HER3 Antigen-Binding Molecules

Abstract

A HER3 antigen-binding molecule is disclosed. Also disclosed are a nucleic acid encoding the HER3 antigen-binding molecule and an expression vector, a composition comprising the same, and a method for using the same.

Description

Technology Field

[0001] This application claims priority to PCT / EP2018 / 058259 filed March 29, 2018, and US 16 / 170,370 filed October 25, 2018, the contents and elements thereof incorporated herein by reference for all purposes.

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

[0003] Increased HER3 expression is associated with a poor prognosis in multiple solid tumors, including breast, gastric, head and neck, pancreatic, ovarian, and lung cancers. HER3-mediated signaling has an adverse effect on tumor progression, HER3 upregulation is associated with resistance to anti-HER2 and anti-EGFR therapies, and solid tumors refractory to anti-PD-1 therapy have been shown to have higher HER3 expression compared to those responsive to anti-PD-1 therapy.

[0004] HER3-binding antibodies are described, for example, in the literature [Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1): 39-48]. The anti-HER3 antibody LJM-716 binds to epitopes on subdomains II and IV of the HER3 extracellular domain, immobilizing HER3 into an inactive conformation (Garner et al., Cancer Res (2013) 73: 6024-6035]). MM-121 (also known as seribantumab) has been shown to inhibit HER3-mediated signaling by blocking the binding of heregulin (HRG) to HER3 (Schoeberl et al., Sci. Signal. (2009) 2 (77): ra31). Patritumab (also known as U-1287 and AMG-888) also blocks the binding of heregulin to HER3 (see, e.g., Shimizu et al. Cancer Chemother Pharmacol. (2017) 79 (3): 489-495). RG7116 (lumretuzumab and RO-5479599) recognizes an epitope in 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 of subdomain III (corresponding to the positions following 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 of subdomain II (see reference [Lee et al., Proc Natl Acad Sci US A. 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 HER3 to its ligand (see [Zhang et al., Mol Cancer Ther (2014) 13: 1345-1355]). RG7597 (duligotuzumab) is a dual-acting Fab (DAF) capable of binding 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 having a HER3-binding arm that inhibits the binding of HER3 to HRG ligands (see McDonagh et al., Mol Cancer Ther (2012) 11:582-593 and Fitzgerald et al., Mol Cancer Ther (2014) 13:410-425).

[0005] In a first aspect, the present invention provides a selectively isolated antigen-binding molecule capable of binding to HER3 in the extracellular region subdomain II.

[0006] In some embodiments, the antigen-binding molecule inhibits the interaction between HER3 and the interaction partner of HER3.

[0007] In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising or composed of the amino acid sequence of SEQ ID NO. 16.

[0008] In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising the amino acid sequence of SEQ ID NO. 229.

[0009] In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising the amino acid sequences of SEQ ID NOs 230 and 231.

[0010] In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising the amino acid sequence of SEQ ID NO. 230.

[0011] In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising the amino acid sequence of SEQ ID NO. 231.

[0012] In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising the amino acid sequence of SEQ ID NO. 23.

[0013] In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising the amino acid sequence of SEQ ID NO. 21.

[0014] In some embodiments, the antigen-binding molecule is

[0015] (i) Heavy chain variable (VH) region containing the following CDR:

[0016] HC-CDR1 having the amino acid sequence of SEQ ID NO. 43

[0017] HC-CDR2 having the amino acid sequence of SEQ ID NO. 46

[0018] HC-CDR3 having the amino acid sequence of SEQ ID NO. 51; and

[0019] (ii) Light chain variable (VL) regions containing the following CDRs:

[0020] LC-CDR1 having the amino acid sequence of SEQ ID NO. 91

[0021] LC-CDR2 having the amino acid sequence of SEQ ID NO. 94

[0022] LC-CDR3 having the amino acid sequence of SEQ ID NO. 99

[0023] Includes

[0024] In some embodiments, the antigen-binding molecule is

[0025] (i) Heavy chain variable (VH) region containing the following CDR:

[0026] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0027] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[0028] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[0029] (ii) Light chain variable (VL) regions containing the following CDRs:

[0030] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0031] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0032] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[0033] Includes

[0034] In some embodiments, the antigen-binding molecule is

[0035] (i) Heavy chain variable (VH) region containing the following CDR:

[0036] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0037] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[0038] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[0039] (ii) Light chain variable (VL) regions containing the following CDRs:

[0040] LC-CDR1 having the amino acid sequence of SEQ ID NO. 89

[0041] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0042] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[0043] Includes

[0044] In some embodiments, the antigen-binding molecule is

[0045] (i) Heavy chain variable (VH) region containing the following CDR:

[0046] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0047] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[0048] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[0049] (ii) Light chain variable (VL) regions containing the following CDRs:

[0050] LC-CDR1 having the amino acid sequence of SEQ ID NO. 90

[0051] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0052] LC-CDR3 having the amino acid sequence of SEQ ID NO. 96

[0053] Includes

[0054] In some embodiments, the antigen-binding molecule is

[0055] (i) Heavy chain variable (VH) region containing the following CDR:

[0056] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0057] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[0058] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[0059] (ii) Light chain variable (VL) regions containing the following CDRs:

[0060] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0061] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0062] LC-CDR3 having the amino acid sequence of SEQ ID NO. 98

[0063] Includes

[0064] In some embodiments, the antigen-binding molecule is

[0065] (i) Heavy chain variable (VH) region containing the following CDR:

[0066] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0067] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0068] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[0069] (ii) Light chain variable (VL) regions containing the following CDRs:

[0070] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0071] LC-CDR2 having the amino acid sequence of SEQ ID NO. 93

[0072] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[0073] Includes

[0074] In some embodiments, the antigen-binding molecule is

[0075] (i) Heavy chain variable (VH) region containing the following CDR:

[0076] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0077] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0078] HC-CDR3 having the amino acid sequence of SEQ ID NO. 49; and

[0079] (ii) Light chain variable (VL) regions containing the following CDRs:

[0080] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0081] LC-CDR2 having the amino acid sequence of SEQ ID NO. 93

[0082] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[0083] Includes

[0084] In some embodiments, the antigen-binding molecule is

[0085] (i) Heavy chain variable (VH) region containing the following CDR:

[0086] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0087] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0088] HC-CDR3 having the amino acid sequence of SEQ ID NO. 50; and

[0089] (ii) Light chain variable (VL) regions containing the following CDRs:

[0090] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0091] LC-CDR2 having the amino acid sequence of SEQ ID NO. 93

[0092] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[0093] Includes

[0094] In some embodiments, the antigen-binding molecule is

[0095] (i) Heavy chain variable (VH) region containing the following CDR:

[0096] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0097] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0098] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[0099] (ii) Light chain variable (VL) regions containing the following CDRs:

[0100] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0101] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0102] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[0103] Includes

[0104] In some embodiments, the antigen-binding molecule is

[0105] (i) Heavy chain variable (VH) region containing the following CDR:

[0106] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0107] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0108] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[0109] (ii) Light chain variable (VL) regions containing the following CDRs:

[0110] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0111] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0112] LC-CDR3 having the amino acid sequence of SEQ ID NO. 97

[0113] Includes

[0114] In some embodiments, the antigen-binding molecule is

[0115] (i) Heavy chain variable (VH) region containing the following CDR:

[0116] HC-CDR1 having the amino acid sequence of SEQ ID NO. 42

[0117] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0118] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[0119] (ii) Light chain variable (VL) regions containing the following CDRs:

[0120] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0121] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0122] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[0123] Includes

[0124] In some embodiments, the antigen-binding molecule is

[0125] (i) Heavy chain variable (VH) region containing the following CDR:

[0126] HC-CDR1 having the amino acid sequence of SEQ ID NO. 158

[0127] HC-CDR2 having the amino acid sequence of SEQ ID NO. 159

[0128] HC-CDR3 having the amino acid sequence of SEQ ID NO. 160; and

[0129] (ii) Light chain variable (VL) regions containing the following CDRs:

[0130] LC-CDR1 having the amino acid sequence of SEQ ID NO. 165

[0131] LC-CDR2 having the amino acid sequence of SEQ ID NO. 166

[0132] LC-CDR3 having the amino acid sequence of SEQ ID NO. 167

[0133] Includes

[0134] In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising the amino acid sequence of SEQ ID NO. 22.

[0135] In some embodiments, the antigen-binding molecule is

[0136] (i) Heavy chain variable (VH) region containing the following CDR:

[0137] HC-CDR1 having the amino acid sequence of SEQ ID NO. 128

[0138] HC-CDR2 having the amino acid sequence of SEQ ID NO. 129

[0139] HC-CDR3 having the amino acid sequence of SEQ ID NO. 130; and

[0140] (ii) Light chain variable (VL) regions containing the following CDRs:

[0141] LC-CDR1 having the amino acid sequence of SEQ ID NO. 136

[0142] LC-CDR2 having the amino acid sequence of SEQ ID NO. 137

[0143] LC-CDR3 having the amino acid sequence of SEQ ID NO. 138

[0144] Includes

[0145] In some embodiments, the antigen-binding molecule is

[0146] (i) Heavy chain variable (VH) region containing the following CDR:

[0147] HC-CDR1 having the amino acid sequence of SEQ ID NO. 144

[0148] HC-CDR2 having the amino acid sequence of SEQ ID NO. 145

[0149] HC-CDR3 having the amino acid sequence of SEQ ID NO. 146; and

[0150] (ii) Light chain variable (VL) regions containing the following CDRs:

[0151] LC-CDR1 having the amino acid sequence of SEQ ID NO. 151

[0152] LC-CDR2 having the amino acid sequence of SEQ ID NO. 152

[0153] LC-CDR3 having the amino acid sequence of SEQ ID NO. 153

[0154] Includes

[0155] In certain embodiments, the antigen-binding molecule is

[0156] (i) Heavy chain variable (VH) region containing the following CDR:

[0157] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0158] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0159] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[0160] (ii) Light chain variable (VL) regions containing the following CDRs:

[0161] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0162] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0163] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95;

[0164] or

[0165] (i) Heavy chain variable (VH) region containing the following CDR:

[0166] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0167] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0168] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[0169] (ii) Light chain variable (VL) regions containing the following CDRs:

[0170] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0171] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0172] LC-CDR3 having the amino acid sequence of SEQ ID NO. 97;

[0173] or

[0174] (i) Heavy chain variable (VH) region containing the following CDR:

[0175] HC-CDR1 having the amino acid sequence of SEQ ID NO. 42

[0176] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0177] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[0178] (ii) Light chain variable (VL) regions containing the following CDRs:

[0179] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0180] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0181] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[0182] Includes

[0183] In some embodiments, the antigen-binding molecule is

[0184] (i) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 24; and

[0185] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 74;

[0186] or

[0187] (ii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 25; and

[0188] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 75;

[0189] or

[0190] (iii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 26; and

[0191] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 76;

[0192] or

[0193] (iv) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 27; and

[0194] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 77;

[0195] or

[0196] (v) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 28; and

[0197] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78;

[0198] or

[0199] (vi) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 29; and

[0200] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78;

[0201] or

[0202] (vii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 30; and

[0203] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78;

[0204] or

[0205] (viii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 31; and

[0206] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 79;

[0207] or

[0208] (ix) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 32; and

[0209] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 79;

[0210] or

[0211] (x) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 33; and

[0212] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 80;

[0213] or

[0214] (xi) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 34; and

[0215] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 81;

[0216] or

[0217] (xii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 35; and

[0218] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 82;

[0219] or

[0220] (xiii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 36; and

[0221] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 83;

[0222] or

[0223] (xiv) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 37; and

[0224] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 84;

[0225] or

[0226] (xv) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 38; and

[0227] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 85;

[0228] or

[0229] (xvi) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 39; and

[0230] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 86;

[0231] or

[0232] (xvii) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 40; and

[0233] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 87;

[0234] or

[0235] (xviii) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 127; and

[0236] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 135;

[0237] or

[0238] (xix) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 143; and

[0239] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 150;

[0240] or

[0241] (xx) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 157; and

[0242] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 164.

[0243] Includes

[0244] In certain embodiments, the antigen-binding molecule is

[0245] (i) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 36; and

[0246] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 83;

[0247] or

[0248] (ii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 37; and

[0249] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 84;

[0250] or

[0251] (iii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 38; and

[0252] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 85.

[0253] Includes

[0254] In some embodiments, the antigen-binding molecule may bind to one or more of human HER3 and mouse HER3, rat HER3 and cynomolgus macaque HER3.

[0255] Additionally, an optionally isolated antigen-binding molecule is provided, comprising (i) an antigen-binding molecule according to the present invention, and (ii) an antigen-binding molecule capable of binding to an antigen other than HER3.

[0256] In some embodiments, the antigen-binding molecule can bind to a cell expressing HER3 on the cell surface.

[0257] In some embodiments, the antigen-binding molecule can inhibit HER3-mediated signaling.

[0258] In some embodiments, the antigen-binding molecule comprises an Fc region, said Fc region comprises a polypeptide having one or more of: (i) C at position 242 and C at position 334, and (ii) A at position 236, D at position 239, E at position 332, L at position 330, K at position 345, and G at position 430. In some embodiments, the Fc region comprises a polypeptide having C at position 242, C at position 334, A at position 236, D at position 239, E at position 332, and L at position 330.

[0259] In addition, a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the present invention is provided.

[0260] In addition, an antigen-binding molecule or an optionally isolated nucleic acid encoding a CAR according to the present invention or a plurality of nucleic acids are provided.

[0261] In addition, an expression vector comprising a nucleic acid or a plurality of nucleic acids or a plurality of expression vectors according to the present invention is provided.

[0262] In addition, a cell comprising an antigen-binding molecule, a CAR, a nucleic acid according to the present invention or a plurality of nucleic acids or an expression vector or a plurality of expression vectors is provided.

[0263] In addition, a method is provided comprising the step of culturing a cell containing the nucleic acid according to the present invention or a plurality of nucleic acids or an expression vector or a plurality of expression vectors under conditions suitable for the expression of an antigen-binding molecule or CAR from the nucleic acid(s) or expression vector(s).

[0264] In addition, a composition comprising an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors or a cell according to the present invention is provided.

[0265] In addition, an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present invention is provided for use in a medical treatment or prevention method.

[0266] In addition, an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present invention is provided for use in a method for treating or preventing cancer.

[0267] In addition, the use of an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present invention is provided in the manufacture of a medicine for use in a method of treating or preventing cancer.

[0268] Additionally, as a method for treating or preventing cancer, a method is provided comprising the step of administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present invention.

[0269] In some embodiments, according to various aspects of the present invention, the method further comprises administering an inhibitor of signal transduction mediated by an EGFR family member, and optionally, the inhibitor of signal transduction mediated by an EGFR family member is an inhibitor of signal transduction mediated by HER2 and / or EGFR.

[0270] In some embodiments, cancer includes cancer comprising cells expressing EGFR family members, cancer comprising cells expressing HER3, solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, 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, ovarian serous adenocarcinoma, renal cancer, renal cell carcinoma, Renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary 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,It is selected from uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma, and thymoma.

[0271] In addition, as a method for inhibiting HER3-mediated signaling, a method comprising the step of contacting a HER3-expressing cell with an antigen-binding molecule according to the present invention is provided.

[0272] In addition, as a method for reducing the number or activity of HER3-expressing cells, the present invention provides a method comprising the step of contacting HER3-expressing cells with an antigen-binding molecule according to the present invention.

[0273] Also, a selectively isolated in vitro complex comprising an antigen-binding molecule according to the present invention bound to HER3 ( in vitro complex) is provided.

[0274] Additionally, a method is provided comprising the steps of contacting a sample containing or presumed to contain HER3 with an antigen-binding molecule according to the present invention, and detecting the formation of a complex of the antigen-binding molecule and HER3.

[0275] Additionally, as a method for selecting or stratifying subjects for treatment using a HER3-targeted agent, the present method comprises the steps of contacting a sample from a subject with an antigen-binding molecule according to the present invention in vitro and detecting the formation of a complex of the antigen-binding molecule and HER3.

[0276] In addition, the use of the antigen-binding molecule according to the present invention as an in vitro or in vivo diagnostic agent or prognostic agent is provided.

[0277] In addition, as an application of the antigen-binding molecule according to the present invention in a method for detecting, localizing, or imaging cancer, selectively cancer includes cancer comprising cells expressing EGFR family members, cancer comprising cells expressing HER3, solid tumors, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, 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, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, papillary renal cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, endometrial carcinoma of the uterine body. Uses selected from thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma, and thymoma are provided.

[0278] details

[0279] The present invention relates to a novel HER3-binding molecule having improved properties compared to a known anti-HER3 antibody.

[0280] The inventors have produced a targeted antigen-binding molecule that binds to a specific target region of the extracellular domain of HER3. The HER3-binding molecule of the present invention provides a combination of appropriate biophysical and / or functional properties compared to antigen-binding molecules disclosed in the prior art.

[0281] In an embodiment of the present invention, the antigen-binding molecule can bind to subdomain II of the extracellular region of HER3 (SEQ ID NO. 16) and inhibits the binding of the bound HER3 molecule and its interacting partner.

[0282] In particular, the antigen-binding molecules described herein, which bind to the epitope of HER3, have been shown to provide potent inhibition of the binding of HER3 and its interacting partners, as well as potent inhibition of downstream signaling, in addition to anticancer activity against a wide range of cancers.

[0283] HER3

[0284] HER3 (also known, for example, as ERBB3 LCCS2, MDA-BF-1) is a protein identified by UniProt P21860. Human ERBB3 Alternative splicing of mRNA encoded by the gene comprises five different isoforms: isoform 1 (UniProt: P21860-1, v1; SEQ ID NO. 1); isoform 2 (UniProt: P21860-2; SEQ ID NO. 2) (containing a sequence different from SEQ ID NO. 1 from position 141, and lacking the amino acid sequence corresponding to positions 183 to 1342 of SEQ ID NO. 1); isoform 3 (UniProt: P21860-3; SEQ ID NO. 3 (containing a substitution C331F relative to SEQ ID NO. 1, and the amino acid sequence corresponding to positions 332 to 1342 of SEQ ID NO. 1 is absent); isoform 4 (UniProt: P21860-4; SEQ ID NO. 4) (the amino acid sequence corresponding to positions 1 to 59 of SEQ ID NO. 1 is absent); and isoform 5 (UniProt: P21860-5; SEQ ID NO. 5) (the amino acid sequence corresponding to positions 1 to 643 of SEQ ID NO. 1 is absent) are produced.

[0285] Since the 19 N-terminal amino acids of SEQ ID NOs 1 to 3 constitute a signal peptide, the mature forms of HER3 isoforms 1, 2, and 3 (i.e., after treatment to remove the signal peptide) have the amino acid sequences presented in SEQ ID NOs 6, 7, and 8, respectively.

[0286] The structure and function of HER3 are described, for example, in the literature [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 Cancers (2005) S17-S27 and Mujoo et al., Oncotarget (2014) 5(21):10222-10236], the full text of each of the above literature is incorporated herein by reference. HER3 is a single-pass transmembrane ErbB receptor tyrosine kinase having an N-terminal extracellular domain (SEQN 9) containing two leucine polyvalent subdomains (domains I and III, presented in SEQN 15 and 17, respectively) and two cysteine ​​polyvalent subdomains (domains II and IV, presented in SEQN 16 and 18, respectively). Domain II contains a β-hairpin dimerization loop (SEQN 19) involved in intermolecular interactions with other HER receptor molecules. The extracellular domain is connected to a cytoplasmic domain (SEQN 11) through a transmembrane domain (SEQN 10). The cytoplasmic domain contains a membrane-proximal fragment (SEQN 12), a protein kinase domain (SEQN 13), and a C-terminal fragment (SEQN 14).

[0287] Signaling via HER3 involves receptor homomerization (i.e., with other HER3 receptors) or heteromerization (with other HER receptors, e.g., HER2) and consequently, autophosphorylation by the protein kinase domain of tyrosine in the cytoplasmic region. The phosphorylated tyrosine residues assemble adapter / effector proteins (e.g., Grb2 and phospholipase Cγ (PLCγ), containing the src homologous domain 2 (SH2) or phosphotyrosine binding (PTB) domain).

[0288] Signaling via HER3 can be activated in a ligand-dependent or ligand-independent manner. In the absence of a ligand, the HER3 receptor molecule is generally expressed on the cell surface as a monomer with a conformation that inhibits receptor dimerization, and the dimerization loop of subdomain II is in intramolecular contact with a pocket on subdomain IV. Binding of a HER3 ligand, e.g., neuregulin (NRG), e.g., NRG1 (also known as heregulin, HRG) or NRG2, to subdomains I and III of the extracellular region induces a conformation change, exposing the dimerization loop of subdomain II, thereby promoting receptor dimerization and signaling. Some cancer-related mutations in HER3 can disrupt the interaction between subdomains II and IV, which is necessary for the formation of an inactive 'closed' conformation, thereby inducing the constancyal presentation of the dimerization loop and the activation of HER3-mediated signaling in the absence of ligand binding (see, e.g., [Jaiswal et al., Cancer Cell (2013) 23 (5): 603-617]).

[0289] In this specification, "HER3" refers to HER3 from any species and includes HER3 isoforms, fragments, variants (including mutants) or homologs from any species.

[0290] As used herein, a “fragment,” “variant,” or “homologous” 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 with respect to the amino acid sequence of a reference protein (e.g., reference isoform). In some embodiments, fragments, variants, isoforms, and homologous of a reference protein may be characterized by their ability to perform the function performed by the reference protein.

[0291] "Fragment" generally refers to a fraction of a reference protein. "Variant" generally refers to a protein that has an amino acid sequence containing one or more amino acid substitutions, insertions, deletions, or other modifications to the amino acid sequence of the reference protein, but possesses a significant degree of sequence identity (e.g., at least 60%) with respect to the amino acid sequence of the reference protein. "Isotype" generally refers to a variant of the reference protein expressed by the same species as the reference protein. (e.g., HER3 isotypes 1 through 5 are all isotypes of each other). "Homologous" generally refers to a variant of the reference protein produced by a different species compared to the reference protein. For example, human HER3 isotype 1 (P21860-1, v1; SEQ ID NO. 1) and Rhesus macaque HER3 (UniProt: F7HEH3-1, v2; SEQ ID NO. 20) are homologous to each other. Homologous bodies include orthologues.

[0292] The “fragment” of the reference protein 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, but may be any length (by the number of amino acids).

[0293] The HER3 fragment can 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.

[0294] In some embodiments, HER3 is HER3 from mammals (e.g., primates (rhesus, cynomolgus, non-human primates or humans) and / or rodents (e.g., rats or rodents) HER3). An isoform, fragment, variant, or homolog of HER3 may optionally be characterized by having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of an immature or mature HER3 isoform from a given species, e.g., humans.

[0295] The isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs having the functional characteristics / activities of reference HER3 (e.g., human HER3 isoform 1), as measured by appropriate analysis of functional characteristics / activities, for example. For example, the isoforms, fragments, variants, or homologs of HER3 may show association with one or more of HER2, NRG1 (types I, II, III, IV, V, or VI), or NRG2 (α or β).

[0296] 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 for one of SEQ ID NOs 1 to 8.

[0297] 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 with respect to one of SEQ ID NOs 9 to 19, e.g., 9, 16, or 19.

