeptides, the polypeptide of interest can be isolated. Any suitable method for separating proteins from cells known in the art can be used. To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. 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 is collected intracellularly, protein isolation can include centrifugation to separate the cells from the cell culture medium, treating the cell pellet with a lysis buffer, and disrupting the cells, for example, by sonication, rapid freeze-thawing, or osmotic lysis.

[0423] It may then be desirable to isolate the polypeptide of interest from the supernatant or culture medium, which may contain other proteins and non-protein components. A common method for separating protein components from the supernatant or culture medium is by precipitation. Proteins with different solubility are precipitated with different concentrations of precipitants, such as ammonium sulfate. For example, low concentrations of precipitants extract water-soluble proteins. Therefore, by adding increasing concentrations of precipitants, proteins with different solubility can be distinguished. Dialysis can then be used to remove ammonium sulfate from the separated proteins.

[0424] Other methods for distinguishing between different proteins are known in the art, such as ion exchange chromatography and size chromatography, which can be used as alternatives to precipitation or can be performed following precipitation.

[0425] Once the polypeptide of interest has been isolated from the culture, it may be desirable or necessary to concentrate the polypeptide. Numerous methods are known in the art for concentrating proteins, such as ultrafiltration or lyophilization.

[0426] composition The present invention also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein.

[0427] 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, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal administration routes, which may include injection or infusion.

[0428] Suitable formulations may include antigen-binding molecules in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in a fluid, including a gel form. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) to a selected area within the human or animal body.

[0429] In some embodiments, the composition is formulated for injection or infusion, for example, into a blood vessel or tumor. Also provided are methods for producing pharmaceutically useful compositions in accordance with the invention described herein, which may include one or more steps selected from: producing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein; and / or mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0430] For example, a further aspect of the invention described herein relates to a method of formulating or producing a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., cancer), comprising the step of formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0431] In aspects and embodiments of the present disclosure, the antigen-binding molecule may be provided in the form of a composition comprising specific chemical moieties at specified concentrations / ratios. In some embodiments, the antigen-binding molecule is provided in the form of a buffer solution. As used herein, "buffer" refers to a buffered solution that is resistant to pH changes due to the action of its acid-base conjugate components. The buffer solution of the present disclosure preferably has a pH in the range of about 4.5 to about 7.0, and preferably in the range of about 5.0 to about 6.5. Examples of buffers that control the pH within this range include acetate, histidine, histidine-arginine, histidine-methionine, and other organic acid buffers.

[0432] In some embodiments, the composition comprising the antigen-binding molecule has a pH of 4.0 to 7.0, for example, one of pH 4.5 to 6.8, pH 4.6 to 6.4, pH 4.8 to 6.2, or pH 5.0 to 6.2. In some embodiments, the composition has a pH of about 5.5. In some embodiments, the composition has a pH of about 5.8. In some embodiments, the composition has a pH of about 6.5.

[0433] In some embodiments, the antigen-binding molecule is provided in the form of an acetate buffer, i.e., a buffer containing acetate ions. In some embodiments, the antigen-binding molecule is provided in the form of a composition containing acetate at a final acetate concentration of 2 mM to 200 mM, for example, at one of a final concentration of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM acetate.

[0434] In some embodiments, the antigen-binding molecule is provided in the form of a histidine buffer, i.e., a buffer containing histidine ions. In some embodiments, the antigen-binding molecule is provided in the form of a composition containing histidine at a final concentration of 2 mM to 200 mM, for example, at one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM histidine.

[0435] In some embodiments, the antigen-binding molecule is provided in the form of a sodium phosphate buffer, i.e., a buffer containing sodium and phosphate ions. In some embodiments, the antigen-binding molecule is provided in the form of a composition containing sodium phosphate at a final sodium phosphate concentration of 2 mM to 200 mM, for example, at one of a final concentration of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM sodium phosphate.

[0436] In some embodiments, the antigen-binding molecule is provided in the form of a sodium acetate buffer, i.e., a buffer containing sodium and acetate ions. In some embodiments, the antigen-binding molecule is provided in the form of a composition containing sodium acetate at a final concentration of 2 mM to 200 mM sodium acetate, for example, at one of a final concentration of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM sodium acetate.

[0437] In some embodiments, the antigen-binding molecule is provided in the form of an arginine buffer, i.e., a buffer containing arginine ions. In some embodiments, the antigen-binding molecule is provided in the form of a composition containing arginine at a final concentration of 1 mM to 250 mM, for example, at one of a final concentration of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM arginine.

[0438] In some embodiments, the antigen-binding molecule is provided in the form of a histidine-arginine buffer, i.e., a buffer containing histidine and arginine ions. In some embodiments, the antigen-binding molecule is provided in the form of a composition containing histidine at a final concentration of 2 mM to 200 mM, e.g., 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM, and arginine at a final concentration of 1 mM to 300 mM, e.g., 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 mM to 175 mM. In some embodiments, the composition may contain about 20 mM histidine and about 150 mM arginine.

