CNX antigen binding molecules

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By designing specific antigen binding molecules, using their binding ability to CNX to inhibit the activity of CNX, the problem of lack of effective CNX antibodies in the prior art is solved, and effective prevention and treatment methods for diseases including cancer are provided.

CN120092020APending Publication Date: 2025-06-03AGENCY FOR SCI TECH & RES +1

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Patent Information

Application Number: CN202380064683.4
Authority / Receiving Office: CN · China
Patent Type: Applications(China)
Current Assignee / Owner
Priority Date: 2022-07-08
Filing Date: 2023-07-07
Publication Date: 2025-06-03

AI Technical Summary

Technical Problem

There is a lack of effective CNX antibodies in the prior art, making it difficult to develop CNX antibodies suitable for medical and preventive methods.

Method used

An optionally isolated antigen binding molecule comprising a specific CDR sequence capable of binding to CNX and inhibiting degradation of extracellular matrix (ECM).

Benefits of technology

By inhibiting CNX activity, antigen-binding molecules can effectively slow the progression of diseases, including cancer, and provide potential medical and preventive methods.

✦ Generated by Eureka AI based on patent content.

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Abstract

Disclosed herein are antigen binding molecules capable of binding to calnectin (CNX). Chimeric antigen receptors, antibody-drug conjugates, and compositions comprising such antigen binding molecules, as well as nucleic acids, vectors, and cells are also disclosed. Uses and methods relating to antigen binding molecules capable of binding to calnectin (CNX) are also disclosed.

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Description

[0001] This application claims priority to US 63 / 359,499, filed Jul. 8, 2022, the contents and elements of which are incorporated herein by reference for all purposes. FIELD OF THE INVENTION

[0002] The present disclosure relates to the field of molecular biology, and more particularly, to antibody technology. The present disclosure also relates to medical and prophylactic methods.

[0003] BACKGROUND

[0004] CNX is a lectin chaperone protein resident in the endoplasmic reticulum (ER) that binds to N-glycoproteins carrying monoglycosylated glycans and recruits various other chaperones, mediating protein disulfide formation, proline isomerization, and protein folding.

[0005] Recent studies have shown that CNX and CNX-containing complexes (such as CNX:ERp57) are associated with diseases / pathologies including cancer, particularly through their activity in degrading the ECM (see Ros et al., Nat. Cell Biol. (2020) 22(11):1371-1381).

[0006] Ros et al., Nat. Cell Biol. (2020) 22(11):1371-1381 disclose antibodies ab10286 and ab22595 against CNX. Ab10286 and ab22595 from Abcam are both rabbit polyclonal antibody preparations against human CNX. There is still a need to develop CNX antibodies suitable for medical and prophylactic methods.

[0007] SUMMARY

[0008] In a first aspect, the present disclosure provides an optionally isolated antigen-binding molecule that binds to CNX.

[0009] In certain embodiments, the antigen-binding molecule can inhibit extracellular matrix (ECM) degradation.

[0010] In certain embodiments, the antigen-binding molecule comprises:

[0011] (a)

[0012] (i) A heavy chain variable (VH) region comprising the following CDRs:

[0013] HC-CDR1 having the amino acid sequence of SEQ ID NO:166

[0014] HC-CDR2 having the amino acid sequence of SEQ ID NO:167

[0015] HC-CDR3 having the amino acid sequence of SEQ ID NO:168; and

[0016] (ii) a light chain variable (VL) region comprising the following CDRs:

[0017] LC-CDR1 having the amino acid sequence of SEQ ID NO:179

[0018] LC-CDR2 having the amino acid sequence of SEQ ID NO:180

[0019] LC-CDR3 having the amino acid sequence of SEQ ID NO:173; or

[0020] (b)

[0021] (i) a heavy chain variable (VH) region comprising the following CDRs:

[0022] HC-CDR1 having the amino acid sequence of SEQ ID NO:33

[0023] HC-CDR2 having the amino acid sequence of SEQ ID NO:34

[0024] HC-CDR3 having the amino acid sequence of SEQ ID NO:35; and

[0025] (ii) a light chain variable (VL) region comprising the following CDRs:

[0026] LC-CDR1 having the amino acid sequence of SEQ ID NO:41

[0027] LC-CDR2 having the amino acid sequence of SEQ ID NO:42

[0028] LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or

[0029] (c)

[0030] (i) a heavy chain variable (VH) region comprising the following CDRs:

[0031] HC-CDR1 having the amino acid sequence of SEQ ID NO:2

[0032] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0033] HC-CDR3 having the amino acid sequence of SEQ ID NO:4; and

[0034] (ii) a light chain variable (VL) region comprising the following CDRs:

[0035] LC-CDR1 having the amino acid sequence of SEQ ID NO:10

[0036] LC-CDR2 having the amino acid sequence of SEQ ID NO:11

[0037] LC-CDR3 having the amino acid sequence of SEQ ID NO:12; or

[0038] (d)

[0039] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0040] HC-CDR1 having the amino acid sequence of SEQ ID NO:18

[0041] HC-CDR2 having the amino acid sequence of SEQ ID NO:19

[0042] HC-CDR3 having the amino acid sequence of SEQ ID NO:20; and

[0043] (ii) The light chain variable (VL) region comprising the following CDRs:

[0044] LC-CDR1 having the amino acid sequence of SEQ ID NO:25

[0045] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0046] LC-CDR3 having the amino acid sequence of SEQ ID NO:27; or

[0047] (e)

[0048] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0049] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0050] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0051] HC-CDR3 having the amino acid sequence of SEQ ID NO:49; and

[0052] (ii) The light chain variable (VL) region comprising the following CDRs:

[0053] LC-CDR1 having the amino acid sequence of SEQ ID NO:53

[0054] LC-CDR2 having the amino acid sequence of SEQ ID NO:54

[0055] The LC-CDR3 having the amino acid sequence of SEQ ID NO:55; or

[0056] (f)

[0057] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0058] The HC-CDR1 having the amino acid sequence of SEQ ID NO:61

[0059] The HC-CDR2 having the amino acid sequence of SEQ ID NO:62

[0060] The HC-CDR3 having the amino acid sequence of SEQ ID NO:63; and

[0061] (ii) The light chain variable (VL) region comprising the following CDRs:

[0062] The LC-CDR1 having the amino acid sequence of SEQ ID NO:68

[0063] The LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0064] The LC-CDR3 having the amino acid sequence of SEQ ID NO:69; or

[0065] (g)

[0066] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0067] The HC-CDR1 having the amino acid sequence of SEQ ID NO:61

[0068] The HC-CDR2 having the amino acid sequence of SEQ ID NO:62

[0069] The HC-CDR3 having the amino acid sequence of SEQ ID NO:63; and

[0070] (ii) The light chain variable (VL) region comprising the following CDRs:

[0071] The LC-CDR1 having the amino acid sequence of SEQ ID NO:73

[0072] The LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0073] The LC-CDR3 having the amino acid sequence of SEQ ID NO:74; or

[0074] (h)

[0075] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0076] HC-CDR1 having the amino acid sequence of SEQ ID NO:61

[0077] HC-CDR2 having the amino acid sequence of SEQ ID NO:62

[0078] HC-CDR3 having the amino acid sequence of SEQ ID NO:63; and

[0079] (ii) a light chain variable (VL) region comprising the following CDRs:

[0080] LC-CDR1 having the amino acid sequence of SEQ ID NO:78

[0081] LC-CDR2 having the amino acid sequence of SEQ ID NO:79

[0082] LC-CDR3 having the amino acid sequence of SEQ ID NO:80; or

[0083] (i)

[0084] (i) a heavy chain variable (VH) region comprising the following CDRs:

[0085] HC-CDR1 having the amino acid sequence of SEQ ID NO:2

[0086] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0087] HC-CDR3 having the amino acid sequence of SEQ ID NO:83; and

[0088] (ii) a light chain variable (VL) region comprising the following CDRs:

[0089] LC-CDR1 having the amino acid sequence of SEQ ID NO:73

[0090] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0091] LC-CDR3 having the amino acid sequence of SEQ ID NO:74; or

[0092] (j)

[0093] (i) a heavy chain variable (VH) region comprising the following CDRs:

[0094] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0095] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0096] an HC-CDR3 having the amino acid sequence of SEQ ID NO:86; and

[0097] (ii) a light chain variable (VL) region comprising the following CDRs:

[0098] an LC-CDR1 having the amino acid sequence of SEQ ID NO:89

[0099] an LC-CDR2 having the amino acid sequence of SEQ ID NO:11

[0100] an LC-CDR3 having the amino acid sequence of SEQ ID NO:90; or

[0101] (k)

[0102] (i) a heavy chain variable (VH) region comprising the following CDRs:

[0103] an HC-CDR1 having the amino acid sequence of SEQ ID NO:61

[0104] an HC-CDR2 having the amino acid sequence of SEQ ID NO:95

[0105] an HC-CDR3 having the amino acid sequence of SEQ ID NO:96; and

[0106] (ii) a light chain variable (VL) region comprising the following CDRs:

[0107] an LC-CDR1 having the amino acid sequence of SEQ ID NO:101

[0108] an LC-CDR2 having the amino acid sequence of SEQ ID NO:102

[0109] an LC-CDR3 having the amino acid sequence of SEQ ID NO:103; or

[0110] (l)

[0111] (i) a heavy chain variable (VH) region comprising the following CDRs:

[0112] an HC-CDR1 having the amino acid sequence of SEQ ID NO:108

[0113] an HC-CDR2 having the amino acid sequence of SEQ ID NO:109

[0114] an HC-CDR3 having the amino acid sequence of SEQ ID NO:110; and

[0115] (ii) a light chain variable (VL) region comprising the following CDRs:

[0116] LC-CDR1 having the amino acid sequence of SEQ ID NO:115

[0117] LC-CDR2 having the amino acid sequence of SEQ ID NO:116

[0118] LC-CDR3 having the amino acid sequence of SEQ ID NO:117; or

[0119] (m)

[0120] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0121] HC-CDR1 having the amino acid sequence of SEQ ID NO:2

[0122] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0123] HC-CDR3 having the amino acid sequence of SEQ ID NO:122; and

[0124] (ii) The light chain variable (VL) region comprising the following CDRs:

[0125] LC-CDR1 having the amino acid sequence of SEQ ID NO:125

[0126] LC-CDR2 having the amino acid sequence of SEQ ID NO:126

[0127] LC-CDR3 having the amino acid sequence of SEQ ID NO:127; or

[0128] (n)

[0129] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0130] HC-CDR1 having the amino acid sequence of SEQ ID NO:132

[0131] HC-CDR2 having the amino acid sequence of SEQ ID NO:133

[0132] HC-CDR3 having the amino acid sequence of SEQ ID NO:134; and

[0133] (ii) The light chain variable (VL) region comprising the following CDRs:

[0134] LC-CDR1 having the amino acid sequence of SEQ ID NO:139

[0135] LC-CDR2 having the amino acid sequence of SEQ ID NO:140

[0136] LC-CDR3 having the amino acid sequence of SEQ ID NO:80; or

[0137] (o)

[0138] (i) A heavy chain variable (VH) region comprising the following CDRs:

[0139] HC-CDR1 having the amino acid sequence of SEQ ID NO:146

[0140] HC-CDR2 having the amino acid sequence of SEQ ID NO:147

[0141] HC-CDR3 having the amino acid sequence of SEQ ID NO:148; and

[0142] (ii) A light chain variable (VL) region comprising the following CDRs:

[0143] LC-CDR1 having the amino acid sequence of SEQ ID NO:41

[0144] LC-CDR2 having the amino acid sequence of SEQ ID NO:42

[0145] LC-CDR3 having the amino acid sequence of SEQ ID NO:153; or

[0146] (p)

[0147] (i) A heavy chain variable (VH) region comprising the following CDRs:

[0148] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0149] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0150] HC-CDR3 having the amino acid sequence of SEQ ID NO:156; and

[0151] (ii) A light chain variable (VL) region comprising the following CDRs:

[0152] LC-CDR1 having the amino acid sequence of SEQ ID NO:158

[0153] LC-CDR2 having the amino acid sequence of SEQ ID NO:159

[0154] LC-CDR3 having the amino acid sequence of SEQ ID NO:160; or

[0155] (q)

[0156] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0157] HC-CDR1 having the amino acid sequence of SEQ ID NO: 166

[0158] HC-CDR2 having the amino acid sequence of SEQ ID NO: 167

[0159] HC-CDR3 having the amino acid sequence of SEQ ID NO: 168; and

[0160] (ii) The light chain variable (VL) region comprising the following CDRs:

[0161] LC-CDR1 having the amino acid sequence of SEQ ID NO: 171

[0162] LC-CDR2 having the amino acid sequence of SEQ ID NO: 172

[0163] LC-CDR3 having the amino acid sequence of SEQ ID NO: 173; or

[0164] (r)

[0165] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0166] HC-CDR1 having the amino acid sequence of SEQ ID NO: 185

[0167] HC-CDR2 having the amino acid sequence of SEQ ID NO: 186

[0168] HC-CDR3 having the amino acid sequence of SEQ ID NO: 187; and

[0169] (ii) The light chain variable (VL) region comprising the following CDRs:

[0170] LC-CDR1 having the amino acid sequence of SEQ ID NO: 73

[0171] LC-CDR2 having the amino acid sequence of SEQ ID NO: 26

[0172] LC-CDR3 having the amino acid sequence of SEQ ID NO: 194; or

[0173] (s)

[0174] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0175] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0176] HC-CDR2 having the amino acid sequence of SEQ ID NO:199

[0177] HC-CDR3 having the amino acid sequence of SEQ ID NO:200; and

[0178] (ii) The light chain variable (VL) region comprising the following CDRs:

[0179] LC-CDR1 having the amino acid sequence of SEQ ID NO:205

[0180] LC-CDR2 having the amino acid sequence of SEQ ID NO:42

[0181] LC-CDR3 having the amino acid sequence of SEQ ID NO:206; or

[0182] (t)

[0183] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0184] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0185] HC-CDR2 having the amino acid sequence of SEQ ID NO:211

[0186] HC-CDR3 having the amino acid sequence of SEQ ID NO:212; and

[0187] (ii) The light chain variable (VL) region comprising the following CDRs:

[0188] LC-CDR1 having the amino acid sequence of SEQ ID NO:216

[0189] LC-CDR2 having the amino acid sequence of SEQ ID NO:172

[0190] LC-CDR3 having the amino acid sequence of SEQ ID NO:217; or

[0191] (u)

[0192] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0193] HC-CDR1 having the amino acid sequence of SEQ ID NO:222

[0194] HC-CDR2 having the amino acid sequence of SEQ ID NO:223

[0195] The HC-CDR3 having the amino acid sequence of SEQ ID NO:224; and

[0196] (ii) The light chain variable (VL) region comprising the following CDRs:

[0197] The LC-CDR1 having the amino acid sequence of SEQ ID NO:229

[0198] The LC-CDR2 having the amino acid sequence of SEQ ID NO:172

[0199] The LC-CDR3 having the amino acid sequence of SEQ ID NO:230; or

[0200] (v)

[0201] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0202] The HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0203] The HC-CDR2 having the amino acid sequence of SEQ ID NO:199

[0204] The HC-CDR3 having the amino acid sequence of SEQ ID NO:200; and

[0205] (ii) The light chain variable (VL) region comprising the following CDRs:

[0206] The LC-CDR1 having the amino acid sequence of SEQ ID NO:235

[0207] The LC-CDR2 having the amino acid sequence of SEQ ID NO:236

[0208] The LC-CDR3 having the amino acid sequence of SEQ ID NO:237; or

[0209] (w)

[0210] (i) The heavy chain variable (VH) region comprising the following CDRs:

[0211] The HC-CDR1 having the amino acid sequence of SEQ ID NO:185

[0212] The HC-CDR2 having the amino acid sequence of SEQ ID NO:243

[0213] The HC-CDR3 having the amino acid sequence of SEQ ID NO:244; and

[0214] (ii) The light chain variable (VL) region comprising the following CDRs:

[0215] LC-CDR1 having the amino acid sequence of SEQ ID NO: 248

[0216] LC-CDR2 having the amino acid sequence of SEQ ID NO: 249

[0217] LC-CDR3 having the amino acid sequence of SEQ ID NO: 250; or

[0218] In some embodiments, the antigen-binding molecule comprises:

[0219] a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 165, 32, 1, 17, 47, 60, 82, 85, 94, 107, 121, 131, 154, 155, 184, 198, 210, 221, or 242; and

[0220] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 178, 40, 9, 24, 52, 67, 72, 77, 88, 100, 114, 124, 138, 152, 157, 170, 191, 204, 215, 228, 234, or 247.

[0221] In some embodiments, the antigen-binding molecule comprises:

[0222] (i) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 165; and

[0223] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 178;

[0224] or

[0225] (ii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 32; and

[0226] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 40;

[0227] or

[0228] (iii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1; and

[0229] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:9;

[0230] or

[0231] (iv) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:17; and

[0232] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:24;

[0233] or

[0234] (v) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:47; and

[0235] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:52;

[0236] or

[0237] (vi) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:60; and

[0238] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:67;

[0239] or

[0240] (vii) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:60; and

[0241] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:72;

[0242] or

[0243] (viii) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:60; and

[0244] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:77;

[0245] or

[0246] (ix) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: amino acid sequence; and

[0247] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

[0248] or

[0249] (x) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 82; and

[0250] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 72;

[0251] or

[0252] (xi) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 85; and

[0253] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 88;

[0254] or

[0255] (xii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 94; and

[0256] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 100;

[0257] or

[0258] (xiii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 107; and

[0259] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 114;

[0260] or

[0261] (xiv) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 121; and

[0262] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 124;

[0263] or

[0264] (xv) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 131; and

[0265] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 138;

[0266] Or

[0267] (xvi) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 145; and

[0268] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 152;

[0269] Or

[0270] (xvii) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 155; and

[0271] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 157;

[0272] Or

[0273] (xviii) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 165; and

[0274] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 170;

[0275] Or

[0276] (xix) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 184; and

[0277] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 191;

[0278] Or

[0279] (xx) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 198; and

[0280] A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 204;

[0281] or

[0282] (xxi) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 210; and

[0283] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 215;

[0284] or

[0285] (xxii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 221; and

[0286] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 228;

[0287] or

[0288] (xxiii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 198; and

[0289] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 234;

[0290] or

[0291] (xxiv) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 242; and

[0292] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 247.

[0293] In some embodiments, the antigen-binding molecule binds to CNX by contacting with: (a) one or more amino acid residues of the CNX region corresponding to the region shown in SEQ ID NO: 363, optionally, wherein the antigen-binding molecule binds to CNX by contacting with one or more amino acid residues of the CNX region corresponding to the region shown in SEQ ID NO: 361 or 362; or (b) one or more amino acid residues of the CNX region corresponding to the region shown in SEQ ID NO: 371, optionally, wherein the antigen-binding molecule binds to CNX by contacting with one or more amino acid residues of the CNX region corresponding to the region shown in SEQ ID NO: 364, 365, 366, 367, 368, 369, 370, 372 or 373.

[0294] In some embodiments, the antigen-binding molecule binds to CRT.

[0295] In some embodiments, the antigen-binding molecule binds to human CNX and murine CNX.

[0296] In some embodiments, the antigen-binding molecule is a multispecific antigen-binding molecule, wherein the antigen-binding molecule further comprises an antigen-binding domain that binds to an antigen other than CNX. In some embodiments, the multispecific antigen-binding molecule is a bispecific T cell engager (BiTE).

[0297] The present disclosure also provides a chimeric antigen receptor (CAR) comprising the antigen-binding molecule of the present disclosure.

[0298] The present disclosure also provides an antibody-drug conjugate (ADC) comprising the antigen-binding molecule of the present disclosure and a drug moiety.

[0299] The present disclosure also provides one or more optionally isolated nucleic acids encoding the antigen-binding molecule or CAR of the present disclosure

[0300] The present disclosure also provides one or more expression vectors comprising one or more nucleic acids of the present disclosure.

[0301] The present disclosure also provides a cell comprising the antigen-binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors of the present disclosure.

[0302] The present disclosure also provides a method comprising culturing a cell of the present disclosure under conditions suitable for the cell to express the antigen-binding molecule or CAR.

[0303] The present disclosure also provides a composition comprising the antigen-binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, or cell of the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0304] The present disclosure also provides an antigen-binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell or composition for use in a method of medicine or prophylaxis.

[0305] The present disclosure also provides an antigen-binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell or composition for use in a method of treating or preventing a disease / condition characterized by extracellular matrix (ECM) degradation.

[0306] The present disclosure also provides an antigen-binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell or composition for use in a method of treating or preventing cancer.

[0307] In some embodiments, the cancer is selected from: liver cancer, breast cancer, oral cancer, oral squamous cell carcinoma, sarcoma, lung cancer, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, pancreatic cancer, endometrial cancer, colorectal cancer, and thyroid cancer.

[0308] The present disclosure also provides an antigen-binding molecule, a CAR, a nucleic acid or multiple nucleic acids, an expression vector or multiple expression vectors, a cell or a composition for treating or preventing cartilage degeneration or a disease / condition characterized by cartilage degeneration.

[0309] In some embodiments, the disease / condition characterized by cartilage degeneration is selected from: joint disorders, arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, juvenile arthritis, post-traumatic arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage injury, and polychondritis.

[0310] The present disclosure also provides the use of the antigen-binding molecule of the present disclosure for depleting cells expressing CNX or increasing the killing of cells expressing CNX.

[0311] The present disclosure also provides an optionally isolated in vitro complex comprising the antigen-binding molecule of the present disclosure that binds to CNX.

[0312] The present disclosure also provides a method for detecting CNX in a sample, comprising contacting a sample containing or suspected of containing CNX with the antigen-binding molecule of the present disclosure and detecting the formation of a complex of the antigen-binding molecule and CNX.

[0313] The present disclosure also provides a method for selecting or sorting a subject for treatment with a CNX-targeting agent, the method comprising contacting a sample of the subject with the antigen-binding molecule of the present disclosure in vitro and detecting the formation of a complex of the antigen-binding molecule and CNX.

[0314] The present disclosure also provides the use of the antigen-binding molecule of the present disclosure as an in vitro or in vivo diagnostic or prognostic agent.

[0315] Description

[0316] The present disclosure provides an antigen-binding molecule that binds to CNX and has new biophysical and / or functional properties compared to the antigen-binding proteins disclosed in the prior art.

[0317] CNX and CRT

[0318] The present disclosure relates to a CNX-specific antigen-binding molecule.

[0319] Human CNX (also known as CNX, CANX, or IP90) is a protein identified by UniProt P27824. Alternative splicing of the mRNA encoded by the human CANX gene produces three major CNX isoforms: isoform 1 (SEQ ID NO:333), isoform 2 (SEQ ID NO:334), and isoform 3 (SEQ ID NO:335). Isoform 2 differs from isoform 1 by the insertion of a 35-amino acid sequence after position 1 of SEQ ID NO:333. Positions 1 to 108 of SEQ ID NO:333 are absent in isoform 3.

[0320] Human CNX isoform 1 contains an N-terminal signal peptide (SEQ ID NO:336), followed by a calcium-binding luminal domain (SEQ ID NO:337), a single-pass transmembrane domain (SEQ ID NO:338), and an acidic cytoplasmic domain at the C-terminus (SEQ ID NO:339). The luminal domain includes a globular lectin domain (SEQ ID NO:340), followed by an arm-like proline-rich P domain (SEQ ID NO:341) and a second lectin domain (SEQ ID NO:342). The mature form of human CNX isoform 1 is shown as SEQ ID NO:343.

[0321] As used herein, "CNX" refers to CNX of any species, including CNX isoforms, fragments, variants, or homologs from any species. In some embodiments, CNX is CNX from a mammal (e.g., a therian, a placental, an epitherian, a preptotheria, an archontan, a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human)). In some embodiments, CNX is human CNX or mouse CNX.

[0322] As used herein, a "fragment", "variant", "isoform", or "homolog" of a given protein can optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher amino acid sequence identity to the amino acid sequence of a reference protein (e.g., a reference isoform).

[0323] "Fragment" generally refers to a part of the protein. "Variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of the protein, but maintains a substantial degree of sequence identity (e.g., at least 60%) with the sequence of the protein. "Isoform" generally refers to a variant of the protein expressed by the same species as the protein species (e.g., human CNX isoform 1, isoform 2, and isoform 3 are all isoforms of each other). "Homolog" generally refers to a variant of the protein produced by a different species compared to the protein species. For example, human CNX isoform 1 (UniProt: P27824-1, v2; SEQ ID NO: 333) and mouse CNX (UniProt: P35564-1, v1; SEQ ID NO: 344) are homologs of each other. Homologs include orthologs.

[0324] The isoforms, fragments, variants, or homologs of CNX of the present disclosure may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of an immature or mature CNX isoform of a given species (e.g., human).

[0325] The isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs, e.g., having the functionality / activity of a reference CNX (e.g., human CNX isoform 1), as determined by analysis using an appropriate functional or activity assay. For example, an isoform, fragment, variant, or homolog of CNX may bind to a monoglucosylated glycan-containing N-glycoprotein and / or bind to ERp57, cyclophilin B, and / or ERp29.

[0326] In some embodiments, CNX 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 SEQ ID NO: 333, 334, 335, or 343.

[0327] In some embodiments, CNX 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 SEQ ID NO: 344 or 352.

[0328] A "fragment" of a reference protein can be of any length (counted by the number of amino acids), but can optionally be at least 25% of the length of the reference protein (i.e., the protein from which the fragment is derived), and the maximum length can be one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of the reference protein.

[0329] The minimum length of the CNX fragment can be one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 amino acids, and the maximum length can be one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 amino acids.

[0330] In some embodiments, the CNX fragment comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 343, 337, 338, 339, 340, 341 or 342.

[0331] In some embodiments, the CNX fragment comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 352, 346, 347, 348, 349, 350 or 351.

[0332] In some embodiments, the antigen-binding molecule of the present disclosure can bind to calreticulin (CRT).

[0333] The inventors observed that CNX-depleted cells can compensate for the loss of CNX through the action of CRT. Therefore, the inventors believe that the antigen-binding molecule is capable of binding to CNX and CRT.

[0334] In some embodiments, the antigen-binding molecule has cross-reactivity to human CNX and CRT. In some embodiments, the antigen-binding molecule reduces one activity of CNX and one activity of CRT. In some embodiments, the antigen-binding molecule reduces CNX activity and CRT activity.

[0335] As used herein, a "cross-reactive" antigen-binding molecule / domain is cross-reactive and thus capable of binding to multiple target antigens. For example, an antigen-binding molecule / domain / polypeptide that is cross-reactive with CNX and CRT binds to CNX and is also capable of binding to CRT. A cross-reactive antigen-binding molecule / domain / polypeptide may exhibit specific binding to each target antigen.