[0298] Specific target area on the target molecule

[0299] The antigen-binding molecule of the present invention is specifically designed to target a specific target region of HER3. In a two-step approach, a HER3 region to be targeted was selected after analysis of predicted antigenicity, function, and safety. Subsequently, a specific monoclonal antibody was generated using a peptide corresponding to the target region as an immunogen, and an antibody specific to the target region of HER3 was prepared by subsequently screening identified antibodies capable of binding to HER3 in their natural state. This approach provides sophisticated control over the antibody epitope.

[0300] The antigen-binding molecules of the present invention can be defined by reference to the HER3 regions to which they bind. The antigen-binding molecules of the present invention may bind to specific target regions of HER3. In some embodiments, the antigen-binding molecules may bind to linear epitopes of HER3 consisting of adjacent amino acid sequences (i.e., primary amino acid sequences). In some embodiments, the antigen-binding molecules may bind to stereotypic epitopes of HER3 consisting of discontinuous amino acid sequences.

[0301] In some embodiments, the antigen-binding molecule of the present invention binds to HER3. In some embodiments, the antigen-binding molecule binds to the extracellular region of HER3 (e.g., the region presented in SEQ ID NO. 9). In some embodiments, the antigen-binding molecule binds to subdomain II of the extracellular region of HER3 (e.g., the region presented in SEQ ID NO. 16).

[0302] In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 229. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 229. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented 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 presented in SEQ ID NOs 230 and 231. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 230. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 230. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 231. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 231.

[0303] In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 23. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 23. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 21. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 21. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 19. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 19. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 22. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 22.

[0304] 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 come into contact with the amino acid residues of 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 presented in SEQ ID NO. 23. In some embodiments, the antigen-binding molecule does not come into contact with the amino acid residues of the region of HER3 presented in SEQ ID NO. 23.

[0305] The region of the peptide / polypeptide to which the antibody binds may be measured by those skilled in the art using various methods widely known in the art, including X-ray co-crystallographic analysis of antibody-antigen complexes, peptide scanning, mutagenic mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and proteolysis-based 'protection' methods. Such methods are described, for example, in the literature [Gershoni et al., BioDrugs, 2007, 21(3): 145-156], the full text of which is incorporated herein by reference.

[0306] In some embodiments, the antigen-binding molecule is the same region of HER3 or an overlapping region of HER3 bound by an antibody comprising the VH and VL sequences of one of the antibody clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6, 4-35-B2, or 4-35-B4 described herein It can bind to a region of HER3. In some embodiments, the antigen-binding molecule may bind to the same region of HER3 or an overlapping region of HER3 to a region of HER3 bound by an antibody containing the VH and VL sequences of one of antibody clones 10D1_c89, 10D1_c90, or 10D1_c91. In some embodiments, the antigen-binding molecule may bind to the same region of HER3 or an overlapping region of HER3 to a region of HER3 bound by an antibody containing the VH and VL sequences of antibody clone 10D1_c89.

[0307] As used herein, "peptide" refers to a chain of two or more amino acid monomers connected by peptide bonds. Peptides typically have a length of about 2 to 50 amino acid regions. "Polypeptide" is a polymer chain of two or more peptides. Polypeptides generally have a length longer than about 50 amino acids.

[0308] In some embodiments, the antigen-binding molecule of the present invention may be bound to a polypeptide comprising or composed of one amino acid sequence of SEQ ID NOs 1, 3, 4, 6, or 8.

[0309] In some embodiments, the antigen-binding molecule may be bound to a polypeptide comprising the amino acid sequence of SEQ ID NO. 9 or composed thereof. In some embodiments, the antigen-binding molecule may be bound to a polypeptide comprising the amino acid sequence of SEQ ID NO. 16 or composed thereof.

[0310] In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 229. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequences of SEQ ID NO. 230 and 231. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 230. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 231. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 23. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 21. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 19. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 22.

[0311] In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of an amino acid sequence corresponding to positions 260 to 279 of SEQ ID NO. 1. In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of an amino acid sequence of SEQ ID NO. 23.

[0312] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods widely 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 (e.g., Lad et al., (2015) J Biomol Screen 20(4): 498-507).

[0313] In an embodiment in which an 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 ends 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 ends of the reference amino acid sequence.

[0314] In some embodiments, additional amino acid(s) provided at one or both ends (i.e., N-terminus and C-terminus) of the reference sequence correspond to terminal positions of the reference sequence in relation to the amino acid sequence of HER3. For 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 may be threonine and lysine, which correspond to positions 278 and 279 of SEQ ID NO. 1.

[0315] In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of one of the antibody clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6, 4-35-B2 or 4-35-B4 described herein. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of one of antibody clones 10D1_c89, 10D1_c90, or 10D1_c91. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of antibody clone 10D1_c89.

[0316] antigen-binding molecules

[0317] The present invention provides an antigen-binding molecule capable of binding to HER3.

[0318] “Antigen-binding molecule” refers to a molecule capable of binding to a target antigen and includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabody, triabody, scFv-Fc, minibody), and single-domain antibodies (e.g., VhH, etc.) insofar as they exhibit binding to the relevant target molecule(s).

[0319] The antigen-binding molecule of the present invention comprises a moiety capable of binding to target antigen(s). In some embodiments, the moiety capable of binding to the 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 the target antigen comprises or consists of an aptamer capable of binding to the target antigen, such as a nucleic acid aptamer (e.g., reviewed in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an antigen-binding peptide / polypeptide, e.g., peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e., single-domain antibody (sdAb)), afilin, armadillo repeat protein (ArmRP), OBody, or fibronectin (e.g., reviewed in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101, the full text of which is incorporated herein by reference) (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0320] The antigen-binding molecule of the present invention generally comprises an antigen-binding domain comprising VH and VL of an antibody capable of specifically binding to a target antigen. The antigen-binding domain formed by VH and VL may also be referred to herein as the Fv region.

[0321] The antigen-binding molecule may be an antigen-binding polypeptide or an antigen-binding polypeptide complex, or may comprise such. The antigen-binding molecule may comprise one or more polypeptides that together form an antigen-binding domain. The polypeptides may be associated covalently or non-covalently. In some embodiments, the polypeptide forms a portion of a larger polypeptide comprising the polypeptide (e.g., in the case of scFv containing VH and VL, or in the case of scFab containing VH-CH1 and VL-CL).

[0322] The antigen-binding molecule may refer to a non-covalent or covalent complex of one or more polypeptides (e.g., 2, 3, 4, 6, or 8 polypeptides), such as an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

[0323] The antigen-binding molecule of the present invention can be designed and manufactured using the sequence of a monoclonal antibody (mAb) capable of binding to HER3. The antigen-binding region of the antibody, for example, a single-chain variable fragment (scFv), Fab, and F(ab')2 fragment may also be used or provided. The "antigen-binding region" is any fragment of an antibody to which a specific antibody can bind to a specific target.

[0324] Antibodies generally contain six complementary-determining regions (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 paratope of the antibody, which is the part of the antibody that binds to the target antigen.

[0325] The VH region and the VL region each comprise a backbone region (FR) of one aspect of the CDR, which provides a scaffold for the CDR. From the N-terminus to the 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.

[0326] There are several different conventions for defining antibody CDRs and FRs, such as those described in the literature [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 antibody CDRs and FRs as described in the literature [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 are defined according to the International IMGT (ImMunoGeneTics) Information System (LeFranc et al., Nucleic Res. (2015) 43 (Database Issue):D413-22), using the IMGT V-DOMAIN numbering rules as described in the literature [Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77].

[0327] In some embodiments, the antigen-binding molecule comprises a CDR of the antigen-binding molecule capable of binding to HER3. In some embodiments, the antigen-binding molecule comprises an FR of the antigen-binding molecule capable of binding to HER3. In some embodiments, the antigen-binding molecule comprises a CDR and an FR of the antigen-binding molecule capable of binding to HER3. That is, in some embodiments, the antigen-binding molecule comprises a VH region and a VL region of the antigen-binding molecule capable of binding to HER3.

[0328] In some embodiments, the antigen-binding molecule is a HER3-binding antibody clone described herein (i.e., anti-HER3 antibody clone 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10D1, 10A6, 4-35-B2 or 4-35-B4; e.g., 10D1_c89, 10D1_c90 or 10D1_c91; e.g., 10D1_c89) is a VH / VL region or includes a VH region and a VL region derived therefrom.

[0329] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (1) to (10) below:

[0330] (1) VH region (derived from 10D1) containing the following CDR:

[0331] HC-CDR1 having the amino acid sequence of SEQ ID NO. 43

[0332] HC-CDR2 having the amino acid sequence of SEQ ID NO. 46

[0333] HC-CDR3 having the amino acid sequence of SEQ ID NO. 51,

[0334] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0335] (2) VH regions (10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c87, 10D1_c92, 10D1_c93) including the following CDRs:

[0336] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0337] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[0338] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47,

[0339] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0340] (3) VH regions (10D1_c85v1, 10D1_c85v2) containing the following CDRs:

[0341] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0342] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0343] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47,

[0344] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0345] (4) VH region (10D1_c85o1) containing the following CDR:

[0346] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0347] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0348] HC-CDR3 having the amino acid sequence of SEQ ID NO. 49,

[0349] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0350] (5) VH region (10D1_c85o2) containing the following CDR:

[0351] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0352] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0353] HC-CDR3 having the amino acid sequence of SEQ ID NO. 50,

[0354] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0355] (6) VH region (10D1_c89, 10D1_c90) containing the following CDR:

[0356] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[0357] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0358] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48,

[0359] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0360] (7) VH region (10D1_c91) containing the following CDR:

[0361] HC-CDR1 having the amino acid sequence of SEQ ID NO. 42

[0362] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[0363] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48,

[0364] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0365] (8) VH region (10A6) containing the following CDR:

[0366] HC-CDR1 having the amino acid sequence of SEQ ID NO. 158

[0367] HC-CDR2 having the amino acid sequence of SEQ ID NO. 159

[0368] HC-CDR3 having the amino acid sequence of SEQ ID NO. 160,

[0369] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0370] (9) VH region (4-35-B2) containing the following CDR:

[0371] HC-CDR1 having the amino acid sequence of SEQ ID NO. 128

[0372] HC-CDR2 having the amino acid sequence of SEQ ID NO. 129

[0373] HC-CDR3 having the amino acid sequence of SEQ ID NO. 130,

[0374] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0375] (10) VH region (4-35-B4) containing the following CDR:

[0376] HC-CDR1 having the amino acid sequence of SEQ ID NO. 144

[0377] HC-CDR2 having the amino acid sequence of SEQ ID NO. 145

[0378] HC-CDR3 having the amino acid sequence of SEQ ID NO. 146,

[0379] or variants thereof in which one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0380] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (11) to (24) below:

[0381] (11) VH region (10D1) containing the following FR:

[0382] HC-FR1 having the amino acid sequence of SEQ ID NO. 55

[0383] HC-FR2 having the amino acid sequence of SEQ ID NO. 58

[0384] HC-FR3 having the amino acid sequence of SEQ ID NO. 69

[0385] HC-FR4 having the amino acid sequence of SEQ ID NO. 73,

[0386] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0387] (12) VH region (10D1_c75, 10D1_c92) containing the following FR:

[0388] HC-FR1 having the amino acid sequence of SEQ ID NO. 52

[0389] HC-FR2 having the amino acid sequence of SEQ ID NO. 56

[0390] HC-FR3 having the amino acid sequence of SEQ ID NO. 61

[0391] HC-FR4 having the amino acid sequence of SEQ ID NO. 70,

[0392] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0393] (13) VH regions (10D1_c76, 10D1_c77, 10D1_c78v1) containing the following FR:

[0394] HC-FR1 having the amino acid sequence of SEQ ID NO. 52

[0395] HC-FR2 having the amino acid sequence of SEQ ID NO. 56

[0396] HC-FR3 having the amino acid sequence of SEQ ID NO. 62

[0397] HC-FR4 having the amino acid sequence of SEQ ID NO. 70,

[0398] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0399] (14) VH region (10D1_c78v2) containing the following FR:

[0400] HC-FR1 having the amino acid sequence of SEQ ID NO. 52

[0401] HC-FR2 having the amino acid sequence of SEQ ID NO. 57

[0402] HC-FR3 having the amino acid sequence of SEQ ID NO. 62

[0403] HC-FR4 having the amino acid sequence of SEQ ID NO. 70,

[0404] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0405] (15) VH region (10D1_11B) containing the following FR:

[0406] HC-FR1 having the amino acid sequence of SEQ ID NO. 224

[0407] HC-FR2 having the amino acid sequence of SEQ ID NO. 60

[0408] HC-FR3 having the amino acid sequence of SEQ ID NO. 63

[0409] HC-FR4 having the amino acid sequence of SEQ ID NO. 70,

[0410] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0411] (16) VH region (10D1_c85v1) containing the following FR:

[0412] HC-FR1 having the amino acid sequence of SEQ ID NO. 52

[0413] HC-FR2 having the amino acid sequence of SEQ ID NO. 56

[0414] HC-FR3 having the amino acid sequence of SEQ ID NO. 64

[0415] HC-FR4 having the amino acid sequence of SEQ ID NO. 70,

[0416] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0417] (17) VH regions (10D1_c85v2, 10D1_c85o1, 10D1_c85o2) containing the following FR:

[0418] HC-FR1 having the amino acid sequence of SEQ ID NO. 52

[0419] HC-FR2 having the amino acid sequence of SEQ ID NO. 57

[0420] HC-FR3 having the amino acid sequence of SEQ ID NO. 64

[0421] HC-FR4 having the amino acid sequence of SEQ ID NO. 70,

[0422] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0423] (18) VH region (10D1_c87, 10D1_c93) containing the following FR:

[0424] HC-FR1 having the amino acid sequence of SEQ ID NO. 52

[0425] HC-FR2 having the amino acid sequence of SEQ ID NO. 56

[0426] HC-FR3 having the amino acid sequence of SEQ ID NO. 65

[0427] HC-FR4 having the amino acid sequence of SEQ ID NO. 70,

[0428] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0429] (19) VH region (10D1_c89) containing the following FR:

[0430] HC-FR1 having the amino acid sequence of SEQ ID NO. 53

[0431] HC-FR2 having the amino acid sequence of SEQ ID NO. 59

[0432] HC-FR3 having the amino acid sequence of SEQ ID NO. 66

[0433] HC-FR4 having the amino acid sequence of SEQ ID NO. 71,

[0434] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0435] (20) VH region (10D1_c90) containing the following FR:

[0436] HC-FR1 having the amino acid sequence of SEQ ID NO. 54

[0437] HC-FR2 having the amino acid sequence of SEQ ID NO. 59

[0438] HC-FR3 having the amino acid sequence of SEQ ID NO. 67

[0439] HC-FR4 having the amino acid sequence of SEQ ID NO. 71,

[0440] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0441] (21) VH region (10D1_c91) containing the following FR:

[0442] HC-FR1 having the amino acid sequence of SEQ ID NO. 53

[0443] HC-FR2 having the amino acid sequence of SEQ ID NO. 59

[0444] HC-FR3 having the amino acid sequence of SEQ ID NO. 68

[0445] HC-FR4 having the amino acid sequence of SEQ ID NO. 72,

[0446] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0447] (22) VH region (10A6) containing the following FR:

[0448] HC-FR1 having the amino acid sequence of SEQ ID NO. 161

[0449] HC-FR2 having the amino acid sequence of SEQ ID NO. 162

[0450] HC-FR3 having the amino acid sequence of SEQ ID NO. 163

[0451] HC-FR4 having the amino acid sequence of SEQ ID NO. 73,

[0452] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0453] (23) VH region (4-35-B2) containing the following FR:

[0454] HC-FR1 having the amino acid sequence of SEQ ID NO. 131

[0455] HC-FR2 having the amino acid sequence of SEQ ID NO. 132

[0456] HC-FR3 having the amino acid sequence of SEQ ID NO. 133

[0457] HC-FR4 having the amino acid sequence of SEQ ID NO. 134,

[0458] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0459] (24) VH region (4-35-B4) containing the following FR:

[0460] HC-FR1 having the amino acid sequence of SEQ ID NO. 147

[0461] HC-FR2 having the amino acid sequence of SEQ ID NO. 148

[0462] HC-FR3 having the amino acid sequence of SEQ ID NO. 149

[0463] HC-FR4 having the amino acid sequence of SEQ ID NO. 73,

[0464] or variants thereof in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have 1 or 2 or 3 amino acids substituted with another amino acid.

[0465] In some embodiments, the antigen-binding molecule comprises a VH region including a CDR according to (1) to (10) and a FR according to one of (11) to (24).

[0466] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (25) to (41) below:

[0467] (25) A VH region including a CDR according to (1) and a FR according to (11), (12), (13), (14), (15), (16), (17), (18), (19), (20) or (21).

[0468] (26) A VH region including CDR according to (2) and FR according to (11).

[0469] (27) A VH region including CDR according to (2) and FR according to (12).

[0470] (28) VH region including CDR according to (2) and FR according to (13).

[0471] (29) A VH region including CDR according to (2) and FR according to (14).

[0472] (30) A VH region including CDR according to (2) and FR according to (15).

[0473] (31) A VH region including CDR according to (2) and FR according to (18).

[0474] (32) VH region including CDR according to (3) and FR according to (16).

[0475] (33) VH region including CDR according to (3) and FR according to (17).

[0476] (34) VH region including CDR according to (4) and FR according to (17).

[0477] (35) VH region including CDR according to (5) and FR according to (17).

[0478] (36) VH region including CDR according to (6) and FR according to (19).

[0479] (37) VH region including CDR according to (6) and FR according to (20).

[0480] (38) VH region including CDR according to (7) and FR according to (21).

[0481] (39) VH region including CDR according to (8) and FR according to (22).

[0482] (40) VH region including CDR according to (9) and FR according to (23).

[0483] (41) A VH region including CDR according to (10) and FR according to (24).

[0484] In some embodiments, the antigen-binding molecule comprises a VH region according to one of the following (42) to (61):

[0485] (42) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 24, 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.

[0486] (43) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 25, 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.

[0487] (44) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 26, 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.

[0488] (45) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 27, 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.

[0489] (46) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 28, 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.

[0490] (47) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 29, 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.

[0491] (48) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 30, 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.

[0492] (49) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 31, 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.

[0493] (50) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 32, 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.

[0494] (51) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 33, 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.

[0495] (52) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 34, 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.

[0496] (53) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 35, 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.

[0497] (54) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 36, 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.

[0498] (55) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 37, 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.

[0499] (56) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 38, 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.

[0500] (57) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 39, 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.

[0501] (58) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 40, 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.

[0502] (59) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 127, 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.

[0503] (60) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 143, 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.

[0504] (61) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 157, 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.

[0505] In some embodiments, the antigen-binding molecule comprises a VL region according to one of the following (62) to (71):

[0506] (62) VL regions (derived from 10D1) containing the following CDR:

[0507] LC-CDR1 having the amino acid sequence of SEQ ID NO. 91

[0508] LC-CDR2 having the amino acid sequence of SEQ ID NO. 94

[0509] LC-CDR39 having the amino acid sequence of SEQ ID NO. 99;

[0510] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0511] (63) VL regions (10D1, 10D1_c75, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c87, 10D1_c89, 10D1_c91, 10D1_c93) containing the following CDRs:

[0512] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0513] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0514] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95;

[0515] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0516] (64) VL region (10D1_c76) containing the following CDR:

[0517] LC-CDR1 having the amino acid sequence of SEQ ID NO. 89

[0518] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0519] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95;

[0520] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0521] (65) VL region (10D1_c77) containing the following CDR:

[0522] LC-CDR1 having the amino acid sequence of SEQ ID NO. 90

[0523] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0524] LC-CDR3 having the amino acid sequence of SEQ ID NO. 96;

[0525] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0526] (66) VL regions (10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2) containing the following CDRs:

[0527] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0528] LC-CDR2 having the amino acid sequence of SEQ ID NO. 93

[0529] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95;

[0530] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0531] (67) VL region (10D1_c90) containing the following CDR:

[0532] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0533] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0534] LC-CDR3 having the amino acid sequence of SEQ ID NO. 97;

[0535] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0536] (68) VL region (10D1_c92) containing the following CDR:

[0537] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[0538] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[0539] LC-CDR3 having the amino acid sequence of SEQ ID NO. 98;

[0540] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0541] (69) (10A6) VL region containing the following CDR:

[0542] LC-CDR1 having the amino acid sequence of SEQ ID NO. 165

[0543] LC-CDR2 having the amino acid sequence of SEQ ID NO. 166

[0544] LC-CDR3 having the amino acid sequence of SEQ ID NO. 167;

[0545] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0546] (70) VL area (4-35-B2) containing the following CDR:

[0547] LC-CDR1 having the amino acid sequence of SEQ ID NO. 136

[0548] LC-CDR2 having the amino acid sequence of SEQ ID NO. 137

[0549] LC-CDR3 having the amino acid sequence of SEQ ID NO. 138;

[0550] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0551] (71) VL area (4-35-B4) containing the following CDR:

[0552] LC-CDR1 having the amino acid sequence of SEQ ID NO. 151

[0553] LC-CDR2 having the amino acid sequence of SEQ ID NO. 152

[0554] LC-CDR3 having the amino acid sequence of SEQ ID NO. 153;

[0555] or variants thereof in which one or more of LC-CDR1, LC-CDR2, or LC-CDR3 have 1, 2, or 3 amino acids substituted with another amino acid.