[0439] In some embodiments, the antigen-binding molecule is provided in the form of a composition comprising sodium chloride. The sodium chloride component of the composition may be provided at a final sodium chloride concentration of 1 mM to 250 mM, for example, at one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 mM to 175 mM. In some embodiments, the composition may contain about 150 mM sodium chloride.

[0440] In some embodiments, the antigen-binding molecule is provided in the form of a composition containing methionine. The methionine component of the composition may be provided at a final concentration of 1 mM to 250 mM methionine, for example, at one of a final concentration of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM. In some embodiments, the composition may contain about 150 mM methionine.

[0441] In some embodiments, a composition comprising an antigen-binding molecule comprises an isotonicity agent. The isotonicity agent can be used to provide an isotonic formulation. Examples of isotonicity agents include salts (e.g., sodium chloride, potassium chloride) and sugars (e.g., sucrose, glucose, trehalose).

[0442] In some embodiments, the antigen-binding molecule is provided in the form of a composition containing sucrose. The sucrose component of the composition may be provided at a final concentration (weight / volume) of 2% to 20%, for example, 2% to 15%, 3% to 12%, or 4% to 10%. In some embodiments, the composition may contain about 2, about 4, about 6, or about 8% (w / v) sucrose. The sucrose component of the composition may be provided at a final concentration of 200 to 300 nM, for example, about 240 mM.

[0443] In some embodiments, a composition comprising an antigen-binding molecule comprises a surfactant. As used herein, "surfactant" refers to a substance that lowers interfacial tension. The surfactant is preferably a nonionic surfactant. Examples of surfactants include polysorbates (polysorbate-20, polysorbate-80), poloxamer (poloxamer-188), and Triton X-100. The surfactant is preferably present in the composition in a range of about 0.001% (w / v) to about 0.5% (w / v).

[0444] In some embodiments, the antigen-binding molecule is provided in the form of a composition comprising polysorbate-20. The polysorbate-20 component of the composition may be provided at a final concentration (weight / volume) of 0.001% to 0.1%, e.g., 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%. In some embodiments, the composition may comprise about 0.02% (w / v) polysorbate-20. In some embodiments, the composition may comprise about 0.05% (w / v) polysorbate-20.

[0445] In some embodiments, the antigen-binding molecule is provided in the form of a composition comprising polysorbate-80. The polysorbate-80 component of the composition may be provided at a final concentration (weight / volume) of 0.001% to 0.1%, e.g., 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%. In some embodiments, the composition may comprise about 0.01% (w / v) polysorbate-80. In some embodiments, the composition may comprise about 0.02% (w / v) polysorbate-80.

[0446] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably a pH of about 5.8.

[0447] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.1.

[0448] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 1% to 20% (e.g., one of 1% to 15%, 2% to 10%, or 3% to 8%) sucrose (w / v), more preferably about 4% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.8.

[0449] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 0.5% to 10% (e.g., one of 1% to 8%, 1.5% to 5%, or 1.8% to 3%) sucrose (w / v), more preferably about 2% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.3.

[0450] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 6.1.

[0451] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-20 (w / v), more preferably about 0.02% (w / v) polysorbate-20; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.8.

[0452] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-20 (w / v), more preferably about 0.02% (w / v) polysorbate-20; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.5.

[0453] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) phosphate, such as sodium phosphate, more preferably about 20 mM phosphate; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 6.5.

[0454] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) acetate, such as sodium acetate, more preferably about 20 mM acetate; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.5.

[0455] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) acetate, more preferably about 20 mM acetate; 1 mM to 250 mM (e.g., one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM) sodium chloride, more preferably about 150 mM sodium chloride; 0.001% to 0.1% (e.g., one of 0.002% to 0.09%, 0.006% to 0.08%, or 0.008% to 0.07%) polysorbate-20 (w / v), more preferably about 0.05% (w / v) polysorbate-20; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.5.

[0456] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 1 mM to 250 mM (e.g., one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM) arginine, more preferably about 150 mM arginine; 0.001% to 0.1% (e.g., one of 0.002% to 0.09%, 0.006% to 0.08%, or 0.008% to 0.07%) polysorbate-20 (w / v), more preferably about 0.05% (w / v) polysorbate-20; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 6.5.

[0457] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 1 mM to 250 mM (e.g., one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM) arginine, more preferably about 150 mM arginine; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 6.5.

[0458] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 1 mM to 250 mM (e.g., one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM) sodium chloride, more preferably about 150 mM sodium chloride; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 6.5.

[0459] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.5.

[0460] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 6.5.

[0461] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) succinate, such as sodium succinate, more preferably about 20 mM succinate; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; It is provided in the form of a composition having a pH of 4.0 to 7.0 (for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.5.