[0336] Human CRT (also known as calreticulin, calmodulin, or ERp60) is the protein identified by UniProt P27797. Human CRT has the amino acid sequence shown in SEQ ID NO:353. Human CRT contains an N-terminal signal peptide (SEQ ID NO:354), followed by a calcium-binding N domain (SEQ ID ID:355), and an acidic C domain (SEQ ID NO:356) at the C-terminus. The N-domain includes a globular lectin domain (SEQ ID NO:357), followed by an arm-like proline-rich P-domain (SEQ ID NO:359) and a second lectin domain (SEQ ID NO:358). The mature form of human CRT is shown in SEQ ID NO:360.

[0337] In this specification, "CRT" refers to CRT of any species, including subtypes, fragments, variants, or homologs from any species. In some embodiments, CRT is from a mammal (e.g., a therian mammal, a placental mammal, an epitherian mammal, a preptotheria, an archontan, a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human)). In some embodiments, CRT is human CRT or mouse CRT.

[0338] An isotype, fragment, variant, or homolog of the CRT of the present disclosure may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature CRT isotype of a given species (e.g., human).

[0339] An isotype, fragment, variant, or homolog of CRT may optionally be a functional isotype, fragment, variant, or homolog, e.g., having the functionality / activity of a reference CRT (e.g., human CRT), as determined by analysis using an appropriate functional or activity assay. For example, an isotype, fragment, variant, or homolog of CRT may bind to a monoglucosylated glycan-containing N-glycoprotein and / or bind to ERp57, cyclophilin B, and / or ERp29.

[0340] In some embodiments, the CRT comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 353 or 360.

[0341] The minimum length of the CRT fragment can be one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids, and the maximum length can be one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids.

[0342] In some embodiments, the CRT fragment comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 360, 355, 356, 357, 358 or 359.

[0343] The structures and functions of CNX and CRT are reviewed, for example, in Kozlov and Gehring, FEBS J. (2020) 287(20):4322 - 4340, the entire content of which is incorporated herein by reference.

[0344] CNX and CRT are lectin chaperone proteins within the endoplasmic reticulum (ER). CNX / CRT binds to monoglucosylated glycan - bearing N - glycoproteins and recruits various other chaperones, mediating protein disulfide bond formation, proline isomerization and protein folding. CNX / CRT is capable of binding to the protein - folding enzyme ERp57 to catalyze the formation of glycoprotein - specific disulfide bonds. The CNX:ERp57 complex has also been shown to translocate to the cancer cell surface, where they reduce disulfide bridges in the extracellular matrix (Ros et al., Nat. Cell Biol. 22:1371 - 1381, 2020). The reduction of disulfide bridges has been shown to be crucial for the effective activity of matrix metalloproteinases (MMPs) and thus for the degradation of the cancer cell extracellular matrix. CNX / CRT also binds to peptidyl - prolyl cis - trans isomerase cyclophilin B (CypB) for proline isomerization of peptide bonds. CNX / CRT has also been reported to bind to ERp29 to form the CNX / CRT:ERp29 complex, which has general chaperone functions. CNX also acts as a chaperone for the folding of MHC class I α - chains in the ER membrane.

[0345] Treatment with glucosidase II removes the glucose residue of the monoglucosylated N-glycan required for the interaction of glycoproteins with CNX / CRT, resulting in the release of mature processed glycoproteins from CNX / CRT. For proteins that have not been correctly folded, UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a checkpoint by re-adding glucose residues to the N-glycan, reconstructing the interaction site with CNX / CRT. In this way, misfolded proteins rebind to CNX / CRT for additional chaperone-mediated folding and are prevented from exiting the ER and progressing to the Golgi. If multiple folding cycles are unsuccessful, terminally misfolded proteins are transported to the cytoplasm for degradation via the ER-associated protein degradation (ERAD) pathway.

[0346] Antigen-binding molecule

[0347] The present disclosure provides antigen-binding molecules capable of binding to CNX. Antigen-binding molecules capable of binding to a given target antigen may also be described as target antigen-binding molecules.

[0348] "Antigen-binding molecule" refers to a molecule that binds to a given target antigen. Antigen-binding molecules include antibodies (i.e., immunoglobulins (Igs)) and antigen-binding fragments thereof. As used herein, "antibody" includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived antigen-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single-domain antibodies (e.g., VhH), etc. Antigen-binding fragments of antibodies include, for example, Fv, Fab, F(ab') 2 and F(ab") fragments. In some embodiments, the antigen-binding molecule can be an antibody or an antigen-binding fragment thereof.

[0349] The antigen-binding molecules of the present disclosure also include antibody-derived molecules, such as molecules comprising an antigen-binding region / domain derived from an antibody. Antibody-derived antigen-binding molecules can comprise an antigen-binding region / domain that comprises or consists of the antigen-binding region of an antibody (such as an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding region / domain of an antibody-derived antigen-binding molecule can be or include the Fv of an antibody (such as provided as a scFv) or a Fab region, or the whole antibody. For example, the antigen-binding molecules of the present disclosure include antibody-drug conjugates (ADCs), which comprise a (cytotoxic) drug moiety (such as described below). The antigen-binding molecules of the present disclosure also include multispecific antigen-binding molecules, such as immunocyte engager molecules comprising a domain for recruiting (effector) immune cells (such as reviewed in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17:418-434 and Ellerman, Methods (2019) 154:102-117, the entire contents of both of which are incorporated herein by reference), including BiTEs, BiKEs, and TriKEs. The antigen-binding molecules of the present disclosure also include chimeric antigen receptors (CARs), which are recombinant receptors that provide antigen-binding and T cell activation functions (the structure, function, and engineering of CARs are reviewed in Dotti et al., Immunol Rev (2014) 257(1), the entire contents of which are incorporated herein by reference).

[0350] The antigen-binding molecules of the present disclosure comprise one or more moieties capable of binding to a target antigen. In some embodiments, the moieties capable of binding to the target antigen include the variable heavy chain (VH) and variable light chain (VL) of an antibody that can specifically bind to the target antigen. In some embodiments, the moieties capable of binding to the target antigen include or consist of aptamers capable of binding to the target antigen, such as nucleic acid aptamers (e.g., reviewed in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). In some embodiments, the moieties capable of binding to the target antigen include or consist of antigen-binding peptides / polypeptides, such as peptide aptamers, thioredoxin, monomers, antitoxins, Kunitz domains, guanylins, knottins, fynomers, trimers, DARPins, affibody molecules, nanobodies (i.e., single-domain antibodies (sdAb)), affilins, armadillo repeat proteins (ArmRP), OBody or fibronectin - e.g., as described in Reverdatto et al., Curr Top MedChem. 2015;15(12):1082-1101, which is hereby incorporated by reference in its entirety herein (see also Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., mabs (2011) 3:38-48).

[0351] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. The length of a peptide is typically in the range of about 2 to 50 amino acids. "Polypeptide" is a polymeric chain composed of two or more peptides. The length of a polypeptide is typically greater than about 50 amino acids.

[0352] The antigen-binding molecules of the present disclosure generally comprise an antigen-binding domain that contains the VH and VL of an antibody that can specifically bind to a target antigen. The antigen-binding domain formed by VH and VL may also be referred to herein as the Fv region.

[0353] The antigen-binding molecule can be or can include an antigen-binding polypeptide or an antigen-binding polypeptide complex. The antigen-binding molecule can contain more than one polypeptide, which together form an antigen-binding domain. The polypeptides can be covalently or non-covalently bound. In some embodiments, the polypeptides form part of a polypeptide that comprises a larger polypeptide (e.g., in the case where an scFv contains VH and VL, or in the case where an scFab contains VH-CH1 and VL-CL).

[0354] The antigen-binding molecule can refer to a non-covalent or covalent complex of more than one polypeptide (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.

[0355] The antigen-binding molecules of the present disclosure can be designed and prepared using monoclonal antibody (mAb) sequences capable of binding to CNX. Antigen-binding regions of antibodies can also be used / provided, such as single-chain variable fragments (scFv), Fab, and F(ab') 2 fragments. An "antigen-binding region" refers to any fragment of an antibody that binds to the target specific to a given antibody.

[0356] Antibodies typically contain six complementarity-determining regions CDRs; three heavy-chain variable regions (VH): HC-CDR1, HC-CDR2, and HC-CDR3, and three light-chain variable regions: LC-CDR1, LC-CDR2, and LC-CDR3. These six CDRs together define the paratope of the antibody, which is the part of the antibody that binds to the target antigen.

[0357] The VH region and the VL region include framework regions (FRs) flanking each CDR, which provide a scaffold for the CDRs. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus - [HC-FR1] - [HC-CDR1] - [HC-FR2] - [HC-CDR2] - [HC-FR3] - [HC-CDR3] - [HC-FR4] - C-terminus; the VL region has the following structure: N-terminus - [LC-FR1] - [LC-CDR1] - [LC-FR2] - [LC-CDR2] - [LC-FR3] - [LC-CDR3] - [LC-FR4] - C-terminus.

[0358] There are several different conventions for defining antibody CDRs and FRs, such as those described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); Chothia et al., Journal of Molecular Biology, 196:901-917 (1987), and in VBASE2, as described by Retter et al. in 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 Acids Res. (2015) 43 (Database issue): D413-22), which uses the IMGT V-DOMAIN numbering rules described by LeFranc et al. in Dev. Comp. Immunol. (2003) 27:55-77. In a preferred embodiment, the CDRs and FRs of the antigen-binding molecules described herein are defined according to the IMGT information system.

[0359] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule that binds to CNX. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds to CNX. In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of an antigen-binding molecule that binds to CNX. That is, in some embodiments, the antigen-binding molecule comprises the VH and VL regions of an antigen-binding molecule that binds to CNX.

[0360] In some embodiments, the antigen-binding molecule comprises the CDRs, FRs, and / or VH and / or VL regions of the CNX-binding antibody clones described herein, or CDRs, FRs, and / or VH and / or VL regions derived from the CNX-binding antibody clones described herein. In some embodiments, the CNX-binding antibody clones are selected from: 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001, C008, C010, C023, C025, C040, C046, and C117.

[0361] In some embodiments, the antigen-binding molecule comprises a VH region of one of the following (1) to (19):

[0362] (1) A VH region comprising the following CDRs:

[0363] HC-CDR1 having the amino acid sequence of SEQ ID NO:2

[0364] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0365] HC-CDR3 having the amino acid sequence of SEQ ID NO:4,

[0366] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0367] (2) A VH region comprising the following CDRs:

[0368] HC-CDR1 having the amino acid sequence of SEQ ID NO:18

[0369] HC-CDR2 having the amino acid sequence of SEQ ID NO:19

[0370] HC-CDR3 having the amino acid sequence of SEQ ID NO:20,

[0371] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0372] (3) A VH region comprising the following CDRs:

[0373] HC-CDR1 having the amino acid sequence of SEQ ID NO:33

[0374] HC-CDR2 having the amino acid sequence of SEQ ID NO:34

[0375] HC-CDR3 having the amino acid sequence of SEQ ID NO:35,

[0376] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0377] (4) A VH region comprising the following CDRs:

[0378] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0379] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0380] HC-CDR3 having the amino acid sequence of SEQ ID NO:49,

[0381] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0382] (5) A VH region comprising the following CDRs:

[0383] HC-CDR1 having the amino acid sequence of SEQ ID NO:61

[0384] HC-CDR2 having the amino acid sequence of SEQ ID NO:62

[0385] HC-CDR3 having the amino acid sequence of SEQ ID NO:63,

[0386] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0387] (6) A VH region comprising the following CDRs:

[0388] HC-CDR1 having the amino acid sequence of SEQ ID NO:2

[0389] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0390] HC-CDR3 having the amino acid sequence of SEQ ID NO:83,

[0391] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0392] (6) A VH region comprising the following CDRs:

[0393] HC-CDR1 having the amino acid sequence of SEQ ID NO:2

[0394] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0395] HC-CDR3 having the amino acid sequence of SEQ ID NO:83,

[0396] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0397] (7) A VH region comprising the following CDRs:

[0398] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0399] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0400] HC-CDR3 having the amino acid sequence of SEQ ID NO:86,

[0401] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0402] (8) A VH region comprising the following CDRs:

[0403] HC-CDR1 having the amino acid sequence of SEQ ID NO:61

[0404] HC-CDR2 having the amino acid sequence of SEQ ID NO:95

[0405] HC-CDR3 having the amino acid sequence of SEQ ID NO:96,

[0406] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0407] (9) A VH region comprising the following CDRs:

[0408] HC-CDR1 having the amino acid sequence of SEQ ID NO:108

[0409] HC-CDR2 having the amino acid sequence of SEQ ID NO:109

[0410] HC-CDR3 having the amino acid sequence of SEQ ID NO:110,

[0411] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0412] (10) A VH region comprising the following CDRs:

[0413] HC-CDR1 having the amino acid sequence of SEQ ID NO:2

[0414] HC-CDR2 having the amino acid sequence of SEQ ID NO:3

[0415] HC-CDR3 having the amino acid sequence of SEQ ID NO:122,

[0416] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0417] (11) A VH region comprising the following CDRs:

[0418] HC-CDR1 having the amino acid sequence of SEQ ID NO: 132

[0419] HC-CDR2 having the amino acid sequence of SEQ ID NO: 133

[0420] HC-CDR3 having the amino acid sequence of SEQ ID NO: 134,

[0421] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0422] (12) A VH region comprising the following CDRs:

[0423] HC-CDR1 having the amino acid sequence of SEQ ID NO: 146

[0424] HC-CDR2 having the amino acid sequence of SEQ ID NO: 147

[0425] HC-CDR3 having the amino acid sequence of SEQ ID NO: 148,

[0426] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0427] (13) A VH region comprising the following CDRs:

[0428] HC-CDR1 having the amino acid sequence of SEQ ID NO: 48

[0429] HC-CDR2 having the amino acid sequence of SEQ ID NO: 3

[0430] HC-CDR3 having the amino acid sequence of SEQ ID NO: 156,

[0431] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0432] (14) A VH region comprising the following CDRs:

[0433] HC-CDR1 having the amino acid sequence of SEQ ID NO:166

[0434] HC-CDR2 having the amino acid sequence of SEQ ID NO:167

[0435] HC-CDR3 having the amino acid sequence of SEQ ID NO:168,

[0436] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0437] (15) A VH region comprising the following CDRs:

[0438] HC-CDR1 having the amino acid sequence of SEQ ID NO:185

[0439] HC-CDR2 having the amino acid sequence of SEQ ID NO:186

[0440] HC-CDR3 having the amino acid sequence of SEQ ID NO:187,

[0441] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or 1 or 2 or 3 amino acids in HC-CDR2, and / or 1 or 2 or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0442] (16) A VH region comprising the following CDRs:

[0443] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0444] HC-CDR2 having the amino acid sequence of SEQ ID NO:199

[0445] HC-CDR3 having the amino acid sequence of SEQ ID NO:200,

[0446] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0447] (17) A VH region comprising the following CDRs:

[0448] HC-CDR1 having the amino acid sequence of SEQ ID NO:48

[0449] HC-CDR2 having the amino acid sequence of SEQ ID NO:211

[0450] HC-CDR3 having the amino acid sequence of SEQ ID NO:212,

[0451] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0452] (18) A VH region comprising the following CDRs:

[0453] HC-CDR1 having the amino acid sequence of SEQ ID NO:222

[0454] HC-CDR2 having the amino acid sequence of SEQ ID NO:223

[0455] HC-CDR3 having the amino acid sequence of SEQ ID NO:224,

[0456] or a variant thereof, wherein 1, 2, or 3 amino acids in HC-CDR1, and / or 1, 2, or 3 amino acids in HC-CDR2, and / or 1, 2, or 3 amino acids in HC-CDR3 are replaced by another amino acid.

[0457] (19) A VH region comprising the following CDRs:

[0458] HC-CDR1 having the amino acid sequence of SEQ ID NO:185

[0459] HC-CDR2 having the amino acid sequence of SEQ ID NO:243

[0460] HC-CDR3 having the amino acid sequence of SEQ ID NO:244,

[0461] or a variant thereof, wherein one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are replaced by another amino acid.

[0462] In some embodiments, the antigen-binding molecule comprises a VH region of one of the following (20) to (37):

[0463] (20) A VH region comprising the following FRs:

[0464] HC-FR1 having the amino acid sequence of SEQ ID NO:5

[0465] HC-FR2 having the amino acid sequence of SEQ ID NO:6

[0466] HC-FR3 having the amino acid sequence of SEQ ID NO:7

[0467] HC-FR4 having the amino acid sequence of SEQ ID NO:8,

[0468] or a variant thereof, wherein one, two or three amino acids in HC-FR1, and / or one, two or three amino acids in HC-FR2, and / or one, two or three amino acids in HC-FR3, and / or one, two or three amino acids in HC-FR4 are replaced by another amino acid.

[0469] (21) A VH region comprising the following FRs:

[0470] HC-FR1 having the amino acid sequence of SEQ ID NO:21

[0471] HC-FR2 having the amino acid sequence of SEQ ID NO:22

[0472] HC-FR3 having the amino acid sequence of SEQ ID NO:23

[0473] HC-FR4 having the amino acid sequence of SEQ ID NO:8,

[0474] or a variant thereof, wherein one, two or three amino acids in HC-FR1, and / or one, two or three amino acids in HC-FR2, and / or one, two or three amino acids in HC-FR3, and / or one, two or three amino acids in HC-FR4 are replaced by another amino acid.

[0475] (22) A VH region comprising the following FRs:

[0476] HC-FR1 having the amino acid sequence of SEQ ID NO:36

[0477] HC-FR2 having the amino acid sequence of SEQ ID NO:37

[0478] HC-FR3 having the amino acid sequence of SEQ ID NO:38

[0479] HC-FR4 having the amino acid sequence of SEQ ID NO:39,

[0480] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0481] (23) The VH region comprising the following FRs:

[0482] HC-FR1 having the amino acid sequence of SEQ ID NO:50

[0483] HC-FR2 having the amino acid sequence of SEQ ID NO:6

[0484] HC-FR3 having the amino acid sequence of SEQ ID NO:7

[0485] HC-FR4 having the amino acid sequence of SEQ ID NO:51,

[0486] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0487] (24) The VH region comprising the following FRs:

[0488] HC-FR1 having the amino acid sequence of SEQ ID NO:64

[0489] HC-FR2 having the amino acid sequence of SEQ ID NO:65

[0490] HC-FR3 having the amino acid sequence of SEQ ID NO:66

[0491] HC-FR4 having the amino acid sequence of SEQ ID NO:39,

[0492] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0493] (25) The VH region comprising the following FRs:

[0494] HC-FR1 having the amino acid sequence of SEQ ID NO:84

[0495] HC-FR2 having the amino acid sequence of SEQ ID NO:6

[0496] HC-FR3 having the amino acid sequence of SEQ ID NO:7

[0497] HC-FR4 having the amino acid sequence of SEQ ID NO:39,

[0498] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0499] (26) The VH region comprising the following FRs:

[0500] HC-FR1 having the amino acid sequence of SEQ ID NO:87

[0501] HC-FR2 having the amino acid sequence of SEQ ID NO:6

[0502] HC-FR3 having the amino acid sequence of SEQ ID NO:7

[0503] HC-FR4 having the amino acid sequence of SEQ ID NO:51,

[0504] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0505] (27) The VH region comprising the following FRs:

[0506] HC-FR1 having the amino acid sequence of SEQ ID NO:97

[0507] HC-FR2 having the amino acid sequence of SEQ ID NO:98

[0508] HC-FR3 having the amino acid sequence of SEQ ID NO:66

[0509] HC-FR4 having the amino acid sequence of SEQ ID NO:39,

[0510] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0511] (28) The VH region comprising the following FRs:

[0512] HC-FR1 having the amino acid sequence of SEQ ID NO:111

[0513] HC-FR2 having the amino acid sequence of SEQ ID NO:112

[0514] HC-FR3 having the amino acid sequence of SEQ ID NO:113

[0515] HC-FR4 having the amino acid sequence of SEQ ID NO:51,

[0516] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0517] (29) The VH region comprising the following FRs:

[0518] HC-FR1 having the amino acid sequence of SEQ ID NO:123

[0519] HC-FR2 having the amino acid sequence of SEQ ID NO:6

[0520] HC-FR3 having the amino acid sequence of SEQ ID NO:7

[0521] HC-FR4 having the amino acid sequence of SEQ ID NO:51,

[0522] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0523] (30) The VH region containing the following FRs:

[0524] HC-FR1 having the amino acid sequence of SEQ ID NO: 135

[0525] HC-FR2 having the amino acid sequence of SEQ ID NO: 136

[0526] HC-FR3 having the amino acid sequence of SEQ ID NO: 137

[0527] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0528] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0529] (31) The VH region containing the following FRs:

[0530] HC-FR1 having the amino acid sequence of SEQ ID NO: 149

[0531] HC-FR2 having the amino acid sequence of SEQ ID NO: 150

[0532] HC-FR3 having the amino acid sequence of SEQ ID NO: 151

[0533] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0534] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0535] (32) The VH region containing the following FRs:

[0536] HC-FR1 having the amino acid sequence of SEQ ID NO: 87

[0537] HC-FR2 having the amino acid sequence of SEQ ID NO: 169

[0538] HC-FR3 having the amino acid sequence of SEQ ID NO: 168

[0539] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0540] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0541] (33) The VH region comprising the following FRs:

[0542] HC-FR1 having the amino acid sequence of SEQ ID NO: 188

[0543] HC-FR2 having the amino acid sequence of SEQ ID NO: 189

[0544] HC-FR3 having the amino acid sequence of SEQ ID NO: 7

[0545] HC-FR4 having the amino acid sequence of SEQ ID NO: 8,

[0546] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0547] (34) The VH region comprising the following FRs:

[0548] HC-FR1 having the amino acid sequence of SEQ ID NO: 201

[0549] HC-FR2 having the amino acid sequence of SEQ ID NO: 202

[0550] HC-FR3 having the amino acid sequence of SEQ ID NO: 203

[0551] HC-FR4 having the amino acid sequence of SEQ ID NO: 8,

[0552] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0553] (35) The VH region comprising the following FRs:

[0554] HC-FR1 having the amino acid sequence of SEQ ID NO: 213

[0555] HC-FR2 having the amino acid sequence of SEQ ID NO: 214

[0556] HC-FR3 having the amino acid sequence of SEQ ID NO: 7

[0557] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0558] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0559] (36) The VH region comprising the following FRs:

[0560] HC-FR1 having the amino acid sequence of SEQ ID NO: 225

[0561] HC-FR2 having the amino acid sequence of SEQ ID NO: 226

[0562] HC-FR3 having the amino acid sequence of SEQ ID NO: 227

[0563] HC-FR4 having the amino acid sequence of SEQ ID NO: 8,

[0564] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or 1 or 2 or 3 amino acids in HC-FR2, and / or 1 or 2 or 3 amino acids in HC-FR3, and / or 1 or 2 or 3 amino acids in HC-FR4 are replaced by another amino acid.

[0565] (37) The VH region comprising the following FRs:

[0566] HC-FR1 having the amino acid sequence of SEQ ID NO: 245

[0567] HC-FR2 having the amino acid sequence of SEQ ID NO: 246

[0568] HC-FR3 having the amino acid sequence of SEQ ID NO: 190

[0569] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0570] or a variant thereof, wherein one, two, or three amino acids in HC-FR1, and / or one, two, or three amino acids in HC-FR2, and / or one, two, or three amino acids in HC-FR3, and / or one, two, or three amino acids in HC-FR4 are replaced by another amino acid.

[0571] In some embodiments, the antigen-binding molecule comprises a VH region that comprises the CDRs of any one of (1) to (19) above and the FRs of any one of (20) to (37) above.

[0572] In some embodiments, the antigen-binding molecule comprises a VH region of one of the following (38) to (56):

[0573] (38) A VH region comprising the CDRs of (1) and the FRs of (20).

[0574] (39) A VH region comprising the CDRs of (2) and the FRs of (21).

[0575] (40) A VH region comprising the CDRs of (3) and the FRs of (22).

[0576] (41) A VH region comprising the CDRs of (4) and the FRs of (23).

[0577] (42) A VH region comprising the CDRs of (5) and the FRs of (24).

[0578] (43) A VH region comprising the CDRs of (6) and the FRs of (25).

[0579] (44) A VH region comprising the CDRs of (7) and the FRs of (26).

[0580] (45) A VH region comprising the CDRs of (8) and the FRs of (27).

[0581] (46) A VH region comprising the CDRs of (9) and the FRs of (28).

[0582] (47) A VH region comprising the CDRs of (10) and the FRs of (29).

[0583] (48) A VH region comprising the CDRs of (11) and the FRs of (30).

[0584] (49) A VH region comprising the CDRs of (12) and the FRs of (31).

[0585] (50) A VH region comprising the CDRs of (13) and the FRs of (26).

[0586] The VH region comprising the CDRs of (14) and the FRs of (32).

[0587] (52) The VH region comprising the CDRs of (15) and the FRs of (33).

[0588] (53) The VH region comprising the CDRs of (16) and the FRs of (34).

[0589] (54) The VH region comprising the CDRs of (17) and the FRs of (35).

[0590] (55) The VH region comprising the CDRs of (18) and the FRs of (36).

[0591] (56) The VH region comprising the CDRs of (19) and the FRs of (37).