[0556] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (72) to (86):

[0557] (72) (10D1) VL region containing the following FR:

[0558] LC-FR1 having the amino acid sequence of SEQ ID NO. 106

[0559] LC-FR2 having the amino acid sequence of SEQ ID NO. 113

[0560] LC-FR3 having the amino acid sequence of SEQ ID NO. 123

[0561] LC-FR4 having the amino acid sequence of SEQ ID NO. 126,

[0562] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0563] (73) VL region (10D1_c75) containing the following FR:

[0564] LC-FR1 having the amino acid sequence of SEQ ID NO. 100

[0565] LC-FR2 having the amino acid sequence of SEQ ID NO. 107

[0566] LC-FR3 having the amino acid sequence of SEQ ID NO. 114

[0567] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0568] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0569] (74) VL region (10D1_c76) containing the following FR:

[0570] LC-FR1 having the amino acid sequence of SEQ ID NO. 101

[0571] LC-FR2 having the amino acid sequence of SEQ ID NO. 108

[0572] LC-FR3 having the amino acid sequence of SEQ ID NO. 115

[0573] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0574] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0575] (75) VL region (10D1_c77) containing the following FR:

[0576] LC-FR1 having the amino acid sequence of SEQ ID NO. 102

[0577] LC-FR2 having the amino acid sequence of SEQ ID NO. 108

[0578] LC-FR3 having the amino acid sequence of SEQ ID NO. 116

[0579] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0580] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0581] (76) VL regions (10D1_c78v1, 10D1_c78v2, 10D1_11B) containing the following FR:

[0582] LC-FR1 having the amino acid sequence of SEQ ID NO. 103

[0583] LC-FR2 having the amino acid sequence of SEQ ID NO. 108

[0584] LC-FR3 having the amino acid sequence of SEQ ID NO. 117

[0585] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0586] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0587] (77) VL regions (10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2) containing the following FR:

[0588] LC-FR1 having the amino acid sequence of SEQ ID NO. 103

[0589] LC-FR2 having the amino acid sequence of SEQ ID NO. 108

[0590] LC-FR3 having the amino acid sequence of SEQ ID NO. 118

[0591] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0592] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0593] (78) VL region (10D1_c87) containing the following FR:

[0594] LC-FR1 having the amino acid sequence of SEQ ID NO. 103

[0595] LC-FR2 having the amino acid sequence of SEQ ID NO. 109

[0596] LC-FR3 having the amino acid sequence of SEQ ID NO. 119

[0597] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0598] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0599] (79) VL region (10D1_c89) containing the following FR:

[0600] LC-FR1 having the amino acid sequence of SEQ ID NO. 104

[0601] LC-FR2 having the amino acid sequence of SEQ ID NO. 110

[0602] LC-FR3 having the amino acid sequence of SEQ ID NO. 120

[0603] LC-FR4 having the amino acid sequence of SEQ ID NO. 125,

[0604] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0605] (80) VL region (10D1_c90) containing the following FR:

[0606] LC-FR1 having the amino acid sequence of SEQ ID NO. 105

[0607] LC-FR2 having the amino acid sequence of SEQ ID NO. 110

[0608] LC-FR3 having the amino acid sequence of SEQ ID NO. 121

[0609] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0610] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0611] (81) VL region (10D1_c91) containing the following FR:

[0612] LC-FR1 having the amino acid sequence of SEQ ID NO. 104

[0613] LC-FR2 having the amino acid sequence of SEQ ID NO. 111

[0614] LC-FR3 having the amino acid sequence of SEQ ID NO. 122

[0615] LC-FR4 having the amino acid sequence of SEQ ID NO. 125,

[0616] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0617] (82) VL region (10D1_c92) containing the following FR:

[0618] LC-FR1 having the amino acid sequence of SEQ ID NO. 100

[0619] LC-FR2 having the amino acid sequence of SEQ ID NO. 112

[0620] LC-FR3 having the amino acid sequence of SEQ ID NO. 114

[0621] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0622] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0623] (83) VL region (10D1_c93) containing the following FR:

[0624] LC-FR1 having the amino acid sequence of SEQ ID NO. 103

[0625] LC-FR2 having the amino acid sequence of SEQ ID NO. 108

[0626] LC-FR3 having the amino acid sequence of SEQ ID NO. 119

[0627] LC-FR4 having the amino acid sequence of SEQ ID NO. 124,

[0628] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0629] (84) (10A6) VL region containing the following FR:

[0630] LC-FR1 having the amino acid sequence of SEQ ID NO. 168

[0631] LC-FR2 having the amino acid sequence of SEQ ID NO. 169

[0632] LC-FR3 having the amino acid sequence of SEQ ID NO. 170

[0633] LC-FR4 having the amino acid sequence of SEQ ID NO. 142,

[0634] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0635] (85) (4-35-B2) VL region containing the following FR:

[0636] LC-FR1 having the amino acid sequence of SEQ ID NO. 139

[0637] LC-FR2 having the amino acid sequence of SEQ ID NO. 140

[0638] LC-FR3 having the amino acid sequence of SEQ ID NO. 141

[0639] LC-FR4 having the amino acid sequence of SEQ ID NO. 142,

[0640] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0641] (86) (4-35-B4) VL region containing the following FR:

[0642] LC-FR1 having the amino acid sequence of SEQ ID NO. 154

[0643] LC-FR2 having the amino acid sequence of SEQ ID NO. 155

[0644] LC-FR3 having the amino acid sequence of SEQ ID NO. 156

[0645] LC-FR4 having the amino acid sequence of SEQ ID NO. 142,

[0646] or variants thereof in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one or two or three amino acids substituted with another amino acid.

[0647] In some embodiments, the antigen-binding molecule comprises a VL region including a CDR according to one of (62) to (71) and a FR according to one of (72) to (86).

[0648] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (87) to (102) below:

[0649] (87) A VL region including a CDR according to (62) and a FR according to (72), (73), (74), (75), (76), (77), (78), (79), (80), (81), (82) or (83).

[0650] (88) VL region including CDR according to (63) and FR according to (72)).

[0651] (89) VL region including CDR according to (63) and FR according to (73).

[0652] (90) VL region including CDR according to (63) and FR according to (76).

[0653] (91) VL region including CDR according to (63) and FR according to (78).

[0654] (92) VL region including CDR according to (63) and FR according to (79).

[0655] (93) A VL region including CDR according to (63) and FR according to (81).

[0656] (94) VL region including CDR according to (63) and FR according to (83).

[0657] (95) VL region including CDR according to (64) and FR according to (74).

[0658] (96) VL region including CDR according to (65) and FR according to (75).

[0659] (97) VL region including CDR according to (66) and FR according to (77).

[0660] (98) VL region including CDR according to (67) and FR according to (80).

[0661] (99) VL region including CDR according to (68) and FR according to (82).

[0662] (100) VL region including CDR according to (69) and FR according to (84).

[0663] (101) A VL region including CDR according to (70) and FR according to (85).

[0664] (102) VL region including CDR according to (71) and FR according to (86).

[0665] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (103) to (119) below:

[0666] (103) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 74, 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.

[0667] (104) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 75, 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.

[0668] (105) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 76, 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.

[0669] (106) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 77, 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.

[0670] (107) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78, 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.

[0671] (108) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 79, 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.

[0672] (109) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 80, 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.

[0673] (110) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 81, 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.

[0674] (111) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 82, 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.

[0675] (112) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 83, 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.

[0676] (113) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 84, 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.

[0677] (114) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 85, 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.

[0678] (115) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 86, 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.

[0679] (116) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 87, 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.

[0680] (117) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 135, 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.

[0681] (118) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 150, 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.

[0682] (119) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 164, 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.

[0683] In some embodiments, the antigen-binding molecule comprises a VH region according to any one of (1) to (61) and a VL region according to any one of (62) to (119).

[0684] In embodiments according to the present invention in which one or more amino acids are substituted with other amino acids, the substitution may be, for example, a conservative substitution according to the following table. In some embodiments, amino acids within the same block in the middle column are substituted. In some embodiments, amino acids within the same line in the far right column are substituted:

[0685]

[0686] In some embodiments, the substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may substantially not affect) one or more functional properties (e.g., target binding) of the antigen-binding molecule containing the substitution compared to an equivalent unsubstituted molecule.

[0687] The VH and VL regions of the antigen-binding region of the antibody together constitute the Fv region. In some embodiments, the 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 Fv are provided as a single polypeptide linked by a linker region, i.e., a single chain Fv (scFv).

[0688] In some embodiments, the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is a heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, or is derived therefrom.

[0689] 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 through 98 of SEQ ID NO. 171 form a CH1 region (SEQ ID NO. 172). Positions 99 through 110 of SEQ ID NO. 171 form a hinge region (SEQ ID NO. 173) between the CH1 region and the CH2 region. Positions 111 through 223 of SEQ ID NO. 171 form a CH2 region (SEQ ID NO. 174). Positions 224 through 330 of SEQ ID NO. 171 form a CH3 region (SEQ ID NO. 175).

[0690] The exemplified antigen-binding molecule can be prepared using pFUSE-CHIg-hG1, which includes the substitution 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 shown in SEQ ID NO. 176. It will be recognized that the CH3 region may be provided with additional substitutions depending on modifications to the Fc region of the antigen-binding molecule as described herein.

[0691] In some embodiments, the CH1 region comprises or consists of a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the sequence of SEQ ID NO. 172 or the amino acid sequence of SEQ ID NO. 172. In some embodiments, the CH1-CH2 hinge region comprises or consists of a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the sequence of SEQ ID NO. 173 or the amino acid sequence of SEQ ID NO. 173. In some embodiments, the CH2 region comprises or consists of a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the sequence of SEQ ID NO. 174 or the amino acid sequence of SEQ ID NO. 174. In some embodiments, the CH3 region comprises or consists of a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the sequence of SEQ ID NO. 175 or 176 or the amino acid sequence of SEQ ID NO. 175 or 176.

[0692] In some embodiments, the antigen-binding molecule of the present invention comprises 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), e.g., IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In some embodiments, the CL region comprises or consists of a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the sequence of SEQ ID NO. 177, or the amino acid sequence of SEQ ID NO. 177.

[0693] The VL and light chain constant (CL) regions of the antigen-binding region of the antibody, and the VH region and heavy chain constant 1 (CH1) region together constitute a Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising VH, CH1, VL, and CL (e.g., Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide), and a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising VL and CH (e.g., a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of Fab or CrossFab are provided as a single polypeptide joined by a linker region, i.e., as a single-chain Fab (scFab) or a single-chain CrossFab (scCrossFab).

[0694] In some embodiments, the antigen-binding molecule of the present invention comprises or is composed of a Fab region that binds to HER3.

[0695] In some embodiments, the antigen-binding molecule described herein comprises or consists 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 the literature [Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52], the full text of which is incorporated herein by reference.

[0696] G-type immunoglobulin (i.e., IgG) is a ~150 kDa glycoprotein comprising two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains a heavy chain constant region containing three constant domains (CH1, CH2, and CH3) following VH, and similarly, the light chain contains CL following VL. 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).

[0697] In some embodiments, the antigen-binding molecules described herein include or consist of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that bind to HER3.

[0698] In some embodiments, the antigen-binding molecule of the present invention is monovalent to HER3. The binding valence refers to the number of binding sites of the antigen-binding molecule for a given antigenic determinant. Accordingly, in some embodiments, the antigen-binding molecule comprises at least one binding site for HER3.

[0699] In some embodiments, the antigen-binding molecule comprises one or more binding sites for HER3, e.g., two, three, or four binding sites. The binding sites may be identical or different. In some embodiments, the antigen-binding molecule is, e.g., divalent, trivalent, or tetravalent for HER3.

[0700] Embodiments of the present invention relate to multispecific antigen-binding molecules. “Multispecific” means that the antigen-binding molecule exhibits specific binding to one or more targets. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains (i.e., at least two antigen-binding domains including, for example, non-identical VH and VL).

[0701] In some embodiments, the antigen-binding molecule binds to HER3 and another target (e.g., an antigen other than HER3), so it is at least bispecific. The term “bispecific” means that the antigen-binding molecule can specifically bind to at least two distinct antigenic determinants.

[0702] An antigen-binding molecule according to the present invention (e.g., a multispecific antigen-binding molecule) may include an antigen-binding molecule to which the antigen-binding molecule can bind to a specific target. For example, an antigen-binding molecule capable of binding to HER3 and antigens other than HER3 comprises: (i) an antigen-binding molecule capable of binding to HER3, and (ii) an antigen-binding molecule capable of binding to antigens other than HER3.

[0703] The antigen-binding molecule according to the present invention (e.g., a multispecific antigen-binding molecule) may comprise an antigen-binding polypeptide or an antigen-binding polypeptide complex to which the antigen-binding molecule can bind to a specific target. For example, the antigen-binding molecule according to the present invention may comprise, for example, (i) an antigen-binding polypeptide complex capable of binding to HER3 comprising a light chain polypeptide (including structure VL-CL) and a heavy chain polypeptide (including structure VH-CH1-CH2-CH3), and (ii) an antigen-binding polypeptide complex capable of binding to an antigen other than HER3 comprising a light chain polypeptide (including structure VL-CL) and a heavy chain polypeptide (including structure VH-CH1-CH2-CH3).

[0704] In some embodiments, a component antigen-binding molecule of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to as an “antigen-binding domain” or “antigen-binding region” of the larger antigen-binding molecule.

[0705] 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 an immune cell surface molecule. In some embodiments, the antigen other than HER3 is a cancer cell antigen. In some embodiments, the antigen other than HER3 is a receptor molecule, e.g., a cell surface receptor. In some embodiments, the antigen other than HER3 is a cell signaling molecule, e.g., a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen other than HER3 is a growth factor or a hormone.

[0706] A cancer cell antigen is an antigen expressed or overexpressed by a cancer cell. A cancer cell antigen may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The expression of a cancer cell antigen may be associated with cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g., the cancer cell antigen may be expressed with abnormal localization) or expressed with an abnormal structure by a cancer cell. A cancer cell antigen may trigger 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, a portion of the antigen that is bound by the antigen-binding molecule described herein appears on the outer surface (i.e., extracellular) of the cancer cell. A cancer cell antigen may be a cancer-associated antigen. In some embodiments, the cancer cell antigen is an antigen whose expression is associated with the development, progression, or severity of symptoms of cancer. Cancer-related antigens may be associated with the cause or pathology of cancer, or may be abnormally expressed as a result of cancer. In some embodiments, cancer cell antigens are antigens whose expression is upregulated by cancer cells (e.g., at the RNA and / or protein level) compared to the level of expression by comparable non-cancer cells (e.g., non-cancer cells derived from the same tissue / cell type). In some embodiments, cancer-related antigens may be preferentially expressed by cancer cells and are not expressed by comparable non-cancer cells (e.g., non-cancer cells derived from the same tissue / cell type). In some embodiments, cancer-related antigens may be products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer-related antigens may be products of overexpressed cell proteins, cancer antigens produced by oncogeneic viruses, oncofetal antigens, or cell surface glycolipids or glycoproteins.

[0707] In some embodiments, antigens other than HER3 are antigens expressed by HER3-associated cancer cells. HER3-associated cancer may be a cancer that expresses HER3 (e.g., expresses HER3 protein on the cell surface); such cancer may be referred to as "HER3-positive" cancer. HER3-associated cancer includes cancers in which HER3 gene / protein expression is a risk factor for the onset, development, progression or severity, and / or metastasis of cancer symptoms, and / or is positively associated with it. HER3-associated cancer includes cancers described in the literature [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], the full text of both of which is incorporated by reference into this specification. In some embodiments, HER3-related cancers may be lung cancer (e.g., NSCLC), melanoma, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, stomach cancer, colon cancer, or oral cancer.

[0708] The immune cell surface molecule may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed on or on the cell surface of an 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 outer surface of the immune cell (i.e., extracellular). The immune cell surface molecule may be expressed on the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, e.g., neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be, e.g., T cell, B cell, natural killer (NK) cell, NKT cell, or innate lymphocyte (ILC), or a precursor thereof (e.g., thymic cell or pre-B cell). In some embodiments, the immune cell surface molecule may be a co-stimulator molecule (e.g., CD28, OX40, 4-1BB, ICOS, or CD27) or a ligand thereof. In some embodiments, the immune cell surface molecule may be a checkpoint molecule (e.g., PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, or BTLA) or a ligand thereof.

[0709] The multispecific antigen-binding molecule according to the present invention may be provided in any suitable form, such as the form described in the literature [Brinkmann and Kontermann MAbs (2017) 9(2): 182-212] (the full text of which is incorporated herein by reference). Suitable forms are those shown in FIG. 2 of the literature [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., IgG, chimeric IgG, κλ-body, general HC; CH1 / CL fusion proteins, e.g., scFv2-CH1 / CL, VHH2-CH1 / CL; 'Variable domain alone' bispecific antigen-binding molecules, e.g., tandem scFv (taFV), triplebody, diabody (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, 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-cytokine 2, 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(SEED body), taFv-Fc(kih), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc (SEED body), 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-Cytokine 2; asymmetric IgG or IgG-like molecules, e.g., IgG(kih), IgG(kih) general LC, ZW1 IgG general LC, Biclonics general 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 general 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, half DVD-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; Attached IgG-HC fusions, e.g., IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CαCβ)Fab, scFv-HC-IgG, Tamdem Fab-IgG (orthogonal Fab), Fab-IgG (CαCβ Fab), Fab-IgG (CR3), Fab-hinge-IgG (CR3); attached IgG-LC fusions, e.g., IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; attached 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 IgG, e.g., DAF (two-in-one-IgG), DutaMab, Mab2; and non-Ig fusion proteins, e.g., DNL-Fab4-IgG.

[0710] A person skilled in the art can design and manufacture bispecific antigen-binding molecules. A method for producing bispecific antigen-binding molecules comprises chemically crosslinking an antigen-binding molecule or antibody fragment with a reducing disulfide or non-reducing thioether bond, for example, as described in the literature [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 full text of which is incorporated herein by reference. For example, a disulfide-linked bispecific F(ab)2 heteromer can be produced by chemically crosslinking a Fab fragment, for example, through a hinge region SH-group using N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP).

[0711] Another method for producing bispecific antigen-binding molecules includes fusing an antibody-producing hybridoma with, for example, polyethylene glycol to produce a quadroma cell capable of secreting a bispecific antibody as described in, for example, the literature [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].

[0712] The bispecific antigen-binding molecule according to the present invention may also be recombinantly produced by expression from a nucleic acid construct encoding a polypeptide for an antigen-binding molecule, as described in, for example, the literature [Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at 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 full contents of both of which are incorporated herein by reference. For example, a DNA construct comprising a sequence encoding light and heavy chain variable domains for two antigen-binding fragments (i.e., a light and heavy chain variable domain for an antigen-binding fragment capable of binding to HER3, and a light and heavy chain variable domain for an antigen-binding fragment capable of binding to another target protein) and encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning technology. A recombinant bispecific antibody can then be generated by the expression of the construct (e.g., in vitro) in a suitable host cell (e.g., mammalian host cell), and the expressed recombinant bispecific antibody can then be selectively purified.

[0713] Fc area

[0714] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region.

[0715] In IgG IgA and IgD isoforms, the Fc region consists of CH2 and CH3 regions from one polypeptide and CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from two polypeptides together form the Fc region. In IgM and IgE isoforms, the Fc region includes three constant domains (CH2, CH3, and CH4), and CH2 to CH4 from two polypeptides together form the Fc region.

[0716] In a preferred embodiment according to various aspects of the present disclosure, the Fc region comprises two polypeptides, each polypeptide comprising a CH2 region and a CH3 region.

[0717] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region containing modifications in one or more of the CH2 and CH3 regions that promote the association of the Fc region. The recombination co-expression and subsequent association of the constituent polypeptides of the antigen-binding molecule lead to several possible combinations. To improve the yield of the desired combination of polypeptides within the antigen-binding molecule upon recombinant production, it is advantageous to introduce modification(s) into the Fc region that promote the association of the desired combination of heavy chain polypeptides. The modifications may, 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 the literature [Ha et al., Front. Immnol (2016) 7:394], the full text of which is incorporated herein by reference.

[0718] In some embodiments, the antigen-antigen-binding molecule of the present invention comprises an Fc region containing paired substitutions within the CH3 region of the Fc region according to one of the following forms, as shown in Table 1 of the literature [Ha et al., Front. Immnol (2016) 7:394: KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED or A107].

[0719] In some embodiments, the Fc region comprises a "knob-into-hole" or "KiH" modification, as described in, for example, the literature [US 7,695,936 and Carter, J Immunol Meth 248, 7-15 (2001)]. In these embodiments, one of the CH3 regions of the Fc region comprises a "knob" modification, and the other CH3 region comprises a "hole" modification. The "knob" and "hole" modifications are located within their respective CH3 regions, so that the "knob" is located within the "hole" to promote (and inhibit) the heteromerization of the polypeptide and / or stabilize the heteromer. The knob is created by substituting an amino acid with a small side chain with an amino acid with a larger side chain (e.g., tyrosine or tryptophan). The hole is created by substituting an amino acid with a large side chain with one having a smaller side chain (e.g., alanine or threonine).

[0720] In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule of the present invention comprises the substitution T366W (the numbering of positions / substitutions within the Fc, CH2 and CH3 regions herein follows the EU numbering system as described in the literature [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991]), and another CH3 region of the Fc region comprises the substitution Y407V. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and another CH3 region of the Fc region comprises the substitutions T366S and L368A. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and another CH3 region of the Fc region comprises the substitutions Y407V, T366S, and L368A.

[0721] In some embodiments, the Fc region comprises a “DD-KK” variant, such as that described in WO 2014 / 131694 A1. In some embodiments, one of the CH3 regions comprises the substitutions K392D and K409D, and the other CH3 region of the Fc region comprises the substitutions E356K and D399K. The variant promotes electrostatic interaction between the CH3 regions.

[0722] In some embodiments, the antigen-binding molecule of the present invention comprises a modified Fc region as described in the literature [Labrijn et al., Proc Natl Acad Sci US A. (2013) 110(13):5145-50], referred to as the “duobody” mode. In some embodiments, one of the CH3 regions comprises a substitution K409R, and another CH3 region of the Fc region comprises a substitution K405L.

[0723] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region containing an "EEE-RRR" variant as described in the literature [Strop et al., J Mol Biol. (2012) 420(3):204-19]. In some embodiments, one of the CH3 regions comprises the substitutions D221E, P228E and L368E, and another CH3 region of the Fc region comprises the substitutions D221R, P228R and K409R.

[0724] In some embodiments, the antigen-binding molecule comprises an Fc region containing "EW-RVT" as described in the literature [Choi et al., Mol Cancer Ther (2013) 12(12):2748-59]. In some embodiments, one of the CH3 regions comprises the substitutions K360E and K409W, and the other CH3 region of the Fc region comprises the substitutions Q347R, D399V and F405T.

[0725] 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 further stabilizes the heteromer by causing the formation of a disulfide bridge between the two CH3 regions of the Fc region (Carter (2001), J Immunol Methods 248, 7-15).

[0726] In some embodiments, the Fc region is "KiH S - S Includes variations. In some embodiments, one of the CH3 regions includes the substitutions T366W and S354C, and the other CH3 regions of the Fc region include the substitutions T366S, L368A, Y407V, and Y349C.

[0727] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region containing a “SEED” modification as described in the literature [Davis et al., Protein Eng Des Sel (2010) 23(4):195-202], wherein the β-strand segments of human IgG1 CH3 and IgA CH3 are exchanged.

[0728] In some embodiments, one of the CH3 regions contains the substitutions S364H and F405A, and the other CH3 region of the Fc region contains the substitutions Y349T and T394F (see: e.g. Moore et al., MAbs (2011) 3(6):546-57).

[0729] 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).

[0730] In some embodiments, one of the CH3 regions comprises the substitutions K360D, D399M, and Y407A, and the other CH3 region of the Fc region comprises the substitutions E345R, Q347R, T366V, and K409V (see: e.g., Leaver-Fay et al., Structure (2016) 24(4):641-51).

[0731] In some embodiments, one of the CH3 regions comprises the substitutions K370E and K409W, and the other CH3 region of the Fc region comprises the substitutions E357N, D399V and F405T (see: e.g., Choi et al., PLoS One (2015) 10(12):e0145349).

[0732] Fc-mediated functions include Fc receptor binding, antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of membrane attack complexes (MACs), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0733] Modifications to the antibody Fc region that affect Fc-mediated function are known in the art, for example, as described in the literature [Wang et al., Protein Cell (2018) 9(1):63-73], the full text of which is incorporated herein by reference. In particular, exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of the literature [Wang et al., Protein Cell (2018) 9(1):63-73].

[0734] The combination of substitutions F243L / R292P / Y300L / V305I / P396L is described in the literature [Stavenhagen et al. Cancer Res. (2007)] as increasing binding to FcγRIIIa and thus improving ADCC. The combination of substitutions S239D / I332E or S239D / I332E / A330L is described in the literature [Lazar et al., Proc Natl Acad Sci USA. (2006)103:4005-4010] as increasing binding to FcγRIIIa and thus increasing ADCC. The combination of substitutions S239D / I332E / A330L is also described as decreasing binding to FcγRIIb and thus increasing ADCC. The combination of substitutions S298A / E333A / K334A is described in the literature [Shields et al., J Biol Chem. (2001) 276:6591-6604] as increasing binding to FcγRIIIa and thus increasing ADCC. The combination of one heavy chain substitution L234Y / L235Q / G236W / S239M / H268D / D270E / S298A and the other heavy chain substitution D270E / K326D / A330M / K334E is described in the literature [Mimoto et al., MAbs. (2013): 5:229-236] as increasing binding to FcγRIIIa and thus increasing ADCC. The combination of substitutions G236A / S239D / I332E is described in the literature [Richards et al., Mol Cancer Ther. (2008) 7:2517-2527] as increasing binding to FcγRIIa, increasing binding to FcγRIIIa, and thus increasing ADCP.