[0462] The composition may contain about 0.5 mg / mL to about 100 mg / mL of the antigen-binding molecule. The composition may contain about 0.5 mg / mL to about 80 mg / mL of the antigen-binding molecule. The composition may contain about 0.75 mg / mL to about 70 mg / mL of the antigen-binding molecule. The composition may contain about 1 mg / mL to about 60 mg / mL of the antigen-binding molecule. The composition may contain about 1.2 mg / mL to about 50 mg / mL of the antigen-binding molecule.

[0463] The antigen-binding molecule may be formulated, for example, in the form of a composition according to the present disclosure, at a concentration of about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, or about 70 mg / mL, or any range of concentrations therein. The antigen-binding molecule may be formulated, for example, in the form of a composition according to the present disclosure, at a concentration of about 50 mg / mL.

[0464] The antigen-binding molecule may be formulated, for example, in the form of a composition according to the present disclosure, at a concentration of about 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, or 2.0 mg / mL. The antigen-binding molecule may be formulated, for example, in the form of a composition according to the present disclosure, at a concentration of about 1.2 mg / mL.

[0465] The 50 mg / mL antigen-binding molecule solution may be diluted using any suitable excipient prior to administration. In some embodiments, the 50 mg / mL antigen-binding molecule solution is diluted with 0.9% sodium chloride (NaCl) for administration. In some embodiments, the 50 mg / mL antigen-binding molecule solution is diluted to a concentration of at least 1.2 mg / mL, e.g., in a volume of 100 to 250 mL, for administration. In some embodiments, the diluted antigen-binding molecule solution is then administered to a subject, e.g., via intravenous administration, using a dosage / administration regimen according to the present disclosure, e.g., to treat a disease / condition according to the present disclosure. In some embodiments, the diluted antigen-binding molecule solution is administered to a subject within 48 hours of the initial dilution step.

[0466] Therapeutic and prophylactic applications The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells, and compositions described herein are used in therapeutic and prophylactic methods.

[0467] 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, 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.

[0468] The method may be effective in reducing the onset or progression of a disease / condition, alleviating symptoms of a disease / condition, or reducing the pathology of a disease / condition. The method may be effective in preventing the progression of a disease / condition, for example, preventing the worsening of a disease / condition or slowing the rate of onset of a disease / condition. In some embodiments, the method may result in an improvement of a disease / condition, for example, a reduction in symptoms of a disease / condition, or a reduction in some other correlates of the severity / activity of a disease / condition. In some embodiments, the method may prevent the onset of a later stage of a disease / condition (e.g., chronic or metastatic).

[0469] It will be understood that the articles of the present invention can be used for the treatment / prevention of any disease / condition that derives therapeutic or preventive benefit from reducing the number and / or activity of cells expressing HER3. For example, the disease / condition can be a disease / condition in which cells expressing HER3 are pathologically involved, such as a disease / condition in which an increase in the number / proportion of cells expressing HER3 is positively associated with the occurrence, development, or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition, or a disease / condition in which an increase in the number / proportion of cells expressing HER3 is a risk factor for the occurrence, development, or progression of the disease / condition.

[0470] In some embodiments, the disease / condition treated / prevented in accordance with the present invention is, for example, a disease / condition characterized by an increase in the number / proportion / activity of cells expressing HER3 compared to the number / proportion / activity of cells expressing HER3 in the absence of the disease / condition.

[0471] In some embodiments, the disease / condition being treated / prevented is cancer. Cancer can be any unwanted cell proliferation (or any disease that manifests itself due to 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. The cancer can be, for example, a cancer of tissue / cells derived from 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, omentum, 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, tonsils, trachea, uterus, vulva, and / or leukocytes.

[0472] The tumors to be treated can be nervous system or non-nervous system tumors.Nervous system tumors, such as glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma, can originate from the central or peripheral nervous system.Non-nervous system cancers / tumors can originate from any other non-nervous tissue, including melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, blood cancer, and sarcoma.

[0473] HER3 and its association with and 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 entireties.

[0474] In some embodiments, the cancer is selected from cancer comprising cells that express HER3, a solid tumor, breast cancer, breast cancer, ductal carcinoma, gastric cancer, gastric adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, serous ovarian adenocarcinoma, renal 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, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma, and thymoma.

[0475] In some embodiments, the cancer treated in accordance with the present invention is selected from a 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 (e.g., triple-negative breast cancer), ovarian cancer (e.g., ovarian cancer), lung cancer (e.g., NSCLC, lung adenocarcinoma, squamous cell lung carcinoma), melanoma, prostate cancer (e.g., castration-resistant prostate cancer), oral cancer (e.g., oropharyngeal cancer), kidney cancer (e.g., renal cell carcinoma), or colorectal cancer (e.g., colorectal carcinoma, RSA wild-type colorectal carcinoma), esophageal cancer, pancreatic cancer, solid tumors, and / or liquid tumors.