[0592] In some embodiments, the antigen-binding molecule comprises a VH region selected from the following (57) to (75):

[0593] (57) The VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:1, more preferably, having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0594] (58) The VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:17, more preferably, having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0595] (59) The VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:32, more preferably, having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0596] (60) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 47, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0597] (61) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 60, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0598] (62) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 82, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0599] (63) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 85, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0600] (64) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 94, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0601] (65) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 107, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0602] (66) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 121, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0603] (67) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 131, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0604] (68) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 145, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0605] (69) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 155, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0606] (70) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 165, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0607] (71) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 184, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0608] (72) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 198, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0609] (73) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 210, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0610] (74) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 221, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0611] (75) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 242, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0612] In some embodiments, the antigen-binding molecule comprises a VL region of one of the following (76) to (97):

[0613] (76) A VL region comprising the following CDRs:

[0614] LC-CDR1 having the amino acid sequence of SEQ ID NO: 10

[0615] LC-CDR2 having the amino acid sequence of SEQ ID NO: 11

[0616] LC-CDR3 having the amino acid sequence of SEQ ID NO: 12,

[0617] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0618] (77) A VL region comprising the following CDRs:

[0619] LC-CDR1 having the amino acid sequence of SEQ ID NO:25

[0620] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0621] LC-CDR3 having the amino acid sequence of SEQ ID NO:27,

[0622] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0623] (78) A VL region comprising the following CDRs:

[0624] LC-CDR1 having the amino acid sequence of SEQ ID NO:41

[0625] LC-CDR2 having the amino acid sequence of SEQ ID NO:42

[0626] LC-CDR3 having the amino acid sequence of SEQ ID NO:43,

[0627] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0628] (79) A VL region comprising the following CDRs:

[0629] LC-CDR1 having the amino acid sequence of SEQ ID NO:53

[0630] LC-CDR2 having the amino acid sequence of SEQ ID NO:54

[0631] LC-CDR3 having the amino acid sequence of SEQ ID NO:55,

[0632] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0633] (80) A VL region comprising the following CDRs:

[0634] LC-CDR1 having the amino acid sequence of SEQ ID NO:68

[0635] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0636] LC-CDR3 having the amino acid sequence of SEQ ID NO:69,

[0637] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0638] (81) A VL region comprising the following CDRs:

[0639] LC-CDR1 having the amino acid sequence of SEQ ID NO:73

[0640] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0641] LC-CDR3 having the amino acid sequence of SEQ ID NO:74,

[0642] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0643] (82) A VL region comprising the following CDRs:

[0644] LC-CDR1 having the amino acid sequence of SEQ ID NO:78

[0645] LC-CDR2 having the amino acid sequence of SEQ ID NO:79

[0646] LC-CDR3 having the amino acid sequence of SEQ ID NO:80,

[0647] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0648] (83) The VL region comprising the following CDRs:

[0649] LC-CDR1 having the amino acid sequence of SEQ ID NO:89

[0650] LC-CDR2 having the amino acid sequence of SEQ ID NO:11

[0651] LC-CDR3 having the amino acid sequence of SEQ ID NO:90,

[0652] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0653] (84) The VL region comprising the following CDRs:

[0654] LC-CDR1 having the amino acid sequence of SEQ ID NO:101

[0655] LC-CDR2 having the amino acid sequence of SEQ ID NO:102

[0656] LC-CDR3 having the amino acid sequence of SEQ ID NO:103,

[0657] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0658] (85) The VL region comprising the following CDRs:

[0659] LC-CDR1 having the amino acid sequence of SEQ ID NO:115

[0660] LC-CDR2 having the amino acid sequence of SEQ ID NO:116

[0661] LC-CDR3 having the amino acid sequence of SEQ ID NO:117,

[0662] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0663] (86) The VL region comprising the following CDRs:

[0664] LC-CDR1 having the amino acid sequence of SEQ ID NO: 125

[0665] LC-CDR2 having the amino acid sequence of SEQ ID NO: 126

[0666] LC-CDR3 having the amino acid sequence of SEQ ID NO: 127,

[0667] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0668] (87) The VL region comprising the following CDRs:

[0669] LC-CDR1 having the amino acid sequence of SEQ ID NO: 139

[0670] LC-CDR2 having the amino acid sequence of SEQ ID NO: 140

[0671] LC-CDR3 having the amino acid sequence of SEQ ID NO: 80,

[0672] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0673] (88) The VL region comprising the following CDRs:

[0674] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0675] LC-CDR2 having the amino acid sequence of SEQ ID NO: 42

[0676] LC-CDR3 having the amino acid sequence of SEQ ID NO: 153,

[0677] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0678] (89) The VL region comprising the following CDRs:

[0679] LC-CDR1 having the amino acid sequence of SEQ ID NO: 158

[0680] LC-CDR2 having the amino acid sequence of SEQ ID NO: 159

[0681] LC-CDR3 having the amino acid sequence of SEQ ID NO: 160,

[0682] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0683] (90) The VL region comprising the following CDRs:

[0684] LC-CDR1 having the amino acid sequence of SEQ ID NO: 171

[0685] LC-CDR2 having the amino acid sequence of SEQ ID NO: 172

[0686] LC-CDR3 having the amino acid sequence of SEQ ID NO: 173,

[0687] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or 1 or 2 or 3 amino acids in LC-CDR2, and / or 1 or 2 or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0688] (91) The VL region comprising the following CDRs:

[0689] LC-CDR1 having the amino acid sequence of SEQ ID NO: 179

[0690] LC-CDR2 having the amino acid sequence of SEQ ID NO: 180

[0691] LC-CDR3 having the amino acid sequence of SEQ ID NO: 173,

[0692] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0693] (92) A VL region comprising the following CDRs:

[0694] LC-CDR1 having the amino acid sequence of SEQ ID NO:73

[0695] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0696] LC-CDR3 having the amino acid sequence of SEQ ID NO:194,

[0697] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0698] (93) A VL region comprising the following CDRs:

[0699] LC-CDR1 having the amino acid sequence of SEQ ID NO:205

[0700] LC-CDR2 having the amino acid sequence of SEQ ID NO:42

[0701] LC-CDR3 having the amino acid sequence of SEQ ID NO:206,

[0702] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0703] (94) A VL region comprising the following CDRs:

[0704] LC-CDR1 having the amino acid sequence of SEQ ID NO:216

[0705] LC-CDR2 having the amino acid sequence of SEQ ID NO:172

[0706] LC-CDR3 having the amino acid sequence of SEQ ID NO:217,

[0707] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0708] (95) The VL region comprising the following CDRs:

[0709] LC-CDR1 having the amino acid sequence of SEQ ID NO: 229

[0710] LC-CDR2 having the amino acid sequence of SEQ ID NO: 172

[0711] LC-CDR3 having the amino acid sequence of SEQ ID NO: 230,

[0712] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0713] (96) The VL region comprising the following CDRs:

[0714] LC-CDR1 having the amino acid sequence of SEQ ID NO: 235

[0715] LC-CDR2 having the amino acid sequence of SEQ ID NO: 236

[0716] LC-CDR3 having the amino acid sequence of SEQ ID NO: 237,

[0717] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-CDR1, and / or 1, 2, or 3 amino acids in LC-CDR2, and / or 1, 2, or 3 amino acids in LC-CDR3 are replaced by another amino acid.

[0718] (97) The VL region comprising the following CDRs:

[0719] LC-CDR1 having the amino acid sequence of SEQ ID NO: 248

[0720] LC-CDR2 having the amino acid sequence of SEQ ID NO: 249

[0721] LC-CDR3 having the amino acid sequence of SEQ ID NO: 250,

[0722] or a variant thereof, wherein one, two, or three amino acids in LC-CDR1, and / or one, two, or three amino acids in LC-CDR2, and / or one, two, or three amino acids in LC-CDR3 are replaced by another amino acid.

[0723] In some embodiments, the antigen-binding molecule comprises a VL region selected from the following (98) to (119):

[0724] (98) A VL region comprising the following FRs:

[0725] LC-FR1 having the amino acid sequence of SEQ ID NO:13

[0726] LC-FR2 having the amino acid sequence of SEQ ID NO:14

[0727] LC-FR3 having the amino acid sequence of SEQ ID NO:15

[0728] LC-FR4 having the amino acid sequence of SEQ ID NO:16,

[0729] or a variant thereof, wherein one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid.

[0730] (99) A VL region comprising the following FRs:

[0731] LC-FR1 having the amino acid sequence of SEQ ID NO:28

[0732] LC-FR2 having the amino acid sequence of SEQ ID NO:29

[0733] LC-FR3 having the amino acid sequence of SEQ ID NO:30

[0734] LC-FR4 having the amino acid sequence of SEQ ID NO:31,

[0735] or a variant thereof, wherein one, two, or three amino acids in LC-FR1, and / or one, two, or three amino acids in LC-FR2, and / or one, two, or three amino acids in LC-FR3, and / or one, two, or three amino acids in LC-FR4 are replaced by another amino acid.

[0736] (100) A VL region comprising the following FRs:

[0737] LC-FR1 having the amino acid sequence of SEQ ID NO:44

[0738] LC-FR2 having the amino acid sequence of SEQ ID NO: 45

[0739] LC-FR3 having the amino acid sequence of SEQ ID NO: 46

[0740] LC-FR4 having the amino acid sequence of SEQ ID NO: 16,

[0741] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0742] (101) The VL region comprising the following FRs:

[0743] LC-FR1 having the amino acid sequence of SEQ ID NO: 56

[0744] LC-FR2 having the amino acid sequence of SEQ ID NO: 57

[0745] LC-FR3 having the amino acid sequence of SEQ ID NO: 58

[0746] LC-FR4 having the amino acid sequence of SEQ ID NO: 59,

[0747] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0748] (102) The VL region comprising the following FRs:

[0749] LC-FR1 having the amino acid sequence of SEQ ID NO: 28

[0750] LC-FR2 having the amino acid sequence of SEQ ID NO: 70

[0751] LC-FR3 having the amino acid sequence of SEQ ID NO: 71

[0752] LC-FR4 having the amino acid sequence of SEQ ID NO: 31,

[0753] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0754] (103) The VL region comprising the following FRs:

[0755] LC-FR1 having the amino acid sequence of SEQ ID NO:28

[0756] LC-FR2 having the amino acid sequence of SEQ ID NO:75

[0757] LC-FR3 having the amino acid sequence of SEQ ID NO:76

[0758] LC-FR4 having the amino acid sequence of SEQ ID NO:31,

[0759] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0760] (104) The VL region comprising the following FRs:

[0761] LC-FR1 having the amino acid sequence of SEQ ID NO:28

[0762] LC-FR2 having the amino acid sequence of SEQ ID NO:81

[0763] LC-FR3 having the amino acid sequence of SEQ ID NO:76

[0764] LC-FR4 having the amino acid sequence of SEQ ID NO:31,

[0765] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0766] (105) The VL region comprising the following FRs:

[0767] LC-FR1 having the amino acid sequence of SEQ ID NO:91

[0768] LC-FR2 having the amino acid sequence of SEQ ID NO:92

[0769] LC-FR3 having the amino acid sequence of SEQ ID NO:93

[0770] LC-FR4 having the amino acid sequence of SEQ ID NO:16,

[0771] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0772] (106) The VL region comprising the following FRs:

[0773] LC-FR1 having the amino acid sequence of SEQ ID NO:104

[0774] LC-FR2 having the amino acid sequence of SEQ ID NO:105

[0775] LC-FR3 having the amino acid sequence of SEQ ID NO:106

[0776] LC-FR4 having the amino acid sequence of SEQ ID NO:31,

[0777] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0778] (107) The VL region comprising the following FRs:

[0779] LC-FR1 having the amino acid sequence of SEQ ID NO:118

[0780] LC-FR2 having the amino acid sequence of SEQ ID NO:119

[0781] LC-FR3 having the amino acid sequence of SEQ ID NO:120

[0782] LC-FR4 having the amino acid sequence of SEQ ID NO:31,

[0783] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0784] (108) The VL region comprising the following FRs:

[0785] LC-FR1 having the amino acid sequence of SEQ ID NO: 128

[0786] LC-FR2 having the amino acid sequence of SEQ ID NO: 129

[0787] LC-FR3 having the amino acid sequence of SEQ ID NO: 130

[0788] LC-FR4 having the amino acid sequence of SEQ ID NO: 16,

[0789] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0790] (109) The VL region comprising the following FRs:

[0791] LC-FR1 having the amino acid sequence of SEQ ID NO: 141

[0792] LC-FR2 having the amino acid sequence of SEQ ID NO: 142

[0793] LC-FR3 having the amino acid sequence of SEQ ID NO: 143

[0794] LC-FR4 having the amino acid sequence of SEQ ID NO: 144,

[0795] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0796] (110) The VL region comprising the following FRs:

[0797] LC-FR1 having the amino acid sequence of SEQ ID NO: 44

[0798] LC-FR2 having the amino acid sequence of SEQ ID NO: 45

[0799] LC-FR3 having the amino acid sequence of SEQ ID NO: 46

[0800] LC-FR4 having the amino acid sequence of SEQ ID NO: 154,

[0801] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0802] (111) The VL region comprising the following FRs:

[0803] LC-FR1 having the amino acid sequence of SEQ ID NO: 161

[0804] LC-FR2 having the amino acid sequence of SEQ ID NO: 162

[0805] LC-FR3 having the amino acid sequence of SEQ ID NO: 163

[0806] LC-FR4 having the amino acid sequence of SEQ ID NO: 164,

[0807] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0808] (112) The VL region comprising the following FRs:

[0809] LC-FR1 having the amino acid sequence of SEQ ID NO: 174

[0810] LC-FR2 having the amino acid sequence of SEQ ID NO: 175

[0811] LC-FR3 having the amino acid sequence of SEQ ID NO: 176

[0812] LC-FR4 having the amino acid sequence of SEQ ID NO: 177,

[0813] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0814] (113) The VL region comprising the following FRs:

[0815] LC-FR1 having the amino acid sequence of SEQ ID NO: 181

[0816] LC-FR2 having the amino acid sequence of SEQ ID NO: 182

[0817] LC-FR3 having the amino acid sequence of SEQ ID NO: 183

[0818] LC-FR4 having the amino acid sequence of SEQ ID NO: 177,

[0819] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0820] (114) The VL region comprising the following FRs:

[0821] LC-FR1 having the amino acid sequence of SEQ ID NO: 28

[0822] LC-FR2 having the amino acid sequence of SEQ ID NO: 75

[0823] LC-FR3 having the amino acid sequence of SEQ ID NO: 197

[0824] LC-FR4 having the amino acid sequence of SEQ ID NO: 177,

[0825] or a variant thereof, wherein 1, 2, or 3 amino acids in LC-FR1, and / or 1, 2, or 3 amino acids in LC-FR2, and / or 1, 2, or 3 amino acids in LC-FR3, and / or 1, 2, or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0826] (115) The VL region comprising the following FRs:

[0827] LC-FR1 having the amino acid sequence of SEQ ID NO: 44

[0828] LC-FR2 having the amino acid sequence of SEQ ID NO: 207

[0829] LC-FR3 having the amino acid sequence of SEQ ID NO: 208

[0830] LC-FR4 having the amino acid sequence of SEQ ID NO: 209,

[0831] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0832] (116) The VL region comprising the following FRs:

[0833] LC-FR1 having the amino acid sequence of SEQ ID NO: 218

[0834] LC-FR2 having the amino acid sequence of SEQ ID NO: 219

[0835] LC-FR3 having the amino acid sequence of SEQ ID NO: 220

[0836] LC-FR4 having the amino acid sequence of SEQ ID NO: 31,

[0837] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0838] (117) The VL region comprising the following FRs:

[0839] LC-FR1 having the amino acid sequence of SEQ ID NO: 231

[0840] LC-FR2 having the amino acid sequence of SEQ ID NO: 232

[0841] LC-FR3 having the amino acid sequence of SEQ ID NO: 233

[0842] LC-FR4 having the amino acid sequence of SEQ ID NO: 31,

[0843] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0844] (118) The VL region comprising the following FRs:

[0845] LC-FR1 having the amino acid sequence of SEQ ID NO:238

[0846] LC-FR2 having the amino acid sequence of SEQ ID NO:239

[0847] LC-FR3 having the amino acid sequence of SEQ ID NO:240

[0848] LC-FR4 having the amino acid sequence of SEQ ID NO:241,

[0849] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0850] (119) The VL region comprising the following FRs:

[0851] LC-FR1 having the amino acid sequence of SEQ ID NO:231

[0852] LC-FR2 having the amino acid sequence of SEQ ID NO:251

[0853] LC-FR3 having the amino acid sequence of SEQ ID NO:252

[0854] LC-FR4 having the amino acid sequence of SEQ ID NO:253,

[0855] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or 1 or 2 or 3 amino acids in LC-FR2, and / or 1 or 2 or 3 amino acids in LC-FR3, and / or 1 or 2 or 3 amino acids in LC-FR4 are replaced by another amino acid.

[0856] In some embodiments, the antigen-binding molecule comprises a VL region that comprises the CDRs of any one of (76) to (97) above and the FRs of any one of (98) to (119) above.

[0857] In some embodiments, the antigen-binding molecule comprises a VL region selected from one of the following (120) to (142):

[0858] (120) A VL region comprising the CDRs of (76) and the FRs of (98).

[0859] (121) A VL region comprising the CDRs of (77) and the FRs of (99).

[0860] (123) A VL region comprising the CDRs of (78) and the FRs of (100).

[0861] (124) A VL region comprising the CDRs of (79) and the FRs of (101).

[0862] (125) A VL region comprising the CDRs of (80) and the FRs of (102).

[0863] (126) A VL region comprising the CDRs of (81) and the FRs of (103).

[0864] (127) A VL region comprising the CDRs of (82) and the FRs of (104).

[0865] (128) A VL region comprising the CDRs of (83) and the FRs of (105).

[0866] (129) A VL region comprising the CDRs of (84) and the FRs of (106).

[0867] (130) A VL region comprising the CDRs of (85) and the FRs of (107).

[0868] (131) A VL region comprising the CDRs of (86) and the FRs of (108).

[0869] (132) A VL region comprising the CDRs of (87) and the FRs of (109).

[0870] (133) A VL region comprising the CDRs of (88) and the FRs of (110).

[0871] (134) A VL region comprising the CDRs of (89) and the FRs of (111).

[0872] (135) A VL region comprising the CDRs of (90) and the FRs of (112).

[0873] (136) A VL region comprising the CDRs of (91) and the FRs of (113).

[0874] The VL region comprising the CDRs of (92) and the FRs of (114).

[0875] (138) The VL region comprising the CDRs of (93) and the FRs of (115).

[0876] (139) The VL region comprising the CDRs of (94) and the FRs of (116).

[0877] (140) The VL region comprising the CDRs of (95) and the FRs of (117).

[0878] (141) The VL region comprising the CDRs of (96) and the FRs of (118).

[0879] (142) The VL region comprising the CDRs of (97) and the FRs of (119).

[0880] In some embodiments, the antigen-binding molecule comprises a VL region of one of the following (143) to (164):

[0881] (143) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:9, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0882] (144) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:24, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0883] (145) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:40, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0884] (146) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:52, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0885] (147) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:67, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0886] (148) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:72, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0887] (149) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:77, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0888] (150) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:88, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0889] (151) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:100, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0890] (152) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 114, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0891] (153) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 124, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0892] (154) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 138, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0893] (155) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 152, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0894] (156) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 157, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0895] (157) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 170, more preferably, an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0896] (158) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 178, more preferably an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0897] (159) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 191, more preferably an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0898] (160) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 204, more preferably an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0899] (161) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 215, more preferably an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0900] (162) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 228, more preferably an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0901] (163) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 234, more preferably having an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0902] (164) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 247, more preferably having an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0903] In some embodiments, the antigen-binding molecule comprises a VH region of any one of (1) to (75) above and a VL region of any one of (76) to (164) above.

[0904] In an embodiment of the present disclosure, one or more amino acids are replaced by another amino acid. One replacement includes replacing an amino acid residue with a non-identical "substitute" amino acid residue. The substitute amino acid residues of the present disclosure can be naturally occurring amino acid residues (i.e., encoded by the genetic code), which are different from the amino acid residues at the corresponding positions of the equivalent, unsubstituted amino acid sequence. The naturally occurring amino acid residues are selected from: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). In some embodiments, the replacing amino acid can be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than the amino acid residues described in the previous sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib and other amino acid residue analogs, such as those described by Ellman et al. in Meth. Enzym. 202 (1991) 301-336.

[0905] In some embodiments, the substitution may be biochemically conservative. In some embodiments, when an alternative amino acid of one of rows 1 to 5 of the following table is provided, the substituted amino acid is another non-identical amino acid provided in the same row:

[0906] Row Shared property Amino acid 1 Hydrophobicity Met, Ala, Val, Leu, Ile, Trp, Tyr, Phe, Norleucine 2 Neutral hydrophilicity Cys, Ser, Thr, Asn, Gln 3 Acidic or negatively charged Asp, Glu 4 Basic or positively charged His, Lys, Arg 5 Orientation effect Gly, Pro

[0907] For example, in some embodiments, if the residue being replaced is a Met residue, the replacement amino acid can be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and norleucine.

[0908] In some embodiments, the replacement amino acid can have the same side-chain polarity as the amino acid residue being replaced. In some embodiments, the replacement amino acid can have the same side-chain charge (at pH 7.4) as the amino acid residue being replaced:

[0909]

[0910]

[0911] That is, in some embodiments, a nonpolar amino acid is replaced by another, different nonpolar amino acid. In some embodiments, a polar amino acid is replaced by another, different polar amino acid. In some embodiments, an acidic polar amino acid is replaced by another, different acidic polar amino acid. In some embodiments, a basic polar amino acid is replaced by another, different basic polar amino acid. In some embodiments, a neutral amino acid is replaced by another, different neutral amino acid. In some embodiments, a positively charged amino acid is replaced by another, different positively charged amino acid. In some embodiments, a negatively charged amino acid is replaced by another, different negatively charged amino acid.

[0912] In some embodiments, the substitute may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may substantially not affect) one or more functional properties (such as target binding) of the antigen-binding molecule containing the substitution as compared to an equivalent unsubstituted molecule.

[0913] The VH and VL regions of the antibody antigen-binding region together form the Fv region. In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an Fv region that binds to CNX. In some embodiments, the VH and VL regions of the Fv are provided as a single polypeptide linked by a linker region, i.e., a single-chain Fv (scFv).

[0914] The VL and light chain constant (CL) regions, as well as the VH and heavy chain constant 1 (CH1) regions of the antigen-binding region of an antibody together constitute the Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region that comprises VH, CH1, VL, and CL (such as Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (such as a VH-CH1 fusion polypeptide), and a polypeptide comprising VL and CL (such as a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising VH and CL (such as a VH-CL fusion polypeptide) and a polypeptide comprising VL and CH1 (such as 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 the Fab or CrossFab are provided as a single polypeptide linked by a linker region, i.e., as a single-chain Fab (scFab) or single-chain CrossFab (scCrossFab).

[0915] In some embodiments, the antigen-binding molecules described herein comprise or consist of a full antibody that binds to CNX. As used herein, "full antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different classes of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41-S52, the entire content of which is incorporated herein by reference.

[0916] Immunoglobulin G (i.e., IgG) is a ~150 kDa glycoprotein composed of two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain comprises VH, followed by a heavy chain constant region comprising three constant domains (CH1, CH2, and CH3). Similarly, the light chain comprises VL, followed by CL. Immunoglobulins can be classified as IgG (such as IgG1, IgG2, IgG3, IgG4), IgA (such as IgA1, IgA2), IgD, IgE, or IgM according to the heavy chain. The light chain can be kappa (κ) or lambda (λ).

[0917] In some embodiments, the antigen-binding molecules described herein comprise or consist of IgG (such as IgG1, IgG2, IgG3, IgG4), IgA (such as IgA1, IgA2), IgD, IgE, or IgM that binds to CNX.

[0918] In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more regions of an immunoglobulin heavy chain constant sequence (e.g., CH1, CH2, CH3, etc.). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE or IgM, such as human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE or hIgM. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3 or G1m17).

[0919] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 254, 259, 260 or 263, more preferably having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0920] In some embodiments, the antigen-binding molecule comprises a CH1 region that comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 255 or 261, 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. In some embodiments, the antigen-binding molecule comprises a CH2 region that comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 257, 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. In some embodiments, the antigen-binding molecule comprises a CH3 region that comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 258 or 262, 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.

[0921] In some embodiments, the antigen-binding molecule comprises a hinge region that comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 256, 264, 265, or 266, 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.

[0922] It should be understood that further substitutions can be provided in the CH2 and / or CH3 regions according to the modifications of the Fc region of the antigen-binding molecule described herein.

[0923] In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin kappa constant (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0924] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 267, 268, 269, 270, 271, or 272, 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.

[0925] In some embodiments, the antigen-binding molecule is or comprises a monoclonal antibody or an antigen-binding fragment thereof.

[0926] In some embodiments, the antigen-binding molecule is or comprises a fully human antibody / antibody fragment. The fully human antibody / antibody fragment can be encoded by a human nucleic acid sequence. The fully human antibody / antibody fragment may not contain non-human amino acid sequences. Commonly used techniques for producing fully human antibodies include (i) phage display, in which human antibody genes are expressed in a phage display library, and (ii) production of antibodies in transgenic mice that have been engineered to have human antibody genes (described in Park and Smolen, Advances in Protein Chemistry (2001) 56:369-421). Briefly, in the human antibody gene phage display technique, genes encoding the VH and VL chains are generated from "naive" human lymphocytes by PCR amplification and cloning, and assembled into a library from which they can be expressed as disulfide-linked Fab fragments or single-chain Fv (scFv) fragments. The genes encoding Fab or scFv are fused to the surface coat protein of a filamentous phage, and then the library can be screened with an antigen to identify Fab or scFv that can bind to the target. Molecular evolution or affinity maturation procedures can be used to enhance the affinity of the Fab / scFv fragments. In the transgenic mouse technique, mice are immunized with an antigen, the endogenous mouse Ig gene loci of which have been replaced by homologous recombination with their human counterparts, and monoclonal antibodies are prepared by conventional hybridoma techniques to produce fully human monoclonal antibodies.

[0927] In some embodiments, the antigen-binding molecule of the present disclosure is a murine antibody / antibody fragment. In some embodiments, the antibody / antibody fragment is obtained from phage display using a human naive antibody gene library.

[0928] In some embodiments, the antigen-binding molecule is a murine / human chimeric antibody / antibody fragment (i.e., an antigen-binding molecule comprising a murine antibody variable domain and a human antibody constant region). In some embodiments, the antigen-binding molecule is a humanized antibody / antibody fragment. In some embodiments, the antigen-binding molecule comprises murine antibody CDRs and human antibody framework regions and constant regions.

[0929] Murine / human chimeric antigen-binding molecules can be prepared from murine antibodies by a chimerization process, for example, as described in Chapter 8, particularly Section 3 of Chapter 8, of Human Monoclonal Antibodies: Methods and Protocols, Methods in Molecular Biology 1060, SpringerProtocols, Humana Press (2014), edited by Michael Steinitz.

[0930] Humanized antigen-binding molecules can be prepared from murine antibodies by a humanization process, for example, as described in Chapter 7, specifically Section 3.1 entitled "Antibody Humanization" of Chapter 7 in Michael Steinitz (ed.), Human Monoclonal Antibodies: Methods and Protocols, Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014). Antibody humanization techniques are also described in Biotechnology and Genetic Engineering Reviews (2013) 29:175-86 by Safdari et al.

[0931] The present disclosure relates to multispecific antigen-binding molecules. "Multispecific" means that the antigen-binding molecule exhibits specific binding to multiple 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 regions, such as comprising non-identical VHs and VLs).

[0932] In some embodiments, the antigen-binding molecule binds to CNX and another target (e.g., an antigen other than CNX), and is thus at least bispecific. The term "bispecific" means that the antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants.

[0933] It should be understood that the antigen-binding molecules of the present disclosure (e.g., multispecific antigen-binding molecules) can include antigen-binding proteins capable of binding to the specific targets of the antigen-binding molecule. For example, an antigen-binding molecule that binds to CNX and an antigen other than CNX can include: (i) an antigen-binding molecule that binds to CNX, and (ii) an antigen-binding molecule that binds to an antigen other than CNX.

[0934] It should also be understood that the antigen-binding molecules of the present disclosure (e.g., multispecific antigen-binding molecules) can include antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to the specific targets of the antigen-binding molecule.

[0935] In some embodiments, the component antigen-binding molecules of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) can be referred to as, for example, "antigen-binding domains" or "antigen-binding regions" of the larger antigen-binding molecule.

[0936] In some embodiments, the antigen other than CNX in the multispecific antigen-binding molecule is an immune cell surface molecule. In some embodiments, the antigen is a cancer cell antigen. In some embodiments, the antigen is a receptor molecule, such as a cell surface receptor. In some embodiments, the antigen is a cell signaling molecule, such as a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen is a growth factor or hormone.