[0735] The combination of substitutions K326W / E333S is described in the literature [Idusogie et al. J Immunol. (2001) 166(4):2571-5] as increasing binding to C1q and thus increasing CDC. The combination of substitutions S267E / H268F / S324T is described in the literature [Moore et al. MAbs. (2010) 2(2):181-9] as increasing binding to C1q and thus increasing CDC. The combination of substitutions described in the literature [Natsume et al., Cancer Res. (2008) 68(10):3863-72] has been reported to increase binding to C1q and thus increase CDC. The combination of substitutions E345R / E430G / S440Y is [Diebolder et al. It is described in Science (2014) 343(6176):1260-3] that it increases hexamerization and thus increases CDC.

[0736] The combination of substitutions M252Y / S254T / T256E is described in the literature [Dall'Acqua et al. J Immunol. (2002) 169:5171-5180] as increasing binding to FcRn at pH 6.0 and thus increasing the antigen-binding molecular half-life. The combination of substitutions M428L / N434S is described in the literature [Zalevsky et al. Nat Biotechnol. (2010) 28:157-159] as increasing binding to FcRn at pH 6.0 and thus increasing the antigen-binding molecular half-life.

[0737] Where the heavy chain invariant region / Fc region / CH2-CH3 region / CH2 region / CH3 region is described in this specification as including a location(s) / substitution(s) that "correspond" to a reference location(s) / substitution(s), an equivalent location(s) / substitution(s) of the homology heavy chain invariant region / Fc region / CH2-CH3 region / CH2 region / CH3 region is considered.

[0738] Where the Fc region is described as containing specific position(s) / substitution(s), the position(s) / substitution(s) may be present on one or both of the polypeptide chains that together form the Fc region.

[0739] Unless otherwise specified, the positions in this specification refer to the positions of the amino acid sequences of the human immunoglobulin constant region numbered according to the EU numbering system as described in the literature [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991]. For example, the human IgG1 substitutions L242C and K334C 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.

[0740] The 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 with respect to the heavy chain constant region of human IgG1 (i.e., the amino acid sequence presented in SEQ ID NO. 171). The homologous Fc region is 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 with respect to the CH2-CH3 region of human IgG1 (i.e., the amino acid sequence presented in SEQ ID NOs 174 and 175). The 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 with respect to the CH2 region of human IgG1 (i.e., the amino acid sequence presented in SEQ ID NO. 174). The 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 with respect to the CH3 region of human IgG1 (i.e., the amino acid sequence presented in SEQ ID NO. 175).

[0741] The location corresponding to the identified position in human IgG1 can be identified by sequence alignment, which can be performed using sequence alignment software such as ClustalOmega (Literature [Soding, J. 2005, Bioinformatics 21, 951-960]).

[0742] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region containing a modification that increases Fc-mediated function. In some embodiments, the Fc region comprises a modification that increases ADCC. In some embodiments, the Fc region comprises a modification that increases ADCP. In some embodiments, the Fc region comprises a modification that increases CDC. An antigen-binding molecule comprising an Fc region containing a modification that increases Fc-mediated function (e.g., ADCC, ADCP, CDC) induces an increased level of the associated effector function compared to an antigen-binding molecule comprising a corresponding non-modified Fc region.

[0743] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region containing a modification that increases affinity for one or more Fc receptors (e.g., FcγRIIa, FcγRIIIa). The modification that increases affinity for Fc receptors may increase Fc-mediated effector functions, e.g., antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP). In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region containing a modification that decreases affinity for C1q; such modification may be desirable for decreasing complement-dependent cytotoxicity (CDC). In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region containing a modification that increases hexamer formation. A modification to the Fc region that increases affinity for one or more Fc receptors and decreases affinity for C1q and / or increases hexamer formation is, for example, [Saxena and Wu Front Immunol. Described in [Saxena and Wu Front Immunol. (2016) 7:580], the full text of which is incorporated herein by reference. In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising CH2 / CH3 including one or more substitutions shown in Table 1 of the document [Saxena and Wu Front Immunol. (2016) 7:580].

[0744] In some embodiments, the antigen-binding molecule of the present invention comprises Fc, which includes a modification that increases binding to the Fc receptor. In some embodiments, the Fc region includes a modification that increases binding to the Fcγ receptor. In some embodiments, the Fc region includes a modification that increases binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIIa. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIa. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIb. In some embodiments, the Fc region includes a modification that increases binding to FcRn. In some embodiments, the Fc region includes a modification that increases binding to a complement protein. In some embodiments, the Fc region includes a modification that increases or decreases binding to C1q. In some embodiments, the Fc region includes a modification that promotes the hexamerization of the antigen-binding molecule. In some embodiments, the Fc region includes a modification that increases the antigen-binding molecule half-life. In some embodiments, the Fc region includes a modification that increases co-binding.

[0745] In this specification, "Fcγ receptor" may be derived from any species and includes isoforms, fragments, variants (including mutations), or homologs from any species. Similarly, "FcγRI," "FcγRIIa," "FcγRIIb," "FcγRIIc," "FcγRIIIa," and "FcγRIIIb" each refer to FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb from any species and include isoforms, fragments, variants (including mutations), or homologs from any species. Humans have six different classes of Fcγ receptors (mouse orthologs are shown in parentheses): FcγRI (mFcγRI), FcγRIIa (mFcγRIII), FcγRIIb (mFcγRIIb), FcγRIIc, FcγRIIIa (mFcγRIV), and FcγRIIIb. Dimorphic Fcγ receptors include, for example, the 158V and 158F polymorphs of human FcγRIIIa and the 167H and 167R polymorphs of human FcγRIIa.

[0746] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising one or more of the following (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) (e.g., comprising more than one polypeptide comprising a heavy chain constant region or a CH2-CH3 region comprising the same): C at position corresponding to position 242; C at position corresponding to position 334; A at position corresponding to position 236; D at position corresponding to position 239; E at position corresponding to position 332; L at position corresponding to position 330; K at position corresponding to position 345; and G at position corresponding to position 430.

[0747] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising one or more of the following substitutions (or such substitutions) (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) (e.g., comprising one or more polypeptides comprising a heavy chain constant region or a CH2-CH3 region comprising the same): L242C, K334C, G236A, S239D, I332E, A330L, E345K, and E430G.

[0748] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a C at a position corresponding to position 242 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it). In some embodiments, the Fc region comprises a C at a position corresponding to position 334 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it). In some embodiments, the Fc region comprises a C at a position corresponding to position 242 and a C at a position corresponding to position 334 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it).

[0749] In some embodiments, the antigen-binding molecule comprises an Fc region comprising A at position 236 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it). In some embodiments, the Fc region comprises D at position 239 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it). In some embodiments, the Fc region comprises A at position 236 and D at position 239 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it).

[0750] In some embodiments, the antigen-binding molecule comprises an Fc region comprising position E corresponding to position 332 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it). In some embodiments, the Fc region comprises position A corresponding to position 236, position D corresponding to position 239, and position E corresponding to position 332 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it).

[0751] In some embodiments, the antigen-binding molecule comprises an Fc region comprising L at position 330 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it). In some embodiments, the Fc region comprises A at position 236, D at position 239, E at position 332, and L at position 330 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it).

[0752] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a K at a position corresponding to position 345 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it). In some embodiments, the Fc region comprises a G at a position corresponding to position 430 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it). In some embodiments, the Fc region comprises a K at a position corresponding to position 345 and a G at a position corresponding to position 430 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising it).

[0753] In some embodiments, the antigen-binding molecule comprises an Fc region comprising C at position 242, C at position 334, A at position 236, and D at position 239 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region including these).

[0754] In some embodiments, the antigen-binding molecule comprises an Fc region comprising C at position 242, C at position 334, A at position 236, D at position 239, and E at position 332 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region including these).

[0755] In some embodiments, the antigen-binding molecule comprises an Fc region comprising C at position 242, C at position 334, A at position 236, D at position 239, E at position 332, and L at position 330 (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region including these).

[0756] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a C at position 242, a C at position 334, a K at position 345, and a G at position 430 (e.g., comprising one or more polypeptides comprising a heavy chain constant region or a CH2-CH3 region comprising these).

[0757] In some embodiments, the antigen-binding molecule comprises an Fc region containing a substitution L242C (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same). In some embodiments, the Fc region comprises a substitution K334C (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same). In some embodiments, the Fc region comprises a substitution L242C (or an equivalent substitution) and a substitution K334C (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same).

[0758] In some embodiments, the antigen-binding molecule comprises an Fc region containing a substitution G236A (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same). In some embodiments, the Fc region comprises a substitution S239D (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same). In some embodiments, the Fc region comprises a substitution G236A (or an equivalent substitution) and a substitution S239D (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same).

[0759] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a substitution I332E (or an equivalent substitution) (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising the same). In some embodiments, the Fc region comprises a substitution G236A (or an equivalent substitution), a substitution S239D (or an equivalent substitution), and a substitution I332E (or an equivalent substitution) (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising the same).

[0760] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a substitution A330L (or an equivalent substitution) (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising the same). In some embodiments, the Fc region comprises a substitution G236A (or an equivalent substitution), a substitution S239D (or an equivalent substitution), a substitution I332E (or an equivalent substitution), and a substitution A330L (or an equivalent substitution) (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region comprising the same).

[0761] In some embodiments, the antigen-binding molecule comprises an Fc region containing a substitution E345K (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same). In some embodiments, the Fc region comprises a substitution E430G (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same). In some embodiments, the Fc region comprises a substitution E345K (or an equivalent substitution) and a substitution E430G (or an equivalent substitution) (e.g., comprising one or more polypeptides containing a heavy chain constant region, a CH2-CH3 region, or a CH2 region containing the same).

[0762] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a substitution L242C (or equivalent substitution), a substitution K334C (or equivalent substitution), a substitution G236A (or equivalent substitution), and a substitution S239D (or equivalent substitution) (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region including the same).

[0763] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a substitution L242C (or equivalent substitution), a substitution K334C (or equivalent substitution), a substitution G236A (or equivalent substitution), a substitution S239D (or equivalent substitution), and a substitution I332E (or equivalent substitution) (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region including the same).

[0764] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a substitution L242C (or equivalent substitution), a substitution K334C (or equivalent substitution), a substitution G236A (or equivalent substitution), a substitution S239D (or equivalent substitution), a substitution I332E (or equivalent substitution), and a substitution A330L (or equivalent substitution) (e.g., comprising one or more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region including the same).

[0765] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a substitution L242C (or equivalent substitution), a substitution K334C (or equivalent substitution), a substitution E345K (or equivalent substitution), and a substitution E430G (or equivalent substitution) (e.g., comprising one or more polypeptides comprising a heavy chain constant region or a CH2-CH3 region comprising the same).

[0766] In some embodiments, the antigen-binding molecule comprises an Fc region comprising one or more of the following (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) (e.g., comprising more than one polypeptide comprising a heavy chain constant region or a CH2-CH3 region comprising therein): L at position corresponding to position 243, P at position corresponding to position 292, L at position corresponding to position 300, I at position corresponding to position 305, and L at position corresponding to position 396; D at position corresponding to position 239 and E at position corresponding to position 332; D at position corresponding to position 239, E at position corresponding to position 332, and L at position corresponding to position 330; A at position corresponding to position 298, A at position corresponding to position 333, and A at position corresponding to position 334; Y for position corresponding to position 234, Q for position corresponding to position 235, W for position corresponding to position 236, M for position corresponding to position 239, D for position corresponding to position 268, E for position corresponding to position 270, and A for position corresponding to position 298; E for position corresponding to position 270, D for position corresponding to position 326, M for position corresponding to position 330, and E for position corresponding to position 334; A for position corresponding to position 236, D for position corresponding to position 239, and E for position corresponding to position 332; W for position corresponding to position 326 and S for position corresponding to position 333; E for position corresponding to position 267, F for position corresponding to position 268, and T for position corresponding to position 324; R for position corresponding to position 345, G for position corresponding to position 430, and Y for position corresponding to position 440; Y of the position corresponding to position 252, T of the position corresponding to position 254, and E of the position corresponding to position 256; and L of the position corresponding to position 428 and S of the position corresponding to position 434.

[0767] In some embodiments, the antigen-binding molecule comprises an Fc region comprising one or more of the following combinations of substitutions (or such substitutions) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) (e.g., comprising more than one polypeptide comprising a heavy chain constant region or a CH2-CH3 region comprising such substitutions): F243L / R292P / Y300L / V305I / P396L; S239D / I332E; S239D / I332E / A330L; S298A / E333A / K334A; L234Y / L235Q / G236W / S239M / H268D / D270E / S298A; D270E / K326D / A330M / K334E; G236A / S239D / I332E; K326W / E333S; S267E / H268F / S324T; E345R / E430G / S440Y; M252Y / S254T / T256E; and M428L / N434S.

[0768] polypeptide

[0769] The present invention also provides a polypeptide component of an antigen-binding molecule. The polypeptide may be provided in an isolated or substantially purified form.

[0770] The antigen-binding molecule of the present invention may be a complex of polypeptides or may include the same.

[0771] In this specification, where a polypeptide comprises one or more domains or regions, the plurality of domains / regions will preferably be recognized as existing within the same polypeptide chain. That is, the polypeptide comprises one or more domains, or the region is a fusion polypeptide comprising domains / regions.

[0772] In some embodiments, the polypeptide according to the present invention comprises or consists of VH as described herein. In some embodiments, the polypeptide according to the present invention comprises or consists of VL as described herein.

[0773] In some embodiments, the polypeptide further comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises the CH1, CH2, and / or CH3 regions of immunoglobulin (Ig).

[0774] In some embodiments, the polypeptide comprises one or more regions of the immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region as described herein. In some embodiments, the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments, the polypeptide comprises a CH2 region as described herein. In some embodiments, the polypeptide comprises a CH3 region as described herein. In some embodiments, the polypeptide comprises a CH2-CH3 region as described herein.

[0775] In some embodiments, the polypeptide comprises a CH3 region containing any one of the following combinations of amino acid substitutions / amino acid substitutions (e.g., as shown in Table 1 of the reference incorporated herein by reference [Ha et al., Front. Immnol (2016) 7:394]): T366W; T366S, L368A, and Y407V; T366W and S354C; T366S, L368A, Y407V, and Y349C; S364H and F405A; Y349T and T394F; T350V, L351Y, F405A, and Y407V; T350V, T366L, K392L, and T394W; K360D, D399M, and Y407A; E345R, Q347R, T366V, and K409V; K409D and K392D; D399K and E356K; K360E and K409W; Q347R, D399V and F405T; K360E, K409W and Y349C; Q347R, D399V, F405T and S354C; K370E and K409W; and E357N, D399V and F405T.

[0776] In some embodiments, the CH2 and / or CH3 regions of the polypeptide include one or more amino acid substitutions to promote association between the polypeptide and another polypeptide comprising the CH2 and / or CH3 regions.

[0777] In some embodiments, the polypeptide comprises one or more regions of the immunoglobulin light chain constant sequence. In some embodiments, the polypeptide comprises a CL region as described herein.

[0778] In some embodiments, the polypeptide according to the present invention comprises a structure from the N-terminus to the C-terminus according to one of the following:

[0779] (i) VH

[0780] (ii) VL

[0781] (iii) VH-CH1

[0782] (iv) VL-CL

[0783] (v) VL-CH1

[0784] (vi) VH-CL

[0785] (vii) VH-CH1-CH2-CH3

[0786] (viii) VL-CL-CH2-CH3

[0787] (ix) VL-CH1-CH2-CH3

[0788] (x) VH-CL-CH2-CH3

[0789] Additionally, an antigen-binding molecule composed of a polypeptide of the present invention is provided by the present invention. In some embodiments, the antigen-binding molecule of the present invention comprises one of the following combinations of polypeptides:

[0790] (A) VH+VL

[0791] (B) VH-CH1+VL-CL

[0792] (C) VL-CH1+VH-CL

[0793] (D) VH-CH1-CH2-CH3+ VL-CL

[0794] (E) VH-CL-CH2-CH3+ VL-CH1

[0795] (F) VL-CH1-CH2-CH3+ VH-CL

[0796] (G) VL-CL-CH2-CH3+ VH-CH1

[0797] (H) VH-CH1-CH2-CH3+ VL-CL-CH2-CH3

[0798] (I) VH-CL-CH2-CH3+ VL-CH1-CH2-CH3

[0799] In some embodiments, the antigen-binding molecule comprises one or more of the polypeptides of the combinations shown in (A) to (I). For example, with reference to (D), in some embodiments, the antigen-binding molecule comprises two polypeptides having the structure VH-CH1-CH2-CH3 and two polypeptides having the structure VL-CL.

[0800] In some embodiments, the antigen-binding molecule of the present invention comprises one of the following combinations of polypeptides:

[0801] (J) VH (anti-HER3)+VL (anti-HER3)

[0802] (K) VH (anti-HER3)-CH1+VL (anti-HER3)-CL

[0803] (L) VL (anti-HER3)-CH1+VH (anti-HER3)-CL

[0804] (M) VH (anti-HER3)-CH1-CH2-CH3+ VL (anti-HER3)-CL

[0805] (N) VH (Anti-HER3)-CL-CH2-CH3+ VL (Anti-HER3)-CH1

[0806] (O) VL (anti-HER3)-CH1-CH2-CH3+ VH (anti-HER3)-CL

[0807] (P) VL (anti-HER3)-CL-CH2-CH3+ VH (anti-HER3)-CH1

[0808] (Q) VH (Anti-HER3)-CH1-CH2-CH3+ VL (Anti-HER3)-CL-CH2-CH3

[0809] (R) VH (anti-HER3)-CL-CH2-CH3+ VL (anti-HER3)-CH1-CH2-CH3

[0810] Herein: "VH(anti-HER3)" refers to VH of an antigen-binding molecule capable of binding to HER3 as described in this specification, e.g., as defined in one of (1) to (61); and "VL(anti-HER3)" refers to VL of an antigen-binding molecule capable of binding to HER3 as described in this specification, e.g., as defined in one of (62) to (119).

[0811] 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 with respect to one of the amino acid sequences of SEQ ID NOs 187 to 223.

[0812] Linker and additional sequence

[0813] In some embodiments, the antigen-binding molecule and polypeptide of the present invention comprise a hinge region. In some embodiments, the hinge region is provided between a CH1 region and a CH2 region. In some embodiments, the hinge region is provided between a CL region and a 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 with respect to the amino acid sequence of SEQ ID NO. 173.

[0814] In some embodiments, the antigen-binding molecule and polypeptide of the present invention comprise one or more linker sequences between amino acid sequence numbers. The linker sequence 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.

[0815] Linker sequences are known to those skilled in the art, for example, as described in the literature [Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369], the full text of which is incorporated herein by reference. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows for relative shifting of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those skilled in the art, and several are identified in the literature [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.

[0816] 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 1-2, 1-3, 1-4, 1-5, or 1-10 amino acids.

[0817] The antigen-binding molecules and polypeptides of the present invention may further comprise additional amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides facilitate the expression, folding, trafficking, processing, purification, or detection of the antigen-binding molecule / polypeptide by comprising amino acid sequence(s). For example, the antigen-binding molecule / polypeptide may optionally comprise a sequence encoding His (e.g., 6XHis), Myc, GST, MBP, FLAG, HA, E, or a biotin tag at the N- or C-terminus of the antigen-binding molecule / polypeptide. In some embodiments, the antigen-binding molecule / polypeptide comprises a detectable moiety, e.g., fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzyme label.

[0818] 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). The signal peptide generally consists of a sequence of 5 to 30 hydrophobic amino acids, which forms a single alpha helix. Secreted proteins and proteins expressed on the cell surface often comprise the signal peptide.

[0819] Signal peptides may be present at the N-terminus of antigen-binding molecules / polypeptides or within newly synthesized antigen-binding molecules / polypeptides. Signal peptides are provided for the efficient trafficking and secretion of antigen-binding molecules / polypeptides. Since signal peptides are often removed by cleavage, they are not included in mature antigen-binding molecules / polypeptides secreted from cells expressing antigen-binding molecules / polypeptides.

[0820] Signal peptides are known for many proteins and are recorded in databases, e.g., GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, or can be identified / predicted using amino acid sequencing tools, e.g., Signal P (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

[0821] In some embodiments, the signal peptide of the antigen-binding molecule / polypeptide of the present invention comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to one of the amino acid sequences of SEQ ID NOs 178 to 186.

[0822] Labels and conjugates

[0823] In some embodiments, the antigen-binding molecule of the present invention further comprises a detectable moiety.

[0824] In some embodiments, the antigen-binding molecule comprises a detectable moiety, e.g., a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radiolabel, a chemical, nucleic acid, or enzymatic label. The antigen-binding molecule may be covalently or non-covalently labeled with a detectable moiety.

[0825] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB; green fluorescent protein (GFP) chelates of rare earth elements, for example, europium (Eu), terbium (Tb), and samarium (Sm); tetramethylrhodamine; Texas Red; 4-methyl umbelliferone; 7-amino-4-methyl coumarin; Cy3; and Cy5. Radiolabels include radioisotopes, for example, 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 l167 , thulium 168 , copper 67 , fluoride 18 , yttrium 90 , palladium 100 , bismuth 217 and antimony 211Includes. Luminescent labels include radioluminescent, chemiluminescent (e.g., acridinium ester, luminol, isoluminol), and bioluminescent labels. Immuno-detectorable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands, e.g., biotin, avidin, streptavidin, or digoxigenin. Nucleic acid labels include aptamers. Enzyme labels include, e.g., peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, and luciferase.

[0826] In some embodiments, the antigen-binding molecule of the present invention is conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect. Antibody-drug conjugates are reviewed, for example, in the literature [Parslow et al., Biomedicines. 2016 Sep; 4(3):14]. In some embodiments, the chemical moiety may be a drug moiety (e.g., a cytotoxic agent). In some embodiments, the drug moiety may be a chemotherapy agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1, DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD.

[0827] Special exemplary embodiments of antigen-binding molecules

[0828] In some embodiments, the antigen-binding molecule is:

[0829] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 187, or two polypeptides composed thereof; and

[0830] (ii) comprises 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 with respect to the amino acid sequence of SEQ ID NO. 188, or comprises two polypeptides made therefrom, or made therefrom.

[0831] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0832] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 189, or two polypeptides composed thereof; and

[0833] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 190, or two polypeptides composed thereof.

[0834] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0835] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 191, or two polypeptides composed thereof; and

[0836] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 192, or two polypeptides composed thereof.

[0837] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0838] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 193, or two polypeptides composed thereof; and

[0839] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 195, or two polypeptides composed thereof.

[0840] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0841] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 194, or two polypeptides composed thereof; and

[0842] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 195, or two polypeptides composed thereof.

[0843] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0844] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 196, or two polypeptides composed thereof; and

[0845] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 195, or two polypeptides composed thereof.

[0846] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0847] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 197, or two polypeptides composed thereof; and

[0848] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 199, or two polypeptides composed thereof.

[0849] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0850] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 198, or two polypeptides composed thereof; and

[0851] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 199, or two polypeptides composed thereof.

[0852] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0853] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 200, or two polypeptides composed thereof; and

[0854] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 201, or two polypeptides composed thereof.

[0855] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0856] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 202, or two polypeptides composed thereof; and

[0857] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 203, or two polypeptides composed thereof.

[0858] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0859] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 204, or two polypeptides composed thereof; and

[0860] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 205, or two polypeptides composed thereof.