[0476] In some embodiments, the cancer to be treated according to the present invention is a HER3-expressing / overexpressing solid cancer selected from gastric cancer (e.g., gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), breast cancer (e.g., triple-negative breast cancer), ovarian cancer (e.g., ovarian cancer), lung cancer (e.g., NSCLC, lung adenocarcinoma, squamous cell lung carcinoma, invasive mucinous adenocarcinoma), melanoma, prostate cancer (e.g., castration-resistant prostate cancer), oral cancer (e.g., oropharyngeal cancer), colorectal cancer (e.g., colorectal carcinoma; RAS wild-type colorectal cancer), esophageal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, or hepatocellular carcinoma (HCC). The cancer may be metastatic.

[0477] Treatment / prevention may aim at one or more of delaying / preventing the onset / progression of cancer symptoms, reducing the severity of cancer symptoms, reducing survival / growth / invasion / metastasis of cancer cells, reducing the number of cancer cells, and / or prolonging the survival of the subject.

[0478] 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 comparable non-cancerous cells / non-tumor tissue.

[0479] In some embodiments, the cancer to be treated / prevented comprises cells that express HER3 and another EGFR family member (e.g., EGFR, HER2, or HER4). In some embodiments, the cancer to be treated / prevented comprises cells that overexpress HER3 and overexpress another EGFR family member (e.g., EGFR, HER2, or HER4). Overexpression of HER3 / another EGFR family member can be determined by detecting a level of expression of HER3 / another EGFR family member that is greater than the level of expression by comparable non-cancerous cells / non-tumor tissue.

[0480] Expression can be determined by any suitable means. Expression can be gene expression (for example, transcriptional upregulation) or protein expression. Gene expression can be determined, for example, by quantitative real-time PCR (qRT-PCR), for example, by detecting the 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.

[0481] 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 an expression level of HER3 that exceeds the level of expression by comparable non-cancerous cells / non-tumor tissues.

[0482] In some embodiments, patients may be selected for the treatments described herein based on, for example, detection of a HER3-expressing or HER3-overexpressing cancer in a sample obtained from the patient.

[0483] In some embodiments, patients can be selected for the treatments described herein based on detection of a cancer that expresses HER3 and another EGFR family member (e.g., EGFR, HER2, or HER4) or overexpresses HER3 and another EGFR family member (e.g., EGFR, HER2, or HER4), e.g., in a sample obtained from the patient.

[0484] In some embodiments, the cancer being treated / prevented comprises cells that express a ligand for HER3 (e.g., NRG1 and / or NRG2). In some embodiments, the cancer being treated / prevented comprises cells that express a level of expression of NRG1 and / or NRG2 that exceeds the level of expression by comparable non-cancerous cells / non-tumor tissues. The cancer may also be described as comprising cells that overexpress NRG1 and / or NRG2.

[0485] The HER3-binding antigen-binding molecules described herein are demonstrated to bind to HER3 with extremely high affinity when HER3 is bound to NRG (i.e., when HER3 is provided in an "open" conformation), and also when HER3 is not bound to NRG (i.e., when HER3 is provided in a "closed" conformation).

[0486] Therefore, the antigen-binding molecules of the present invention are particularly useful for treating / preventing cancers characterized by HER3 ligand expression / overexpression, for example, cancers / tumors comprising cells that express / overexpress a ligand for HER3.

[0487] In some embodiments, patients can be selected for treatment as described herein based on detection of a cancer characterized by HER3 ligand expression / overexpression, e.g., a cancer comprising cells that express / overexpress NRG1 and / or NRG2, e.g., in a sample obtained from the subject. Selection based on detection of HER3 ligand expression / overexpression can be combined with selection based on detection of HER3 and / or another EGFR family member (e.g., EGFR, HER2, or HER4).

[0488] In some embodiments, cancers treated according to the present invention comprise cells harboring a genetic variant (e.g., a mutation) that causes increased (gene and / or protein) expression of a ligand to HER relative to comparable cells harboring 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 larger translocation / rearrangement of a nucleotide sequence relative to the reference allele.

[0489] Mutations that "result in" increased expression of a ligand for HER3 may be known or predicted to cause, or may be associated with, increased gene / protein expression of a ligand for HER3. Mutations that result in increased expression of a ligand for HER3 may be referred to as "activating" mutations.

[0490] 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 and / or encoded by the genomic nucleic acid of a comparable cell that does not carry the mutation. That is, the "increased expression" may not be derived from expression, as the ligand for HER3 may be a neoantigen that arises as a result of the mutation. By way of example, cells containing a CD74-NRG1 gene fusion exhibit increased expression of the CD74-NRG1 fusion polypeptide encoded by the gene fusion compared to cells lacking the CD74-NRG1 gene fusion.