[0937] A cancer cell antigen is an antigen expressed or overexpressed by cancer cells. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The expression of a cancer cell antigen may be associated with cancer. A cancer cell antigen can be abnormally expressed by cancer cells (e.g., a cancer cell antigen can be expressed with abnormal localization), or can be expressed by cancer cells with an abnormal structure. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the antigenic moiety bound by the antigen-binding molecules described herein is displayed on the outer surface (i.e., extracellular) of cancer cells. A cancer cell antigen may be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the development, progression, or severity of cancer symptoms. A cancer-associated antigen may be related to the etiology or pathology of cancer, or may be abnormally expressed due to cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells, e.g., compared to the expression level of similar non-cancer cells (e.g., non-cancer cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be preferentially expressed by cancer cells and not by similar non-cancer cells (e.g., non-cancer cell nuclei derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen can be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, a cancer-associated antigen can be an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, a carcinoembryonic antigen, or the product of a cell surface glycolipid or glycoprotein.

[0938] An immune cell surface molecule can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof that is expressed on or at the cell surface of an immune cell. In some embodiments, a portion of the immune cell surface molecule that is bound by an antigen-binding molecule of the present disclosure is located on the outer surface (i.e., extracellular) of the immune cell. An immune cell surface molecule can be expressed on the cell surface of any immune cell. In some embodiments, the immune cell can be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte can be, for example, a T cell, B cell, natural killer (NK) cell, NKT cell, or innate lymphoid cell (ILC), or a precursor thereof (e.g., a thymocyte or pre-B cell).

[0939] In some embodiments, the antigen-binding molecule is an immune cell engager. Immune cell engagers are reviewed, for example, in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17:418–434 and Ellerman, Methods (2019) 154:102-117, the entire contents of which are incorporated herein by reference. An immune cell engager molecule includes an antigen-binding region for a target antigen and an antigen-binding region for recruiting / engaging a target immune cell. An immune cell engager recruits / engages an immune cell through an antigen-binding region specific for an immune cell surface molecule.

[0940] The best-studied immune cell engagers are bispecific T cell engagers (BiTEs), which contain a target antigen-binding domain and a CD3 polypeptide (usually CD3ε) binding domain through which the BiTE recruits T cells. Binding of the BiTE to its target antigen and the CD3 polypeptide expressed on T cells results in activation of the T cells and ultimately directs the effector activity of the T cells against cells expressing the target antigen. Other types of immune cell engagers are well known in the art, including natural killer cell engagers, such as bispecific killer cell engagers (BiKEs), which recruit and activate NK cells.

[0941] In some embodiments, the multispecific antigen-binding molecule described herein exhibits at least monovalent binding to CNX and also exhibits monovalent binding to a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3ε). In some embodiments, the antigen-binding molecule comprises one CNX-binding site and one CD3 polypeptide-binding site.

[0942] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule that binds a CD3 polypeptide (such as CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3κ). In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds a CD3 polypeptide (such as CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3κ). In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of an antigen-binding molecule that binds a CD3 polypeptide (such as CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3κ). That is, in some embodiments, the antigen-binding molecule comprises the VH and VL regions of an antigen-binding molecule that binds to a CD3 polypeptide (such as CD3ε, CD3δ, CD3γ, or CD3ζ; preferably CD3ε, CD3δ, or CD3γ; or more preferably CD3κ).

[0943] In some embodiments, the antigen-binding molecule comprises the CDRs, FRs, and / or VH and / or VL regions of a CD3 polypeptide-binding antibody clone, or CDRs, FRs, and / or VH and / or VL regions derived from a CD3 polypeptide-binding antibody clone.

[0944] In some embodiments, the CD3 polypeptide-binding antibody clones are selected from: OKT3 (described in Kjer-Nielsen et al., PNAS (2004) 101(20):7675-80), SP34 (such as described in WO 2014 / 122143 A1), UCHT1 (such as described in WO2000 / 041474A1), HIT3a (Invitrogen Cat#16-0039-85), and clone SK7 (Invitrogen Cat#106-0036-81).

[0945] In some embodiments, the immune cell conjugated by the immune cell conjugate is a T cell or an NK cell. In some embodiments, the immune cell conjugate is a T cell conjugate.

[0946] The multispecific antigen-binding molecules of the present disclosure can be provided in any suitable form, such as those described in Brinkmann and Kontermann, MAbs (2017) 9(2):182-212, the entire content of which is incorporated herein by reference. Suitable formats include those shown in Brinkmann and Kontermann, MAbs (2017) 9(2):182-212: antibody conjugates, such as IgG Figure 2 as shown in: antibody conjugates, such as IgG 2 、F(ab')2 or CovX-Body; IgG or IgG-like molecules such as IgG, chimeric IgG, common HC of kappa-lambda body; CH1 / CL fusion proteins such as scFv2-CH1 / CL, VHH2-CH1 / CL; "variable domain only" bispecific antigen-binding molecules such as tandem scFv (taFV), trisomes, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triheads, tandem dAb / VHH, trivalent dAb.VHH; non-Ig fusion proteins such as scFv 2 albumin, scDb albumin, taFv-albumin, taFv-toxin, nanobody, DNL-Fab 2 , DNL-Fab 2 -scFv, DNL-Fab 2 -IgG-cytokine 2 , ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins such as scFv-Fc(kih), scFv-Fc(CH3 charge pair), scFv Fc(EW-RVT), scFv-Fc(HA-TF), scFv-Fc(SEEDbody), taFv Fc(kih), scFv Fc(kih)-Fv, Fab Fc(kih)-scFv, Fab scFv Fc, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc(SEEDbody), DART-Fc, scFv-CH3(kih), TriFabs; Fc fusion proteins such as diabodies, scDb-Fc, taFv-Fc, scFv-Fc scFv, HCAb VHH, Fab scFv-Fc, scFv 4 -Ig, scFv 2 Fcab; CH3 fusion proteins such as diabodies, scDb-CH3; IgE / IgM CH2 fusion proteins such as scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins such as Fab scFv (diabody), Fab-scFv 2 (trisome), Fab Fv, Fab dsFv, Fab VHH, orthogonal Fab-Fab; non-Ig fusion proteins such as DNL-Fab 3 , DNL-Fab 2 -scFv, DNL-Fab 2-IgG-cytokine 2; asymmetric IgG or IgG-like molecules such as IgG(kih), IgG(kich) common LC, ZW1 IgG common LC, bispecific common LC, CrossMab, CrossMab(kih), scFab IgG(kih), Fab-scFab IgG(kih), orthogonal Fab-IgG(kih), DuetMab, CH3 charge pair + CH1 / CL charge pair, hinge / CH3 charge pair, SEEDbody, Duobody, quadravalent CrossMab(kih); LUZ-Y common LC; LUZ-Y scFab IgG, FcFc*; additional and Fc-modified IgG such as IgG(kih)-Fv, IgG-HA-TF-Fv, IgG(kich)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, semi-DVD-Ig, DVI-Ig (quadravalent), CrossMab-Fab; modified Fc and CH3 fusion proteins such as Fab Fc(kih)-scFv, Fab scFv-Fc(kih), FabscFv Fc(BEAT), Fab-scFv-Fc SEEDbody, TriFab; additional IgG-HC fusions such as IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CαCβ)Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab), Fab-IgG(CαCβFab), Fab-IgG(CR3), Fab-hinge-IgG(CR3); additional IgG-LC fusions such as IgG-scFv(LC), scFv(LC-IgG), dAb-IgG; additional IgG-HC and LC fusions such as DVD-Ig, TVD-Ig, CODV-Ig, scFv 4 -IgG, Zybody; Fc fusion proteins such as Fab-scFv-Fc, scFv 4 -Ig; F(ab’)2 fusion proteins such as F(ab‘) 2 -scFv 2 ; CH1 / CL fusion proteins such as scFv 2 -CH1-hinge / CL; modified IgGs such as DAF (bivalent IgG), DutaMab, Mab 2 ; and non-Ig fusions such as DNL-Fab 4 -IgG.

[0947] Those skilled in the art are able to design and prepare bispecific antigen-binding molecules. Methods for producing multispecific antigen-binding molecules include chemically crosslinking antigen-binding molecules or antibody fragments, such as with reducible disulfide or non-reducible thioether bonds, as described, for example, in "Segal and Bast, 2001, Production of Bispecific Antigen-Binding Molecules, Current Protocols in Immunology, 14:IV:2.13:2.13.1-2.13.16", the entire content of which is incorporated herein by reference. For example, N-succinimidyl 3-(-2-pyridyldithio)propionate (SPDP) can be used, for example, to chemically crosslink Fab fragments via hinge region SH-groups to produce disulfide-linked bispecific F(ab) 2 heterodimers.

[0948] Other methods for producing multispecific antigen-binding molecules include fusing antibody-producing hybridomas with polyethylene glycol to produce tetraomas capable of secreting bispecific antibodies, as described, for example, in "D.M. and Bast, B.J. 2001, Production of Bispecific Antigen-Binding Molecules, Current Protocols in Immunology, 14:IV:2.13:2.13.1-2.13.16".

[0949] The multispecific antigen-binding molecules of the present disclosure can also be recombinantly produced, for example, by expression of a nucleic acid construct encoding an antigen-binding molecule polypeptide, as described, for example, in "Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), Chapter 40: Production of Bispecific Antigen-Binding Molecules: Bispecific Antibodies and Tandem scFv (Hornig and -Schwarz)", or "French, How to Prepare Bispecific Antibody-Binding Molecules, Methods Mol. Med. 2000, 40:333-339", the entire content of both of which is incorporated herein by reference.

[0950] For example, a DNA construct encoding the light and heavy chain variable domains of two antigen-binding fragments (i.e., an antigen-binding fragment capable of binding CNX, and the light or heavy chain variable domain of an antigen-binding fragment capable of binding another target protein) can be prepared by molecular cloning techniques and include sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments. Thereafter, a recombinant bispecific antibody can be produced by expressing (e.g., in vitro) the construct in a suitable host cell (such as a mammalian host cell), and the expressed recombinant bispecific antibody can then be optionally purified.

[0951] Fc region

[0952] In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region.

[0953] The Fc region is composed of the CH2 and CH3 regions of one polypeptide and the CH2 and CH3 regions of another polypeptide. The CH2 and CH3 regions of the two polypeptides together form the Fc region.

[0954] Fc-mediated functions include Fc receptor binding, antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, production of cytokines and / or chemokines, and antigen processing and presentation. Modifications of the antibody Fc region that affect Fc-mediated functions are known in the art, such as those described by Wang et al. in Protein Cell (2018) 9(1):63-73, the entire content of which is incorporated herein by reference. Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73 summarizes exemplary Fc region modifications known to affect antibody effector functions. In some embodiments, the antigen-binding proteins of the present disclosure comprise an Fc region that contains modifications to increase or decrease Fc-mediated functions as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

[0955] When the Fc region / CH2 / CH3 is described as containing a modification “corresponding to” a reference substitution, equivalent substitutions in homologous Fc / CH2 / CH3 may be considered. For example, the L234A / L235A substitution in human IgG1 (numbered according to the EU numbering system described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991) corresponds to the L to A substitution at positions 117 and 118 in the C region of the mouse Igγ-2A chain (UniProtKB:P01863-1, v1).

[0956] When the Fc region is described as containing a modification, the modification may be present in one or both of the polypeptide chains that together form the Fc region.

[0957] In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region that contains a modification. In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region that contains a modification, and the Fc region contains a modification in one or more of the CH2 and / or CH3 regions.

[0958] In some embodiments, the Fc region comprises modifications to increase Fc-mediated functions. In some embodiments, the Fc region comprises modifications to increase ADCC. In some embodiments, the Fc region comprises modifications to increase ADCP. In some embodiments, the Fc region comprises modifications to increase CDC. Compared to an antigen-binding molecule comprising a corresponding unmodified Fc region, the Fc region of the antigen-binding molecule comprises modifications to increase Fc-mediated functions (such as ADCC, ADCP, CDC), thereby inducing an increase in the level of associated effector functions.

[0959] In some embodiments, the Fc region comprises modifications to increase binding to Fc receptors. In some embodiments, the Fc region comprises modifications to increase binding to Fcγ receptors. In some embodiments, the Fc region comprises modifications to increase binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises modifications to increase binding to FcγRIIIa. In some embodiments, the Fc region comprises modifications to increase binding to FcγRIIa. In some embodiments, the Fc region comprises modifications to increase binding to FcγRIIb. In some embodiments, the Fc region comprises modifications to increase binding to FcRn. In some embodiments, the Fc region comprises modifications to increase binding to complement proteins. In some embodiments, the Fc region comprises modifications to increase binding to C1q. In some embodiments, the Fc region comprises modifications to promote hexamerization of the antigen-binding molecule. In some embodiments, the Fc region comprises modifications to increase the half-life of the antigen-binding molecule. In some embodiments, the Fc region comprises modifications to increase co-engagement.

[0960] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of F243L / R292P / Y300L / V305I / P396L as described in Stavenhagen et al., Cancer Res. (2007) 67:8822–8890. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of S239D / I332E or S239D / I332E / A330L as described in Lazar et al., Proc Natl Acad Sci USA (2006) 103:4005–4010. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of S298A / E333A / K334A as described in Shields et al., J Biol Chem. (2001) 276:6591–6604. In some embodiments, the Fc region comprises a modification to one of the heavy chain polypeptides corresponding to the substitution combination of L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and a modification to another heavy chain polypeptide corresponding to the substitution combination of D270E / K326D / A330M / K334E, as described in Mimot et al., MAbs. (2013):5:229–236. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of G236A / S239D / I332E as described in Richards et al., Mol Cancer Ther. (2008) 7:2517–2527.

[0961] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of K326W / E333S as described in Idusogie et al., J Immunol. (2001) 166(4):2571-5. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of S267E / H268F / S324T as described in Moore et al., Mabs (2010) 2(2):181-9. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination as described in Natsume et al., Cancer Res. (2008) 68(10):3863-72. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of E345R / E430G / S440Y as described in Dieboler et al., Science (2014) 343(6176):1260-3.

[0962] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of M252Y / S254T / T256E as described in Dall'Acqua et al., J Immunol. (2002) 169:5171–5180. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of M428L / N434S as described in Zalevsky et al., Nat Biotechnol. (2010) 28:157–159.

[0963] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of S267E / L328F as described in Chu et al., Mol Immunol (2008) 45:3926–3933. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination of N325S / L328F as described in Shang et al., Biol Chem. (2014) 289:15309–15318.

[0964] In some embodiments, the Fc region comprises modifications to reduce / prevent Fc-mediated functions. In some embodiments, the Fc region comprises modifications to reduce / prevent ADCC. In some embodiments, the Fc region comprises modifications to reduce / prevent ADCP. In some embodiments, the Fc region comprises modifications to reduce / prevent CDC. Compared to an antigen-binding molecule comprising the corresponding unmodified Fc region, the Fc region of the antigen-binding molecule comprises modifications to reduce / prevent Fc-mediated functions (e.g., ADCC, ADCP, CDC), thereby inducing a reduced level of the relevant effector functions.

[0965] In some embodiments, the Fc region comprises modifications to reduce / prevent binding to Fc receptors. In some embodiments, the Fc region comprises modifications to reduce / prevent binding to Fcγ receptors. In some embodiments, the Fc region comprises modifications to reduce / prevent binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises modifications to reduce / prevent binding to FcγRIIIa. In some embodiments, the Fc region comprises modifications to reduce / prevent binding to FcγRIIa. In some embodiments, the Fc region comprises modifications to reduce / prevent binding to FcγRIIb. In some embodiments, the Fc region comprises modifications to reduce / prevent binding to complement proteins. In some embodiments, the Fc region comprises modifications to reduce / prevent binding to C1q. In some embodiments, the Fc region comprises modifications to reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0966] In some embodiments, the Fc region is unable to induce one or more Fc-mediated functions (i.e., lacks the ability to trigger the relevant Fc-mediated functions). Accordingly, an antigen-binding molecule comprising such an Fc region also lacks the ability to induce the relevant functions. Such an antigen-binding molecule can be described as lacking the relevant functions.

[0967] In some embodiments, the Fc region is unable to induce ADCC. In some embodiments, the Fc region is unable to induce ADCP. In some embodiments, the Fc region is unable to induce CDC. In some embodiments, the Fc region is unable to induce ADCC and / or is unable to induce ADCP and / or is unable to induce CDC.

[0968] In some embodiments, the Fc region is unable to bind to an Fc receptor. In some embodiments, the Fc region is unable to bind to an Fcγ receptor. In some embodiments, the Fc region is unable to bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region is unable to bind to FcγRIIIa. In some embodiments, the Fc region is unable to bind to FcγRIIa. In some embodiments, the Fc region is unable to bind to FcγRIIb. In some embodiments, the Fc region is unable to bind to FcRn. In some embodiments, the Fc region is unable to bind to a complement protein. In some embodiments, the Fc region is unable to bind to C1q. In some embodiments, the Fc region is not glycosylated at the amino acid residue corresponding to N297.

[0969] In some embodiments, the Fc region comprises a modification corresponding to N297A or N297Q or N297G as described in Leabman et al., MAbs (2013) 5:896–903. In some embodiments, the Fc region comprises a modification corresponding to L235E as described in Alegre et al., Journal of Immunology (1992) 148:3461–3468. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination of L234A / L235A or F234A / L235A as described in Xu et al., Cellular Immunology (2000) 200:16–26. In some embodiments, the Fc region comprises a modification corresponding to P329A or P329G as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination of L234A / L235A / P329G as described in Lo et al., J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination as described in Rother et al., Nat Biotechnol (2007) 25:1256–1264. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination of S228P / L235E as described in Newman et al., Clin. Immunol. (2001) 98:164–174. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination of H268Q / V309L / A330S / P331S as described in An et al., MAbs. (2009) 1:572–579. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination of V234A / G237A / P238S / H268A / V309L / A330S / P331S as described in Vafa et al., Methods (2014) 65:114–126. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination of L234A / L235E / G237A / A330S / P331S as described in US2015 / 0044231 A1.

[0970] The known substitution combination 'L234A / L235A' and corresponding substitutions (e.g., F234A / L235A in human IgG4) disrupt the binding of Fc to Fcγ receptors, inhibit ADCC and ADCP, and also reduce C1q binding, thereby reducing CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457 - 466, which is incorporated herein by reference in its entirety). The "P329G" and "P329A" substitutions reduce C1q binding (and thus reduce CDC). Substituting "N297" with "A", "G", or "Q" is known to abolish glycosylation, thereby reducing the binding of Fc to C1q and Fcγ receptors, and thus reducing CDC and ADCC. Lo et al., J. Biol. Chem (2017) 292(9):3900 - 3908 (which is incorporated herein by reference in its entirety) reported that the substitution combination of L234A / L235A / P329G abolished complement binding and fixation and Fcγ receptor - dependent, antibody - dependent, cell - mediated cytotoxicity in murine IgG2a and human IgG1.

[0971] The substitution combination of L234A / L235E / G237A / A330S / P331S in IgG1 Fc was disclosed in US2015 / 0044231 A1 to abolish the induction of phagocytosis, ADCC, and CDC.

[0972] In some embodiments, the Fc region comprises a modification corresponding to the S228P substitution as described in Silva et al., J Biol Chem (2015) 290(9):5462 - 5469. The S228P substitution in IgG4 - Fc reduces Fab - arm exchange (which may be undesirable).

[0973] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination of L234A / L235A. In some embodiments, the Fc region comprises a modification corresponding to the P329G substitution. In some embodiments, the Fc region comprises a modification corresponding to the N297Q substitution.

[0974] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination of L234A / L235A / P329G.

[0975] In some embodiments, the Fc comprises a modification corresponding to the substitution combination of L234A / L235A / P329G / N297Q.

[0976] In some embodiments, the Fc comprises a modification corresponding to the substitution combination of L234A / L235E / G237A / A330S / P331S.

[0977] In some embodiments, the Fc region comprises a modification corresponding to the S228P substitution in, for example, IgG4.

[0978] In some embodiments, particularly in embodiments where the antigen-binding molecule is a multispecific (e.g., bispecific) antigen-binding molecule - the antigen-binding molecule comprises an Fc region that comprises one or more modifications of the CH2 and CH3 regions that promote binding of the Fc region. Recombinant co-expression of the component polypeptides of the antigen-binding molecule and subsequent binding results in several possible combinations. To increase the yield of the desired polypeptide combinations in the recombinant production of antigen-binding molecules, it is advantageous to introduce modifications in the Fc region that promote binding of the desired heavy-chain polypeptide combinations. The modifications can promote hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described, for example, in Ha et al., Front. Immunol (2016) 7:394, the entire content of which is incorporated herein by reference.

[0979] In some embodiments, the antigen-antibody binding molecules of the present disclosure comprise an Fc region that comprises paired substitutions in the CH3 region in one of the following formats, as shown in Table 1 of Ha et al., Front. Immunol (2016) 7:394: KiH, KiH s-s , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT s-s , SEED or A107》.

[0980] Polypeptides and specific exemplary antigen-binding molecules

[0981] The present invention also provides polypeptide components of the antigen-binding molecule. The polypeptides can be provided in isolated or substantially purified form.

[0982] The antigen-binding molecules of the present disclosure can be or can comprise polypeptide complexes.

[0983] In the present specification, when a polypeptide comprises more than one domain or region, it should be understood that the multiple domains / regions are preferably present in the same polypeptide chain. That is, a polypeptide comprising more than one domain or region is a fusion polypeptide comprising these domains / regions.

[0984] In some embodiments, the polypeptides of the present disclosure comprise a VH as described herein or consist of a VH. In some embodiments, the polypeptides of the present disclosure comprise a VL as described herein or consist of a VL.

[0985] In some embodiments, the polypeptide further comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide further comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises the CH1, CH2 region, and / or CH3 region of an immunoglobulin (Ig).

[0986] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region 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.

[0987] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the polypeptide comprises a CL region as described herein.

[0988] In some embodiments, the polypeptides of the present disclosure comprise any of the following structures from N-terminus to C-terminus:

[0989] (i) VH

[0990] (ii) VL

[0991] (iii) VH-CH1

[0992] (iv) VL-CL

[0993] (v) VL-CH1

[0994] (vi) VH-CL

[0995] (vii) VH-CH1-CH2-CH3

[0996] (viii) VL-CL-CH2-CH3

[0997] (ix) VL-CH1-CH2-CH3

[0998] (x) VH-CL-CH2-CH3

[0999] The present disclosure also provides an antigen-binding molecule composed of the polypeptide of the present invention. In some embodiments, the antigen-binding molecules of the present disclosure include one of the following polypeptide combinations:

[1000] (A) VH + VL

[1001] (B) VH-CH1 + VL-CL

[1002] (C) VL-CH1 + VH-CL

[1003] (D) VH-CH1-CH2-CH3 + VL-CL

[1004] (E) VH-CL-CH2-CH3 + VL-CH1

[1005] (F) VL-CH1-CH2-CH3 + VH-CL

[1006] (G) VL-CL-CH2-CH3 + VH-CH1

[1007] (H) VH-CH1-CH2-CH3 + VL-CL-CH2-CH3

[1008] (I) VH-CL-CH2-CH3 + VL-CH1-CH2-CH3

[1009] In some embodiments, the antigen-binding molecule comprises more than one of the polypeptide combinations shown in (A) to (I) above. For example, with reference to (D) above, in some embodiments, the antigen-binding molecule comprises two polypeptides comprising the structure VH-CH1-CH2-CH3, and two polypeptides comprising the structure VL-CL.

[1010] In some embodiments, the antigen-binding molecules of the present disclosure include one of the following polypeptide combinations:

[1011] (J) VH(anti-CNX) + VL(anti-CNX)

[1012] (K) VH(anti-CNX)-CH1 + VL(anti-CNX)-CL

[1013] (L) VL(anti-CNX)-CH1 + VH(anti-CNX)-CL

[1014] (M) VH(anti-CNX)-CH1-CH2-CH3 + VL(anti-CNX)-CL

[1015] (N) VH(anti-CNX)-CL-CH2-CH3 + VL(anti-CNX)-CH1

[1016] (O) VL(anti-CNX)-CH1-CH2-CH3 + VH(anti-CNX)-CL

[1017] (P) VL(anti-CNX)-CL-CH2-CH3 + VH(anti-CNX)-CH1

[1018] (Q)VH(anti-CNX)-CH1-CH2-CH3 + VL(anti-CNX)-CL-CH2-CH3

[1019] Wherein: "VH(anti-CNX)" refers to the VH of an antigen-binding molecule capable of binding CNX as described herein, such as defined in any one of (1) to (75); and "VL(anti-CNX)" refers to the VL of an antigen-binding molecule capable of binding CNX as described herein, such as defined in any one of (76) to (164).

[1020] In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide that comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1, 17, 32, 47, 60, 82, 85, 94, 107, 121, 131, 145, 155, 165, 184, 198, 210, 221 or 242.

[1021] In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide that comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 9, 24, 40, 52, 67, 72, 77, 88, 100, 114, 124, 138, 152, 157, 170, 178, 191, 204, 215, 228, 234 or 247.

[1022] In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide that comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 273, 276, 279, 282, 285, 290, 292, 295, 298, 301, 304, 307, 310, 313, 317, 320, 323, 326, 330, 274, 277, 280, 283, 286, 291, 293, 296, 299, 302, 305, 308, 311, 314, 318, 321, 324, 327 or 331.

[1023] In some embodiments, the antigen-binding molecules of the present disclosure comprise a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 275, 278, 281, 284, 287, 288, 289, 294, 297, 300, 303, 306, 309, 312, 315, 316, 319, 322, 325, 328, 329 or 332 or consisting of the same.

[1024] In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more polypeptides comprising a VH region comprising heavy chain CDRs and a VL region comprising light chain CDRs cloned from those selected from 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001, C008, C010, C023, C025, C040, C046 and C117, as shown in Table A herein. That is, in some embodiments, the antigen-binding molecule comprises one or more polypeptides comprising: (i) a VH region comprising HC-CDR1, HC-CDR2 and HC-CDR3 as shown in Column A of Table A, and (ii) a VL region comprising LC-CDR1, LC-CDR2 and LC-CDR3 as shown in Column B of Table A, wherein the sequences of Column A and B are selected from the same row of Table A.

[1025] In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more polypeptides comprising a VH region comprising heavy chain FRs and a VL region comprising light chain FRs cloned from those selected from 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001, C008, C010, C023, C025, C040, C046 and C117, as shown in Table B herein. That is, in some embodiments, the antigen-binding molecule comprises one or more polypeptides comprising: (i) a VH region comprising HC-FR1, HC-FR2, HC-FR3 and HC-FR4 as shown in Column A of Table B, and (ii) a VL region comprising LC-FR1, LC-FR2, LC-FR3 and LC-FR4 as shown in Column B of Table B, wherein the sequences of Column A and B are selected from the same row of Table B.