[0861] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0862] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 206, or two polypeptides composed thereof; and

[0863] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 207, or two polypeptides composed thereof.

[0864] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0865] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 208, or two polypeptides composed thereof; and

[0866] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 209, or two polypeptides composed thereof.

[0867] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0868] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 210, or two polypeptides composed thereof; and

[0869] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 211, or two polypeptides composed thereof.

[0870] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0871] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 212, or two polypeptides composed thereof; and

[0872] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 213, or two polypeptides composed thereof.

[0873] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0874] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 214, or two polypeptides composed thereof; and

[0875] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 215, or two polypeptides composed thereof.

[0876] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0877] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 216, or two polypeptides composed thereof; and

[0878] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 217, or two polypeptides composed thereof.

[0879] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0880] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 218, or two polypeptides composed thereof; and

[0881] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 219, or two polypeptides composed thereof.

[0882] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0883] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 220, or two polypeptides composed thereof; and

[0884] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 221, or two polypeptides composed thereof.

[0885] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0886] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 222, or two polypeptides composed thereof; and

[0887] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 223, or two polypeptides composed thereof.

[0888] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0889] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 225, or two polypeptides composed thereof; and

[0890] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 207, or two polypeptides composed thereof.

[0891] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0892] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 226, or two polypeptides composed thereof; and

[0893] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 207, or two polypeptides composed thereof.

[0894] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0895] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 227, or two polypeptides composed thereof; and

[0896] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 217, or two polypeptides composed thereof.

[0897] In some embodiments, the antigen-binding molecule comprises or consists of the following:

[0898] (i) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 228, or two polypeptides composed thereof; and

[0899] (ii) an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO. 217, or two polypeptides composed thereof.

[0900] Functional characteristics of antigen-binding molecules

[0901] The antigen-binding molecules described herein may be characterized by specific functional properties. In some embodiments, the antigen-binding molecules described herein may have one or more of the following properties:

[0902] Binds to HER3 (e.g., human, mouse, rat, or Synomolgus macaqui HER3);

[0903] Does not bind to EGFR and / or HER2;

[0904] Binds to HER3-expressing cells;

[0905] It binds to subdomain II of the extracellular region of HER3;

[0906] When HER3 is in an open or closed stereotype, it binds to HER3;

[0907] Binds to HER3 independently of NRG;

[0908] It does not compete with MM-121 and / or LJM-716 for binding to HER3;

[0909] For binding to HER3, it does not compete with M-05-74 and / or M-08-11;

[0910] [01259 Inhibits the interaction between HER3 and HER3's interaction partners (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R and / or cMet);

[0911] Inhibits HER3-mediated signaling;

[0912] Inhibits the proliferation of HER3-expressing cells (e.g., in response to stimulation by NRG);

[0913] Inhibits PI3K / AKT / mTOR and / or MAPK signaling by HER3-expressing cells (e.g., response to stimulation by NRG);

[0914] It binds to an active Fcγ receptor (e.g., FcγRIIIa);

[0915] Increased binding to active Fcγ receptors;

[0916] Increased binding to an active Fcγ receptor 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 to 175;

[0917] Reduced binding to an inhibitory Fcγ receptor 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 to 175;

[0918] Increased binding to an active Fcγ receptor compared to an inhibitory Fcγ receptor, compared to an equivalent antigen-binding molecule having a CH2-CH3 Fc region having the amino acid sequences of SEQ ID NOs 174 to 175;

[0919] Increased or decreased binding to a complement protein (e.g., C1q) compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs 174 to 175;

[0920] Increased hexamerization compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs 174 to 175;

[0921] 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 to 175;

[0922] 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 to 175;

[0923] Increased or decreased CDC activity compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs 174 to 175;

[0924] Similar or increased thermal stability compared to an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs 174 to 175;

[0925] Increases the death of HER3-expressing cells;

[0926] Reduces the number / ratio of HER3-expressing cells;

[0927] and

[0928] It inhibits the development and / or progression of cancer in vivo.

[0929] The antigen-binding molecules described herein preferably exhibit a specific binding to HER3. As used herein, "specific binding" refers to a binding that is selective for an antigen and can be distinguished from a non-specific binding to a non-target antigen. An antigen-binding molecule that specifically binds to a target molecule preferably binds to the target with high affinity and / or for a longer period than it binds to other non-target molecules.

[0930] The ability of a specific polypeptide to specifically bind to a specific molecule can be measured by analysis according to methods known in the art, e.g., ELISA, surface plasmon resonance (SPR; see e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometer (see e.g., Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or radiolabeled antigen-binding assay (RIA) enzyme-labeled immunosorbent assay. Through such analysis, binding to the specific molecule can be measured and quantified. In some embodiments, binding may be a reaction detected in the specific analysis.

[0931] In some embodiments, the degree 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 interferometer, or RIA. Alternatively, binding specificity may be reflected in the binding affinity modulus, where the antigen-binding molecule has a KD of at least 0.1 orders of magnitude (i.e., 0.1 x 10⁻¹⁰) greater than that of the antigen-binding molecule to the non-target molecule. n, where n is an integer representing the number of digits) combine with a larger dissociation constant (KD). This can 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.

[0932] In some embodiments, the antigen-binding molecule is human HER3, mouse HER3, rat HER3 and / or Synomolgus macaqui (Macaqui faciculariis ( Macaca fascicularis )) Indicates binding with HER3. That is, in some embodiments, the antigen-binding molecule is cross-reactive to human HER3, mouse HER3, rat HER3, and / or Synomolgus macaqui HER3. In some embodiments, the antigen-binding molecule of the present invention exhibits cross-reactivity with HER3 of non-human primates. Through cross-reactivity to HER3 in model species, efficacy in synonyms can be explored in vivo without relying on surrogate molecules.

[0933] In some embodiments, the antigen-binding molecule binds to human HER3, mouse HER3, rat HER3 and / or synomolgus macaqui HER3; and does not bind to HER2 and / or EGFR (e.g., human HER2 and / or human EGFR).

[0934] In some embodiments, the antigen-binding molecule does not exhibit specific binding to EGFR (e.g., human EGFR). In some embodiments, the antigen-binding molecule does not exhibit specific binding to HER2 (e.g., human HER2). In some embodiments, the antigen-binding molecule does not exhibit specific binding to members of the EGFR protein family other than HER3 (i.e., does not cross-react with them). In some embodiments, the antigen-binding molecule does not exhibit specific binding to EGFR, HER2, and / or HER4.

[0935] In some embodiments, the antigen-binding molecule of the present invention binds to HER3 (e.g., human HER3), and K D is 10 μM or less, preferably ≤5 μM, ≤2 μM, ≤1 μM, ≤500 nM, ≤400 nM, ≤300 nM, ≤200 nM, ≤100 nM, ≤95 nM, ≤90 nM, ≤85 nM, ≤80 nM, ≤75 nM, ≤70 nM, ≤65 nM, ≤60 nM, ≤55 nM, ≤50 nM, ≤45 nM, ≤40 nM, ≤35 nM, ≤30 nM, ≤25 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM ≤3 nM, ≤2 nM, ≤1 nM, ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤90 pM, ≤80 pM, ≤70 pM, ≤60 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM, ≤9 pM, ≤8 pM, ≤7 pM, ≤6 pM, ≤5 pM, ≤4 pM, ≤3 pM, ≤2 pM, ≤1 pM.

[0936] The antigen-binding molecule of the present invention can bind to a specific region of a desired HER3. The antigen-binding region of the antigen-binding molecule according to the present invention can bind to a linear epitope of HER3 consisting of a continuous sequence of amino acids (i.e., a primary sequence of amino acids). In some embodiments, the antigen-binding molecule can bind to a structural epitope of HER3 consisting of a discontinuous sequence of amino acids.

[0937] In some embodiments, the antigen-binding molecule of the present invention binds to HER3. In some embodiments, the antigen-binding molecule binds to the extracellular region of HER3 (e.g., the region presented in SEQ ID NO. 9). In some embodiments, the antigen-binding molecule binds to subdomain II of the extracellular region of HER3 (e.g., the region presented in SEQ ID NO. 16).

[0938] In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 229. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 229. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented 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 presented in SEQ ID NOs 230 and 231. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 230. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 230. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 231. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 231. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 23. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 23. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 21. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 21. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 19. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 19. In some embodiments, the antigen-binding molecule binds to the region of HER3 presented in SEQ ID NO. 22. In some embodiments, the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 presented in SEQ ID NO. 22.

[0939] In some embodiments, the antigen-binding molecule of the present invention may bind to a polypeptide comprising or consisting of one of the amino acid sequences of SEQ ID NOs 1, 3, 4, 6, or 8. In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 9. In some embodiments, the antigen-binding molecule may bind to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 16. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 229. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequences of SEQ ID NOs 230 and 231. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 230. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 231. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 23. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 21. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 19. In some embodiments, the antigen-binding molecule may bind to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO. 22.

[0940] 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 come into contact with the amino acid residues of 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 presented in SEQ ID NO. 23. In some embodiments, the antigen-binding molecule does not come into contact with the amino acid residues of the region of HER3 presented in SEQ ID NO. 23. In some embodiments, the antigen-binding molecule cannot 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 cannot bind to a peptide consisting of the amino acid sequence of SEQ ID NO. 23.

[0941] As used herein, "peptide" refers to a chain of two or more amino acid monomers connected by peptide bonds. Peptides typically have a length within the range of about 2 to 50 amino acids. "Polypeptide" is a polymer chain of two or more peptides. Polypeptides typically have a length of about 50 amino acids or more.

[0942] 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, e.g., ELISA, immunoblot (e.g., Western blot), immunoprecipitation, surface plasmon resonance, and biolayer interference.

[0943] Ligand binding to HER3 promotes conformational changes that enable the homodimerization or heterodimerization of HER3, leading to the activation of downstream pathways. HER3 exhibits 'closed' and 'open' conformations. The closed conformation means that HER3 is in a tethered form and cannot be used for receptor homodimerization or heterodimerization. The open conformation means that HER3 is in an extended form and is available for receptor homodimerization or heterodimerization.

[0944] In some embodiments, the antigen-binding molecule may bind to HER3 when HER3 is in an open stereotype. In some embodiments, the antigen-binding molecule may bind to HER3 when HER3 is in a closed stereotype. In some embodiments, the antigen-binding molecule may bind to HER3 when HER3 is in an open and / or closed stereotype. In some embodiments, the antigen-binding molecule may bind to the HER3 ectododomain when HER3 is in an open and / or closed stereotype. In some embodiments, the antigen-binding molecule may bind to the HER3 dimerizing arm when HER3 is in an open and / or closed stereotype. For example, as described herein, by binding to the dimerizing arm, the antigen-binding molecule may inhibit the interaction between HER3 and its interaction partner.

[0945] In some embodiments, the antigen-binding molecule may bind to HER3 in the presence and / or absence of a ligand for HER3. In some embodiments, the antigen-binding molecule may bind to HER3 independently of a ligand for HER3. In some embodiments, the ligand is NRG, NRG-1, and / or NRG-2. HER3 is activated by ligand binding to an extracellular domain that promotes conformational changes enabling the homodimerization or heterodimerization of HER3. Binding of the antigen-binding molecule to HER3 independently of ligand binding allows the antigen-binding molecule to inhibit the action of HER3 in both ligand-absent and ligand-present conformational states. In some embodiments, the antigen-binding molecule does not compete with the ligand binding to HER3. In some embodiments, the antigen-binding molecule does not bind to HER3 at the ligand binding site.

[0946] In some embodiments, the antigen-binding molecule may bind to the same region of HER3 or an overlapping region of HER3 bound by an antibody comprising the VH and VL sequences of one of 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. There are. In some embodiments, the antigen-binding molecule may bind to the same region of HER3 or an overlapping region of HER3 bound by an antibody containing the VH and VL sequences of one of clone 10D1_c89, 10D1_c90, or 10D1_c91. In some embodiments, the antigen-binding molecule may bind to the same region of HER3 or an overlapping region of HER3 bound by an antibody containing the VH and VL sequences of clone 10D1_c89.

[0947] The region of the peptide / polypeptide to which the antibody binds can be measured by those skilled in the art using various methods well known in the art, e.g., X-ray co-crystallographic analysis of antibody-antigen complexes, peptide scanning, mutagenic mapping, hydrogen-deuterium-hydrogen exchange analysis by mass spectrometry, phage display, competitive ELISA, and proteolysis-based 'protection' methods. Such methods are described, for example, in the literature [Gershoni et al., BioDrugs, 2007, 21(3):145-156], the full text of which is incorporated herein by reference. Such methods can also be used to measure whether an antigen-binding molecule can bind to proteins of different conformations.

[0948] In some embodiments, the antigen-binding molecule of the present invention does not bind to HER3 in the same region of HER3 or in an overlapping region of HER3 as the antibody containing the VH and VL sequences of the anti-HER3 antibody clone MM-121 (e.g., described in [Schoeberl et al., Sci. Signal. (2009) 2(77): ra31]) and / or LJM-716 (e.g., described in [Garner et al., Cancer Res (2013) 73: 6024-6035]). In some embodiments, the antigen-binding molecule of the present invention does not exhibit competition for binding to HER3 with the antibody containing the VH and VL sequences of the anti-HER3 antibody clone MM-121 and / or LJM-716, as measured, for example, by SPR analysis.

[0949] In some embodiments, the antigen-binding molecule of the present invention binds to HER3 within a region accessible to the antigen-binding molecule (i.e., extracellular antigen-binding molecule) when HER3 is expressed on the cell surface (i.e., within or on the cell membrane). In some embodiments, the antigen-binding molecule may bind to HER3 expressed on the cell surface of a cell expressing HER3. In some embodiments, the antigen-binding molecule may bind to a HER3-expressing cell (e.g., a HER3+ cell, e.g., a HER3+ cancer cell).

[0950] The ability of an antigen-binding molecule to bind to a specific cell type can be analyzed by contacting the cell with the antigen-binding molecule and, for example, by detecting the antigen-binding molecule bound to the cell 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.

[0951] The antigen-binding molecule of the present invention may be an antagonist of HER3. In some embodiments, the antigen-binding molecule may inhibit a function or process (e.g., interaction, signaling, or other activity) mediated by HER3 and / or HER3's binding partners (e.g., HER3 (i.e., in the case of homomerization), HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet). As specified herein, 'inhibition' refers to a reduction, reduction, or attenuation compared to a control state.

[0952] In some embodiments, the antigen-binding molecule of the present invention may inhibit the interaction between HER3 and its interaction partner. The interaction partner of HER3 may be expressed by the same cell as HER3. The interaction partner or HER3 may be expressed on the cell surface (i.e., within or on the cell membrane). In some embodiments, the interaction partner of HER3 may be a member of the EGFR protein family, for example, HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet. In some embodiments, the interaction partner of HER3 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. If multimerization occurs between polypeptide monomers, multimerization may be referred to as dimerization.

[0953] In some embodiments, the antigen-binding molecule may inhibit the interaction between HER3 monomers. In some embodiments, the antigen-binding molecule may inhibit the interaction between HER3 and HER2. In some embodiments, the antigen-binding molecule may inhibit the interaction between HER3 and EGFR. In some embodiments, the antigen-binding molecule may inhibit the interaction between HER3 and HER4. In some embodiments, the antigen-binding molecule may inhibit the interaction between HER3 and HGFR. In some embodiments, the antigen-binding molecule may inhibit the interaction between HER3 and IGF1R. In some embodiments, the antigen-binding molecule may inhibit the interaction between HER3 and cMet.

[0954] Inhibition of the interaction may be achieved by binding of an antigen-binding molecule to a HER3 region required for the interaction between HER3 and its interaction partner (e.g., the dimerization loop of HER3 shown in SEQ ID NO. 19). In some embodiments, the antigen-binding molecule contacts one or more residues of HER3 required for the interaction between HER3 and its interaction partner; in this way, the antigen-binding molecule inhibits the interaction by rendering the corresponding region unavailable. In some embodiments, the antigen-binding molecule binds to a binding to HER3, thereby inhibiting / preventing the interaction between HER3 and its interaction partner. In some embodiments, the antigen-binding molecule inhibits / prevents access of the HER3 interaction partner to a HER3 region required for the interaction between HER3 and its interaction partner; This can be achieved, for example, by sterically inhibiting the access of the HER3 interaction partner to the HER3 region required for the interaction between HER3 and the interaction partner, even when the antigen-binding molecule does not come into contact with the HER3 region required for the interaction between HER3 and the HER3 interaction partner.

[0955] In some embodiments, the antigen-binding molecule may inhibit the homomerization of HER3 monomers. In some embodiments, the antigen-binding molecule may inhibit dimerization between HER3 and HER2. In some embodiments, the antigen-binding molecule may inhibit dimerization between HER3 and EGFR. In some embodiments, the antigen-binding molecule may inhibit dimerization between HER3 and HER4. In some embodiments, the antigen-binding molecule may inhibit dimerization between HER3 and HGFR. In some embodiments, the antigen-binding molecule may inhibit dimerization between HER3 and IGF1R. In some embodiments, the antigen-binding molecule may inhibit dimerization between HER3 and cMet.

[0956] The ability of an antigen-binding molecule to inhibit the interaction between two factors can be measured, for example, by analyzing the interaction in the presence of an antibody / fragment, or by incubating the antibody / fragment with one or both of the interaction partners. A competitive ELISA assay is used to determine whether a given antigen-binding molecule can inhibit the interaction between two interaction partners. In some embodiments, the antigen-binding molecule is a competitive inhibitor of the interaction between HER3 and its interaction partners.

[0957] In some embodiments, the antigen-binding molecule of the present invention, in the presence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule) in a suitable assay, the interaction between HER3 and its interaction partner (e.g., HER3, HER2, EGFR, HER4, HGFR, IGF1R, and / or cMet) is less than 1 times the level of interaction between HER3 and its interaction partner, e.g., ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, It can be increased by ≤0.05 times or ≤0.01 times.

[0958] The ability of an antigen-binding molecule to inhibit interactions between interaction partners can also be measured by analyzing the downstream functional consequences of such interactions. For example, downstream functional consequences of interactions between HER3 and its interaction partners include PI3K / AKT / mTOR and / or MAPK signals. For example, the ability of an antigen-binding molecule to inhibit interactions between HER3 and its interaction partners can be measured 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 using antibodies capable of detecting phosphorylated members of the signaling pathway.

[0959] The ability of an antigen-binding molecule to inhibit the interaction between HER3 and its interaction partners can also be measured by analyzing the proliferation of HER3-expressing cells after treatment with NRG in the presence of the antigen-binding molecule. Cell proliferation can be, for example, detected by the detection of changes in cell number over time, 3The incorporation of H-thymidine can be measured by in vitro analysis or CFSE dilution analysis (e.g., described in the literature [Fulcher and Wong, Immunol Cell Biol (1999) 77 (6): 559-564], the full text of which is incorporated herein by reference).

[0960] In some embodiments, the antigen-binding molecule of the present invention can inhibit the proliferation of cells having a mutation for, for example, BRAF V600, such as cells containing BRAF V600E or V600K mutations (see Example 10).

[0961] In some embodiments, the antigen-binding molecule inhibits HER3-mediated signaling. HER3-mediated signaling can be analyzed, for example, using correlation analysis of HER3-mediated signaling, for example, phosphorylation of one or more signaling molecules of the PI3K / AKT / mTOR and / or MAPK signaling pathways and / or cell proliferation.

[0962] In some embodiments, the antigen-binding molecule of the present invention may inhibit PI3K / AKT / mTOR and / or MAPK signaling by HER3-expressing cells. Levels of PI3K / AKT / mTOR and / or MAPK signaling may be analyzed by detecting and quantifying the phosphorylation levels of one or more components of the PI3K / AKT / mTOR and / or MAPK pathways after stimulation with NRG (see Example 4.3).

[0963] In some embodiments, the antigen-binding molecule of the present invention can inhibit the proliferation of HER3-expressing cells in response to stimulation by, for example, NRG. In some embodiments, the antigen-binding molecule of the present invention can inhibit the proliferation of HER3-expressing cells in a suitable assay to less than 1x the proliferation level of HER3-expressing cells in the presence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), e.g., ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x.

[0964] In some embodiments, the antigen-binding molecule of the present invention in a suitable assay reduces PI3K / AKT / mTOR and / or MAPK signaling by HER3-expressing cells and the proliferation of HER3-expressing cells in the presence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule) the signaling level of HER3-expressing cells to less than 1, e.g., ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, ≤0.5, ≤0.45, ≤0.4, ≤0.35, ≤0.3, ≤0.25, ≤0.2, ≤0.15, ≤0.1, ≤0.05, or It can be increased by ≤0.01 times.

[0965] HER3-mediated signaling can be investigated in vitro, for example as described in Example 8.9, or in vivo, for example as described in Example 11.

[0966] ADCC activity may be determined by, for example, according to the method described in the literature [Yamashita et al., Scientific Reports (2016) 6:19772] (the full text of which is incorporated by reference into this specification), or, for example, according to the literature [Jedema et al., Blood (2004) 103: 2677-82 (the full text of which is incorporated by reference into this specification)]. 51 It can be analyzed by Cr release assay. ADCC activity can also be analyzed using the Pierce LDH cytotoxicity assay kit according to the manufacturer's instructions (described in Example 5 of this specification).

[0967] ADCP can be analyzed according to the method described in, for example, the literature [Kamen et al., J Immunol (2017) 198 (1 Supplement) 157.17 (the full text of the above literature is incorporated by reference into this specification)].

[0968] The ability to induce CDC can be analyzed, for example, using C1q binding analysis, as described in the literature [Schlothauer et al., Protein Engineering, Design and Selection (2016), 29 (10): 457-466] (the full text of which is incorporated by reference into this specification).

[0969] The thermal stability of antigen-binding molecules can be analyzed by methods well known to those skilled in the art, including, for example, differential scanning fluorescence and differential scanning calorimetry (DSC) as described in the literature [He et al., J Pharm Sci. (2010)], the full text of which is incorporated herein by reference. Thermal stability is determined by the dissolution temperature (T m ), can be reflected in terms of non-folding temperature or decomposition temperature (e.g., indicated as °C or F°).

[0970] In some embodiments, an antigen-binding molecule comprising an Fc region as described herein binds to an active Fcγ receptor (e.g., hFcγRIIa (e.g., hFcγRIIa167H, hFcγRIIa167R), hFcγRIIIa (e.g., hFcγRIIIa158V, hFcγRIIIa158F), mFcγRIV, mFcγRIII) with a binding affinity greater than 1, e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or greater than 20 times the binding affinity to the active Fcγ receptor by an equivalent antigen-binding molecule comprising a CH2-CH3 Fc region having the amino acid sequences of SEQ ID NOs. 174 to 175. In some embodiments, K for binding of an antigen-binding molecule comprising an Fc region as described herein to an active Fcγ receptor D K for an active Fcγ receptor 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 D Less than 1 time of, for example, 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.

[0971] In some embodiments, an antigen-binding molecule comprising an Fc region as described herein is present at an amount of 1000 nM or less, preferably ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤20 nM, or 1 One of K less than or equal to nM D Combines as.

[0972] In some embodiments, an antigen-binding molecule comprising an Fc region as described herein binds to FcRn (e.g., hFcRn, mFcRn) with a binding affinity greater than 1 times the binding affinity to FcRn by an equivalent antigen-binding molecule comprising a CH2-CH3 Fc region having the amino acid sequences of SEQ ID NOs. 174 to 175, e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or greater than 20 times. In some embodiments, K for binding to FcRn of an antigen-binding molecule comprising an Fc region as described herein. D is K for FcRn 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 D Less than 1 time of, 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 or less than 0.05 times.