[0491] A mutation that causes increased expression of a ligand for HER3 can result in increased gene or protein expression of a ligand for HER3 expressed and / or encoded by genomic nucleic acid of a comparable cell that does not contain the mutation. By way of example, a cell can contain a mutation that results in an increased level of transcription of a nucleic acid encoding NRG1 compared to the level of transcription of the nucleic acid encoding NRG1 by a comparable cell that does not contain the mutation.

[0492] In some embodiments, a mutation that causes increased expression of a ligand for HER3 may cause increased gene expression of the ligand for HER3 compared to a comparable cell that does not contain the mutation. In some embodiments, a mutation that causes increased expression of a ligand for HER3 may cause increased protein expression of the ligand for HER3 compared to a comparable cell that does not contain the mutation.

[0493] In some embodiments, a mutation that causes increased expression of a ligand for HER3 may cause increased levels of a ligand for HER3 on or at the cell surface of cells containing the mutation compared to comparable cells that do not contain the mutation. In some embodiments, a mutation that causes increased expression of a ligand for HER3 may cause increased levels of secretion of a ligand for HER3 from cells containing the mutation compared to comparable cells that do not contain the mutation.

[0494] Cells that have increased expression of a ligand for HER3 compared to the level of expression of a ligand for HER3 by a reference cell (e.g., as a result of a mutation) can be described as "overexpressing" the ligand for HER3 or having "upregulated expression" of the ligand for HER3. For example, a cancer containing cells that harbor a mutation that results in increased expression of a ligand for HER3 compared to comparable cells lacking the mutation can be described as a cancer containing cells that exhibit overexpression / upregulated expression of a ligand for HER3. In some embodiments, the reference cells lacking the mutation can be non-cancerous cells (e.g., of a comparable cell type) or cancerous cells (e.g., of a comparable cancer type).

[0495] As used herein, a "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, a ligand for HER3 binds to HER3 by interacting with domains I and / or III of HER3. Exemplary ligands for HER3 include neuregulins, such as NRG1 and NRG2, which bind to HER3 through interaction of their EGF-like domains with the ligand-binding region of HER3.

[0496] The HER3 ligand preferably binds to the HER3 receptor and / or a receptor complex containing HER3 and can induce signal transduction therethrough. As is apparent from the present disclosure, the receptor complex containing HER3 may further include an interaction partner for HER3 described herein, such as HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet.

[0497] In some embodiments, the ligand for HER3 can bind to a HER3 receptor / receptor complex expressed by a cell other than a cell having increased expression of the HER3 ligand. For example, in some embodiments, the ligand for HER3 can bind to a HER3-expressing cancer cell.

[0498] In some embodiments, the ligand for HER3 is capable of binding to a HER3 receptor / receptor complex expressed by cells that have increased expression of a HER3 ligand.

[0499] In some embodiments, the cancer to be treated comprises (i) cells that express HER3 and (ii) cells that express a ligand for HER3 (e.g., have increased expression of a ligand for HER3 as a result of a mutation that results in increased expression of the ligand for HER3).

[0500] In some embodiments, the cancer to be treated comprises cells that (i) express HER3 and (ii) also express a ligand for HER3 (e.g., have increased expression of a ligand for HER3 as a result of a mutation that results in increased expression of the ligand for HER3).

[0501] 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 may comprise 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.

[0502] In some embodiments, the ligand for HER3 comprises an EGF-like domain capable of binding to HER3, or a HER3-binding fragment thereof. In some embodiments, the HER3-binding EGF-like domain / fragment is or is derived from 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).

[0503] EGF family members contain one or more repeats of the conserved amino acid sequence set forth in SEQ ID NO: 240, which contains six cysteine ​​residues that form three intramolecular disulfide bonds that result in three structural loops required for high-affinity binding to their cognate receptors (see Harris et al., Experimental Cell Research (2003) 284(1):2-13). In some embodiments, a ligand for HER3 contains one or more copies of the amino acid sequence that matches the consensus sequence set forth in SEQ ID NO: 240.

[0504] Exemplary ligands for HER3 include neuregulins (NRGs). Neuregulins include NRG1, NRG2, NRG3, and NRG4. The amino acid sequence of human NRG1 (alpha isoform) is set forth 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), contain an EGF-like domain set forth in SEQ ID NO: 233, through which they bind to HER3. The amino acid sequence of human NRG2 (isoform 1) is set forth 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), contain an EGF-like domain as set forth in SEQ ID NO: 235, through which they bind to HER3. The amino acid sequence of human NRG3 is set forth in SEQ ID NO: 236, and the EGF-like domain of human NRG3 is set forth in SEQ ID NO: 237. The amino acid sequence of human NRG4 is set forth in SEQ ID NO: 238, and the EGF-like domain of human NRG3 is set forth in SEQ ID NO: 239. In some embodiments, the NRG is selected from NRG1, NRG2, NRG3, and NRG4. In some embodiments, the NRG is selected from NRG1 and NRG2.