[1026] In some embodiments, the antigen-binding molecules of the invention comprise one or more polypeptides, comprising: (i) an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequences shown in column A of Table C, and (ii) an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequences shown in column B of Table C, wherein the sequences of column A and column B are from the same row selected from Table C.

[1027] In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more polypeptides, the polypeptides comprising VH and VL regions cloned from those selected from 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001, C008, C010, C023, C025, C040, C046 and C117, as shown in Table C. That is, in some embodiments, the antigen-binding molecule comprises one or more polypeptides, which comprise: (i) the amino acid sequence shown in column A of Table C, and (ii) the amino acid sequence shown in column B of Table C, wherein the sequences of column A and column B are from the same row selected from Table C.

[1028] In some embodiments, the antigen-binding molecules of the present disclosure comprise: (i) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequences shown in column A of Table D, and (ii) a polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85, 90%, 91, 92%, 93, 94%, 95, 96%, 97, 98%, 99 or 100% amino acid sequence identity to the amino acid sequences shown in column B of Table D, wherein the sequences of column A and column B are from the same row selected from Table D.

[1029] In some embodiments, the antigen-binding molecules of the present disclosure comprise polypeptides of the antigen-binding molecules shown in any of Tables D[1] to

[46] herein. That is, in some embodiments, the antigen-binding molecule comprises: (i) a polypeptide comprising or consisting of the amino acid sequence shown in column A of Table D, and (ii) a polypeptide comprising or consisting of the amino acid sequence shown in column B of Table D, wherein the sequences of column A and column B are from the same row selected from Table D.

[1030] Linkers and additional sequences

[1031] In some embodiments, the antigen-binding molecules and polypeptides of the present disclosure include one or more linker sequences between amino acid sequences. Linker sequences 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.

[1032] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences linked by the linker sequence. Flexible linkers are known to those skilled in the art, and several flexible linkers are identified in Chen et al., Adv Drug delivery Rev (2013) 65(10):1357-1369. Flexible linker sequences typically contain a high proportion of glycine and / or serine residues.

[1033] In some embodiments, the linker sequence includes at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of or comprises glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n (SEQ ID NO; 410 and 411) or (GxS)nGm (SEQ ID NO: 412 and 413); where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1, 2 or 3. In some embodiments, the linker sequence includes the sequence motif G 4 S (SEQ ID NO: 414) one or more (e.g., 1, 2, 3, 4, 5 or 6) copies (e.g., in tandem). In some embodiments, the linker sequence consists of or comprises (G 4 S) 4 (SEQ ID NO: 415) or (G 4 S) 6 (SEQ ID NO: 416). In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-30 amino acids.

[1034] The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise further amino acids or amino acid sequences. For example, the antigen-binding molecules and polypeptides may comprise amino acid sequences to facilitate the expression, folding, transport, processing, purification or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecules and polypeptides of the present disclosure may additionally comprise amino acid sequences that form a detectable fragment, as described below, for example.

[1035] The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a signal peptide (also referred to as a leader sequence or signal sequence). Signal peptides typically consist of a 5-30 amino acid sequence of hydrophobic amino acids that form a single α-helix. Secreted proteins and proteins expressed on the cell surface typically comprise a signal peptide.

[1036] The signal peptide may be present at the N-terminus of the antigen-binding molecule / polypeptide or may be present in the newly synthesized antigen-binding molecule / polypeptide. The signal peptide provides for the efficient transport and secretion of the antigen-binding molecule / polypeptide. The signal peptide is typically cleaved off and thus is not included in the mature antigen-binding molecule / polypeptide secreted by the cell expressing the antigen-binding molecule / polypeptide.

[1037] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl and InterPro, and / or can be identified / predicted, for example using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nat Methods 8:785-786) or Signal-blast (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[1038] Tags and conjugates

[1039] In some embodiments, the antigen-binding molecules of the present disclosure additionally comprise a detectable moiety.

[1040] In some embodiments, the antigen-binding molecule comprises a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immuno-detectable label (e.g., an epitope tag), a radioactive label, a chemical, nucleic acid or enzyme label. The antigen-binding molecule may be labeled covalently or non-covalently with the detectable moiety.

[1041] Fluorescent labels include fluorescein, rhodamine, allophycocyanin, eosin and NDB, green fluorescent protein (GFP), rare earth chelates such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3 and Cy5. Radioactive labels include radioisotopes such as hydrogen3 , sulfur 35 , carbon 14 , phosphorus 32 , iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 , bromine 77 , technetium 99m , indium 111 , indium 113m , gallium 67 , gallium 68 , ruthenium 95 , ruthenium 97 , ruthenium 103 , ruthenium 105 , mercury 207 , mercury 203 , rhenium 99m , rhenium 101 , rhenium 105 , scandium 47 , tellurium 121m , tellurium 122m , tellurium 125m , thulium 165 , thulium 167 , thulium 168 , copper 67 , fluorine 18 , yttrium 90 , palladium 100 , bismuth 217 and antimony 211 . Luminescent labels include radioluminescence, chemiluminescence (such as acridinium esters, luminol, isoluminol) and bioluminescent labels. Immuno-detectable labels include haptens, polypeptides, antibodies, receptors and ligands, such as biotin, avidin, streptavidin or digoxin. Nucleic acid labels include aptamers.

[1042] In some embodiments, the antigen-binding molecule / polypeptide comprises an epitope tag, such as His (e.g., 6XHis), FLAG, C-Myc, StrepTag, hemagglutinin, calmodulin-binding protein (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin, s-peptide, T7 peptide, SH2 domain, avidin, streptavidin and haptens (such as biotin, digoxigenin, dinitrophenol), optionally located at the N-terminus or C-terminus of the antigen-binding molecule / polypeptide.

[1043] In some embodiments, the antigen-binding molecule / polypeptide comprises a moiety having detectable activity, such as an enzyme moiety. Enzyme moieties include, for example, luciferase, glucose oxidase, galactosidase (e.g., β-galactosidase), glucosidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase) and cholinesterase.

[1044] In some embodiments, the antigen-binding molecules of the present disclosure are conjugated to a chemical moiety. The chemical moiety can be a moiety for providing a therapeutic effect, i.e., a drug moiety. The drug moiety can be a small molecule (e.g., an organic compound with a low molecular weight (<1000 daltons, typically between ~300 - 700 daltons)). The drug moiety is described, for example, in Parslow et al., Biomedicines 2016 Sep;4(3):14 (which is hereby incorporated by reference in its entirety). In some embodiments, the drug moiety can be or include a cytotoxic agent. In some embodiments, the drug moiety can be or include a chemotherapeutic agent. The drug moiety includes, for example, calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD.

[1045] The antigen-binding molecules of the present disclosure also include antibody-derived molecules, such as molecules comprising an antigen-binding region / domain derived from an antibody. The antibody-derived antigen-binding molecule can comprise an antigen-binding region / domain that comprises or consists of the antigen-binding region of an antibody (e.g., an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding region / domain of the antibody-derived antigen-binding molecule can be or include the Fv (e.g., provided as a scFv) or Fab region of the antibody, or the whole antibody. For example, the antigen-binding molecules of the present disclosure include antibody-drug conjugates (ADCs) comprising a (cytotoxic) drug moiety. The antigen-binding molecules of the present disclosure also include multispecific antigen-binding molecules, such as immunocyte engager molecules comprising a domain for recruiting (effector) immune cells (e.g., reviewed in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17:418 - 434 and Ellerman, Methods (2019) 154:102 - 117, the entire contents of which are incorporated by reference), including BiTE, BiKEs, and TriKEs. The antigen-binding molecules of the present disclosure also include chimeric antigen receptors (CARs), which are recombinant receptors that provide antigen-binding and T cell activation functions (the CAR structure, function, and engineering are reviewed in Dotti et al., Immunol Rev (2014) 257(1), the entire contents of which are incorporated by reference).

[1046] In some embodiments, the antigen-binding molecules of the present disclosure include a drug moiety. The antigen-binding molecule can be conjugated to the drug moiety. Antibody-drug conjugates are reviewed in Parslow et al., Biomedicines 2016 Sep;4(3):14 (incorporated herein by reference in its entirety). Currently available FDA-approved ADCs are described in Tong et al., Molecules 2021 Oct;26(19):5847 (incorporated herein by reference in its entirety).

[1047] In some embodiments, an antibody-drug conjugate comprises an antigen-binding molecule moiety, a drug moiety (or payload moiety), and a linker that attaches the drug moiety to the antibody. In some embodiments, an antibody-drug conjugate consists of an antibody moiety, a drug moiety (or payload moiety), and a linker moiety that attaches the drug moiety to the antibody.

[1048] The antigen-binding molecule moiety can be a molecule that binds to a given target antigen. Antigen-binding molecules include antibodies (i.e., immunoglobulins (Igs)) and antigen-binding fragments thereof. As used herein, "antibody" includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived antigen-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single-domain antibodies (e.g., VhH), etc. Antigen-binding fragments of an antibody include, for example, Fv, Fab, F(ab')2, and F(ab') fragments.

[1049] The linker can be cleavable or non-cleavable. The linker can be based on chemical motifs such as disulfide, hydrazone, or peptide (cleavable) or thioether (non-cleavable). The type of linker, cleavable or non-cleavable, imparts specific properties to the cytotoxic drug. For example, a non-cleavable linker keeps the drug inside the cell. As a result, the whole antibody, linker, and cytotoxic (anticancer) agent enter the targeted cancer cell, where the antibody degrades into amino acids. The resulting complex - amino acids, linker, and cytotoxic agent - is considered the active drug. In contrast, in cancer cells, a cleavable linker is separated by an enzyme.

[1050] The drug moiety (or payload) can be a small molecule or nucleic acid drug. In some embodiments, the drug moiety (or payload) is or includes a cytotoxic agent. In some embodiments, the drug moiety is or includes a chemotherapeutic agent. In some embodiments, the drug moiety is or includes an anti-arthritis drug. In some embodiments, the drug moiety is or includes a steroid.

[1051] Functional properties of the antigen-binding molecule

[1052] The antigen-binding molecules described herein can be characterized by reference to certain functional properties. In some embodiments, the antigen-binding molecules described herein can have one or more of the following properties:

[1053] Bind to CNX (e.g., human CNX and / or mouse CNX);

[1054] Bind to CRT (e.g., human CRT);

[1055] Bind cross-reactively to CNX (e.g., human CNX and / or mouse CNX) and CRT (e.g., human CRT);

[1056] Reduce the function of CNX / CRT and / or the function of complexes including CNX / CRT;

[1057] Reduce or inhibit extracellular matrix degradation (e.g., collagen and / or gelatin degradation);

[1058] Reduce or inhibit the extracellular matrix degradation activity of cells characterized by CNX expression;

[1059] Reduce or inhibit the extracellular matrix degradation activity of cancer cells;

[1060] Reduce or inhibit the extracellular matrix degradation activity of fibroblasts;

[1061] Reduce or inhibit the extracellular matrix degradation activity of synovial fibroblasts;

[1062] Reduce or inhibit the degradation of the extracellular matrix by cells characterized by CNX expression;

[1063] Reduce or inhibit the degradation of the extracellular matrix by cancer cells;

[1064] Reduce or inhibit the degradation of the extracellular matrix by fibroblasts;

[1065] Reduce or inhibit the degradation of the extracellular matrix by synovial fibroblasts;

[1066] Reduce oxygen reductase activity;

[1067] Reduce disulfide reductase activity;

[1068] Reduce cartilage degradation;

[1069] Increase killing of cells expressing CNX / CRT;

[1070] Increase ADCC of cells expressing CNX / CRT;

[1071] Inhibit tumor growth;

[1072] Reduce or prevent cancer metastasis;

[1073] Increase the survival rate of subjects with cancer; and / or

[1074] Reduce the pathology of diseases / conditions characterized by ECM degradation in a subject;

[1075] Reduce the pathology of diseases / conditions characterized by cartilage degradation (such as arthritis) in a subject.

[1076] It should be understood that a given antigen-binding molecule can exhibit more than one of the properties described in the preceding paragraph. A given antigen-binding molecule can be evaluated for the properties described in the preceding paragraph using a suitable assay. For example, the assay can be, for example, an in vitro assay, an optional cell-based assay, or a cell-free assay. In some embodiments, the assay can be, for example, an in vivo assay, i.e., an assay performed in a non-human animal. In some embodiments, the assay can be, for example, an ex vivo assay, i.e., an assay performed using cells / tissues / organs obtained from a subject.

[1077] When the detection is a cell-based detection, they can include treating the cells with a given antigen-binding molecule to determine whether the antigen-binding molecule exhibits one or more of the said properties. The detection can use a species labeled with a detectable entity to facilitate its detection. The assay can include evaluating the said property after treating the cells separately with a given amount / concentration range (such as a dilution series) of the given antigen-binding molecule. It should be understood that the cells preferably express the target antigen (i.e., CNX / CRT) of the said antigen-binding molecule.

[1078] Analysis of the results of such an assay can include determining the concentration at which 50% of the maximum level of the relevant activity is reached. The concentration of a given reagent at which 50% of the maximum level of the relevant activity is reached can be referred to as the "half-maximal effective concentration" of the said reagent with respect to the relevant activity, and can also be referred to as "EC 50 50". For example, the EC50 of a given antigen-binding molecule binding to human CNX can be the concentration of the antigen-binding molecule at which 50% of the maximum level of binding to human CNX is reached.

[1079] Depending on the nature, EC 50 can also be referred to as the "half-maximal inhibitory concentration" or "IC 50 50", which is the concentration of the reagent at which 50% of the maximum inhibitory level of the given property is observed.

[1080] The antigen-binding molecules described herein bind to CNX. In some embodiments, the antigen-binding molecules bind to CRT. The antigen-binding molecules and antigen-binding domains described herein preferably exhibit specific binding to a relevant target antigen (e.g., CNX). As used herein, "specific binding" refers to binding that is selective for an antigen and can be distinguished from non-specific binding to non-target antigens. An antigen-binding molecule / domain that specifically binds a target molecule preferably binds the target with greater affinity and / or for a longer duration than it binds other non-target molecules.

[1081] The ability of a given polypeptide to specifically bind a given molecule can be determined by assays known in the art, such as by ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. By such assays, binding to a given molecule can be measured and quantified. In some embodiments, binding can be a reaction detected in a given assay.

[1082] In some embodiments, the degree of binding of the antigen-binding molecule to non-target molecules is less than about 10% of the degree of binding of the antibody to the target molecule, as measured, for example, by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, binding specificity can be reflected in terms of binding affinity, wherein the dissociation constant (K D ) of the antigen-binding molecule for binding to the target is at least 0.1 orders of magnitude greater than the K D of the antigen-binding molecule for binding to non-target molecules (i.e., 0.1 x 10 n , where n is an integer representing the order of magnitude). 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.

[1083] The affinity of the antigen-binding molecules described herein for binding to a given target antigen can be determined by biolayer interferometry, such as as described in the examples of the present disclosure.

[1084] In some embodiments, the antigen-binding molecules described herein bind to CNX with an affinity in the micromolar range, i.e., K D = 9.9 x 10 -4 to 1 x 10 -6 M. In some embodiments, the antigen-binding molecules described herein bind to CNX with an affinity in the sub-micromolar range, i.e., K D < 1 x 10-6 M. In some embodiments, the antigen-binding molecules described herein bind to CNX with an affinity in the nanomolar range, i.e., K D = 9.9x 10 -7 to 1x 10 -9 M. In some embodiments, the antigen-binding molecules described herein bind to CNX with an affinity in the sub-nanomolar range, i.e., K D < 1x 10 -9 M. In some embodiments, the antigen-binding molecules described herein bind to CNX with an affinity in the picomolar range, i.e., K D = 9.9x 10 -10 to 1x 10 -12 M. In some embodiments, the antigen-binding molecules described herein bind to CNX with an affinity in the sub-picomolar range, i.e., K D < 1x10 -12 M.

[1085] In some embodiments, the antigen-binding molecules described herein bind to human CNX with a K D of 10 μM or lower, preferably ≤5 μM, ≤2 μM, ≤1 μM, ≤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, ≤1 nM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 cM, ≤10 pM or ≤1 pM. (e.g., determined by the assay described in Example 2 herein). In some embodiments, the antigen-binding molecules described herein bind to human CNX with a K D of 100 nM or lower, preferably ≤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, ≤1 nM, ≤500 pM, ≤400 pM, ≤300 pM, ≥200 pM, ≤100 pM, ≤50 pM, ≤10 pM, ≤30 pM, ≤20 pM, ≤10 pM or ≤1 pM (e.g., determined by the assay described in Example 2 herein).

[1086] In some embodiments, the antigen-binding molecules described herein bind human CNX with an EC50 of 10 μM or less, preferably ≤5 μM, ≤2 μM, ≤1 μM, ≤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, ≤1 nM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM, or ≤1 pM. (e.g., determined by the assays described in Example 2 herein).

[1087] In some embodiments, the antigen-binding molecule is cross-reactive with human CNX and its homologs (e.g., mouse CNX). In some embodiments, the antigen-binding molecule is cross-reactive with CNX and CRT. As used herein, a "cross-reactive" antigen-binding molecule / domain binds to a target antigen that is cross-reactive with the antigen-binding molecule or domain. For example, an antigen-binding molecule / domain / polypeptide that is cross-reactive with human CNX and mouse CNX binds to human CNX and is also capable of binding to mouse CNX. Similarly, an antigen-binding molecule / domain / polypeptide that is cross-reactive with human CNX and human CRT binds to CNX and is also capable of binding to CRT. A cross-reactive antigen-binding molecule / domain / polypeptide may exhibit specific binding to each target antigen.

[1088] In some embodiments, the antigen-binding molecule binds human CNX (e.g., isoform 1) and mouse CNX. In some embodiments, the antigen-binding molecule binds human CNX (e.g., isoform 1) and human CRT.

[1089] The antigen-binding molecules of the present disclosure can bind to specific target regions of CNX. The antigen-binding molecules of the present disclosure can bind to linear epitopes of CNX, which are composed of contiguous amino acid sequences (i.e., amino acid primary sequences). In some embodiments, the antigen-binding molecules can bind to conformational epitopes of CNX, which are composed of discontinuous amino acid sequences of the amino acid sequence.

[1090] The region of a given target molecule to which an antigen-binding molecule binds can be determined by those skilled in the art using a variety of methods well known in the art, including X-ray co-crystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. For example, these methods as described in Gershoni et al., BioDrugs, 2007, 21(3):145-156, the entire content of which is incorporated herein by reference. In a preferred embodiment, the region of the peptide / polypeptide to which the antigen-binding molecule binds is determined by hydrogen-deuterium exchange analysis of mass spectrometry, substantially as described in Example 2 herein.

[1091] In some embodiments, the antigen-binding molecules of the present disclosure bind to the CNX domains described herein, such as the luminal domains (e.g., lectin domain 1, P domain, lectin domain 2), transmembrane domains, or cytoplasmic domains.

[1092] In some embodiments, the antigen-binding molecules of the present disclosure bind to the luminal domain of CNX. In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 337. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 337.

[1093] In some embodiments, the antigen-binding molecules of the present disclosure bind to the lectin domain of CNX. In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 340. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 340.

[1094] In some embodiments, the antigen-binding molecules of the present disclosure bind to the P domain of CNX. In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 341. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 341.

[1095] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 361. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 361. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 361. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 361. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 361.

[1096] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 362. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 362. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 362. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 362. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 362.

[1097] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 363. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 363. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 363. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 363. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 363.

[1098] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 364. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 364. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 364. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 364. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 364.

[1099] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 365. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 365. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 365. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 365. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 365.

[1100] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 366. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 366. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 366. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 366. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 366.

[1101] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 367. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 367. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 367. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 367. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 367.

[1102] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 368. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 368. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 368. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 368. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 368.

[1103] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 369. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 369. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 369. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 369. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 369.

[1104] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 370. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 370. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 370. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 370. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 370.

[1105] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 371. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 371. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids in the region shown in SEQ ID NO: 371. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 371. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 371.

[1106] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 372. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 372. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids of the region shown in SEQ ID NO: 372. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 372. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 372.

[1107] In some embodiments, the antigen-binding molecule binds to the CNX region shown in SEQ ID NO: 373. In some embodiments, the antigen-binding molecule contacts the CNX region shown in SEQ ID NO: 373. In some embodiments, the antigen-binding molecule binds to CNX by contacting one or more amino acids of the region shown in SEQ ID NO: 373. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 373. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 373.

[1108] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (such as Western blotting), immunoprecipitation, surface plasmon resonance, and biolayer interferometry.

[1109] In some embodiments, the antigen-binding molecule is capable of binding the same region of CNX or an overlapping region of CNX to the region of CNX that is bound by an antibody comprising the VH and VL regions of one of clone 1D3, 1D6, 1E1, 1E6, 2C6, 2H6, 3D1, 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001, C008, C010, C023, C025, C040, C046, and C117 (see, for example, Table C). In some embodiments, the antigen-binding molecule is capable of binding the same region of CNX or an overlapping region of CNX to the region of CNX that is bound by an antibody comprising the VH and VL regions of C008 or 1E1.

[1110] Whether the antigen-binding molecule to be tested binds to the same or overlapping regions of a given target as a reference antigen-binding molecule can be evaluated by the following assays, for example, by analyzing (i) the interaction between the antigen-binding molecule to be tested and the target in the absence of the reference binding molecule, and (ii) the interaction between the antigen-binding molecule to be tested and the target in the presence of the reference antigen-binding molecule, or after incubation of the target with the reference antigen-binding molecule. A determination of a reduced level of interaction between the antigen-binding molecule to be tested and the target upon analysis according to (ii) compared to (i) may support the inference that the antigen-binding molecules to be tested and the reference bind to the same or overlapping regions of the target. Suitable assays for such analysis include, for example, competitive ELISA assays and epitope binning assays.

[1111] In some embodiments, the antigen-binding molecule is an antagonist of CNX, CRT, and / or a complex comprising CNX or CRT. In some embodiments, the antigen-binding molecule is capable of inhibiting a function or process mediated by CNX and / or CRT or by a complex comprising CNX / CRT. In some embodiments, the antigen-binding molecule is capable of inhibiting a function or process mediated by a polypeptide complex comprising CNX or CRT. Here, "inhibit" means a reduction, decrease, or alleviation relative to a control group. Suitable assays for studying the functions of CNX and / or CRT and complexes comprising CNX / CRT are well known to those skilled in the art.

[1112] In some embodiments, the complex comprising CNX is selected from: CNX:ERp57 complex, CNX:ERM29 complex, and CNX:CypB complex. In some embodiments, the complex comprising CNX may comprise CNX and a glycopeptide. In some embodiments, the complex comprising CRT is selected from: CRT:ERp57 complex, CRT:ERp29 complex, and CRT:CypB complex. In some embodiments, the complex comprising CRT may comprise CRT and a glycopeptide.

[1113] In a preferred embodiment, the complex comprising CNX is the CNX:ERp57 complex. In a preferred embodiment, the complex comprising CRT is the CRT:ERp57 complex.

[1114] Assays for identifying antigen-binding molecules capable of reducing / inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT may include treating cells / tissues expressing CNX / CRT or a complex comprising CNX / CRT with the antigen-binding molecule to be tested and subsequently comparing the level of the relevant function to that observed under appropriate control conditions (e.g., untreated / carrier-treated / control-treated cells / tissues).

[1115] Antigen-binding molecules that can reduce / inhibit the function of CNX / CRT and / or complexes comprising CNX / CRT can be identified using assays that detect the relevant functional levels of CNX / CRT and / or complexes comprising CNX / CRT (e.g., gene and / or protein expression and / or activity of one or more proteins whose expression is directly / indirectly upregulated or downregulated as a result of the function of CNX / CRT or complexes comprising CNX / CRT). Such assays can include treating cells / tissues that express CNX / CRT and / or complexes comprising CNX / CRT with the antigen-binding molecule, and subsequently (e.g., after an appropriate period of time, i.e., a period sufficient to observe the active functional consequences of CNX / CRT and / or complexes comprising CNX / CRT) comparing the relevant functional levels of CNX / CRT and / or complexes comprising CNX / CRT in such cells / tissues to the relevant functional levels under appropriate control conditions (e.g., untreated / carrier-treated / control-treated cells / groups).

[1116] In some embodiments, the antigen-binding molecules of the invention are capable of reducing / inhibiting the function of CNX / CRT or complexes comprising CNX / CRT to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the relevant functional levels observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

[1117] In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or complexes comprising CNX / CRT by a mechanism that does not require or involve Fc-mediated function. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or complexes comprising CNX / CRT independently of Fc-mediated function. That is, in some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or complexes comprising CNX / CRT in a manner that is independent of the Fc region.

[1118] The ability of an antigen-binding molecule to inhibit the function of CNX / CRT and / or a complex containing the CNX / CRT complex is evaluated by a mechanism that does not require / involve Fc-mediated functions. For example, by analyzing an antigen-binding molecule provided in a form lacking a functional Fc region, the ability to inhibit the function of CNX / CRT and / or a complex containing the CNX / CRT complex. For example, the effect on the function of CNX / CRT and / or a complex containing the CNX / CRT complex can be studied using an antigen-binding molecule containing a "silent" Fc region (e.g., containing the LALAPG substitution), or using an antigen-binding molecule provided in a form lacking an Fc region (e.g., scFv, Fab, etc.).

[1119] In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex containing CNX / CRT by a mechanism that does not involve ADCC. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex containing CNX / CRT by a mechanism that does not involve ADCP. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex containing CNX / CRT by a mechanism that does not involve CDC.

[1120] In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to an Fc receptor. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to an Fcγ receptor. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding of the antigen-binding molecule to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding to FcγRIIIa. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding to FcγRIIa. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding to FcγRIIb. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding to a complement protein. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require binding to C1q. In some embodiments, the antigen-binding molecule is capable of inhibiting the function of CNX / CRT and / or a complex comprising CNX / CRT by a mechanism that does not require N297 glycosylation.

[1121] It should be understood that in some embodiments, the antigen-binding molecules of the present disclosure achieve their functional effects by mechanisms that do not involve Fc-mediated functions. In some embodiments, the antigen-binding molecules of the present invention achieve their functional effects by mechanisms that do not involve killing / consuming cells expressing CNX / CRT or cells expressing a complex comprising CNX / CRT, such as Fc-mediated killing / consuming of such cells.

[1122] In some embodiments, the function of CNX / CRT, or the function of a complex comprising CNX / CRT, can be selected from: extracellular matrix (ECM) degradation, collagen degradation, gelatin degradation, oxidoreductase activity, and disulfide reductase activity. Correlates of the function of CNX / CRT or a complex comprising CNX / CRT can be, for example, products of ECM / collagen / gelatin degradation, or products of oxidoreductase / disulfide reductase activity.