[0973] In some embodiments, an antigen-binding molecule comprising an Fc region as described herein is present in an amount of 1000 nM or less, preferably ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, or ≤1 nM of one of K D Combines as.

[0974] In some embodiments, an antigen-binding molecule comprising an Fc region as described herein binds to an inhibitory Fcγ receptor (e.g., hFcγRIIb mFcγRIIb) with a binding affinity less than 1 times that of an equivalent antigen-binding molecule comprising a CH2-CH3 Fc region having the amino acid sequences of SEQ ID NOs. 174 to 175 to the inhibitory Fcγ receptor, e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or less than 0.1 times that of the binding affinity to the inhibitory Fcγ receptor. In some embodiments, K for binding of an antigen-binding molecule comprising an Fc region as described herein to an inhibitory Fcγ receptor. D K for an inhibitory Fcγ receptor 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 D It is more than 1 time, for example, more than 2, 3, 4, 5, 6, 7, 8, 9 times, or more than 10 times.

[0975] In some embodiments, an antigen-binding molecule comprising an Fc region as described herein is present at a concentration of 1 nM or more, preferably ≥ 5 nM, ≥ 10 nM, ≥ 50 nM, ≥ 100 nM, ≥ 500 nM, ≥ 1000 nM, ≥ 2000 nM, ≥ 3000 nM, ≥ 4000 nM, or ≥ 5000 nM, at a concentration of K D Combines as.

[0976] In some embodiments, the binding selectivity for an active Fcγ receptor (e.g., hFcγRIIa) compared to an inhibitory Fcγ receptor (e.g., hFcγRIIb) for an antigen-binding molecule having an Fc region as described herein is greater than 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times the binding selectivity exhibited by an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequences of SEQ ID NOs 174 to 175.

[0977] In some embodiments, an antigen-binding molecule comprising an Fc region as described herein exhibits an ADCC greater than 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times the ADCC represented by 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.

[0978] In some embodiments, the EC50 (ng / ml) measured for an antigen-binding molecule having an Fc region as described herein in the analysis of ADCC activity is, for example, less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or less than 0.1 of the EC50 (ng / ml) measured for an equivalent antigen-binding molecule having an Fc region composed of CH2-CH3 having the amino acid sequence of SEQ ID NOs 174 to 175.

[0979] In some embodiments, in the analysis of ADCC activity, the EC50 (ng / ml) of an antigen-binding molecule containing an Fc region as described herein is 500 ng / ml or less, preferably ≤400 ng / ml, ≤300 ng / ml, ≤200 ng / ml, ≤100 ng / ml, ≤90 ng / ml, ≤80 ng / ml, ≤70 ng / ml, ≤60 ng / ml, ≤50 ng / ml, ≤40 ng / ml, ≤30 ng / ml, ≤20 ng / ml, or ≤10 ng / ml.

[0980] In some embodiments, an antigen-binding molecule comprising an Fc region as described herein is ≥0.75 times and ≤1.25 times, e.g., ≥0.8 times and ≤1.2 times, ≥0.85 times and ≤1.15 times, ≥0.9 times and ≤1.1 times, ≥0.91 times and ≤1.09 times, ≥0.92 times and ≤1.08 times, ≥0.93 times and ≤1.07 times, ≥0.94 times and ≤1.06 times, ≥0.95 times and ≤1.05 times, ≥0.96 times and ≤1.04 times, ≥0.97 times and ≤ It may have a dissolution temperature, non-folding temperature, or decomposition temperature of 1.03 times, ≥0.98 times and ≤1.02 times, or ≥0.99 times and ≤1.01 times.

[0981] In some embodiments, the antigen-binding molecule of the present invention may increase the death of HER3-expressing cells. The death of HER3-expressing cells may be increased through the effector function of the antigen-binding molecule. In embodiments where the antigen-binding molecule comprises an Fc region, the antigen-binding molecule may increase the death of HER3-expressing cells through one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP).

[0982] Antigen-binding molecules capable of increasing the death of HER3-expressing cells are identified by observing increased levels of HER3-expressing cell death in the presence of the antigen-binding molecule compared to the level of cell death detected in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule) in an appropriate assay, or by subsequently incubating HER3-expressing cells with the antigen-binding molecule. The assays of CDC, ADCC, and ADCP are widely known to those skilled in the art. The level of HER3-expressing cell death can also be measured by measuring the number / characteristics of surviving and / or non-surviving HER3-expressing cells after exposure to different treatment conditions.

[0983] In some embodiments, the antigen-binding molecule of the present invention can increase the death of HER3-expressing cells (e.g., HER3-expressing cancer cells) by more than 1x the level of death observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), e.g., ≥1.01x, ≥1.02x, ≥1.03x, ≥1.04x, ≥1.05x, ≥1.1x, ≥1.2x, ≥1.3x, ≥1.4x, ≥1.5x, ≥1.6x, ≥1.7x, ≥1.8x, ≥1.9x, ≥2x, ≥3x, ≥4x, ≥5x, ≥6x, ≥7x, ≥8x, ≥9x, or ≥10x.

[0984] In some embodiments, the antigen-binding molecule of the present invention is less than 1 times the number of HER3-expressing cells (e.g., HER3-expressing cancer cells) detected after incubation in the presence of the antigen-binding molecule (or incubation in the presence of an appropriate control antigen-binding molecule) in a comparable analysis, e.g., ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, It can be reduced to ≤0.05 times or ≤0.01 times.

[0985] In some embodiments, the antigen-binding molecule of the present invention inhibits the development and / or progression of cancer in vivo.

[0986] In some embodiments, the antigen-binding molecule causes, for example, an increase in cancer cell death by effector immune cells. In some embodiments, the antigen-binding molecule causes, for example, a decrease in the number of cancer cells in vivo compared to appropriate control conditions. In some embodiments, the antigen-binding molecule inhibits tumor growth, which is determined, for example, by measuring tumor size / volume over time.

[0987] The antigen-binding molecule of the present invention may be analyzed for its ability to inhibit the development and / or progression of cancer in a suitable in vivo model, e.g., a cell line-derived xenograft model. The cell line-derived xenograft model may be derived from HER3-expressing cancer cells. In some embodiments, the model is an N87 cell-derived model, an SNU16 cell-derived model, a FaDu cell-derived model, an OvCAR8 cell-derived model, an HCC95 cell-derived model, an A549 cell-derived model, an ACHN cell-derived model, or an HT29 cell-derived model.

[0988] The cancer may be a HER3-related cancer as described herein (i.e., a cancer in which HER3 gene / protein expression is a risk factor for the onset, development, progression, or severity and / or endosis of symptoms of the cancer, or is benignly associated). The cancer may comprise HER3-expressing cells. In some embodiments, the cancer comprises a HER3+ tumor.

[0989] In some embodiments, administration of an antigen-binding molecule according to the present invention may induce one or more of the following: inhibition of cancer development / progression, delay / prevention of cancer development, reduction / delay / prevention of tumor growth, reduction / delay / prevention of metastasis, reduction in the severity of cancer symptoms, reduction in the number of cancer cells, reduction in tumor size / volume, and / or increase in survival (e.g., progression-free survival), as measured, for example, in a suitable HER3-expressing cancer cell line-derived xenograft model.

[0990] In some embodiments, the antigen-binding molecule of the present invention can inhibit tumor growth in a HER3-expressing cancer cell line-derived xenograft model to less than 1x of the tumor growth observed in the absence of treatment with the antigen-binding molecule (or after treatment with an appropriate negative control antigen-binding molecule), e.g., ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x.

[0991] Chimeric Antigen Receptor (CAR)

[0992] The present invention also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule or polypeptide of the present invention.

[0993] A CAR is a recombinant receptor that provides both antigen-binding and T cell activation functions. The structure and engineering of a CAR are reviewed, for example, in the literature [Dotti et al., Immunol Rev (2014) 257(1)], the full text of which is incorporated herein by reference. A CAR comprises an antigen-binding region and a signaling region connected to a cell membrane anchor region. Any hinge region may provide separation between the antigen-binding region and the cell membrane anchor region and may act as a flexible linker.

[0994] The CAR of the present invention comprises or is composed of the antigen-binding molecule of the present invention, or comprises an antigen-binding region comprising or composed of a polypeptide according to the present invention.

[0995] A cell membrane anchor region is provided between the antigen-binding region and the signaling region of the CAR and is provided to anchor the CAR to the cell membrane of a cell expressing the CAR using the antigen-binding region in the extracellular space and the signaling region in the cell. In some embodiments, the CAR comprises a cell membrane anchor region comprising, or consisting of, an amino acid sequence of a transmembrane region for one of CD3-ζ, CD4, CD8, or CD28, or an amino acid derived therefrom. As used herein, the region 'derived from reference amino acid sequence' comprises an amino acid sequence having 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 with respect to the reference sequence.

[0996] The signaling domain of a CAR allows for the activation of T cells. The CAR signaling domain may contain the amino acid sequence of the intracellular domain of CD3-ζ, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for the phosphorylation and activation of CAR-expressing T cells. Signaling domains containing other ITAM-containing proteins, e.g., FcγRI, have also been used in CARs (Haynes et al., 2001 J Immunol 166(1):182-187). The CAR signaling domain can also promote the activation of CAR-expressing T cells upon binding to a target protein by containing co-stimulatory sequences derived from the signaling domains of co-stimulatory molecules. Suitable co-stimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, the CAR is processed to provide for the co-stimulation of different intracellular signaling pathways. For example, signaling associated with CD28 co-stimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1BB-mediated signaling is mediated via TNF receptor-associated factor (TRAF) adapter proteins. Therefore, the signaling domain of a CAR sometimes contains a co-stimulatory sequence derived from the signaling domain of one or more co-stimulatory molecules. In some embodiments, the CAR of the present invention comprises one or more co-stimulatory sequences comprising, consisting of, or derived from amino acids of one or more intracellular domains of CD28, OX40, 4-1BB, ICOS, and CD27.

[0997] Any hinge region may provide separation between the antigen-binding domain and the transmembrane domain and may act as a flexible linker. The hinge region may be derived from IgG1. In some embodiments, the CAR of the present invention comprises, or consists of, an amino acid sequence of the hinge region of IgG1, or comprises, or consists of, an amino acid sequence derived therefrom.

[0998] In addition, a cell comprising a CAR according to the present invention is provided. A CAR-expressing immune cell, e.g., a CAR-T or CAR-NK cell, can be generated using the CAR according to the present invention. The processing of the CAR into an immune cell can be performed in vitro during culture.

[0999] The antigen-binding region of the CAR of the present invention may be provided with any suitable form, such as scFv, scFab, etc.

[1000] Nucleic acids and vectors

[1001] The present invention provides an antigen-binding molecule, polypeptide, or nucleic acid encoding a CAR according to the present invention, or a plurality of nucleic acids.

[1002] In some embodiments, the nucleic acid is purified or isolated, for example, from other nucleic acids or naturally occurring biological material. In some embodiments, the nucleic acid(s) comprise or consist of DNA and / or RNA.

[1003] The present invention also provides a vector comprising a nucleic acid or a plurality of nucleic acids according to the present invention, or a plurality of vectors.

[1004] A nucleotide sequence may be contained within a vector, e.g., an expression vector. A “vector” as used herein is a nucleic acid molecule used as a vehicle for delivering exogenous nucleic acids into a cell. The vector may be a vector for the expression of nucleic acids within a cell. Such a vector may include a promoter sequence operatively linked to a nucleotide sequence encoding the sequence to be expressed. The vector also includes a termination codon and an expression enhancer. A peptide or polypeptide may be expressed from a vector according to the present invention using any suitable vector, promoter, enhancer, and termination codon known in the art.

[1005] The term "operationally linked" means that a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a way that the expression of the nucleic acid sequence is replaced (by forming an expression cassette) under the influence or control of the regulatory sequence. Thus, if the regulatory sequence can influence the transcription of the nucleic acid sequence, the regulatory sequence is operationally linked to the selected nucleic acid sequence. Subsequently, the obtained transcript(s) can be deciphered into the desired peptide(s) / polypeptide(s).

[1006] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus vectors (e.g., rodent leukemia virus (MLV)-derived vectors), lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[1007] In some embodiments, the vector may be a eukaryotic vector, for example, a vector containing components essential for the expression of a protein from the vector within a eukaryotic cell. In some embodiments, the vector may be a mammalian vector that drives protein expression by containing, for example, a cytomegalovirus (CMV) or an SV40 promoter.

[1008] The constitutive polypeptide of the antigen-binding molecule according to the present invention may be encoded by different nucleic acids of a plurality of nucleic acids or by different vectors of a plurality of vectors.

[1009] Cells containing / expressing antigen-binding molecules and polypeptides

[1010] The present invention also provides a cell comprising or expressing an antigen-binding molecule, polypeptide, or CAR according to the present invention. Additionally, a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present invention is provided, or a cell expressing such a nucleic acid.

[1011] The cell may be a eukaryotic cell, e.g., a mammalian cell. The mammal may be a primate (rhesus, cynomolgus, non-human primate or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any animal of the order Rodentia), cat, dog, pig, sheep, goat, cattle (cow, e.g., dairy cow, or any animal of the order Bos), horse (including any animal of the order Equidae), donkey, and non-human primate).

[1012] The present invention also provides a method for producing a cell comprising nucleic acid(s) or vector(s) according to the present invention, comprising the step of introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present invention into a cell. In some embodiments, the step of introducing the isolated nucleic acid(s) or vector(s) according to the present invention into a cell comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[1013] The present invention also provides a method for generating a cell expressing / containing an antigen-binding molecule, polypeptide, or CAR according to the present invention, comprising the step of introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present invention into a cell. In some embodiments, the method further comprises the step of culturing the cell under conditions suitable for the expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the method is performed in vitro.

[1014] The present invention also provides cells that are obtained or obtainable by the method according to the present invention.

[1015] Production of antigen-binding molecules and polypeptides

[1016] The antigen-binding molecule and polypeptide according to the present invention can be prepared according to methods for producing polypeptides known to those skilled in the art.

[1017] Polypeptides can be prepared by chemical synthesis, e.g., liquid or solid-phase synthesis. For example, peptides / polypeptides can be synthesized using methods described in the literature [Chandrudu et al., Molecules (2013), 18: 4373-4388], the full text of which is incorporated herein by reference.

[1018] Alternatively, antigen-binding molecules and polypeptides can be generated by recombinant expression. Molecular biology techniques suitable for the recombinant generation of polypeptides are well known in the art, as set forth in the literature [Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and Nat Methods. (2008); 5(2): 135-146], the full text of which is incorporated herein by reference. Methods for the recombinant generation of antigen-binding molecules are also described in the literature [Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461], the full text of which is incorporated herein by reference.

[1019] In some cases, the antigen-binding molecule of the present invention consists of one or more polypeptide chains. In such cases, the generation of the antigen-binding molecule may involve the transcription and decoding of one or more polypeptides, and the subsequent assembly of polypeptide chains to form the antigen-binding molecule.

[1020] For recombination generation according to the present invention, any cell suitable for expressing the polypeptide may be used. The cell may be a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell, for example, an archaea or a bacterium. In some embodiments, the bacterium may be a Gram-negative bacterium, for example, a bacterium of the Enterobacteriaceae family, for example, Escherichia coli. In some embodiments, the cell is a eukaryotic cell, for example, a yeast cell, a plant cell, an insect cell, or a mammalian cell, such as a CHO, HEK (e.g., HEK293), HeLa, or COS cell. In some embodiments, the cell is a CHO cell that transiently or stably expresses the polypeptide.

[1021] In some cases, since certain prokaryotic cells do not allow the same folding or post-translation modifications as eukaryotic cells, the cells are not prokaryotic cells. Furthermore, very high expression levels are possible within eukaryotic cells, and proteins may be easier to purify from eukaryotic cells using appropriate tags. Specific plasmids that enhance the secretion of proteins into the medium can also be utilized.

[1022] In some embodiments, polypeptides may be produced by cell-free protein synthesis (CFPS) by using a system described in the literature [Zemella et al. Chembiochem (2015) 16(17): 2420-2431], the full text of which is incorporated herein by reference.

[1023] Production may involve the culture or fermentation of eukaryotic cells modified to express the desired polypeptide(s). Culture or fermentation may be carried out in a bioreactor that provides an appropriate supply of nutrients, air / oxygen, and / or growth factors. The secreted proteins may be collected by the steps of dispensing culture medium / fermentation broth from the cells, extracting protein contents, and isolating the secreted polypeptide(s) by separating individual proteins. The techniques of culture, fermentation, and secretion are well known to those skilled in the art and are described, for example, in the literature [Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition)]; incorporated herein by reference.

[1024] A bioreactor comprises one or more vessels in which cells can be cultured. Culture within the bioreactor can occur continuously with a continuous flow of reactants into the reactor and a continuous flow of cultured cells from the reactor. Additionally, culture can occur in batches. The bioreactor provides optimal conditions for the cultured cells by monitoring and controlling environmental conditions, such as pH, oxygen, flow rates into and out of the vessel, and stirring within the vessel.

[1025] After culturing cells expressing the target antigen-binding molecule / polypeptide(s), the target polypeptide(s) can be isolated. Any suitable method known in the art for isolating proteins from cells may be used. To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide(s) are secreted from the cells, it may be necessary to separate the cells by centrifugation from the culture medium containing the secreted target polypeptide(s). If the target polypeptide(s) are collected from within the cells, protein isolation may involve centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with a digestion buffer, and cell destruction by, for example, sonification, rapid freeze-thaw, or osmotic separation.

[1026] It may be desirable to isolate the desired polypeptide(s) from the supernatant or culture medium, which may contain other protein and non-protein components. A general approach for separating protein components from the supernatant or culture medium is by precipitation. Proteins with different solubility are precipitated in different concentrations of a precipitating agent, such as ammonium sulfate. For example, at a low concentration of the precipitating agent, water-soluble proteins are extracted. Thus, proteins with different solubility can be distinguished by adding different increasing concentrations of the precipitating agent. Ammonium sulfate can be removed from the separated proteins by subsequent dialysis.

[1027] Other methods for distinguishing different proteins, for example, ion exchange chromatography and size chromatography, are known in the art. These can be used as an alternative to precipitation or can be performed subsequently to precipitation.

[1028] Once the desired polypeptide(s) are isolated from the culture, it may be desirable or necessary to concentrate the polypeptide(s). A number of methods for concentrating proteins, such as ultrafiltration or freeze-drying, are known in the art.

[1029] composition

[1030] The present invention also provides a composition comprising the antigen-binding molecule, polypeptide, CAR, nucleic acid, expression vector, and cell described herein.

[1031] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein may be formulated as pharmaceutical compositions or medicines for clinical use and may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The composition may be formulated for local, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intravertebral, intraocular, conjunctival, intratumoral, subcutaneous, intradermal, intravertebral, oral, or transdermal administration routes, which may include injection or infusion.

[1032] Suitable formulations may contain antigen-binding molecules in a sterile or isotonic medium. Medicinal and pharmaceutical compositions may be formulated in fluid forms including gels. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) to selected areas of the human or animal body.

[1033] In some embodiments, the composition is formulated for injection or infusion into, for example, a blood vessel or a tumor.

[1034] According to the method described herein, a method is also provided for producing a pharmaceutically useful composition, and such method of production may comprise one or more steps selected from the following: producing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number of the same), expression vector (or a number of the same) or cell described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number of the same), expression vector (or a number of the same) or cell described herein; and / or mixing the antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number of the same), expression vector (or a number of the same) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[1035] For example, additional embodiments of the invention described herein relate to a method for formulating or producing a pharmaceutical or medicinal composition for use in treating a disease / pathological condition (e.g., cancer), and such a method comprises the step of formulating the pharmaceutical composition or medicinal composition by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or many thereof), expression vector (or many thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[1036] Therapeutic and preventive applications

[1037] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells, and compositions described in this specification are found to have uses in therapeutic and prophylactic methods.

[1038] The present invention provides an antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number thereof), expression vector (or a number thereof), cell, or composition described herein for use in a method of medical treatment or prevention. Additionally, the use of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number thereof), expression vector (or a number thereof), cell, or composition described herein in the manufacture of a medicine for the treatment or prevention of a disease or condition is provided. A method for the treatment or prevention of a disease or condition is also provided, comprising administering to a subject an amount of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number thereof), expression vector (or a number thereof), cell, or composition described herein that is therapeutically or prophylactically effective.

[1039] The method may be effective in reducing the development or progression of a disease / condition, alleviating the symptoms of the disease / condition, or reducing the pathology of the disease / condition. The method may be effective in preventing the progression of the disease / condition, for example, in preventing the worsening of the disease / condition or slowing the rate of its development. In some embodiments, the method may result in an improvement in the disease / condition, for example, a reduction in the symptoms of the disease / condition or a reduction in some other related aspects of the severity / activity of the disease / condition. In some embodiments, the method may prevent the development of the disease / condition to a later stage (e.g., chronic stage or transition).

[1040] It will be recognized that the subject of the present invention may be used for the treatment or prevention of any disease / pathology in which therapeutic or preventive benefits may be derived from a reduction in the number and / or activity of HER3-expressing cells. For example, the disease / pathology may be a disease / pathology in which HER3-expressing cells are pathologically associated, e.g., an increased number / proportion of HER3-expressing cells is positively associated with the onset, development, or progression of the disease / pathology and / or the severity of one or more symptoms of the disease / pathology, or an increased number / proportion of HER3-expressing cells is a risk factor for the onset, development, or progression of the disease / pathology.

[1041] In some embodiments, the disease / pathology to be treated / prevented according to the present invention is, for example, a disease / pathology characterized by an increase in the number / ratio / activity of cells expressing HER3 compared to the number / ratio / activity of cells expressing HER3 in the absence of the disease / pathology.

[1042] In some embodiments, the disease / pathological condition to be treated / prevented is cancer.

[1043] Cancer may be any unwanted cell proliferation (or any disease that manifests itself through unwanted cell proliferation), a neoplasm, or a tumor. Cancer may be benign or malignant, and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located within any tissue. Cancer may be tissue / cells derived from, for example, the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, appendix, central nervous system (including or excluding 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, uterine muscle, nasopharynx, serosa, 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.

[1044] The tumor to be treated may be a neurological or non-neurological tumor. Neurological tumors may originate from the central or peripheral nervous system, such as gliomas, medulloblastomas, meningiomas, neurofibromas, ependymomas, schwannomas, neurofibrosarcomas, astrocytomas, and oligodendromas. Non-neurological cancers / tumors may originate from any other non-neurological tissue and include, for example, melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), subcutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver tumors, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[1045] The relationship and role of HER3 with cancer can be reviewed, for example, in the literature [Karachaliou et al., BioDrugs. (2017) 31(1):63-73 and Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1): 39-48], the full text of both of the above literature is incorporated by reference into this specification.

[1046] In some embodiments, cancer is a cancer including cells expressing HER3, a solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, 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, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, papillary renal cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, endometrial carcinoma of the uterine body, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate It is selected from adenocarcinoma, sarcoma, and thymoma.

[1047] In some embodiments, the cancer to be treated according to the present invention is selected from HER3-expressing cancer, gastric cancer (e.g., gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), breast cancer, ovarian cancer (e.g., ovarian carcinoma), lung cancer (e.g., NSCLC, lung adenocarcinoma, squamous lung cell carcinoma), melanoma, prostate cancer, oral cavity cancer (e.g., oropharyngeal cancer), kidney cancer (e.g., renal cell carcinoma) or colorectal cancer (e.g., colorectal carcinoma), esophageal cancer, pancreatic cancer, solid tumors and / or liquid tumors.