[0505] 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 an 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 NOs: 233, 235, 237, or 239.

[0506] 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).

[0507] 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., an 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 an NRG (e.g., NRG1, NRG2, NRG3, or NRG4, e.g., NRG1 or NRG2).

[0508] In some embodiments, the ligand for HER3 is not an EGFR family protein (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, cMet).

[0509] In some embodiments, the mutation that results in increased expression of a ligand for HER3 is an NRG gene fusion. In some embodiments, the ligand for HER3 is a product of an NRG gene fusion (i.e., a polypeptide encoded by an NRG gene fusion). In some embodiments, the cancer comprises cells that have an NRG gene fusion.

[0510] As used herein, "NRG gene fusion" refers to a genetic variant that encodes a polypeptide that includes (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.

[0511] It will be understood that the NRG gene fusion preferably encodes a 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 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 EGF-like domain of the NRG protein.

[0512] In some embodiments, the NRG gene fusion encodes a fusion polypeptide that includes a transmembrane domain. In some embodiments, the NRG gene fusion encodes a fusion polypeptide that includes a transmembrane domain of a protein other than an NRG protein.

[0513] 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 can bind to HER3 and 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 EGF-like domain of NRG1.

[0514] 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 incorporated by reference in their entirety. The diversity of NRG1 gene fusions may be due to NRG1, which is located on chromosome 8, which is particularly prone to genomic translocation events (Adelaide et al., Genes Chromosomes Cancer. (2003) 37(4):333-45).

[0515] In some embodiments, the NRG1 gene fusion is selected from the group consisting of 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 In some embodiments, the NRG1 gene fusion is selected from the group consisting of 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. CD74-NRG1 gene fusions are 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. DOC4-NRG1 gene fusions are described, for example, in Liu et al., Oncogene. (1999) 18(50):7110-4 and Wang et al., Oncogene. (1999) 18(41):5718-21. SLC3A2-NRG1 gene fusions are 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. RBPMS-NRG1, WRN-NRG1, RAB2IL1-NRG1, and SDC4-NRG1 gene fusions are described, for example, in Dhanasekaran et al., Nat Commun. (2014) 5:5893. VAMP2-NRG1 gene fusions are 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.KIF13B-NRG1 gene fusions are described, for example, in Xia et al., Int J Surg Pathol. (2017) 25(3):238-240. 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. 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. ATP1B1-NRG1 gene fusions are 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. CLU-NRG1 gene fusions are 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.

[0516] 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 can bind to HER3 and 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 EGF-like domain of NRG2.

[0517] 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.

[0518] Cancers comprising cells with mutations that result in increased expression of a ligand for HER3 (e.g., comprising cells with 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 cancers can be cancers of tissues / cells derived from the lung, breast, head, neck, kidney, ovary, pancreas, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue, or nasopharynx.

[0519] In some embodiments, the cancer comprising cells with a mutation resulting in increased expression of a ligand for HER3 (e.g., comprising cells with 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, breast cancer, invasive breast cancer, head and neck cancer, head and neck squamous cell carcinoma, renal 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, bile duct carcinoma, colorectal cancer, bladder cancer, urothelial bladder cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor, and nasopharyngeal neuroendocrine tumor.

[0520] In certain embodiments, the cancer treated according to the present invention is a lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, or lung squamous cell carcinoma) comprising cells with NRG1 gene fusions.

[0521] It will be understood that in embodiments herein, a cancer comprising cells with particular characteristics may be or may comprise a tumor comprising cells with these characteristics. As is common in the art, a cancer / tumor containing cells with particular characteristics may be referred to herein simply as a cancer / tumor having these characteristics. For illustrative purposes, a cancer / tumor containing cells with an NRG1 gene fusion may be referred to simply as an "NRG1 gene fusion-containing cancer / tumor" or an "NRG1 gene fusion cancer / tumor."

[0522] Administration of the articles of the present invention is preferably in a "therapeutically effective" or "prophylactically effective" amount, that is, an amount sufficient to provide a therapeutic or prophylactic benefit to the subject. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular article being administered. Prescribing treatment, such as determining dosage, is within the responsibility of general practitioners and other medical professionals 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 practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0523] Administration may be alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated. The antigen-binding molecules or compositions described herein and the therapeutic agents may be administered simultaneously or sequentially.

[0524] In some embodiments, the method includes further therapeutic or prophylactic intervention, for example, for the treatment / prevention of cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiation therapy, surgery, vaccination, and / or hormonal therapy. In some embodiments, the therapeutic or prophylactic intervention includes leukapheresis. In some embodiments, the therapeutic or prophylactic intervention includes stem cell transplantation.