[1123] In some embodiments, the antigen-binding molecule reduces / inhibits extracellular matrix (ECM) degradation. In some embodiments, the antigen-binding molecule reduces / inhibits collagen degradation. In some embodiments, the antigen-binding molecule reduces / inhibits gelatin degradation. In some embodiments, the antigen-binding molecule decreases / inhibits oxidoreductase activity. In some embodiments, the antigen-binding molecule decreases / inhibits disulfide reductase activity. In some embodiments, the antigen-binding molecule reduces / inhibits ECM degradation mediated by CNX / CRT or a complex comprising CNX / CRT (e.g., the CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits collagen degradation mediated by CNX / CRT or a complex containing CNX / CRT (e.g., the CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits gelatin degradation mediated by CNX / CRT or a complex containing CNX / CRT (e.g., the CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule decreases / inhibits oxidoreductase activity mediated by CNX / CRT or a complex comprising CNX / CRT (e.g., the CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule decreases / inhibits disulfide reductase activity mediated by CNX / CRT or a complex comprising CNX / CRT (e.g., the CNX / CRT:ERp57 complex).

[1124] The ability of the antigen-binding molecule to inhibit ECM / collagen / gelatin degradation can be determined by analyzing ECM / collagen / gelatin degradation in the presence of the antigen-binding molecule or after incubation with the antigen-binding molecule. An antigen-binding molecule capable of inhibiting ECM / collagen / gelatin degradation can be identified by observing a decrease / reduction in the level of ECM / collagen / gelatin degradation in the presence of or after incubation with the antigen-binding molecule compared to the level of ECM / collagen / gelatin degradation in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1125] Antigen-binding molecules capable of reducing / inhibiting (e.g., via CNX / CRT and / or complexes comprising CNX / CRT) ECM / collagen / gelatin degradation can be identified by detecting ECM / collagen / gelatin levels or levels related to ECM / collagen / gelatin degradation (e.g., products of degraded ECM / collagen / gelatin), e.g., using an antibody / reporter-based method. Collagen / gelatin degradation assays are described, for example, in Hollander, Methods Mol. Biol. (2010) 622:367-78 and Vandooren et al., World J. Biol. Chem. (2011) 2(1):14–24. In a preferred embodiment, ECM / collagen / gelatin degradation can be evaluated by detection substantially as described in Example 4 herein.

[1126] For example, a commercial solution of gelatin (2%) can be labeled with 5-carboxy-X-rhodamine succinimidyl ester. The labeled gelatin is then transferred to a sterile lid to form a thin layer and stabilized by fixation with glutaraldehyde. A solution of rat tail collagen can be applied to the lid to form a thin layer of collagen over the gelatin. The coverslip can then be transferred to a culture vessel, and cells with appropriate degradation activity (e.g., human hepatocellular carcinoma Huh7 cells) can be seeded on the coverslip in the presence of the antigen-binding molecule to be tested and incubated for 48 hours to allow degradation to occur. The lid can then be fixed, subsequently stained with Hoescht to allow cell counting, and then analyzed by confocal microscopy. The images obtained can be analyzed using ImageJ to determine the surface area of the degraded gelatin and the total area of each region. At the same time, the number of cell nuclei can be counted, and the final result normalized to the number of cells in each field.

[1127] For example, a mixture of rat tail collagen and quenched fluorescent DQ-type collagen can be coated and polymerized on the bottom of a 384-well optical grade plate. 3t3-vSrc mouse cell line cells are then inoculated on top of the collagen layer in the presence of the antigen-binding molecule to be tested and incubated for 48 h to 72 h. The fluorescent area of the live cell DQ signal can subsequently be evaluated by high content imaging and normalized by nuclear counting to determine the degradation area / cell.

[1128] In some embodiments, the antigen-binding molecule of the present invention is capable of reducing / inhibiting ECM degradation, collagen degradation, or gelatin degradation to less than 1-fold, such as ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level of ECM degradation / collagen degradation / gelatin degradation observed in a given assay in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1129] The ability of the antigen-binding molecule to inhibit oxidoreductase activity can be determined, for example, by assaying the oxidoreductase activity in the presence of or after incubation with the antigen-binding molecule. An antigen-binding molecule capable of inhibiting oxidase activity is determined by observing a decrease / reduction in the level of oxidase activity in the presence of or after incubation with the antigen-binding molecule, compared to the level of oxidase activity in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1130] Oxidase activity can be evaluated using any of a number of methods known to those skilled in the art. For example, oxidase activity can be evaluated in an insulin reduction assay, as described in Hirano et al., Eur J Biochem (1995) 234(1):336-42.

[1131] In some embodiments, the antigen-binding molecule of the present invention is capable of reducing / inhibiting oxidase activity to less than 1-fold, such as: ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level of oxidase activity observed in a given assay in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1132] The ability of an antigen-binding molecule to inhibit disulfide reductase activity can be determined, for example, by analyzing disulfide reductase activity in the presence of or after incubation with the antigen-binding molecule. An antigen-binding molecule capable of inhibiting disulfide reductase activity is determined by observing a decrease / reduction in the level of disulfide reductase activity in the presence of or after incubation with the antigen-binding molecule compared to the level of disulfide reductase activity in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

[1133] Disulfide reductase activity can be evaluated using any of a number of methods known to those of skill in the art. For example, a disulfide reductase assay can use an antibody to detect reduced disulfide bonds in a protein, such as the antibody clone OX133, which recognizes polypeptides with N-ethylmaleimide (NEM)-modified cysteine residues (see Holbrook et al., mab (2016) 8(4):672-677).

[1134] In some embodiments, the antigen-binding molecules of the present invention are capable of reducing / inhibiting disulfide reductase activity to less than 1-fold, for example: ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level of disulfide reductase activity observed in a given assay in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

[1135] In some embodiments, the antigen-binding molecules of the present disclosure reduce / inhibit cartilage degradation. Antigen-binding molecules capable of reducing / inhibiting cartilage degradation (e.g., via CNX / CRT and / or complexes comprising CNX / CRT) can be identified using detection methods that include detecting the level of cartilage or a cartilage degradation correlate (e.g., a product of degraded cartilage), for example, using an antibody / reporter-based method. Cartilage degradation can be evaluated substantially as described in Example 6 herein. In vitro experiments for cartilage degradation are also described in Neidlin et al., PLoS One (2019) 14(10):e0224231.

[1136] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing / inhibiting cartilage degradation to less than 1-fold, such as ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level of cartilage degradation observed in a given assay in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1137] In some embodiments, the antigen-binding molecules of the present invention can enhance (i.e., upregulate, potentiate) the cytotoxic ability of cells containing / expressing CNX / CRT or complexes containing CNX / CRT. In some embodiments, the antigen-binding molecules of the present invention can inhibit the growth or reduce the metastasis of cancers in cells containing / expressing CNX / CRT or complexes containing CNX / CRT. In some embodiments, the antigen-binding molecules of the present invention can enhance (i.e., upregulate, potentiate) the cytotoxic ability of cells containing / expressing CNX / CRT or complexes containing CNX / CRT. In some embodiments, the antigen-binding molecules of the present invention can inhibit the growth or reduce the metastasis of cancers in cells containing / expressing CNX / CRT or complexes containing CNX / CRT.

[1138] For example, any method described in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616 can be used to investigate cell killing, which is hereby incorporated by reference in its entirety. Examples of in vitro cytotoxicity / cell killing assays include release assays such as the 51Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay, and calcein-acetoxymethyl (calcein-AM) release assay. These assays determine cell killing based on the detection of factors released from lysed cells. The killing effect of specific effector immune cell types on specific cells to be tested can be analyzed, for example, by co-culturing the cells to be tested with effector immune cells and detecting the number / proportion of the cells to be tested that are alive / dead (e.g., lysed) after an appropriate time. Other suitable detection methods include the xCELLigence real-time cell lysis in vitro potency assay described in Cerignoli et al., PLoS One (2018) 13(3):e0193498 (which is hereby incorporated by reference in its entirety). By detecting a decrease in the number / proportion of the cells to be tested that are dead (e.g., lysed) and / or an increase in the number / proportion of the cells to be tested that are alive (e.g., live, unlysed) over a period of time, an increased resistance to cell killing by cells expressing granzyme b (such as effector immune cells) and / or a decreased susceptibility to cell killing by cells expressing granzyme b can be determined relative to a reference level of cell killing (such as for that cell type).

[1139] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the number / proportion of cells expressing CNX / CRT or complexes comprising CNX / CRT. In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the number / proportion of cells expressing CNX / CRT or complexes comprising CNX / CRT. In some embodiments, the antigen-binding molecules of the present disclosure are capable of depleting / enhancing the depletion of the above-mentioned cells.

[1140] The antigen-binding molecules of the present disclosure may include one or more fragments for enhancing the reduction in the number / proportion of cells expressing CNX / CRT or complexes comprising CNX / CRT. For example, the antigen-binding molecules of the present disclosure may include, for example, an Fc region and / or a drug moiety.

[1141] The Fc region provides interactions with Fc receptors and other immune system molecules, resulting in functional effects. IgG Fc-mediated effector functions, as described in Jefferis et al., Immunol Rev 1998 163:59-76 (incorporated herein by reference in its entirety), are generated through the interaction between the Fc region and Fc receptors expressed on immune cells, through the recruitment and activation of Fc-mediated immune cells such as macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells. Complement pathway components are recruited through the binding of the Fc region to complement protein C1q, and the complement cascade is subsequently activated. Fc-mediated functions include Fc receptor binding, antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, production of cytokines and / or chemokines, and antigen processing and presentation.

[1142] In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region that is capable of enhancing / directing one or more of ADCC, ADCP, CDC, against and / or enhancing the formation of MAC or cell degranulation on cells expressing CNX / CRT or comprising a CNX / CRT complex (e.g., cells expressing CNX / CRT, or complexes comprising CNX / CRT on the cell surface).

[1143] In some embodiments, the antigen-binding molecules of the present disclosure are capable of enhancing / directing ADCC against cells expressing CNX / CRT or comprising a CNX / CRT complex.

[1144] The ability and extent to which a given antigen-binding molecule can induce ADCC of a given target cell type can be analyzed according to the method described in Yamashita et al., Scientific Reports (2016) 6:20772 (incorporated herein by reference in its entirety), or by the 51Cr release assay described in Jedema et al., Blood (2004) 103:2677-82 (incorporated herein by reference in its entirety). The ability and extent to which a given antigen-binding molecule can induce ADCP can be analyzed, for example, according to the method described in Carmen et al., J Immunol (2017) 198(1 Supplement) 157.17 (incorporated herein by reference in its entirety). The ability and extent to which a given antigen-binding molecule can induce CDC can be analyzed by a C1q binding assay as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466 (incorporated herein by reference in its entirety).

[1145] In some embodiments, the antigen-binding molecules of the present disclosure do not induce ADCC of cells that express CNX / CRT or a complex comprising CNX / CRT on the cell surface. In some embodiments, the antigen-binding molecule does not induce ADCP of cells that express CNX / CRT or a complex comprising CNX / CRT on the cell surface. In some embodiments, the antigen-binding molecule does not induce CDC of cells that express CNX / CRT or a complex comprising CNX / CRT on the cell surface. In some embodiments, the antigen-binding molecule does not induce ADCC, ADCP, or CDC of cells that express CNX / CRT or a complex comprising CNX / CRT on the cell surface.

[1146] An antigen-binding molecule that does not induce (i.e., is unable to induce) ADCC / ADCP / CDC will substantially not elicit ADCC / ADCP / CDC activity against relevant cell types, e.g., as determined by analysis in a suitable relevant activity assay. "Substantially no ADCC / ADCP / CDC activity" means that the level of said ADCC / ADCP / IDC is not significantly higher in a given assay than the ADCC / ADCP / CDC level determined for a suitable negative control molecule (e.g., an antigen-binding molecule lacking an Fc region, or an antigen-binding molecule containing a "silent" Fc region (e.g., as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, incorporated herein by reference in its entirety)). "Substantially inactive" may mean that the relevant activity level is ≤5-fold, e.g., ≤4-fold, ≤3-fold, ≤2.5-fold, ≤2-fold, or ≤1.5-fold, compared to the activity level determined for a suitable negative control molecule in a given assay.

[1147] In some embodiments, the antigen-binding molecules of the present disclosure include a drug moiety. The antigen-binding molecule can be conjugated to the drug moiety. Antibody-drug conjugates are reviewed in Parslow et al., Biomedicine September 2016; 4(3):14 (incorporated herein by reference in its entirety). In some embodiments, the drug moiety is or comprises a cytotoxic agent such that the antigen-binding molecule exhibits cytotoxicity against cells expressing CNX / CRT or a complex comprising CNX / CRT (e.g., cells expressing CNX / CRT, or a complex comprising CNX / CR on the cell surface). In some embodiments, the drug moiety is or includes a chemotherapeutic agent.

[1148] In some embodiments, the antigen-binding molecules of the present disclosure include an immune cell engaging moiety. In some embodiments, the antigen-binding molecule comprises a CD3 polypeptide binding moiety (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide).

[1149] In some embodiments, the antigen-binding molecules of the present disclosure are capable of enhancing / directing T cell-mediated cytolytic activity against cells expressing CNX / CRT or a complex comprising CNX / CRT.

[1150] In some embodiments, the antigen-binding molecules of the present disclosure exhibit anti-cancer activity. In some embodiments, the antigen-binding molecules of the present disclosure increase the killing of cancer cells. In some embodiments, the antigen-binding molecules of the present disclosure result in a decrease in the number of cancer cells in vivo, e.g., compared to appropriate control conditions. The cancer can be a cancer expressing CNX / CRT or a complex comprising CNX / CRT.

[1151] In some embodiments, the antigen-binding molecules of the present disclosure reduce / inhibit the growth of cancer and / or cancer tumors. In some embodiments, the antigen-binding molecules reduce the invasion of cancer cells into tissues. In some embodiments, the antigen-binding molecules reduce the metastasis of cancer. In some embodiments, the antigen-binding molecules exhibit anti-cancer activity. In some embodiments, the antigen-binding molecules reduce the growth / proliferation of cancer cells. In some embodiments, the antigen-binding molecules decrease the survival rate of cancer cells. In some embodiments, the antigen-binding molecules increase the killing of cancer cells. In some embodiments, the antigen-binding molecules of the present disclosure result in, for example, a decrease in the number of cancer cells in vivo. The cancer can be a cancer comprising cells expressing CNX and / or CRT.

[1152] The antigen-binding molecules of the present disclosure can be assayed for the properties described above in suitable assays. Such assays include, for example, in vivo models, such as performed substantially as described in Example 5 herein.

[1153] In some embodiments, administration of the antigen-binding molecules of the present disclosure can cause one or more effects: inhibiting the development / progression of cancer, delaying / preventing the onset of cancer, reducing / delaying / preventing tumor growth, reducing / delaying / preventing tissue invasion, reducing / delaying / preventing metastasis, reducing / delaying / preventing the severity of cancer symptoms, reducing the number of cancer cells, reducing tumor size / volume, and / or increasing survival rate (e.g., progression-free survival rate or overall survival rate), e.g., as determined in a suitable model.

[1154] In some embodiments, the antigen-binding molecules of the present invention are capable of reducing / inhibiting tumor growth (e.g., in an in vivo model, such as liver cancer) to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to tumor growth in the given assay in the absence of the antigen-binding molecule being treated (or, after treatment, a suitable control antigen-binding molecule known not to affect tumor growth).

[1155] In some embodiments, the antigen-binding molecules of the present invention are capable of reducing / suppressing metastasis (e.g., in an in vivo model, such as breast cancer metastasis to the lung) to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level of metastasis observed in a given assay in the absence of treatment with the antigen-binding molecule (or after treatment with a suitable control antigen-binding molecule known not to affect metastasis).

[1156] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the survival rate of a subject having cancer (e.g., in an in vivo model, such as cancer or cancer) to more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold or ≥10-fold, compared to the survival level observed in a given assay in the absence of treatment with the antigen-binding molecule (or after treatment with a suitable control antigen-binding molecule known not to affect survival).

[1157] In some embodiments, the antigen-binding molecules of the present disclosure reduce / suppress the pathology of a disease / condition characterized by ECM degradation in a subject.

[1158] In some embodiments, the antigen-binding molecules of the present disclosure reduce / suppress the pathology of a disease / condition characterized by cartilage degradation (e.g., arthritis) in a subject. In some embodiments, the antigen-binding molecules of the present disclosure reduce the arthritis score of a subject having arthritis. Arthritis pathology can be evaluated in an assay performed in a suitable in vivo model well known to those skilled in the art. Such models include, for example, the murine collagen antibody-induced arthritis (CAIA) model described in Khachigian, Nat-Protoc (2006) 1(5):2512-6. And such assays can be performed substantially as described in Example 5 or Example 6 herein. In some embodiments, a subject treated with the antigen-binding molecules of the present disclosure is determined to have a lower arthritis score (e.g., on day 7, 8, 9, or 10) compared to a subject not treated with the antigen-binding molecule (or compared to a subject treated with a suitable control antigen-binding molecule known not to affect arthritis pathology).

[1159] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing / suppressing the pathology of a disease / condition characterized by ECM degradation or cartilage degradation (e.g., arthritis) in a subject (e.g., in the CAIA model, e.g., as determined by arthritis score) to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level observed in a given assay in the absence of treatment with the antigen-binding molecule (or after treatment with a suitable control antigen-binding molecule known not to affect the pathology of the disease / condition).

[1160] Compared to known antigen-binding molecules that bind to CNX, the antigen-binding molecules of the present disclosure preferably have novel and / or improved properties. Known CNX antibodies include monoclonal antibody clone AF18 (Thermo Fisher Scientific catalog number MA3-027), clone AF8 (Merck catalog number MAFF2067), clone TO-5 (Merck catalog number C7617), clone 3H4A7 (Thermo Fisher Scientific catalog number.MA5-15389), clone ARC0648 (Thermo Fisher Scientific catalog number MA5-35588), clone GT1563 (GeneTex catalog number GTX629976), clone CANX / 1541 (GeneTex catalog number GTX34446), clone IE2.1C12 (Novus Biologicals catalog number.NBP2-36571), clone 1C2.2D11 (Novus Biologicals catalog number.NBP2-36570SS), clone 2A2C6 (Proteintech catalog number.66903-1-Ig), clone C5C9 (Cell Signaling Technology catalog number.2679), clone E-10 (Santa Cruz Biotechnology catalog number.sc-46669), polyclonal antibodies ab10286 and ab22595 (Abcam), and the anti-CNX antibodies disclosed in CN 101659702 A (e.g., the antibody produced by hybridoma CGMCC No. 3240). In some embodiments, the known CNX antibody is polyclonal antibody ab10286.

[1161] In some embodiments, the antigen-binding molecules according to the present disclosure:

[1162] Bind to CNX (such as human CNX and / or mouse CNX) with a higher affinity than known CNX antibodies;

[1163] has a higher affinity for binding to CRT (such as human CRT) than known CNX antibodies;

[1164] reduces the function of CNX / CRT and / or the function of complexes containing CNX / CRT with greater potency / to a greater extent compared to known CNX antibodies;

[1165] reduces extracellular matrix degradation (such as collagen and / or gelatin degradation) with greater potency / to a greater extent compared to known CNX antibodies;

[1166] reduces oxidoreductase activity with greater potency / to a greater extent compared to known CNX antibodies;

[1167] reduces disulfide reductase activity with greater potency / to a greater extent compared to known CNX antibodies;

[1168] reduces cartilage degradation with greater potency / to a greater extent compared to known CNX antibodies;

[1169] enhances the killing of cells expressing CNX / CRT with greater potency / to a greater extent compared to known CNX antibodies;

[1170] enhances the ADCC of cells expressing CNX / CRT with greater potency / to a greater extent compared to known CNX antibodies;

[1171] inhibits tumor growth with greater potency / to a greater extent compared to known CNX antibodies;

[1172] reduces the metastasis of cancer with greater potency / to a greater extent compared to known CNX antibodies;

[1173] increases the survival rate of cancer - bearing subjects to a greater extent compared to known CNX antibodies; and / or

[1174] alleviates the pathological conditions of diseases / conditions characterized by ECM degradation in subjects to a greater extent compared to known CNX antibodies;

[1175] alleviates the pathological conditions of diseases / conditions characterized by cartilage degradation (such as arthritis) in subjects to a greater extent compared to known CNX antibodies.

[1176] In some embodiments, the antigen - binding molecule according to the present disclosure binds to CNX (such as human CNX and / or mouse CNX) in a specific assay with an EC 50 that is less than 1 - fold of the EC of known CNX antibodies, for example, EC 50 50 ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or any multiple of ≤0.01.

[1177] In some embodiments, the K of the antigen-binding molecule according to the present disclosure that binds CNX (such as human CNX and / or mouse CNX) as determined by a specific assay D is the K of a known CNX antibody D less than 1-fold, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or any multiple of ≤0.01.

[1178] In some embodiments, the EC of the antigen-binding molecule according to the present disclosure that binds CRT (such as human CRT) as determined by a specific assay 50 is the EC of a known CNX antibody 50 less than 1-fold, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or any multiple of ≤0.01.

[1179] In some embodiments, the K of the antigen-binding molecule according to the present disclosure that binds CRT (such as human CRT) as determined by a specific assay D is the K of a known CNX antibody D less than 1-fold, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or any multiple of ≤0.01.

[1180] In some embodiments, the IC of an antigen-binding molecule according to the present disclosure for a particular assay that reduces the function of CNX / CRT and / or the function of a complex comprising CNX / CRT 50 is less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the IC 50 of a known CNX antibody.

[1181] In some embodiments, the IC of an antigen-binding molecule according to the present disclosure for a particular assay that reduces the function of CNX / CRT and / or the function of a complex comprising CNX / CRT is less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the IC

[1182] In some embodiments, the IC of an antigen-binding molecule according to the present disclosure for a particular assay that reduces extracellular matrix degradation, collagen degradation, and / or gelatin degradation 50 is less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the IC 50 of a known CNX antibody.

[1183] In some embodiments, the level of extracellular matrix degradation, collagen degradation, and / or gelatin degradation by an antigen-binding molecule according to the present disclosure as determined by a specific assay is reduced to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, of the level of ECM / collagen / gelatin degradation reduced by a known CNX antibody at the same concentration.

[1184] In some embodiments, the IC of an antigen-binding molecule according to the present disclosure that reduces oxidoreductase activity as determined by a specific assay 50 is less than 1-fold of the IC of a known CNX antibody 50 and is, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, of the IC of a known CNX antibody at the same concentration.

[1185] In some embodiments, the level of oxidoreductase activity is reduced by an antigen-binding molecule according to the present disclosure as determined by a specific assay to less than 1-fold of the level of oxidoreductase activity reduced by a known CNX antibody at the same concentration, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold.

[1186] In some embodiments, the IC of an antigen-binding molecule according to the present disclosure that reduces disulfide reductase activity 50 is less than 1-fold of the IC of a known CNX antibody 50 and is, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, of the IC of a known CNX antibody at the same concentration.

[1187] In some embodiments, the antigen-binding molecules according to the present disclosure as determined by a specific assay reduce the dithiothreitol reductase activity to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the level of dithiothreitol reductase activity reduction by a known CNX antibody at the same concentration.

[1188] In some embodiments, the antigen-binding molecules according to the present disclosure as determined by a specific assay reduce cartilage degradation by an IC 50 that is less than 1-fold of the IC 50 of a known CNX antibody, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold.

[1189] In some embodiments, the antigen-binding molecules according to the present disclosure as determined by a specific assay reduce cartilage degradation to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the level of cartilage degradation reduction by a known CNX antibody at the same concentration.

[1190] In some embodiments, the antigen-binding molecules according to the present disclosure as determined by a specific assay enhance cell killing or ADCC of cells expressing CNX / CRT and / or complexes containing CNX / CRT to greater than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold of the level of killing / ADCC achieved by a known CNX antibody at the same concentration.

[1191] In some embodiments, an antigen-binding molecule according to the present disclosure, as determined by a particular assay, reduces or inhibits tumor growth (e.g., in an in vivo model, such as liver cancer) to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the level of tumor growth inhibition by a known CNX antibody at the same concentration.

[1192] In some embodiments, an antigen-binding molecule according to the present disclosure, as determined by a particular assay, reduces metastasis (e.g., in an in vivo model, such as breast cancer to lung metastasis) to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the level of metastasis reduction by a known CNX antibody at the same concentration.

[1193] In some embodiments, an antigen-binding molecule according to the present disclosure, as determined by a particular assay, increases the survival rate of a cancer-bearing subject to greater than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold of the level of increase in the survival rate of a cancer-bearing subject by a known CNX antibody at the same concentration.

[1194] In some embodiments, an antigen-binding molecule according to the present disclosure, as determined by a particular assay, alleviates the pathological condition (e.g., as determined by arthritis score in a CAIA model) of a disease / condition characterized by cartilage degradation (e.g., arthritis) in a subject to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.50-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the level of inhibition of the pathological condition by a known CNX antibody treatment at the same concentration.

[1195] Chimeric antigen receptor (CAR)

[1196] The present disclosure also provides a chimeric antigen receptor (CAR) comprising the antigen-binding polypeptide or polypeptide described herein.

[1197] The CAR is a recombinant receptor that provides both antigen-binding and T cell activation functions. The structure and engineering of CARs have been reviewed, for example, as described by Dotti et al. in Immunol Rev (2014) 257(1), which is incorporated herein by reference. The CAR comprises an antigen-binding region linked to a cell membrane anchoring region and a signaling region. An optional hinge region can provide a spacer between the antigen-binding region and the cell membrane anchoring region and can serve as a flexible linker.

[1198] The CARs of the present disclosure comprise an antigen-binding region that comprises or consists of the antigen-binding molecules described herein, or that comprises or consists of a polypeptide according to the present disclosure.

[1199] The cell membrane anchoring region is located between the antigen-binding region and the signaling region of the CAR and serves to anchor the CAR to the cell membrane of the cell expressing the CAR, with the antigen-binding region in the extracellular space and the signaling region in the intracellular space. In some embodiments, the CAR comprises a cell membrane anchoring region that comprises or consists of an amino acid sequence that comprises, is derived from, or consists of the transmembrane region amino acid sequence of one of CD3-ζ, CD4, CD8, or CD28. As used herein, a region “derived from” a reference amino acid sequence comprises an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference sequence.

[1200] The signaling region of the CAR can activate T cells. The CAR signaling region can include the amino acid sequence of the CD3-ζ intracellular domain, which provides immunoreceptor tyrosine activation motifs (ITAMs) for phosphorylation and activates T cells expressing the CAR. The CAR also utilizes signaling regions of other ITAM-containing protein sequences such as FcγRI (Haynes et al., J Immunol 166(1):182-187, 2001). The signaling region of the CAR can also include a coactivation sequence derived from the signaling region of a costimulatory molecule, which promotes the activation of T cells expressing the CAR after binding to the target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some instances, the CAR is designed to co-stimulate different intracellular signaling pathways. For example, signals related to CD28 co-stimulation preferentially activate the phosphatidylinositol 3-kinase (PI3K) pathway, while 4-1BB-mediated signals are through TNF receptor-associated factor (TRAF) adapter proteins. Thus, the signaling region of the CAR sometimes contains coactivation sequences derived from more than one costimulatory molecule signaling region. In some embodiments, the CARs of the present disclosure include one or more coactivation sequences that include or consist of an amino acid sequence that includes, is derived from, or consists of the amino acid sequence of the intracellular domain of one or more of CD28, OX40, 4-1BB, ICOS, and CD27.