[1048] Treatment / prevention may aim at one or more of the following: delay / prevention of the onset / progression of cancer symptoms, reduction of the severity of cancer symptoms, reduction of the survival / growth / invasion / metastasis of cancer cells, reduction of the number of cancer cells and / or increase in the survival of the subject.

[1049] In some embodiments, the cancer to be treated / prevented comprises cells expressing an EGFR family member (e.g., HER3, EGFR, HER2, or HER4) and / or cells expressing a ligand of 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 of an EGFR family member. Overexpression may be measured by detecting an expression level greater than the expression level by an equivalent non-cancer cell / non-tumor tissue.

[1050] Expression can be measured by any suitable means. Expression may be gene expression or protein expression. Gene expression may be measured, for example, by the detection of mRNA encoding HER3, for example, by quantitative real-time PCR (qRT-PCR). Protein expression may be measured, for example, by antibody-based methods, for example, Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[1051] In some embodiments, the cancer to be treated / prevented comprises cells expressing 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 may be measured by detecting a level of HER3 expression greater than the expression level by an equal non-cancer cell / non-tumor tissue.

[1052] In some embodiments, a patient may be selected for the treatment described herein, for example, based on the detection of a cancer expressing HER3 or a cancer overexpressing HER3 from a sample obtained from a subject.

[1053] In some embodiments, the cancer to be treated / prevented comprises cells expressing a HER3 ligand (e.g., NRG1 and / or NRG2). In some embodiments, the cancer to be treated / prevented comprises cells exhibiting a greater expression level of NRG1 and / or NRG2 than the expression level by an equivalent non-cancer cell / non-tumor tissue.

[1054] The administration of the article of the present invention is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to demonstrate therapeutic or prophylactic benefit to the subject. The actual amount administered, as well as the rate and duration of administration, will depend on the characteristics and severity of the disease / condition and the specific article administered. The prescription of treatment, e.g., the determination of the dosage, is under the responsibility of a general practitioner and other physicians, taking into account the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the aforementioned techniques and protocols can be found in the literature [Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins].

[1055] Administration may be performed alone or in combination with other treatments, or simultaneously or sequentially depending on the condition to be treated. The antigen-binding molecules or compositions and therapeutic agents described herein may be administered simultaneously or sequentially.

[1056] In some embodiments, the method includes, for example, additional therapeutic or prophylactic intervention for the treatment or prevention of cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiation therapy, surgery, vaccination, and / or hormone therapy. In some embodiments, the therapeutic or prophylactic intervention includes leukocyte apheresis. In some embodiments, the therapeutic or prophylactic intervention includes stem cell transplantation.

[1057] In some embodiments, the antigen-binding molecule is administered in combination with an agent capable of inhibiting signal transduction mediated by EGFR family members.

[1058] Accordingly, the present invention comprises a composition comprising a target according to the present invention (e.g., an antigen-binding molecule according to the present invention) and a preparation capable of inhibiting signal transduction mediated by another EGFR family member (e.g., EGFR, HER2, HER3, or HER4). Additionally, the use of such a composition in the medical treatment and prevention of the disease / pathological condition described herein is provided.

[1059] In addition, as a method for treating or preventing a disease / pathological condition described in this specification, a method is provided comprising the step of administering a target according to the present invention (e.g., an antigen-binding molecule according to the present invention) and a preparation capable of inhibiting signal transduction mediated by another EGFR family member.

[1060] 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 their antigen-binding fragments), peptide / polypeptide inhibitors (e.g., attracting ligand / receptor or peptide aptamers), and nucleic acids (e.g., antisense nucleic acids, splice-exchange nucleic acids, or nucleic acid aptamers). Inhibitors of signal transduction mediated by EGFR family members include agents that inhibit signal transduction through direct influence on EGFR family members, interaction partners, and / or downstream factors involved in signal transduction mediated by EGFR family members.

[1061] In some embodiments, an antagonist of signaling mediated by EGFR family members inhibits signaling mediated by one or more of EGFR, HER2, HER4, and HER3. Inhibitors of signaling mediated by EGFR family members are described, for example, in the literature [Yamaoka et al., Int. J. Mol. Sci. (2018), 19, 3491], the full text of which is incorporated herein by reference. In some embodiments, the antagonist is a pan-ErbB inhibitor. In some embodiments, the antagonist is an inhibitor of signaling mediated by EGFR (e.g., cetuximab, panitumumab, gefitinib, erlotinib, lapatinib, afatinib, brigatinib, icotinib, osimertinib, zalutumumab, vandetanib, necitumumab, nimotuzumab, dacomitinib, duligotuzumab, 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., serivantumab, rumletuzumab, elgemtumab, KTN3379, AV-203, GSK2849330, REGN1400, MP-RM-1, EV20, duligotuzumab, MM-111, istiratumab, MCLA-128, patritumab, EZN-3920, RB200, or U3-1402). In some embodiments, the antagonist is an inhibitor of HER4-mediated signaling (e.g., lapatinib, ibrutinib, afatinib, dacomitinib, or neratinib).

[1062] In some embodiments, the antagonist of signaling mediated by an EGFR family member inhibits downstream effectors of signaling mediated by the EGFR family member. Downstream effectors of signaling mediated by the EGFR family member include, for example, PI3K, AKT, KRAS, BRAF, MEK / ERK, and mTOR. In some embodiments, the antagonist of signaling mediated by an EGFR family member is an inhibitor of the MAPK / ERK pathway. In some embodiments, the antagonist of signaling mediated by an EGFR family member is an inhibitor of the PI3K / ATK / mTOR pathway. In some embodiments, the antagonist is a PI3K inhibitor (e.g., pictilisib, buparisib, idellarisib, 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).

[1063] In some embodiments, the cancer to be treated according to an aspect of the present 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), e.g., an antagonist as described in paragraph 3 above. In some embodiments, the subject to be treated is a cancer that is resistant to treatment with an antagonist of signaling mediated by an EGFR family member. In some embodiments, the subject to be treated is a cancer that has developed resistance to treatment with an antagonist of signaling mediated by an EGFR family member. In some embodiments, the subject to be treated is a cancer that previously responded to treatment with an antagonist of signaling mediated by an EGFR family member and is currently resistant to treatment with the antagonist. In some embodiments, the subject to be treated is a cancer that has relapsed or / or progressed after treatment with an antagonist of signaling mediated by an EGFR family member. In some embodiments, the subject to be treated initially responded to treatment with an antagonist of signaling mediated by an EGFR family member, but subsequently, there is cancer that progressed during said treatment.

[1064] A person skilled in the art can reliably identify cancers and subjects in accordance with the above paragraph. Such cancers and subjects may be identified, for example, by monitoring the development / progression of cancer (and / or its correlation) over time during a course of treatment with an antagonist of signaling mediated by EGFR family members. In some embodiments, identification of such subjects / cancers may include, for example, analysis of a sample in vitro (e.g., biopsy). In some embodiments, cancer may be measured as comprising cells having mutations associated with reduced responsiveness and / or resistance to treatment with an antagonist. In some embodiments, cancer may be measured as comprising cells in which the expression of EGFR family members is upregulated.

[1065] In certain embodiments, the cancer to be treated is a cancer that is resistant to treatment with an antagonist of signaling mediated by EGFR and / or HER2. In some embodiments, the subject to be treated is a cancer that is resistant to treatment with an antagonist of signaling mediated by EGFR and / or HER2. In some embodiments, the subject to be treated is a cancer that has developed resistance to treatment with an antagonist of signaling mediated by EGFR and / or HER2. In some embodiments, the subject to be treated is a cancer that previously responded to treatment with an antagonist of signaling mediated by EGFR and / or HER2 and is now resistant to treatment with the antagonist. In some embodiments, the subject to be treated is a cancer that has relapsed or / or progressed after treatment with an antagonist of signaling mediated by EGFR and / or HER2. In some embodiments, the subject to be treated initially responded to treatment with an antagonist of signaling mediated by EGFR and / or HER2, but subsequently had cancer that progressed during said treatment.

[1066] In certain embodiments, the cancer to be treated contains a mutation that confers resistance to treatment with an inhibitor of BRAF. In some embodiments, the mutation is a mutation of BRAF V600. In some embodiments, the mutation is BRAF V600E or V600K.

[1067] In a specific embodiment, the cancer to be treated contains a mutation that confers resistance to treatment with a BRAF inhibitor (e.g., a mutation in BRAF V600), and said treatment includes the administration of vemurafenib or darafenib.

[1068] In some embodiments, the antigen-binding molecule is administered in combination with an agent capable of inhibiting signal transduction mediated by an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is, for example, PD-1, CTLA-4, LAG-3, VISTA, TIM-3, TIGIT, or BTLA. In some embodiments, the antigen-binding molecule is administered in combination with an agent capable of promoting signal transduction mediated by a co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is, for example, CD28, CD80, CD40L, CD86, OX40, 4-1BB, CD27, or ICOS.

[1069] Accordingly, the present invention provides a composition comprising a target according to the present invention (e.g., an antigen-binding molecule according to the present invention) and an agent capable of inhibiting signal transduction mediated by immune checkpoint molecules. Additionally, a composition comprising a target according to the present invention and an agent capable of promoting signal transduction mediated by co-stimulatory receptors is provided. Furthermore, the use of such composition in the medical treatment and prevention of diseases / pathological conditions described herein is provided.

[1070] In addition, a method for treating or preventing a disease / pathological condition described herein is provided, comprising the step of administering a target according to the present invention (e.g., an antigen-binding molecule according to the present invention) which is the subject of the present invention, and an agent capable of inhibiting signal transduction mediated by immune checkpoint molecules. In addition, a method for treating or preventing a disease / pathological condition described herein is provided, comprising the step of administering a target according to the present invention (e.g., an antigen-binding molecule according to the present invention) which is the subject of the present invention, and an agent capable of promoting signal transduction mediated by co-stimulatory receptors.

[1071] Agents capable of inhibiting signal transduction mediated by immune checkpoint molecules are known in the art and include, for example, antibodies capable of inhibiting signal transduction mediated by immune checkpoint molecules by binding to an immune checkpoint molecule or its ligand. Other agents capable of inhibiting signal transduction mediated by immune checkpoint molecules include agents capable of reducing gene / protein expression of an immune checkpoint molecule or its ligand (e.g., by inhibiting transcription of the gene(s) encoding the immune checkpoint molecule / ligand, inhibiting post-transcriptional processing of the RNA encoding the immune checkpoint molecule / ligand, reducing stability of the RNA encoding the immune checkpoint molecule / ligand, promoting degradation of the RNA encoding the immune checkpoint molecule / ligand, inhibiting post-transcriptional processing of the immune checkpoint molecule / ligand, reducing stability of the immune checkpoint molecule / ligand, or promoting degradation of the immune checkpoint molecule / ligand) and small molecule inhibitors.

[1072] Agents capable of promoting signal transduction mediated by co-stimulatory receptors are known in the art and include, for example, agonist antibodies that bind to a co-stimulatory receptor and can trigger or increase signal transduction mediated by the co-stimulatory receptor. Other agents capable of promoting signal transduction mediated by co-stimulatory receptors include agents capable of increasing gene / protein expression of a co-stimulatory receptor or a ligand of a co-stimulatory receptor (e.g., by promoting transcription of gene(s) encoding the co-stimulatory receptor / ligand, promoting post-transcriptional processing of RNA encoding the co-stimulatory receptor / ligand, increasing stability of RNA encoding the co-stimulatory receptor / ligand, inhibiting degradation of RNA encoding the co-stimulatory receptor / ligand, promoting post-transcriptional processing of the co-stimulatory receptor / ligand, increasing stability of the co-stimulatory receptor / ligand, or inhibiting degradation of the co-stimulatory receptor / ligand) and small molecule agonists.

[1073] In a specific embodiment, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting PD-1-mediated signaling. The agent capable of inhibiting PD-1-mediated signaling is a PD-1- or PD-L1-targeted agent. The agent capable of inhibiting PD-1-mediated signaling may be, for example, an antibody capable of inhibiting PD-1-mediated signaling by binding to PD-1 or PD-L1.

[1074] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signal transduction mediated by CTLA-4. The agent capable of inhibiting signal transduction mediated by CTLA-4 may be a CTLA-4-targeted agent or an agent targeted to a ligand of CTLA-4, e.g., CD80 or CD86. In some embodiments, the agent capable of inhibiting signal transduction mediated by CTLA-4 may be an antibody capable of inhibiting CTLA-4-mediated signal transduction by binding, e.g., to CTLA-4, CD80 or CD86.

[1075] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signal transduction mediated by LAG-3. The agent capable of inhibiting signal transduction mediated by LAG-3 may be a LAG-3-targeted agent or a LAG-3 ligand, e.g., an agent targeted to MHC class II. In some embodiments, the agent capable of inhibiting signal transduction mediated by LAG-3 may be an antibody capable of inhibiting LAG-3-mediated signal transduction by binding, e.g., to LAG-3 or MHC class II.

[1076] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting VISTA-mediated signaling. The agent capable of inhibiting VISTA-mediated signaling may be a VISTA-targeted agent or an agent targeted to a VISTA ligand, e.g., VSIG-3 or VSIG-8. In some embodiments, the agent capable of inhibiting VISTA-mediated signaling may be an antibody capable of inhibiting VISTA-mediated signaling by binding to, e.g., VISTA, VSIG-3 or VSIG-8.

[1077] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signal transduction mediated by TIM-3. The agent capable of inhibiting signal transduction mediated by TIM-3 may be a TIM-3-targeted agent or an agent targeted to a TIM-3 ligand, for example, Galectin 9. In some embodiments, the agent capable of inhibiting signal transduction mediated by TIM-3 may be an antibody capable of inhibiting TIM-3-mediated signal transduction by binding, for example, to TIM-3 or Galectin 9.

[1078] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signal transduction mediated by TIGIT. The agent capable of inhibiting signal transduction mediated by TIGIT may be a TIGIT-targeted agent or an agent targeted to a ligand of TIGIT, e.g., CD113, CD112, or CD155. In some embodiments, the agent capable of inhibiting signal transduction mediated by TIGIT may be an antibody capable of inhibiting TIGIT-mediated signal transduction by binding, e.g., to TIGIT, CD113, CD112, or CD155.

[1079] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signal transduction mediated by BTLA. The agent capable of inhibiting signal transduction mediated by BTLA may be a BTLA-targeted agent or an agent targeted to a BTLA ligand, e.g., HVEM. In some embodiments, the agent capable of inhibiting signal transduction mediated by BTLA may be an antibody capable of inhibiting BTLA-mediated signal transduction by binding, e.g., to BTLA or HVEM.

[1080] In some embodiments, a method using a combination of the antigen-binding molecule of the present invention and a formulation capable of inhibiting signal transduction mediated by an immune checkpoint molecule (e.g., PD-1) provides an improved therapeutic effect compared to the effect observed when the formulation is used as a monotherapy. In some embodiments, a combination of the antigen-binding molecule of the present invention and a formulation capable of inhibiting signal transduction mediated by an immune checkpoint molecule (e.g., PD-1) provides a synergistic (i.e., superadditive) therapeutic effect.

[1081] Concurrent administration refers to administration of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number thereof), expression vector (or a number thereof), cell, or composition and a therapeutic agent together, for example, as a pharmaceutical composition containing both of the preparations (combined preparations), or immediately after administration via the same and optionally the same route of administration, for example, the same artery, vein, or different blood vessel. Sequential administration refers to administration of one of the antigen-binding molecule / composition or therapeutic agent after a predetermined time interval following a separate administration of another preparation. This is the case in some embodiments, but it is not necessary to administer the two preparations via the same route. The time interval may be any time interval.

[1082] Chemotherapy and radiotherapy, respectively, refer to the treatment of cancer using drugs or ionizing radiation (e.g., radiotherapy using X-rays or γ-rays). The drugs may be chemical entities, e.g., small molecule agents, antibiotics, DNA intercalators, protein inhibitors (e.g., kinase inhibitors), or biological agents, e.g., antibodies, antibody fragments, aptamers, nucleic acids (e.g., DNA, RNA), peptides, polypeptides, or proteins. The drugs may be formulated as pharmaceutical compositions or medicines. A formulation may comprise one or more pharmaceutically acceptable diluents, excipients, or carriers together with one or more drugs (e.g., one or more active agents).

[1083] Treatment may involve the administration of one or more drugs. Drugs may be administered alone or in combination with other treatments, either simultaneously or subsequently, depending on the condition to be treated. For example, chemotherapy may be a co-therapy involving the administration of two drugs, one or more of which may be intended for the treatment of cancer.

[1084] Chemotherapy may be administered via one or more routes of administration, e.g., parenteral, intravenous injection, oral, subcutaneous, intradermal, or intratumoral.

[1085] Chemotherapy regimens may be administered according to a therapeutic regimen. A therapeutic regimen may be a scheduled timetable, plan, design, or schedule of chemotherapy administration that can be formulated by a physician or medical professional and adjusted to suit the patient requiring treatment. A therapeutic regimen may represent one or more of the following: the type of chemotherapy regimen to be administered to the patient; the dosage of each drug or radiation; the time interval between administrations; the length of each treatment; the number and characteristics of any treatment holidays, etc. In the case of concurrent therapy regimens, a single therapeutic regimen may be provided indicating how each drug should be administered.

[1086] Chemotherapy drugs may be selected from the following: abemaciclib, abiraterone acetate, avitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Adcetris (brentuximab vedotin), ADE, ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Akinzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Allekensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Alicupa (copanlisib hydrochloride), injectable Alkeran (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Alloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Amvochlorin (Chlorambucil), Amvochlorin (Chlorambucil), Amifostine, Aminlevulinic Acid, Anastrozole, Aprepitan, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Aranon (Nellarabine), Arsenic Trioxide, Arzera (Ofatumumab), Asparaginase, Erwinia Chrysantemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axicabtagen, Silolucel, Axitinib, Azatitidine, Bavenchio (Avelumab), BEACOPP, Besenum (Carmustine), Beleodoc (Vellinostat), Velinostat, Bendamustine Hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, Bexarotene, Bexxar (tocitumomab and Iodine I 131 nocitumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, bosulip (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-maleate), cabozantinib-S-maleate, CAF, Calquence (acalabrutinib), Campath (alemtuzumab),Camptosar (irinotecan hydrochloride), capexitabine, CAPOX, Karac (fluorouracil-topical), carboplatin, carboplatin-taxol, carfilzomib, carmubris (carmustine), carmustine, carmustine graft, Casodex (bicalutamide), CEM, ceritinib, cerubidin (doxorubicin hydrochloride), cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), cloparabine, Cloparex (cloparabine), Chlorar (cloparabine), CMF, cobimetinib, cometriq (cabozantinib-S-maleate), Copenlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotelic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposomes, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darzelex (daranumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomes, dexitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diptytox, denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxate Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil-Topical), Elrictec (Rasburicase), Ellens (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin),Eltrombopag Olamine, Emend (Aprepitan), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzulutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Bismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia Chrysantemi), Etiol (Amifostine), Etopofos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA (Roxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluoruracil Injection), 5-FU (Fluoruracil-Local), Pareston (Toremifene), Paridac (Panovinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroflex (Fluoruracil-Local), Fluoruracil Injection, Fluoruracil-Local, Flutamide, Pollex (Methotrexate), Pollex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-Cetuximab, FOLFIRINOX, FOLFOX, Folotin (Pralatrexate), FU-LV, Fulvestrant, Gardasil (recombinant HPV 4-valent vaccine), Gardasil 9 (recombinant HPV 9-valent vaccine), Gaziva (obinutuzumab), Gefitinib, Gemcitabine hydrochloride, Gemcitabine-cisplatin, Gemcitabine-oxaliplatin, Gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Guillotrip (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine graft), Gliadel wafer (carmustine graft), Glucarpidase, Goserelin acetate,Halaven (Eribulin mesylate), Hemangeol (Profranolol hydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonvalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palvokiclip), Ibritumomab Tiuxetane, Ibrutinib, ICE, Iclusig (Ponatinib hydrochloride), Idamycin (Idarubicin hydrochloride), Idarubicin hydrochloride, Idelalisib, Idhipa (Enasidenib mesylate), Ephex (Ifosfamide), Ifosfamide, Ifosfamideum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (axitinib), inotuzunab ozogamicin, interferon alpha-2b, recombinant, interleukin-2 (aldesleukin), intron A (recombinant interferon alpha-2b), iodine I 131 tocitumomab and tocitumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomes, Estodax (romidepsin), ixabefilone, ixazonib citrate, Ixempra (ixabefilone), Jakafi (ruxolitinib phosphate), JEB, Zevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxyfen (Raloxifene Hydrochloride), Kepivans (Palipermin), Keytruda (Pembrolizumab), Kisqali (Ribokiclip), Kymriah (Tisagen Recleucel), Kaiprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine),Levulan (aminolevulinic acid), Linpoligene (chlorambucil), Lipodox (doxorubicin hydrochloride liposomes), Lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marquibo (vincristine sulfate liposomes), Matulan (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), Melphalan, Melphalan hydrochloride, mercaptopurine, Mesna, Mesnex (Mesna), Metazolastone (temozolomide), methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurine, Mitomycin C, Mitoxantrone Hydrochloride, Mitozitrex (Mitomycin C), MOPP, Mojobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamicin (Mitomycin C), Maileran (Busulfan), Myrosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-Stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Nicitumumab, Nelarabine, Neosar (Cyclosfofamide), Neratinib Maleate, Nerlynx (neratinib maleate), netupitant and palonosetron hydrochloride, Nulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nylandron (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiprostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omasetaxin mefesuccinate, Oncaspar (pegaspargase),Ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomes), Ontax (denileukin diptitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplatin (carboplatin), paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, pegylated interferon alpha-2b, PEG-intron (pegylated interferon alpha-2b), pembrolizumab, Pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), Plerixifor, Pomalidomide, Pomalist (pomalidomide), Ponatinib hydrochloride, Portrazza (necitumumab), Pralatrexate, Prednisone, Procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopac olamine), Propropanolol hydrochloride, Provenge (cipulucel-T), Purinetol (mercaptopurin), Furic acid (mercaptopurin), [No Entries], Radium 223 dichloride, Raloxifene hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonvalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alpha-2b, Regorafenib, Relistor (Methylnalprexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rhumatrex (Methotrexate), Rivociclip, R-ICE, Rituxan (Rituximab), Rituxan Hycelara (Human Rituximab and Hyaluronidase), Rituximab, Human Rituximab and Hyaluronidase, Lolapitant Hydrochloride, Romidepsin, Romiplostim,Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurine), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Cipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), Stanford V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Maleate, Sutent (Sunitinib Maleate), Cilatron (Peginterferon Alpha-2b), Sylvant (Ciltuximab), Synribor (Omasetaxin Mefesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Thalimogen Laherparefvec, Tamoxifen Citrate, Tarabin PFS (Cytarabine), Tarceva (Erlonitib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temozolomide, Temozolomide, Temsirolimus, Thalidomide, Talomide (Thalidomide), Thioguanine, Thiotepa, Tisagen Reclucel, Tolac (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tocitumomab and Iodine I 131 Tocitumomab, Totect (dexrazoxate hydrochloride), TPF, Travectedin, Trametinib, Trastuzumab, Treanda (bendamustine hydrochloride), Trifluridine and Tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), Uridine triacetate, VAC, Valubicin, Valsta (Valubicin), Vandetanib, VAMP, Varubi (lolapitant hydrochloride), Vectibix (panitumumab), VeIP, Belvan (vinblastine sulfate), Velcade (bortezomib), Belsar (vinblastine sulfate),Vemurafenib, Venclecta (Venetoclax), Venetoclax, Verzenio (Abemaciclip), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposomes, Vinorelbine Tartrate, VIP, Bismodegib, Vistogard (Uridine Triacetate), Boraxase (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Saatarabine Liposomes), Welcoborin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capexitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (axicabtagen ciloleucel), Yondelis (travectedin), Zaltrap (ziv-aflivercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zavelin (ibritumomab tiuxetane), Zinecard (dexrazoxate hydrochloride), ziv-aflivercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zolendronic acid), Zydelig (idellalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[1087] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with one or more of trastuzumab, cetuximab, cisplatin, 5-FU, or capecitabine. In some embodiments, the antigen-binding molecule of the present invention is administered in combination with trastuzumab and cisplatin, and 5-FU or capecitabine.