[0525] In some embodiments, the antigen binding molecule is administered in combination with an agent capable of inhibiting signaling mediated by an EGFR family member. Accordingly, the present invention provides compositions comprising an article according to the invention (e.g., an antigen-binding molecule according to the invention) and another agent capable of inhibiting signaling mediated by an EGFR family member (e.g., EGFR, HER2, HER3, or HER4). Also provided is the use of such compositions in methods for the medical treatment and prevention of the diseases / conditions described herein.

[0526] Also provided are methods for treating / preventing the diseases / conditions described herein, comprising administering an article of the invention (e.g., an antigen-binding molecule of the invention) and another agent capable of inhibiting signaling mediated by an EGFR family member.

[0527] Agents capable of inhibiting signal transduction mediated by EGFR family members are known in the art 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 EGFR family members include agents that inhibit signal transduction through a direct effect on an interacting partner that is an EGFR family member, and / or that also inhibit downstream factors involved in signal transduction mediated by EGFR family members.

[0528] In some embodiments, the antagonist of signal transduction mediated by EGFR family members 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, the entire contents of which are incorporated herein by reference. 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, durigotuzumab, or matuzumab). In some embodiments, the antagonist is an inhibitor of HER2-mediated signaling (e.g., trastuzumab, pertuzumab, lapatinib, neratinib, afatinib, dacomitinib, MM-111, MCLA-128, or margetuximab). In some embodiments, the antagonist is an inhibitor of HER3-mediated signaling (e.g., seribantumab, lumletuzumab, elgemtumab, KTN3379, AV-203, GSK2849330, REGN1400, MP-RM-1, EV20, durigotuzumab, MM-111, istiratumab, MCLA-128, patritumab, EZN-3920, RB200, or U3-1402). In some embodiments, the antagonist is an inhibitor of HER4-mediated signaling (eg, lapatinib, ibrutinib, afatinib, dacomitinib, or neratinib).

[0529] In some embodiments, the antagonist of EGFR family member-mediated signal transduction inhibits downstream effectors of EGFR family member-mediated signal transduction. Downstream effectors of EGFR family member-mediated signal transduction include, for example, PI3K, AKT, KRAS, BRAF, MEK / ERK, and mTOR. In some embodiments, the antagonist of EGFR family member-mediated signal transduction is an inhibitor of the MAPK / ERK pathway. In some embodiments, the antagonist of EGFR family member-mediated signal transduction is an inhibitor 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 sapanisertib).

[0530] In some embodiments, the cancer treated according to aspects of the invention (including monotherapy or combination therapy) is a cancer that is resistant to treatment with an antagonist of signaling mediated by an EGFR family member (e.g., EGFR, HER2, HER4, and / or HER3), such as an antagonist described in the preceding three paragraphs. In some embodiments, the subject being treated has a cancer that is resistant to treatment with an antagonist of signaling 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 signaling mediated by an EGFR family member. In some embodiments, the subject being treated has a cancer that previously responded to treatment with an antagonist of signaling 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 signaling mediated by an EGFR family member. In some embodiments, the subject being treated has cancer that initially responded to treatment with an antagonist of signaling mediated by an EGFR family member, but has subsequently progressed on said treatment.

[0531] In some embodiments, a subject treated according to the present invention may have been determined to have (i.e., diagnosed with) a cancer comprising cells with a mutation that causes increased expression of a ligand for HER3 (e.g., as described herein). In some embodiments, the methods of the present invention may include determining whether the subject has a cancer comprising cells with a mutation that causes increased expression of a ligand for HER3. In some embodiments, the methods include analyzing nucleic acid from cells of the cancer. In some embodiments, the methods include detecting a mutation that causes increased expression of a ligand for HER3.

[0532] Those skilled in the art can easily identify the cancers and subjects described herein. Such cancers and subjects can be identified, for example, by monitoring the onset / progression of cancer (and / or its correlates) over time during the course of treatment with, for example, an antagonist of EGFR family member-mediated signal transduction. In some embodiments, identifying such subjects / cancers can include, for example, analyzing in vitro samples (e.g., biopsies). In some embodiments, the cancer can be determined to include cells with mutations associated with reduced sensitivity and / or resistance to treatment with the antagonist. In some embodiments, the cancer can be determined to include cells with upregulated expression of EGFR family members.

[0533] In certain embodiments, the cancer being treated is a cancer that is resistant to treatment with an antagonist of EGFR and / or HER2-mediated signaling. In some embodiments, the subject being treated has a cancer that is resistant to treatment with an antagonist of EGFR and / or HER2-mediated signaling. In some embodiments, the subject being treated has a cancer that has developed resistance to treatment with an antagonist of EGFR and / or HER2-mediated signaling. In some embodiments, the subject being treated has a cancer that previously responded to treatment with an antagonist of EGFR and / or HER2-mediated signaling 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 EGFR and / or HER2-mediated signaling. In some embodiments, the subject being treated has a cancer that initially responded to treatment with an antagonist of EGFR and / or HER2-mediated signaling but subsequently progressed with said treatment. In some embodiments, the subject to be treated has a cancer associated with amplification of signaling of an EGFR family member, eg, EGFR and / or HER2.