[1201] An optional hinge region can provide a spacer between the antigen-binding domain and the cell membrane domain and can serve as a flexible linker. The hinge region can be derived from IgG1. In some embodiments, the CARs of the present disclosure include a hinge region that includes or consists of an amino acid sequence that includes, is derived from, or consists of the amino acid sequence of the IgG1 hinge region.

[1202] Cells comprising a CAR according to the present disclosure are also provided. The CARs according to the present disclosure can be used to generate immune cells expressing the CAR, such as CAR-T or CAR-NK cells. Immune cells can be engineered with the CAR in in vitro culture.

[1203] The antigen-binding region of the CARs of the present disclosure can be in any suitable form, such as scFv, scFab, etc.

[1204] Nucleic Acids and Vectors

[1205] The present disclosure provides nucleic acids or plural nucleic acids encoding an antigen-binding molecule, polypeptide, or CAR according to the present disclosure. In some embodiments, the nucleic acid includes or consists of DNA and / or RNA.

[1206] In some embodiments, the nucleic acid can, or can be comprised in, a vector or vectors. This means that the nucleotide sequence of the nucleic acid can be comprised in a vector. The antigen-binding molecule, polypeptide, or CAR according to the present disclosure can be produced intracellularly by transcription of a vector encoding the antigen-binding molecule, polypeptide, or CAR, followed by translation of the transcribed RNA.

[1207] Accordingly, the present disclosure also provides a vector or vectors comprising the nucleic acid or nucleic acids of the present disclosure. The vector is capable of delivering the nucleic acid encoding the antigen-binding molecule, polypeptide, or CAR of the present disclosure. The vector can be an expression vector comprising the elements required for expression of the nucleic acid comprising / encoding the antigen-binding molecule, polypeptide, or CAR according to the invention.

[1208] The nucleic acid and vector according to the invention can be in a pure or isolated form, e.g., from other nucleic acids, or from naturally-occurring biological material.

[1209] The nucleotide sequence can be comprised in a vector, such as an expression vector. As used herein, a "vector" is a nucleic acid molecule that serves as a vehicle for transferring an exogenous nucleic acid into a cell. The vector can be a vector for expressing a nucleic acid in a cell. Such a vector can comprise a promoter sequence that is operably linked to the nucleic acid sequence encoding the sequence to be expressed. The vector can also comprise a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known to those of skill in the art can be used to express a peptide or polypeptide from a vector according to the present disclosure.

[1210] The term "operably linked" can include the situation where a particular nucleic acid sequence and a regulatory nucleic acid sequence (such as a promoter and / or enhancer) are covalently linked such that the expression of the nucleic acid sequence is affected or controlled by the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a particular nucleic acid sequence if the regulatory sequence can affect the transcription of the nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.

[1211] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (such as retroviral vectors, such as gamma-retroviral vectors (such as murine leukemia virus (MLV)-derived vectors, such as the SFG vector), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (such as yeast artificial chromosomes), e.g., as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biopharmaceutics (2016) 4:9, the contents of which are incorporated herein by reference.

[1212] In some embodiments, the vector can be a eukaryotic vector, such as a vector comprising elements necessary for protein expression by the vector in eukaryotic cells. In some embodiments, the vector can be a mammalian vector, such as a vector comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[1213] The component polypeptides of the antigen-binding molecules according to the present disclosure can be encoded by different nucleic acids of multiple nucleic acids, or different vectors in multiple vectors.

[1214] Cells comprising / expressing the antigen-binding molecule and the polypeptide

[1215] The present disclosure also provides cells comprising or expressing the antigen-binding molecule, polypeptide or CAR according to the present disclosure. Also provided are nucleic acids, multiple nucleic acids, vectors or multiple vectors comprising or expressing the present disclosure.

[1216] The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus macaque, non-human primate or human) or a non-human mammal (such as a rabbit, guinea pig, rat, mouse or other rodent (including any rodent), cat, dog, pig, sheep, goat, cow (including cows, such as dairy cows, or any bovine), horse (including any equine), donkey and non-human primate).

[1217] In some embodiments, the cell is or is derived from a cell type commonly used for expressing polypeptides for human therapy. Example cells are as described by Kunert and Reinhart in Appl Microbiol Biotechnol (2016) 100:3451-3461 (the content of which is incorporated herein by reference), and include, for example, CHO, HEK 293, PER.C6, NS0 and BHK cells. In a preferred embodiment, the cell is or is derived from a CHO cell.

[1218] The present disclosure also provides a method for producing a cell comprising a nucleic acid or vector according to the present disclosure, comprising introducing a nucleic acid, multiple nucleic acids, vector or multiple vectors according to the present disclosure into a cell. In some embodiments, introducing the isolated nucleic acid or vector according to the present disclosure into a cell includes transformation, transfection, electroporation or transduction (such as retroviral transduction).

[1219] The present disclosure also provides a method for producing a cell expressing / comprising the antigen-binding molecule, polypeptide or CAR according to the present disclosure, comprising introducing a nucleic acid, multiple nucleic acids, vector or multiple vectors according to the present disclosure into a cell. In some embodiments, the method further comprises culturing the cell under suitable conditions to enable the cell to express the nucleic acid or vector. In some embodiments, the method is carried out in vitro.

[1220] The present disclosure also provides cells obtainable or obtained by the methods according to the present disclosure.

[1221] Production of antigen-binding molecules and polypeptides

[1222] The antigen-binding molecules and polypeptides according to the present disclosure can be prepared according to polypeptide production methods known to those skilled in the art.

[1223] Polypeptides can be prepared by chemical synthesis, such as lipid or solid-phase synthesis. For example, peptides / polypeptides are synthesized using the methods described in Chandrudu et al., "Molecules" (2013), 18:4373 - 4388, the content of which is incorporated herein by reference.

[1224] Optionally, antigen-binding molecules and polypeptides can be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are known in the art, such as "Molecular Cloning: A Laboratory Manual" (4th Edition) by Green and Sambrook, Cold Spring Harbor Press, 2012, and "Nat Methods" (2008); 5(2):135 - 146, the entire contents of both of which are incorporated herein by reference. Methods for recombinant production of antigen-binding molecules are also described in Frenzel et al., "Front Immunol." (2013); 4:217 and Kunert and Reinhart, "Appl Microbiol Biotechnol." (2016) 100:3451–3461, the entire contents of both of which are incorporated herein by reference.

[1225] In some instances, the antigen-binding molecules of the present disclosure include more than one polypeptide chain. In this instance, production of the antigen-binding molecule can include transcription and translation of more than one polypeptide chain, and subsequent linkage of the polypeptide chains to form the antigen-binding molecule.

[1226] For recombinant production according to the present disclosure, any cell suitable for expressing polypeptides can be used. The cell can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell, such as an archaeal or bacterial cell. In some embodiments, the bacterium can be a Gram-negative bacterium, such as a bacterium of the Enterobacteriaceae family, such as Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, such as the cells described above.

[1227] In some instances, the cells are not prokaryotic cells because some prokaryotic cells cannot perform the same folding or post-translational modifications as eukaryotic cells. Additionally, extremely high expression levels may be present in eukaryotic cells, and it is easier to purify proteins from eukaryotic cells using appropriate tags. Specific plasmids can be used to facilitate the secretion of proteins into the culture medium.

[1228] In some embodiments, a polypeptide can be prepared by cell-free protein synthesis (CFPS), such as according to the system described in Zemella et al., Chembiochem (2015) 16(17):2420-2431, the contents of which are incorporated herein by reference.

[1229] Production can include culturing or fermenting the modified eukaryotic cells to express the polypeptide of interest. The culturing or fermenting can be carried out in a bioreactor, providing appropriate nutrients, air / oxygen, and / or growth factors. The secreted protein can be collected by separating the culture medium / fermentation broth from the cells, extracting the protein component therefrom, and isolating the individual proteins to isolate the secreted polypeptide. Culturing, fermenting, and separation techniques are well known to those skilled in the art, for example, as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition; incorporated by reference above).

[1230] A bioreactor includes one or more containers in which cells can be cultured. The culturing in the bioreactor can be carried out continuously, with reactants continuously flowing into the reactor and cultured cells continuously flowing out of the reactor. Alternatively, the culturing can also be carried out batchwise. The bioreactor can monitor and control environmental conditions, such as pH, oxygen, the flow rates of the inflow and outflow, and the agitation within the container, so as to provide optimal conditions for culturing the cells.

[1231] After culturing the cells expressing the / polypeptide, the polypeptide of interest can be isolated. Any suitable method known in the art for separating proteins from cells can be used. To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide is secreted by the cells, then the cells may be separated from the culture medium containing the secreted polypeptide of interest by centrifugation. If the polypeptide of interest aggregates within the cells, protein separation can include centrifuging to separate the cells from the cell culture medium, treating the cell pellet with lysis buffer, and disrupting the cells by methods such as sonication, rapid freeze-thaw, or osmotic lysis.

[1232] Then, it may be necessary to isolate the target polypeptide from the supernatant or culture medium, as the supernatant or culture medium may contain other proteins and non-protein components. A common method for separating protein components from the supernatant or culture medium is precipitation. Precipitants at different concentrations (such as ammonium sulfate) can precipitate proteins with different solubilities. For example, when the concentration of the precipitant is low, water-soluble proteins will be extracted. Therefore, by adding precipitants with gradually increasing concentrations, proteins with different solubilities can be distinguished. Subsequently, dialysis can be used to remove ammonium sulfate from the separated proteins.

[1233] Other methods for differentiating different proteins are known in the art, such as ion exchange chromatography and size chromatography. These methods can be used as alternatives to precipitation or can be carried out after precipitation.

[1234] After isolating the target polypeptide from the culture broth, it may be necessary to concentrate the polypeptide. Methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[1235] Composition

[1236] The present disclosure also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein.

[1237] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein can be formulated into pharmaceutical compositions or drugs for clinical use and may comprise pharmaceutically acceptable carriers, diluents, excipients, or adjuvants.

[1238] The compositions of the present disclosure may include one or more pharmaceutically acceptable carriers (such as liposomes, micelles, microspheres, nanoparticles), diluents / excipients (such as starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (such as vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants (such as vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (such as magnesium stearate, talc, silica, stearic acid, vegetable stearates), binders (such as sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (such as wetting agents), masking agents, or colorants (such as titanium dioxide).

[1239] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc. within the scope of reasonable medical judgment that are suitable for contact with the tissues of a relevant subject (such as a human subject) without excessive toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, coloring agent, flavoring agent, or sweetening agent in the compositions according to the present disclosure must also be "acceptable", i.e., compatible with the other ingredients in the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, cosolvents, surfactants, masking agents, coloring agents, flavoring agents, or sweetening agents can be found in standard pharmaceutical texts, such as Remington: The Science and Practice of Pharmacy (edited by A. Adejare), 23rd Edition (2020), Academic Press.

[1240] The compositions can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intracorneal, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal routes of administration. In some embodiments, the pharmaceutical composition / drug can be formulated for administration by injection or infusion, or for oral administration.

[1241] Suitable formulations can include the relevant articles in a sterile or isotonic medium. The drugs and pharmaceutical compositions can be formulated in fluid (including gel) form. The fluid formulations can be formulated for administration by injection or infusion (such as through a catheter) to a selected area in a human or animal body.

[1242] In some embodiments, the composition is formulated for injection or infusion into, e.g., a blood vessel, a target tissue / organ, or a tumor.

[1243] The present disclosure also provides methods for producing pharmaceutical compositions, and such production methods can include one or more steps selected from the group consisting of: producing the antigen-binding molecules, polypeptides, CARs, nucleic acids (or multiple nucleic acids), expression vectors (or multiple expression vectors), or cells described herein; isolating the antigen-binding molecules, polypeptides, CARs, nucleic acids (or multiple nucleic acids), expression vectors (or multiple expression vectors), or cells described herein; and / or combining the antigen-binding molecules, polypeptides, CARs, nucleic acids (or multiple nucleic acids), expression vectors (or multiple expression vectors), or cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[1244] For example, another aspect of the present disclosure relates to a method of formulating or manufacturing a medicament or pharmaceutical composition for treating a disease / condition, such as cancer, the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[1245] Therapeutic and prophylactic applications

[1246] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells, and compositions described herein can be used in therapeutic and prophylactic methods.

[1247] The present disclosure provides a method for the medical treatment or prophylaxis using an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition as described herein. Also provided is a method for treating or preventing a disease or condition as described herein using an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition as described herein. Also provided is the use of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition as described herein in the manufacture of a medicament for treating or preventing a disease or condition as described herein. Also provided is a method for treating or preventing a disease or condition as described herein, comprising administering to a subject a therapeutically or prophylactically effective dose of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition as described herein.

[1248] The method can effectively slow down the development or progression of the disease / condition, relieve the symptoms of the disease / condition, or reduce the pathological changes of the disease / condition. The method can effectively prevent the progression of the disease / condition, such as preventing the exacerbation of the disease / condition or delaying the development rate of the disease / condition. In some embodiments, the method can improve the disease / condition, such as alleviating the symptoms of the disease / condition or reducing some other severity / activity associated with the disease / condition. In some embodiments, the method can prevent the disease / condition from progressing to a subsequent stage (such as a chronic stage or metastasis).

[1249] It will be appreciated that the articles of the present disclosure can be used to treat / prevent any disease / condition that derives a therapeutic or prophylactic benefit by reducing the level / activity of CNX, CRT, the complex comprising CNX / CRT, or by reducing the number or activity of cells comprising / expressing CNX, CRT, or the complex comprising CNX / CRT.

[1250] For example, the disease / condition can be a disease / condition pathologically related to CNX, CRT, a complex comprising CNX / CRT, or a cell expressing / expressing CNX, CRT, or a complex comprising CNX / CRT, such as a disease / condition in which the level / activity of CNX, CRT, or a complex comprising CNX / CRT is increased, the number / proportion of cells containing / expressing CNX, CRT, or a complex comprising CNX / CRT is increased, and is positively correlated with the occurrence, development, or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition. In some embodiments, an increase in the level / activity of CNX, CRT, or a complex comprising CNX / CRT, or an increase in the number / proportion of cells containing / expressing CNX, CRT, or a complex comprising CNX / CRT, may be a risk factor for the occurrence, development, or progression of the disease / condition.

[1251] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition characterized by an increased expression level of CNX, CRT, or a complex comprising CNX / CRT, such as compared to the expression level / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterized by an increased number / proportion / activity of cells expressing CNX, CRT, or a complex comprising CNX or CRT, such as compared to the level / number / proportion / activity in the absence of the disease / condition.

[1252] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition described in WO 2020 / 159445A1 (the entire content of which is incorporated herein by reference). In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition described in PCT / EP2022 / 051297 (the entire content of which is incorporated herein by reference).

[1253] Treatment according to the methods of the present disclosure can achieve one or more of the following effects in a subject (compared to an equivalent subject not receiving treatment or a subject receiving appropriate control treatment): a decrease in the level of CNX, CRT, or a complex comprising CNX or CRT; a decrease in the activity of CNX, CRT, or a complex comprising CNX or CRT; and / or a decrease in the number / proportion of cells containing / expressing CNX, CRT, or a complex comprising CNX or CRT.

[1254] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is characterized by elevated O-glycosylation activity. For example, when the disease / condition is cancer, the cancer may include cells having high O-glycosylation activity. As used herein, "O-glycosylation activity" refers to the addition of O-linked sugars to the hydroxyl groups on the side chains of residues such as serine, threonine, tyrosine, hydroxylysine, or hydroxyproline of a protein. An "elevation" in the level of O-glycosylation activity may refer to a level of O-glycosylation activity that is higher than the level of O-glycosylation activity in the absence of the disease / condition (e.g., in a healthy subject, or in equivalent non-diseased tissue). When the disease / condition is cancer, the level of O-glycosylation activity may be higher than the level of O-glycosylation activity in equivalent non-cancerous cells / non-cancerous tissue. The cancer / its cells may include one or more mutations that result in upregulation of O-glycosylation activity (e.g., relative to equivalent non-cancerous cells / non-cancerous tissue).

[1255] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is characterized by elevated Src activity. For example, when the disease / condition is cancer, the cancer may include cells having high Src activity. As used herein, "Src activity" refers to Src-mediated phosphorylation of tryptophan residues. An "elevation" in the level of Src activity may refer to a level of Src activity that is higher than the level of Src activity in the absence of the disease / condition (e.g., in a healthy subject, or in equivalent non-diseased tissue). When the disease / condition is cancer, the level of Src activity may be higher than the level of Src activity in equivalent non-cancerous cells / non-cancerous tissue. The cancer / its cells may include one or more mutations that result in upregulation of Src activity (e.g., relative to equivalent non-cancerous cells / non-cancerous tissue).

[1256] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is characterized by elevated GalNAc transferase (GALNT) activity. For example, when the disease / condition is cancer, the cancer may include cells having high GALNT activity. As used herein, "GALNT activity" refers to GALNT-mediated transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc to the hydroxyl group on the side chain of residues such as serine or threonine. An "elevation" in the level of GALNT activity may refer to a level of GALNT activity that is higher than the level of GALNT activity in the absence of the disease / condition (e.g., in a healthy subject, or in equivalent non-diseased tissue). When the disease / condition is cancer, the level of GALNT activity may be higher than the level of GALNT activity in equivalent non-cancerous cells / non-cancerous tissue. The cancer / its cells may include one or more mutations that result in upregulation of GALNT activity (e.g., relative to equivalent non-cancerous cells / non-cancerous tissue).

[1257] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is characterized by elevated O-glycosylation. For example, when the disease / condition is cancer, the cancer may include cells that express proteins with high levels of O-glycosylation. An "elevation" in the level of O-glycosylation may refer to a level of O-glycosylation that is higher than the level of O-glycosylation in the absence of the disease / condition (e.g., in a healthy subject, or in equivalent non-diseased tissue). When the disease / condition is cancer, the level of O-glycosylation may be higher than the level of O-glycosylation in equivalent non-cancerous cells / non-cancerous tissue. The cancer / its cells may include one or more mutations that cause upregulation of O-glycosylation (e.g., relative to equivalent non-cancerous cells / non-cancerous tissue).

[1258] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is characterized by elevated Tn-glycosylation. For example, when the disease / condition is cancer, the cancer may include cells that express proteins with Tn-glycosylation. As used herein, "Tn-glycosylation" refers to N-acetylgalactosamine (GalNAc) linked by a glycosidic bond to the hydroxyl group of the side chain of a serine or threonine residue in a protein. A "Tn-glycosylated" protein includes at least one Tn polysaccharide and may also refer to the Tn antigen.

[1259] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is characterized by elevated Tn-glycosylation. For example, when the disease / condition is cancer, the cancer may include cells that express proteins with high levels of Tn-glycosylation. An "elevation" in the level of Tn-glycosylation may refer to a level of Tn-glycosylation that is higher than the level of Tn-glycosylation in the absence of the disease / condition (e.g., in a healthy subject, or in equivalent non-diseased tissue). When the disease / condition is cancer, the level of Tn-glycosylation may be higher than the level of Tn-glycosylation in equivalent non-cancerous cells / non-cancerous tissue. The cancer / its cells may include one or more mutations that cause upregulation of Tn-glycosylation (e.g., relative to equivalent non-cancerous cells / non-cancerous tissue). A protein with an "elevated" level of Tn-glycosylation, compared to a reference protein, may have more Tn polysaccharides than the reference protein.

[1260] The anti-CNX antibodies of the present disclosure have been shown to be useful for inhibiting ECM degradation. Thus, in some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition characterized by extracellular matrix (ECM) degradation. A disease / condition "characterized by ECM degradation" may be a disease / condition in which ECM degradation is a symptom of the disease / condition.

[1261] The disease / condition to be treated / prevented according to the present disclosure may be a disease / condition in which ECM degradation is pathologically relevant. For example, the disease / condition may be a disease / condition in which ECM degradation, and / or an elevated level of ECM degradation, is pathologically relevant to the disease / condition.

[1262] Diseases / conditions characterized by ECM degradation include, for example, cancer, and diseases / conditions characterized by cartilage degradation.

[1263] It is known that ECM degradation is involved in the development or progression of cancer and has been reviewed in, for example, Walker et al., Int. J. Mol. Sci. (2018) 19(10):3028, Najafi et al., J. Cell Biochem. (2019) 120(3):2782-2790, and Winkler et al., Nat. Commun. (2020) 11(1):5120, the entire contents of which are incorporated herein by reference.

[1264] In some embodiments, the disease / condition to be treated / prevented is cancer. Cancer can refer to any harmful cell proliferation (or any disease manifested by harmful cell proliferation), tumor or neoplasm. The cancer can be benign or malignant, primary or secondary (metastatic). A tumor or neoplasm can be any abnormally growing or proliferating cells and can be located in any tissue. Cancer can be tissue / cells from the following sites, such as adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or not including the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (such as renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, and / or white blood cells.

[1265] The tumor can be a neural or non-neural system tumor. Neural system tumors can originate from the central nervous system or the peripheral nervous system, such as glioma, medulloblastoma, meningioma, neurofibroma, epithelioma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-neural system cancers / tumors can originate from any other non-neural tissue, for example, melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, squamous cell carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, hematologic malignancies, and sarcoma.

[1266] This article, as well as Ros et al., Nat. Cell Biol. (2020) 22(11):1371-1381 and WO 2020 / 159445A1, have shown that anti-CNX antibodies are used to inhibit tumor growth and metastasis, including breast cancer and liver cancer. Ryan et al. proposed in J. Transl. Med. (2016) 14:196 to use CNX as a therapeutic target for rectal cancer.

[1267] Chen et al., Cancer Immunol. Res. (2019) 7(1):123-135 demonstrated that CNX expression was upregulated in oral squamous cell carcinoma, and knockdown of CNX in melanoma models improved control of tumor growth. The authors also demonstrated that CNX inhibits the proliferation and effector functions of CD4+ and CD8+ T cells through a mechanism involving upregulation of the immune checkpoint molecule PD-1 expression. Thus, Chen et al. showed that CNX-targeted interventions can be used to treat / prevent multiple cancers by indirectly antagonizing PD-1 / PD-L1-mediated anti-cancer response inhibition.

[1268] In some embodiments, the cancer is liver cancer, breast cancer, oral cancer (such as oral squamous cell carcinoma), sarcoma, lung cancer, prostate cancer, bladder cancer, kidney cancer, melanoma, pancreatic cancer, endometrial cancer, rectal cancer, and thyroid cancer.

[1269] In some embodiments, the liver cancer is primary liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), fibrolamellar carcinoma, cholangiocarcinoma (cholangiocellular carcinoma), angiosarcoma, or hepatoblastoma.

[1270] In some embodiments, the breast cancer is primary breast cancer. In some embodiments, the breast cancer is ductal carcinoma, lobular carcinoma, breast cancer in situ (such as ductal carcinoma in situ (DCIS)), invasive breast cancer (such as invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), triple-negative breast cancer, or inflammatory breast cancer), Paget's disease, angiosarcoma, or phyllodes tumor.

[1271] In some embodiments, the cancer is a cancer from which a therapeutic or prophylactic benefit can be obtained by reducing the expression or activity of CNX, CRT, or a complex comprising CNX or CRT. In some embodiments, the cancer is a cancer caused or exacerbated by the expression / overexpression or activity of CNX, CRT, or a complex comprising CNX or CRT. In some embodiments, the cancer is a cancer in which the expression / overexpression or activity of CNX, CRT, or a complex comprising CNX or CRT is a risk factor for cancer development or progression. In some embodiments, the expression / overexpression or activity of CNX, CRT, or a complex comprising CNX or CRT is positively correlated with the occurrence, development, progression, severity, or metastasis of cancer.

[1272] As used herein, overexpression of a specific protein / protein complex (such as CNX, CRT, a complex comprising CNX or CRT) refers to a gene or protein expression level of the relevant protein / protein complex that is higher than the expression level in equivalent cancer-free cells / tissues.

[1273] In some embodiments, the cancer may be a cancer characterized by the expression / overexpression of CNX or CRT (i.e., "CNX-positive" cancer and "CRT-positive" cancer, respectively), or the expression / overexpression of a polypeptide complex comprising CNX / CRT. The cancer may include cells that express / overexpress CNX, CRT, or a polypeptide complex comprising CNX / CRT.

[1274] CNX / CRT expression can be determined by any suitable method. The expression can be gene expression or protein expression. Gene expression can be determined by methods such as detecting the mRNA encoding CNX / CRT, for example, by real-time quantitative PCR (qRT-PCR). Protein expression can be determined by methods such as detecting CNX / CRT, for example, by antibody-based methods, such as by Western blotting, immunohistochemical techniques, immunocytochemical techniques, flow cytometry, or ELISA.

[1275] In some embodiments, the cancer may be a cancer characterized by surface expression of CNX / CRT. In some embodiments, the cancer may include cells with cell surface expression of CNX / CRT. CNX / CRT may be present within or on the cell membrane surface of cancer cells.

[1276] In some embodiments, the cancer may be a cancer characterized by the expression / overexpression of O-glycosylated CNX / CRT. The cancer may include cells that express / overexpress O-glycosylated CNX / CRT. In some embodiments, the cancer may be a cancer characterized by the expression of CNX / CRT with a high level of O-glycosylation. The cancer may include cells that express CNX / CRT with a high level of O-glycosylation.

[1277] In some embodiments, the cancer can be a cancer characterized by the expression / overexpression of Tn-glycosylated CNX / CRT. The cancer can include cells that express / overexpress Tn-glycosylated CNX / CRT. In some embodiments, the cancer can be a cancer characterized by the expression of CNX / CRT with a high level of Tn-glycosylation. The cancer can include cells that express CNX / CRT with a high level of Tn-glycosylation.

[1278] Treating a subject with an antigen-binding molecule according to the present disclosure can: delay / prevent the occurrence of one or more symptoms of cancer; alleviate the severity of one or more symptoms of cancer; improve the survival of the subject; reduce / inhibit the survival of cancer cells; reduce the number of the cancer cells in the subject; reduce the size / volume of the tumor; alleviate the cancer / tumor burden of the subject; reduce / inhibit the growth of cancer cells; reduce / inhibit tumor growth; reduce / inhibit cancer cell infiltration; and / or reduce / inhibit cancer metastasis.

[1279] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is cartilage degradation, or a disease / condition characterized by cartilage degradation.

[1280] As used herein, "cartilage degradation" refers to the degradation / deterioration / loss / damage of cartilage tissue. Cartilage tissue consists of chondrocytes and an extracellular matrix rich in glycosaminoglycans, proteoglycans, collagen, and certain elastin.