[1088] In some embodiments, the antigen-binding molecule of the present invention is administered in combination with cetuximab. Combination administration with cetuximab is particularly considered for the treatment of head and neck cancer (e.g., head and neck squamous cell carcinoma).

[1089] Multiple doses of antigen-binding molecules, polypeptides, CARs, nucleic acids (or many of these), expression vectors (or many of these), cells, or compositions may also be provided. One or more of the doses, or each of them, may be accompanied by concomitant or subsequent administration of other therapeutic agents.

[1090] Multiple doses may be separated into scheduled time intervals, which may be selected to be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. For example, doses may be provided once every 7, 14, 21, or 28 days (plus (+) or minus (-) 3 minutes, 2, or 1 day).

[1091] Detection method

[1092] The present invention also provides an article of the present invention for use in a method of detecting, localizing, or imaging HER3 or cells expressing HER3.

[1093] The antigen-binding molecule described herein is used in a method comprising an antigen-binding molecule for HER3. Such a method may include the detection of a complex of the bound antigen-binding molecule and HER3.

[1094] Thus, a method is provided comprising the steps of contacting a sample containing or presumed to contain HER3, and detecting the formation of a complex of an antigen-binding molecule and HER3. Additionally, a method is provided comprising the steps of contacting a sample containing or presumed to contain a cell expressing HER3, and detecting the formation of a complex of an antigen-binding molecule and a cell expressing HER3.

[1095] Suitable methods are well known in the art, including immunoassays, e.g., sandwich analyses, e.g., ELISA. Such methods may include the step of labeling an antigen-binding molecule, or a target(s), or both, with a detectable moiety as described herein, e.g., a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label, a radiolabel, a chemical, a nucleic acid, or an enzyme label. Detection techniques are well known to those skilled in the art and may be selected to correspond with the labeling agent.

[1096] This type of method may be provided based on a method for the diagnosis and / or prognostic evaluation of a disease or condition, e.g., cancer. Such a method may be performed in vitro on a patient sample or after processing of the patient sample. Once the sample is collected, the patient does not need to be present for the in vitro method to be performed, so such a method may not be performed in a human or animal body. In some embodiments, the method is performed in vivo.

[1097] Detection in a sample may be used for the purpose of diagnosing a disease / condition (e.g., cancer), for the purpose of predicting a tendency for a disease / condition, or to provide a prognosis (prediction) for a disease / condition, e.g., a disease / condition described herein. The diagnosis or prognosis may relate to a pre-existing (already diagnosed) disease / condition.

[1098] Such methods may include, for example, a step of detecting or quantifying HER3 or cells expressing HER3 in a patient sample. If the method includes a step of quantifying a relevant factor, such methods may further include a step of comparing a predetermined amount to a standard or reference value as part of a diagnosis or prognostic evaluation. By using other diagnostic / prognostic tests in conjunction with those described herein, the accuracy of the diagnosis or prognosis may be improved, or the results obtained by using the tests described herein may be verified.

[1099] Samples may be taken from any tissue or body fluid. Samples may include an amount of blood; an amount of serum derived from a person's blood that may include a fluid portion of blood obtained after the removal of fibrin clots and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said person or derived therefrom. In some embodiments, samples may be obtained or derived from tissues or tissues affected by a disease / pathological condition (e.g., tissues or tissues in which symptoms of the disease appear or are involved in the onset of the disease / pathological condition).

[1100] The present invention also provides a method for selecting / stratifying a subject for treatment with a HER3-targeted agent. In some embodiments, the subject is selected for treatment / prevention according to the present invention, or is identified as an individual who can benefit from such treatment / prevention, for example, based on the detection / quantification of HER3 or cells expressing HER3 in a sample obtained from the subject.

[1101] object

[1102] The subject according to the embodiments of the invention described herein may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient. The subject may be diagnosed with a disease or condition requiring treatment (e.g., cancer), may be predicted to have such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[1103] In embodiments according to the present invention, the subject is preferably a human subject. In some embodiments, the subject to be treated according to the therapeutic or prophylactic method of the invention of this specification is a subject who has cancer or is at risk of developing cancer. In embodiments according to the present invention, the subject may be selected for treatment according to a method based on characterization of specific markers of such disease / pathological condition.

[1104] kit

[1105] In some embodiments of the invention described herein, a kit of parts is provided. In some embodiments, the kit may have at least one container having a predetermined amount of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number thereof), expression vector (or a number thereof), cell, or composition described herein.

[1106] In some embodiments, the kit may include a material for producing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or many of the same), expression vector (or many of the same), cell, or composition as described herein.

[1107] The kit may provide an antigen-binding molecule, polypeptide, CAR, nucleic acid (or a number thereof), expression vector (or a number thereof), cell, or composition, along with a description for administration to a patient to treat a specified disease / pathological condition.

[1108] In some embodiments, the kit may further comprise at least one container having a predetermined amount of another therapeutic agent (e.g., an anti-infective or chemotherapy agent). In these embodiments, the kit may also comprise a second medicinal or pharmaceutical composition so that two medicinal or pharmaceutical compositions may be administered simultaneously or separately to provide a combined treatment for a specific disease or condition. The therapeutic agent may also be formulated to be suitable for injection or infusion into a tumor or blood.

[1109] Sequence identity

[1110] As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in a target sequence that are identical to nucleotide / amino acid residues in a reference sequence in order to achieve the maximum percentage of sequence identity between sequences after aligning the sequences and, if necessary, introducing a gap. Pairwise and multiple sequence alignments for the purpose of measuring the percentage of sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to those skilled in the art, for example, using publicly available computer software, for example, ClustalOmega (reference [Soeding, J. 2005, Bioinformatics 21, 951-960]), T-coffee (reference [Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217]), Kalign (reference [Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)]) and MAFFT (reference [Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780]). When using such software, for example, default parameters for gap penalty and extension penalty are preferably used.

[1111] order

[1112]

[1113]

[1114]

[1115]

[1116]

[1117]

[1118]

[1119]

[1120]

[1121]

[1122]

[1123]

[1124]

[1125]

[1126]

[1127]

[1128]

[1129]

[1130]

[1131] A series of numbered items

[1132] The following series of numbered items (paragraphs) provide further description of features and combinations of features considered in relation to the present invention:

[1133] 1. A selectively isolated antigen-binding molecule capable of binding to HER3 in the extracellular domain subdomain II.

[1134] 2. In claim 1, the antigen-binding molecule is an antigen-binding molecule that inhibits the interaction between HER3 and the interaction partner of HER3.

[1135] 3. In claim 1 or 2, the antigen-binding molecule is an antigen-binding molecule capable of binding to a polypeptide comprising or composed of the amino acid sequence of SEQ ID NO. 16.

[1136] 4. In any one of claims 1 to 3, the antigen-binding molecule is an antigen-binding molecule capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO. 23 or SEQ ID NO. 229.

[1137] 5. In any one of claims 1 to 4, the antigen-binding molecule is an antigen-binding molecule capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO. 21 or SEQ ID NO. 229.

[1138] 6. In any one of paragraphs 1 to 5, the antigen-binding molecule

[1139] (i) Heavy chain variable (VH) region containing the following CDR:

[1140] HC-CDR1 having the amino acid sequence of SEQ ID NO. 43

[1141] HC-CDR2 having the amino acid sequence of SEQ ID NO. 46

[1142] HC-CDR3 having the amino acid sequence of SEQ ID NO. 51; and

[1143] (ii) Light chain variable (VL) regions containing the following CDRs:

[1144] LC-CDR1 having the amino acid sequence of SEQ ID NO. 91

[1145] LC-CDR2 having the amino acid sequence of SEQ ID NO. 94

[1146] LC-CDR3 having the amino acid sequence of SEQ ID NO. 99

[1147] Antigen-binding molecule containing

[1148] 7. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1149] (i) Heavy chain variable (VH) region containing the following CDR:

[1150] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1151] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[1152] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[1153] (ii) Light chain variable (VL) regions containing the following CDRs:

[1154] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[1155] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[1156] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[1157] Antigen-binding molecule containing

[1158] 8. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1159] (i) Heavy chain variable (VH) region containing the following CDR:

[1160] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1161] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[1162] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[1163] (ii) Light chain variable (VL) regions containing the following CDRs:

[1164] LC-CDR1 having the amino acid sequence of SEQ ID NO. 89

[1165] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[1166] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[1167] Antigen-binding molecule containing

[1168] 9. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1169] (i) Heavy chain variable (VH) region containing the following CDR:

[1170] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1171] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[1172] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[1173] (ii) Light chain variable (VL) regions containing the following CDRs:

[1174] LC-CDR1 having the amino acid sequence of SEQ ID NO. 90

[1175] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[1176] LC-CDR3 having the amino acid sequence of SEQ ID NO. 96

[1177] Antigen-binding molecule containing

[1178] 10. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1179] (i) Heavy chain variable (VH) region containing the following CDR:

[1180] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1181] HC-CDR2 having the amino acid sequence of SEQ ID NO. 44

[1182] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[1183] (ii) Light chain variable (VL) regions containing the following CDRs:

[1184] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[1185] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[1186] LC-CDR3 having the amino acid sequence of SEQ ID NO. 98

[1187] Antigen-binding molecule containing

[1188] 11. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1189] (i) Heavy chain variable (VH) region containing the following CDR:

[1190] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1191] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[1192] HC-CDR3 having the amino acid sequence of SEQ ID NO. 47; and

[1193] (ii) Light chain variable (VL) regions containing the following CDRs:

[1194] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[1195] LC-CDR2 having the amino acid sequence of SEQ ID NO. 93

[1196] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[1197] Antigen-binding molecule containing

[1198] 12. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1199] (i) Heavy chain variable (VH) region containing the following CDR:

[1200] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1201] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[1202] HC-CDR3 having the amino acid sequence of SEQ ID NO. 49; and

[1203] (ii) Light chain variable (VL) regions containing the following CDRs:

[1204] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[1205] LC-CDR2 having the amino acid sequence of SEQ ID NO. 93

[1206] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[1207] Antigen-binding molecule containing

[1208] 13. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1209] (i) Heavy chain variable (VH) region containing the following CDR:

[1210] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1211] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[1212] HC-CDR3 having the amino acid sequence of SEQ ID NO. 50; and

[1213] (ii) Light chain variable (VL) regions containing the following CDRs:

[1214] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[1215] LC-CDR2 having the amino acid sequence of SEQ ID NO. 93

[1216] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[1217] Antigen-binding molecule containing

[1218] 14. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1219] (i) Heavy chain variable (VH) region containing the following CDR:

[1220] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1221] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[1222] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[1223] (ii) Light chain variable (VL) regions containing the following CDRs:

[1224] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[1225] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[1226] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[1227] Antigen-binding molecule containing

[1228] 15. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1229] (i) Heavy chain variable (VH) region containing the following CDR:

[1230] HC-CDR1 having the amino acid sequence of SEQ ID NO. 41

[1231] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[1232] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[1233] (ii) Light chain variable (VL) regions containing the following CDRs:

[1234] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[1235] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[1236] LC-CDR3 having the amino acid sequence of SEQ ID NO. 97

[1237] Antigen-binding molecule containing

[1238] 16. In any one of paragraphs 1 to 6, the antigen-binding molecule

[1239] (i) Heavy chain variable (VH) region containing the following CDR:

[1240] HC-CDR1 having the amino acid sequence of SEQ ID NO. 42

[1241] HC-CDR2 having the amino acid sequence of SEQ ID NO. 45

[1242] HC-CDR3 having the amino acid sequence of SEQ ID NO. 48; and

[1243] (ii) Light chain variable (VL) regions containing the following CDRs:

[1244] LC-CDR1 having the amino acid sequence of SEQ ID NO. 88

[1245] LC-CDR2 having the amino acid sequence of SEQ ID NO. 92

[1246] LC-CDR3 having the amino acid sequence of SEQ ID NO. 95

[1247] Antigen-binding molecule containing

[1248] 17. In any one of paragraphs 1 to 5, the antigen-binding molecule

[1249] (i) Heavy chain variable (VH) region containing the following CDR:

[1250] HC-CDR1 having the amino acid sequence of SEQ ID NO. 158

[1251] HC-CDR2 having the amino acid sequence of SEQ ID NO. 159

[1252] HC-CDR3 having the amino acid sequence of SEQ ID NO. 160; and

[1253] (ii) Light chain variable (VL) regions containing the following CDRs:

[1254] LC-CDR1 having the amino acid sequence of SEQ ID NO. 165

[1255] LC-CDR2 having the amino acid sequence of SEQ ID NO. 166

[1256] LC-CDR3 having the amino acid sequence of SEQ ID NO. 167

[1257] Antigen-binding molecule containing

[1258] 18. In any one of claims 1 to 4, the antigen-binding molecule may bind to a polypeptide comprising the amino acid sequence of SEQ ID NO. 22.

[1259] 19. In any one of paragraphs 1 to 4 or paragraph 18, the antigen-binding molecule

[1260] (i) Heavy chain variable (VH) region containing the following CDR:

[1261] HC-CDR1 having the amino acid sequence of SEQ ID NO. 128

[1262] HC-CDR2 having the amino acid sequence of SEQ ID NO. 129

[1263] HC-CDR3 having the amino acid sequence of SEQ ID NO. 130; and

[1264] (ii) Light chain variable (VL) regions containing the following CDRs:

[1265] LC-CDR1 having the amino acid sequence of SEQ ID NO. 136

[1266] LC-CDR2 having the amino acid sequence of SEQ ID NO. 137

[1267] LC-CDR3 having the amino acid sequence of SEQ ID NO. 138

[1268] Antigen-binding molecule containing

[1269] 20. In any one of paragraphs 1 to 4 or paragraph 18, the antigen-binding molecule

[1270] (i) Heavy chain variable (VH) region containing the following CDR:

[1271] HC-CDR1 having the amino acid sequence of SEQ ID NO. 144

[1272] HC-CDR2 having the amino acid sequence of SEQ ID NO. 145

[1273] HC-CDR3 having the amino acid sequence of SEQ ID NO. 146; and

[1274] (ii) Light chain variable (VL) regions containing the following CDRs:

[1275] LC-CDR1 having the amino acid sequence of SEQ ID NO. 151

[1276] LC-CDR2 having the amino acid sequence of SEQ ID NO. 152

[1277] LC-CDR3 having the amino acid sequence of SEQ ID NO. 153

[1278] Antigen-binding molecule containing

[1279] 21. In any one of paragraphs 1 to 4, the antigen-binding molecule

[1280] (i) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 24; and

[1281] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 74;

[1282] or

[1283] (ii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 25; and

[1284] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 75;

[1285] or

[1286] (iii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 26; and

[1287] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 76;

[1288] or

[1289] (iv) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 27; and

[1290] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 77;

[1291] or

[1292] (v) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 28; and

[1293] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78;

[1294] or

[1295] (vi) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 29; and

[1296] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78;

[1297] or

[1298] (vii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 30; and

[1299] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78;

[1300] or

[1301] (viii) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 31; and

[1302] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 79;

[1303] or

[1304] (ix) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 32; and

[1305] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 79;

[1306] or

[1307] (x) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 33; and

[1308] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 80;

[1309] or

[1310] (xi) a VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 34; and

[1311] A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino aci...

Claims

Claim 1 An antigen-binding molecule comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the antigen-binding molecule specifically binds to HER3 and binds to the region of HER3 presented in SEQ ID NO 229, and comprises a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO 43, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO 46, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO 51; and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO 91, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO 94, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO 99. Claim 2 In claim 1, the antigen-binding molecule comprises (a) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 41, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 44, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 47; and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 88, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 92, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 95, or (b) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 41, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 44, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 47; and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 89, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 92, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 95, or (c) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 41, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 44, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 47; and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 90, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 92, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 96, or (d) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 41, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 45, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 47;and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 88, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 93, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 95, or (e) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 41, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 45, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 49; and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 88, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 93, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 95, or (f) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 41, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 45, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 50; and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 88, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 93, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 95, or (g) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 41, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 45, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 48;and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 88, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 92, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 97, or (h) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 42, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 45, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 48; An antigen-binding molecule comprising: a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 88, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 92, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 95; or (i) a VH region comprising the following CDRs: HC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 41, HC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 44, and HC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 47; and a VL region comprising the following CDRs: LC-CDR1 consisting of the amino acid sequence of SEQ ID NO. 88, LC-CDR2 consisting of the amino acid sequence of SEQ ID NO. 92, and LC-CDR3 consisting of the amino acid sequence of SEQ ID NO. 98.; Claim 3 In claim 1, (a) the amino acid sequence of the VH region has an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 24, and the amino acid sequence of the VL region has an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 74; or (b) the amino acid sequence of the VH region has an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 25, and the amino acid sequence of the VL region has an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 75; or (c) the amino acid sequence of the VH region has an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 26, and the amino acid sequence of the VL region has an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 76; or (d) the amino acid sequence of the VH region has an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 27, and the amino acid sequence of the VL region is SEQ ID NO (e) having an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of 77, or (e) having an amino acid sequence in the VH region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 28, and having an amino acid sequence in the VL region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78, or (f) having an amino acid sequence in the VH region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 29, and having an amino acid sequence in the VL region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78;(g) the amino acid sequence of the VH region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 30, and the amino acid sequence of the VL region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 78; or (h) the amino acid sequence of the VH region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 31, and the amino acid sequence of the VL region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 79; or (i) the amino acid sequence of the VH region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 32, and the amino acid sequence of the VL region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 79; or (j) the amino acid sequence of the VH region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 33, and the amino acid sequence of the VL region has at least 70% with respect to the amino acid sequence of SEQ ID NO. 80 (k) having an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 34, and having an amino acid sequence of the VL region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 81; (l) having an amino acid sequence of the VH region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 35, and having an amino acid sequence of the VL region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 82; (m) having an amino acid sequence of the VH region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 36, and having an amino acid sequence of the VL region having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 83;(n) the amino acid sequence of the VH region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 37, and the amino acid sequence of the VL region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 84; or (o) the amino acid sequence of the VH region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 38, and the amino acid sequence of the VL region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 85; or (p) the amino acid sequence of the VH region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 39, and the amino acid sequence of the VL region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 86; or (q) the amino acid sequence of the VH region has at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO. 40, and the amino acid sequence of the VL region has at least 70% with respect to the amino acid sequence of SEQ ID NO. 87 Antigen-binding molecule having an amino acid sequence having sequence identity.; Claim 4 In claim 1, the antigen-binding molecule comprises (a) a VH region comprising the following FR: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 55, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 58, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 69, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 73; or (b) a VH region comprising the following FR: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 52, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 56, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 61, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 70; or (c) a VH region comprising the following FR: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 52, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 56, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 62, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 70; or (d) a VH region comprising the following FRs: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 52, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 57, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 62, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 70; or (e) a VH region comprising the following FRs: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 224, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 60, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 63, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 70; or (f) a VH region comprising the following FRs: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 52, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 56, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 64, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 70;or (g) a VH region comprising the following FRs: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 52, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 57, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 64, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 70; or (h) a VH region comprising the following FRs: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 52, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 56, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 65, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 70; or (i) a VH region comprising the following FRs: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 53, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 59, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 66, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 71; or (j) a VH region comprising the following FRs: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 54, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 59, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 67, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 71; or (k) a VH region comprising the following FRs: HC-FR1 consisting of the amino acid sequence of SEQ ID NO. 53, HC-FR2 consisting of the amino acid sequence of SEQ ID NO. 59, HC-FR3 consisting of the amino acid sequence of SEQ ID NO. 68, and HC-FR4 consisting of the amino acid sequence of SEQ ID NO. 72, comprising an antigen-binding molecule.; Claim 5 In claim 1, the antigen-binding molecule comprises (a) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 106, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 113, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 123, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 126; or (b) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 100, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 107, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 114, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124; or (c) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 101, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 108, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 115, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124; or (d) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 102, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 108, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 116, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124; or (e) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 103, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 108, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 117, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124; or (f) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 103, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 108, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 118, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124;or (g) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 103, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 109, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 119, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124; or (h) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 104, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 110, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 120, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 125; or (i) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 105, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 110, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 121, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124; or (j) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 104, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 111, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 122, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 125; or (k) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 100, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 112, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 114, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124; or (l) a VL region comprising the following FRs: LC-FR1 consisting of the amino acid sequence of SEQ ID NO. 103, LC-FR2 consisting of the amino acid sequence of SEQ ID NO. 108, LC-FR3 consisting of the amino acid sequence of SEQ ID NO. 119, and LC-FR4 consisting of the amino acid sequence of SEQ ID NO. 124, comprising an antigen-binding molecule.; Claim 6 The antigen-binding molecule of claim 1, wherein the antigen-binding molecule comprises a heavy chain invariant region consisting of the amino acid sequence of SEQ ID NO.

171. Claim 7 The antigen-binding molecule of claim 1, wherein the antigen-binding molecule comprises a light chain invariant region consisting of the amino acid sequence of SEQ ID NO.

177. Claim 8 Isolated nucleic acid or a plurality of isolated nucleic acids encoding an antigen-binding molecule according to any one of claims 1 to 7. Claim 9 An expression vector or multiple expression vectors comprising a nucleic acid or multiple nucleic acids according to paragraph 8. Claim 10 An isolated cell comprising: an antigen-binding molecule according to any one of claims 1 to 7; a nucleic acid or a plurality of nucleic acids encoding said antigen-binding molecule; or an expression vector or a plurality of expression vectors comprising said nucleic acid or said plurality of nucleic acids. Claim 11 A method for producing an antigen-binding molecule, comprising the step of culturing a cell, wherein the cell comprises a nucleic acid or a plurality of nucleic acids encoding an antigen-binding molecule according to any one of claims 1 to 7, or an expression vector comprising said nucleic acid or said plurality of nucleic acids, or a plurality of expression vectors. Claim 12 A pharmaceutical composition for use in a method for treating or preventing cancer, comprising: an antigen-binding molecule according to any one of claims 1 to 7; a nucleic acid or a plurality of nucleic acids encoding said antigen-binding molecule; an expression vector or a plurality of expression vectors comprising said nucleic acid or said plurality of nucleic acids; or a cell comprising said antigen-binding molecule, said nucleic acid or said plurality of nucleic acids, or said expression vector or said plurality of expression vectors. Claim 13 In paragraph 12, cancer comprising cells expressing an EGFR family member, cancer comprising cells expressing HER3, solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, 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, ovarian serous adenocarcinoma, renal cancer, renal cell carcinoma (renal cell carcinoma), renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary 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,A pharmaceutical composition selected from cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma, and thymoma. Claim 14 A method for detecting HER3, comprising a sample containing or presumed to contain HER3 and an antigen-binding molecule according to any one of claims 1 to 7 in a test tube ( in vitro A method comprising the step of contacting in ); and the step of detecting the formation of a complex of an antigen-binding molecule and HER3. Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete

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