[0534] In certain embodiments, the cancer to be treated comprises a mutation that confers resistance to treatment with a BRAF inhibitor.In some embodiments, the mutation is a mutation in the V600 of BRAF.In some embodiments, the mutation is V600E or V600K of BRAF.The cancer can be thyroid or colon cancer, for example, RAS wild-type colorectal cancer. 【05...

Claims

1. A composition comprising an antigen-binding molecule capable of binding to HER3, The antigen-binding molecule is (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 (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 Contains, and The composition comprises: (i) 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate-80, and having a pH of 4.0 to 7.0; or (ii) 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate-20, and having a pH of 4.0 to 7.0; or (iii) 2 mM to 200 mM histidine, 1 mM to 250 mM sodium chloride, 0.001% to 0.1% (w / v) polysorbate-80, and having a pH of 4.0 to 7.0; or (iv) 2 mM to 200 mM histidine, 1 mM to 250 mM sodium chloride, 0.001% to 0.1% (w / v) polysorbate-20, and having a pH of 4.0 to 7.0; or (v) 2 mM to 200 mM histidine, 1 mM to 250 mM arginine, 0.001% to 0.1% (w / v) polysorbate-80, and having a pH of 4.0 to 7.0; or (vi) 2 mM to 200 mM histidine, 1 mM to 250 mM arginine, 0.001% to 0.1% (w / v) polysorbate-20, and having a pH of 4.0 to 7.0; or (vii) 2 mM to 200 mM acetate, 1 mM to 250 mM sodium chloride, 0.001% to 0.1% (w / v) polysorbate-20, and having a pH of 4.0 to 7.0; The composition.

2. (i) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or (ii) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.1; or (iii) 20 mM histidine, 4% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or (iv) 20 mM histidine, 2% (w / v) sucrose; 0.02% (w / v) polysorbate-80, having a pH of 5.3; or (v) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, having a pH of 6.1; or (vi) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-20, having a pH of 5.8; or (vii) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-20, having a pH of 5.5; or (viii) 20 mM histidine, 150 mM sodium chloride; 0.02% (w / v) polysorbate-80, having a pH of 6.5; or 9. The composition of claim 1, comprising: (ix) 20 mM acetate, 150 mM sodium chloride; 0.05% (w / v) polysorbate-20, and having a pH of 5.

5.

3. 3. The composition of claim 1, comprising 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.

8.

4. 3. The composition of claim 1 or 2, comprising at least 1.2 mg / mL of the antigen-binding molecule.

5. 3. The composition of claim 1 or 2, comprising up to 50 mg / mL of the antigen-binding molecule.

6. The composition of claim 1, wherein 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 and a VH region incorporating (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 The composition comprising:

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.

3. The composition of claim 1 or 2, comprising:

8. 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 and / or 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 A VL region incorporating Including, The composition according to claim 1 or 2.

9. The composition of claim 1 or 2, wherein the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 171, and / or the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:

177.

10. A medicament for treating or preventing cancer in a subject, the medicament comprising the composition of claim 1 or 2.

11. The medicament of claim 10, wherein the cancer comprises cells expressing HER3, EGFR, HER2, HER4, NRG1, NRG2, and / or a ligand for HER3.

12. The method of claim 10, wherein the cancer comprises cells with a mutation that results in increased expression of a ligand for HER3.

13. the cancer comprises cells with NRG gene fusions, and optionally the NRG gene fusions are selected from the group consisting of 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, 11. The pharmaceutical composition of claim 10, wherein the medicament is selected from the group consisting of ATP1B1-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.

14. the cancer originates in the lung, breast, head, neck, kidney, ovary, cervix, pancreas, stomach, liver, esophagus, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue, or nasopharynx, and optionally the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, triple-negative breast cancer, breast cancer, invasive breast cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, renal clear cell carcinoma, ovarian cancer, and ovarian serous cyst. The pharmaceutical agent of claim 10, which is selected from adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, castration-resistant prostate cancer, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, bile duct carcinoma, colorectal cancer, RAS wild-type colorectal cancer, gastric cancer, hepatocellular carcinoma (HCC), esophageal cancer, bladder cancer, urothelial bladder cancer, cervical cancer, endometrial cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor, and nasopharyngeal neuroendocrine tumor.

15. The pharmaceutical composition of claim 10, wherein the composition is administered in combination with one or more of a HER2 targeted therapy, an EGFR targeted therapy, and / or an androgen receptor targeted therapy, and optionally, the composition is administered in combination with one or more of cetuximab, enzalutamide, and / or trastuzumab.

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