[1281] Cartilage is an avascular, aneural, and lymphatic-free connective tissue that is present in synovial joints, the spine, ribs, the outer ear, the nose, the respiratory tract, and the growth plates of children and adolescents. There are mainly three types of cartilage in humans: hyaline cartilage, fibrocartilage, and elastic cartilage (Wachsmuth et al., Histol Histopathol. May 2006; 21(5):477-85). Hyaline cartilage is the most common type of cartilage and is the type that makes up the embryonic skeleton. In human adults, it exists as articular cartilage at the bone ends of freely movable joints, at the ends of the ribs, and in the nose, larynx, trachea, and bronchi.

[1282] Fibrocartilage is a tough and strong tissue that is mainly present in the intervertebral discs and at the insertions of ligaments and tendons; it is similar to other fibrous tissues but contains cartilage matrix and chondrocytes. Elastic cartilage is more flexible than the other two forms of cartilage because, in addition to collagen, it also contains elastic fibers. In the human body, it forms the outer ear, the auditory tube of the middle ear, and the epiglottis.

[1283] In some embodiments, the cartilage degradation can be hyaline cartilage, fibrocartilage, and / or elastic cartilage.

[1284] In most tissues, fibroblasts are the main cell type that produces the extracellular matrix. However, fibroblasts can also degrade the matrix, renewing this important component of the tissue. How fibroblasts regulate these two opposing activities remains unclear. Synovial fibroblasts (SF), also known as synoviocytes, are typical bifunctional cells. In healthy individuals, SF increases the viscosity of synovial fluid by secreting proteins such as hyaluronic acid and lubricin (Jay et al., J. Rheumatol. 27, 594–600, 2000). In arthritis diseases, SF adheres to and degrades cartilage, especially the extracellular matrix (ECM) of cartilage. Understanding this activity change during arthritis has been the focus of recent research (Ospelt. RMD Open. 3, e000471, 2017). The GALNT activation pathway (GALA) regulates ECM degradation in cancer cells through the glycosylation of MMP14 and CNX. The inventors demonstrate herein that cartilage degradation, the dysregulation associated with cartilage degradation, and joint disorders are also associated with elevated levels of GALA and O-glycosylation.

[1285] GALA induces matrix degradation through at least two mechanisms. First, it stimulates the glycosylation of MMP14, which is essential for its proteolytic activity (Nguyen et al., Cancer Cell 32, 639-653.e6, 2017). Second, GALA induces the glycosylation of the ER-resident protein CNX, which forms a complex with ERp57 (Ros et al., Nat. Cell Biol. Vol. 22, pp. 1371-1381, 2020). After GALA-glycosylation, a portion of the CNX:ERp57 complex is transported to the cancer cell surface. The CNX:ERp57 complex aggregates in invadopodia, reducing the disulfide bonds in the ECM (Ros et al., Nat. Cell Biol. Vol. 22, pp. 1371-1381, 2020). The reduction of these disulfide bonds is crucial for the efficient degradation of the ECM (Ros et al., Nat. Cell Biol. Vol. 22, pp. 1371-1381, 2020).

[1286] The inventors demonstrate again the treatment of cartilage degradation using anti-CNX antibodies. Anti-CNX antibodies have shown inhibition of ECM degradation, which is a key factor in cartilage degeneration, cartilage degradation, and diseases / conditions characterized by cartilage degradation. The anti-CNX antibodies herein have also shown alleviation of the pathological changes of in vivo arthritis characterized by cartilage degradation.

[1287] Aspects and embodiments of the present disclosure relate to the treatment / prevention of diseases / conditions characterized by cartilage degradation. A disease / condition "characterized by cartilage degradation" is a disease / condition in which cartilage degradation is a symptom of the disease / condition. The disease / condition to be treated / prevented according to the present disclosure may be a disease / condition in which cartilage degradation is pathologically relevant. For example, the disease / condition may be a disease / condition in which cartilage degradation, and / or an elevated level of cartilage degradation, is pathologically relevant to the disease / condition.

[1288] Cartilage degradation can occur and / or contribute to the development, progression, or exacerbation of diseases such as osteoarthritis, psoriatic arthritis, rheumatoid arthritis, juvenile arthritis, post-traumatic arthritis, bursitis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage injury, and polychondritis. Cartilage degradation can also be the result of physical trauma / mechanical injury, such as sports injuries (e.g., collisions or overstretching) or surgery. Subjects with cartilage degradation typically experience joint pain, stiffness, and inflammation, which can affect quality of life.

[1289] In some embodiments, the disease / condition characterized by cartilage degradation according to the present disclosure may be selected from: joint diseases, arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, juvenile arthritis, post-traumatic arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage injury, and polychondritis.

[1290] Arthritis is a class of diseases that affect joints (Barbour et al., Morbidity and Mortality Weekly Report, Vol. 65, 2016, pp. 1052-1056). Rheumatoid arthritis (RA) and osteoarthritis (OA) are the two most common types (Murphy and Nagase, Nat. Clin. Pract. Rheumatol., Vol. 4, pp. 128-135, 2008). It is generally believed that mechanical damage to cartilage leads to low-grade inflammation, which mediates the gradual loss of cartilage in arthritis (Kapoor et al., Nat. Rev. Rheumatol., Vol. 7, pp. 33-42, 2011; Pap and Korb-Pap, Rheumatol., Vol. 11, pp. 606-615, 2015). Post-traumatic arthritis (PTA) occurs after acute direct trauma to a joint. PTA accounts for approximately 12% of all cases of osteoarthritis, and patients with chronic inflammatory arthritis may also have a history of physical trauma.

[1291] In healthy synovial joints, the synovium surrounds and isolates the joint cavity and secretes extracellular matrix proteins into the synovial fluid. Synovial fibroblasts are the main stromal cells of the synovium, interspersed with resident macrophages (Barbour et al., Morbidity and Mortality Weekly Report, Vol. 65, 2016, pp. 1052-1056). During the active phase of RA, SF cells are activated, express fibroblast activation protein α and proliferate. Like other stromal cells, SF cells also express innate immune receptors such as Toll-like receptors. These can detect local pathogens and molecular damage and secrete cytokines to activate immune cells (Ospelt et al., Arthritis Rheum., Vol. 58, pp. 3684–3692, 2008). During inflammation, SF proliferates and, together with infiltrating immune cells, forms an enlarged synovium called a "pannus" (Choy, Rheumatology, Vol. 51, Appendix 5, v3–11, 2012). The pannus invades the joint cavity and degrades cartilage (Pap and Korb-Pap, Rheumatol., Vol. 11, pp. 606–615, 2015). In particular, SF in the inner synovial layer has been shown to mediate cartilage degradation, while SF in the sub-intimal layer tends to mediate inflammation (Croft et al., Nature, Vol. 570, pp. 246–251, 2019). ECM degradation activity is caused by increased production of matrix metalloproteinases (MMPs), A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), and cathepsins (Rengel and Ospelt, Arthritis Res. Ther., Vol. 9, p. 221, 2007). Arthritic synovial fibroblasts express both secreted MMPs (Jay et al., J. Rheumatol., Vol. 27, pp. 594–600, 2000; Barbour et al., Morbidity and Mortality Weekly Report, Vol. 65, 2016, pp. 1052-1056; Smolen et al., Nature Reviews Disease Primers, Vol. 4, 2018, doi:10.1038 / nrdp.2018.1) and cell surface MMPs (Lange-Brokaar et al., Osteoarthritis Cartilage, Vol. 20, pp. 1484–1499, 2012; Nygaard and Firestein, Nat. Rev. Rheumatol., Vol. 16, pp. 316–333, 2020; Bauer et al., Arthritis Res. Ther., Vol. 8, R171, 2006). MMP14 (MT1-MMP) is particularly crucial for the invasive properties of SF.

[1292] Obtaining abnormal matrix degradation is also a characteristic of SF in OA (Fuchs et al., Osteoarthritis Cartilage, Vol. 12, pp. 409-418, 2004). Although the number of immune cells in the OA synovium is usually less than that in RA, it can also cause cartilage degradation like RA. It is still unclear what substances control the conversion of SF to the ECM degradation mode. Changes in gene expression are obviously suspicious, and similar transcriptional profiles have been detected in both diseases (Cai et al., J Immunol Res. 2019, 4080735. 2019). Epigenetic changes have been detected and are considered to be the driving factors for the arthritis SF phenotype (Nakano et al., Ann. Rheum. Dis., Vol. 72, pp. 110-117, 2013). Whether these changes are sufficient is still unclear.

[1293] The phenotype of SF during arthritis has been compared with that of malignant cancer cells. In fact, the growth of cancer cells requires deep remodeling of the ECM of the primary tissue and degradation of the ECM of the primary tissue (Hotary et al., Cell, Vol. 114, pp. 33–45, 2003). In malignant tumor cells, MMPs and other matrix-degrading enzymes are abnormally active (Castro-Castro et al., Cell Dev. Biol., Vol. 32, pp. 555–576, 2016).

[1294] Gout is an inflammatory arthritis, also known as gouty arthritis. Gout is the most common inflammatory arthritis, with an incidence of 2.5% in the UK. Although gout can be cured, the treatment effect is still not ideal (Abhishek et al., Clin Med (Lond). February 2017; 17(1):54–59). The ultrasound findings of gout include the double contour sign (deposition of MSU crystals on the surface of transparent articular cartilage). Normal adult articular cartilage is composed of abundant ECM, mainly composed of type II collagen fibers interspersed with type IX and type XI collagen. Cartilage loss is often a late feature of gouty arthropathy. Similar to bone erosion, cartilage loss is local rather than diffuse. Cartilage damage is usually associated with erosion and is described as occurring in the biomechanical stress area.

[1295] Chondrocalcinosis or cartilage calcification refers to the calcification (calcium salt deposition) of hyaline cartilage and / or fibrocartilage. Calcium phosphate deposition is found in the ankle joints of approximately 50% of the general population, which may be associated with osteoarthritis (Hubert et al., BMC Musculoskelet Disord. 2018; 19:169). It is commonly present in weight-bearing joints such as the hip, ankle, and knee joints. The molecular structure of calcium pyrophosphate has the potential to trigger an inflammatory response. The presence of chondrocalcinosis is associated with the degeneration of cartilage menisci and synovial tissues. It has been reported that calcium-containing crystals associated with chondrocalcinosis are related to a higher incidence of cartilage and meniscus injuries (Gersing et al., Eur Radiol. June 2017; 27(6):2497-2506. doi:10.1007 / s00330-016-4608-8. Epub 2016 Oct 4).

[1296] Fibromyalgia (FM) is a disorder characterized by chronic widespread pain and enhanced pain response to pressure. Fibromyalgia is common in rheumatoid arthritis, axial spondyloarthritis, and psoriatic arthritis and may thus affect the treatment of these rheumatic conditions. FM is also associated with costochondritis.

[1297] Costochondritis is an inflammation of the rib cartilage. This disorder typically affects the cartilage of the upper ribs where they join the breastbone or sternum, an area known as the costosternal joint or costosternal junction. Costochondritis can be caused by mechanical stress, leading to cartilage loss and / or ECM degradation.

[1298] Osteochondritis dissecans (OCD or OD) is a disorder in which cracks form in the articular cartilage and subchondral bone. OCD typically causes pain during and after movement. In the late stages of the disease, the affected joint becomes swollen and catches and locks during movement. Early physical examination can detect pain symptoms, while in the late stages, there may be effusion, tenderness, and crepitus during joint movement. Treatments that prevent, reduce, or reverse ECM degradation and / or cartilage loss may benefit patients with osteochondritis dissecans. In some cases, osteochondritis dissecans that requires treatment is associated with ECM degradation and / or cartilage loss.

[1299] Polychondritis or relapsing polychondritis (RP) is an immune-mediated systemic disease characterized by recurrent inflammation of tissues rich in cartilage and proteoglycans, including the elastic cartilage of the ear and nose, the hyaline cartilage of the peripheral joints, the fibrocartilage of the axial sites, and the tracheobronchial cartilage, leading to progressive anatomical deformation and dysfunction of the affected structures (Borgio et al., Biomedicines 2018 Sep;6(3):84). Unilateral or more commonly bilateral auricular chondritis is the most common feature in RP, occurring in up to 90% of patients during the course of the disease and being the initial symptom in 20% of cases. The onset is sudden, with painful erythema and edema that are red to violaceous in the auricular cartilage, and the earlobe, which lacks cartilage, is usually not affected. Acute inflammatory episodes tend to resolve spontaneously within a few days or weeks, but the intervals between relapses are not fixed. The long-term consequences of recurrent attacks are severe destruction of the cartilage matrix and replacement by fibrous connective tissue (Borgio et al., Biomedicines 2018 Sep;6(3):84).

[1300] In some embodiments, the disease / condition to be treated according to the present disclosure is a joint disease. A joint refers to the connection between two bones in the skeletal system. Joints can be classified according to the type of tissue present (fibrous tissue, cartilaginous tissue, or synovial tissue) or the degree of movement permitted (synarthrosis, syndesmosis, or diarthrosis). Thus, joint disease is defined as a condition that affects the connection between two bones in the skeletal system. Definitions of specific joints and related anatomical aspects can be found in Netter, F.H., Atlas of Human Anatomy, Philadelphia, PA: Saunders / Elsevier, 2006, the contents of which are incorporated herein by reference. Joint diseases can affect fibrous joints, cartilaginous joints, or synovial joints.

[1301] Fibrous joints are connected by dense connective tissue consisting mainly of collagen. These joints are also known as synarthroses or immovable joints because they do not move. Fibrous joints do not have a joint cavity and are connected by fibrous connective tissue. The skull is connected by fibrous joints called cranial sutures.

[1302] Cartilaginous joints are joints in which the bones are connected entirely by cartilage (hyaline cartilage or fibrocartilage). These joints generally permit more movement than fibrous joints but less movement than synovial joints.

[1303] Synovial joints are characterized by a fluid-filled joint cavity within a fibrous capsule. Synovial joints are the most common type of joint in the human body and contain structures not found in many fibrous or cartilaginous joints. The three main features of synovial joints are: (i) the joint capsule, (ii) articular cartilage, and (iii) synovial fluid. The joint capsule surrounds the joint and is attached to the periosteum of the articulating bones. The articular surfaces of synovial joints (i.e., the surfaces that directly contact each other during bone movement) are covered by a thin layer of hyaline cartilage. Articular cartilage has two main functions: (i) to minimize friction during joint movement; and (ii) to absorb shock. Synovial fluid is located within the joint capsule of synovial joints. It has three basic functions. Synovial joints can include accessory structures such as tendons, ligaments, bursae, and blood vessels. There are various types of synovial joints. In some cases, joint diseases belong to gliding joints, hinge joints, pivot joints, ellipsoid joints, saddle joints, or ball-and-socket joints. Gliding joints, also known as plane joints or flat joints, are a common type of synovial joint formed between bones with flat or nearly flat articular surfaces. Gliding joints allow the bones to slide past each other in any direction (up and down, left and right, and diagonally) along the joint plane. These joints can also undergo slight rotation, but are limited by the shape of the bones and the elasticity of the surrounding joint capsule. Hinge joints (ginglymi) are a type of synovial joint in which the articular surfaces are shaped to allow movement in only one plane. According to one classification system, they are called uniaxial joints (with one degree of freedom) (Color Atlas of Human Anatomy, Volume 1, by Platzer, Werner (2008)). The direction of movement of the distal bone rarely lies in the same plane as the axis of the proximal bone; during flexion, there is usually a certain degree of deviation in a straight line. The articular surfaces of the bones are connected by strong collateral ligaments. The interphalangeal joints of the hand, the interphalangeal joints of the foot, and the joint between the humerus and the ulna are the best examples of hinge joints. The knee joint and the ankle joint are less typical, as they allow slight rotation or lateral movement of the limb in certain positions. The knee joint is the largest hinge joint in the human body. Pivot joints (trochoid joints, rotary joints, or lateral ginglymi) are a type of synovial joint in which the axis of movement is parallel to the long axis of the proximal bone and usually has a convex articular surface. According to one classification system, pivot joints have one degree of freedom (Color Atlas of Human Anatomy, Volume 1, by Platzer, Werner (2008)). Ellipsoid joints (also known as condyloid joints) refer to an oval articular surface or condyle that is received within an elliptical cavity. This allows movement in two planes, such as flexion, extension, adduction, abduction, and external rotation in the wrist joint. Saddle joints are a type of synovial joint in which the opposing surfaces are reciprocally concave and convex. They are found in the thumb, chest, middle ear, and heel. Ball-and-socket joints (or spheroidal joints) are a type of synovial joint in which the spherical surface of one rou...

Claims

1. An optionally isolated antigen-binding molecule that binds to CNX.

2. The antigen-binding molecule according to claim 1, wherein, the antigen-binding molecule inhibits extracellular matrix (ECM) degradation.

3. The antigen-binding molecule according to claim 1 or 2, wherein, the antigen-binding molecule comprises: (a) (i) A variable heavy chain (VH) region comprising the following CDRs: HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 166 HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 167 HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 168; and (ii) A variable light chain (VL) region comprising the following CDRs: LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 179 LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 180 LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 173; or (b) (i) A variable heavy chain (VH) region comprising the following CDRs: HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 33 HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 34 HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 35; and (ii) A variable light chain (VL) region comprising the following CDRs: LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41 LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 42 LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43; or (c) (i) A variable heavy chain (VH) region comprising the following CDRs: HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 2 HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 3 HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 4; and (ii) A variable light chain (VL) region comprising the following CDRs: LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 10, LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 11, LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 12; or (d) (i) A variable heavy chain (VH) region comprising the following CDRs: HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 18, HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 19, having the amino acid sequence shown in SEQ ID NO: 20; and (ii) A variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO:25, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO:26, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO:27; or (e) (i) A variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO:48, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO:3, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO:49; and (ii) a variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO:53, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO:54, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO:55; or (f) (i) A variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO:61, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO:62, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO:63; and (ii) a variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO:68, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO:26, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO:69; or (g) (i) A variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO:61, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO:62, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO:63; and (ii) a variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO:73, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO:26, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO:74; or (h) (i) A variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO:61, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO:62, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO:63; and (ii) a variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO:78, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO:79, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO:80; or (i) (i) A variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO:2, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO:3, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO:83; and (ii) A variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO:73, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO:26, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO:74; or (j) (i) A variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO:48, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO:3, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO:86; and (ii) A variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO:89, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO:11, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO:90; or (k) (i) A variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO:61, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO:95, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO:96; and (ii) A variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO:101, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO:102, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO:103; or (l) (i) A variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO:108, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO:109, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO:110; and (ii) A variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 115, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 116, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 117; or (m) (i) The variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 2, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 3, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 122; and (ii) the variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 125, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 126, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 127; or (n) (i) The variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 132, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 133, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 134; and (ii) the variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 139, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 140, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 80; or (o) (i) The variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 146, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 147, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 148; and (ii) the variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 42, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 153; or (p) (i) The variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 48, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 3, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 156; and (ii) the variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 158, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 159, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 160; or (q) (i) The variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 166, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 167, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 168; and (ii) the variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 171, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 172, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 173; or (r) (i) The variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 185, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 186, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 187; and (ii) the variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 73, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 26, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 194; or (s) (i) The variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 48, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 199, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 200; and (ii) the variable light chain (VL) region comprising the following CDRs: The LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 205, the LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 42, and the LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 206; or (t) (i) The variable heavy chain (VH) region comprising the following CDRs: The HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 48, the HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 211, and the HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 212; and (ii) the variable light chain (VL) region comprising the following CDRs: An LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 216, an LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 172, and an LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 217; or (u) (i) A variable heavy chain (VH) region comprising the following CDRs: An HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 222 An HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 223 An HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 224; and (ii) A variable light chain (VL) region comprising the following CDRs: An LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 229 An LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 172 An LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 230; or (v) (i) A variable heavy chain (VH) region comprising the following CDRs: An HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 48 An HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 199 An HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 200; and (ii) A variable light chain (VL) region comprising the following CDRs: An LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 235 An LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 236 An LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 237; or (w) (i) A variable heavy chain (VH) region comprising the following CDRs: An HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 185 An HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 243 An HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 244; and (ii) A variable light chain (VL) region comprising the following CDRs: An LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 248 An LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 249 An LC-CDR3 having the amino acid sequence shown in SEQ ID NO:

250.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein, the antigen-binding molecule comprises: A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NO: 165, 32, 1, 17, 47, 60, 82, 85, 94, 107, 121, 131, 154, 155, 184, 198, 210, 221 or 242; and A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 178, 40, 9, 24, 52, 67, 72, 77, 88, 100, 114, 124, 138, 152, 157, 170, 191, 204, 215, 228, 234 or 247; optionally, wherein the antigen-binding molecule comprises: (i) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 165; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 178; or (ii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 32; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 40; or (iii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; or (iv) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 24; or (v) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 47; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 52; or (vi) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 67; or (vii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 72; or (viii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 60; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 77; or (ix) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: ; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: ; or (x) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:82; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:72; or (xi) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:85; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:88; or (xii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:94; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:100; or (xiii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:107; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:114; or (xiv) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:121; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:124; or (xv) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:131; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:138; or (xvi) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:145; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:152; or (xvii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:155; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:157; or (xviii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:165; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:170; or (xix) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:184; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:191; or (xx) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 198; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 204; or (xxi) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 210; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 215; or (xxii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 221; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 228; or (xxiii) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 198; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 234; or (xxiv) a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 242; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:

247.

5. The antigen-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding molecule binds to CNX by contacting the following positions: (a) one or more amino acid residues of the CNX region corresponding to the region shown in SEQ ID NO: 363, optionally, wherein the antigen-binding molecule binds to CNX by contacting one or more amino acid residues of the CNX region corresponding to the region shown in SEQ ID NO: 361 or 362; or (b) one or more amino acid residues of the CNX region corresponding to the region shown in SEQ ID NO: 371, optionally, wherein the antigen-binding molecule binds to CNX by contacting one or more amino acid residues of the CNX region corresponding to the regions shown in SEQ ID NO: 364, 365, 366, 367, 368, 369, 370, 372, or 373.

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule binds to CRT.

7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule binds to human CNX and mouse CNX.

8. The antigen-binding molecule according to any one of claims 1 to 8, wherein the antigen-binding molecule is a multispecific antigen-binding molecule, wherein the antigen-binding molecule further comprises an antigen-binding domain that binds to an antigen other than CNX.

9. The antigen-binding molecule according to claim 8, wherein The multispecific antigen-binding molecule is a bispecific T cell engager (BiTE).

10. A chimeric antigen receptor (CAR) comprising the antigen-binding molecule according to any one of claims 1 to 9.

11. An antibody-drug conjugate (ADC) comprising the antigen-binding molecule according to any one of claims 1 to 9 and a drug moiety.

12. One or more optionally isolated nucleic acids encoding the antigen-binding molecule according to any one of claims 1 to 9, the CAR according to claim 10.

13. One or more expression vectors comprising one or more of the nucleic acids according to claim 12.

14. A cell comprising the antigen-binding molecule according to any one of claims 1 to 9, the CAR according to claim 10, the ADC according to claim 11, one or more of the nucleic acids according to claim 12, or one or more of the expression vectors according to claim 13.

15. A method comprising culturing the cell according to claim 14 under conditions suitable for the cell to express the antigen-binding molecule or CAR.

16. A composition comprising the antigen-binding molecule according to any one of claims 1 to 9, the CAR according to claim 10, the ADC according to claim 11, one or more of the nucleic acids according to claim 12, one or more of the expression vectors according to claim 13, or the cell according to claim 14, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

17. The antigen-binding molecule according to any one of claims 1 to 9, the CAR according to claim 10, the ADC according to claim 11, one or more of the nucleic acids according to claim 12, one or more of the expression vectors according to claim 13, the cell according to claim 14, or the composition according to claim 16, for use in a method of medical treatment or prophylaxis.

18. The antigen-binding molecule according to any one of claims 1 to 9, the CAR according to claim 10, the ADC according to claim 11, one or more of the nucleic acids according to claim 12, one or more of the expression vectors according to claim 13, the cell according to claim 14, or the composition according to claim 16, for use in a method of treating or preventing a disease / condition characterized by extracellular matrix (ECM) degradation.

19. The antigen-binding molecule according to any one of claims 1 to 9, the CAR according to claim 10, the ADC according to claim 11, one or more of the nucleic acids according to claim 12, one or more of the expression vectors according to claim 13, the cell according to claim 14, or the composition according to claim 16, for use in a method of treating or preventing cancer.

20. The antigen-binding molecule, CAR, ADC, one or more nucleic acids, one or more expression vectors, cell or composition for use according to claim 19, wherein, The cancer is selected from: liver cancer, breast cancer, oral cancer, oral squamous cell carcinoma, sarcoma, lung cancer, prostate cancer, bladder cancer, kidney cancer, melanoma, pancreatic cancer, endometrial cancer, colorectal cancer, ovarian cancer, cervical cancer, brain cancer, cholangiocarcinoma, testicular cancer, and thyroid cancer.

21. The antigen-binding molecule according to any one of claims 1 to 9, the CAR according to claim 10, the ADC according to claim 11, the one or more nucleic acids according to claim 12, the one or more expression vectors according to claim 13, the cell according to claim 14, or the composition according to claim 16, for use in a method of treating or preventing cartilage degradation, or a disease / condition characterized by cartilage degradation.

22. The antigen-binding molecule, CAR, ADC, one or more nucleic acids, one or more expression vectors, cell or composition for use as defined in claim 21, wherein, the disease / condition characterized by cartilage degradation is selected from: joint disease, arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, juvenile arthritis, post-traumatic arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage injury, and polychondritis.

23. Use of the antigen-binding molecule according to any one of claims 1 to 9 for eliminating cells expressing CNX or increasing the killing of cells expressing CNX.

24. An optionally isolated in vitro complex comprising the antigen-binding molecule according to any one of claims 1 to 9 that binds to CNX.

25. A method for detecting CNX in a sample, comprising contacting a sample containing or suspected of containing CNX with the antigen-binding molecule according to any one of claims 1 to 9, and detecting the complex formed between the antigen-binding molecule and CNX.

26. A method for selecting or stratifying a subject for treatment with a CNX-targeted drug, the method comprising contacting a sample of the subject with the antigen-binding molecule according to any one of claims 1 to 9 in vitro, and detecting the complex formed between the antigen-binding molecule and CNX.

27. Use of the antigen-binding molecule according to any one of claims 1 to 9 as a drug for in vitro or in vivo diagnosis or prognosis.

Citation Information

Patent Citations

Monoclonal antibody of anti-human calnexin protein, preparation method and application thereof
CN101659702A
Therapeutic Antibodies
US20150044231A1
Anti-CD3 immunotoxins and therapeutic uses therefor
WO2000041474A2
Method for the selection of antibodies against bcma
WO2014122143A1
Virtual multiphase flow metering and sand detection
WO2016003985A1

Patent Information

AI Technical Summary

Abstract

Description

Patent Citations

Monoclonal antibody of anti-human calnexin protein, preparation method and application thereof

Therapeutic Antibodies

Anti-CD3 immunotoxins and therapeutic uses therefor

Method for the selection of antibodies against bcma

Virtual multiphase flow metering and sand detection