Anti-il12p35 antibodies and uses thereof

Abstract

The present disclosure relates to anti-IL12p35 (interleukin 12 subunit alpha) antibodies, antigen-binding fragments, and uses thereof. In some embodiments, the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein can be used to treat systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren syndrome (SjS).

Description

[0001] Cross-reference to related applications

[0002] This disclosure claims priority and benefit to U.S. Provisional Patent Application Serial No. 63 / 627,553, filed January 31, 2024, which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to antibodies that can bind to IL12p35 (interleukin 12 subunit alpha) and their uses. Background Technology

[0004] Autoimmune diseases occur when the immune system malfunctions and mistakenly attacks healthy cells. There are over 80 recognized autoimmune conditions affecting approximately one in ten individuals (Conrad, N., et al. “Incidence, prevalence, and co-occurrence of autoimmune disorders over time and by age, sex, and socioeconomic status: a population-based cohort study of 22 million individuals in the UK.” The Lancet 401.10391 (2023): 1878–1890). In the United States alone, over 24 million people suffer from one or more of these diseases.

[0005] Current treatment options for autoimmune diseases primarily involve immunosuppressive drugs and biologics / small molecule drugs that target inflammatory cytokines, immune cells, and intracellular kinases. Effective treatment strategies focus on the IL12 / IL23 pathway, resulting in numerous drug approvals for various autoimmune diseases. However, evidence suggests that IL12 / IL23 pathway-specific genes are effective targets for a group of autoimmune diseases, but not for others. Therefore, there is a need to develop more therapies to treat autoimmune diseases with different pathological mechanisms. Summary of the Invention

[0006] This disclosure relates to anti-IL12p35 antibodies, their antigen-binding fragments, and their uses. This disclosure also demonstrates that the development of IL12p35 / IL12Rβ2 antagonists will be useful in treating a group of autoimmune diseases, such as systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS).

[0007] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to the interleukin 12 subunit alpha (IL12p35), comprising: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, wherein in some embodiments, the selected VH CDR... The amino acid sequences 1, 2, and 3 and the selected VL CDR are selected from... Figure 26 or Figure 27 The VH CDR 1, 2, 3 and VL CDR 1, 2, 3 are listed in the table.

[0008] In some embodiments, the selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 47, 48, and 49, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 50, 51, and 52, respectively; (2) the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 53, 54, and 55, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 56, 57, and 58, respectively; (3) the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 59, 60, and 61, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 62, 63, and 64, respectively; (4) the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 59, 60, and 61, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 59, 60, and 61, respectively; NO: 65, 66, 67, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 68, 69, 70, respectively; (5) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 71, 72, 73, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 74, 75, 76, respectively; (6) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 77, 78, 79, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 80, 81, 82, respectively; (7) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 83, 84, 85, and the selected VLCDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 86, 87, 88, respectively; (8) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 65, 66, 67, 68, respectively. (9) The selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 92, 93, and 94, respectively; (10) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 95, 96, and 97, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 98, 99, and 100, respectively; (11) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 101, 102, and 103, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 104, 105, and 106, respectively;(11) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 107, 108, and 109, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 110, 111, and 112, respectively; (12) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 113, 114, and 115, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 116, 117, and 118, respectively; (13) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 119, 120, and 121, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 122, 123, and 124, respectively; (14) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 107, 108, and 109, respectively. NO: 125, 126, 127, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 128, 129, 130, respectively; (15) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 131, 132, 133, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 134, 135, 136, respectively; (16) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 137, 138, 139, and the selected VLCDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 140, 141, 142, respectively; (17) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 143, 144, 145, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 128, 129, 130, respectively. ID NO: 146, 147, 148; (18) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 149, 150, 151, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 152, 153, 154, respectively; (19) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 155, 156, 157, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 158, 159, 160, respectively; (20) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 161, 162, 163, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 164, 165, 166, respectively;(21) The selected VH CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 167, 168, and 169, respectively, and the selected VL CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 170, 171, and 172, respectively; (22) The selected VH CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 173, 174, and 175, respectively, and the selected VL CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 176, 177, and 178, respectively; and (23) The selected VH CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 179, 180, and 181, respectively, and the selected VLCDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 182, 183, and 184, respectively. In some embodiments, the CDR is defined by Kabat.

[0009] In some embodiments, the selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 185, 186, and 187, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 188, 189, and 190, respectively; (2) the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 191, 192, and 193, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 194, 195, and 196, respectively; (3) the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 197, 198, and 199, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 200, 201, and 202, respectively; (4) the selected VHCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 197, 198, and 199, respectively; and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 200, 201, and 202, respectively; The amino acid sequences of the selected VL CDRs are shown in SEQ ID NO: 203, 204, 205, and the amino acid sequences of the selected VL CDRs are shown in SEQ ID NO: 206, 207, 208, respectively; (5) The amino acid sequences of the selected VH CDRs are shown in SEQ ID NO: 209, 210, 211, and the amino acid sequences of the selected VL CDRs are shown in SEQ ID NO: 212, 213, 214, respectively; (6) The amino acid sequences of the selected VH CDRs are shown in SEQ ID NO: 215, 216, 217, and the amino acid sequences of the selected VL CDRs are shown in SEQ ID NO: 218, 219, 220, respectively; (7) The amino acid sequences of the selected VH CDRs are shown in SEQ ID NO: 221, 222, 223, and the amino acid sequences of the selected VL CDRs are shown in SEQ ID NO: 206, 207, 208, respectively. (8) The amino acid sequences of the selected VH CDR are shown in SEQ ID NO: 224, 225, 226, respectively; and the amino acid sequences of the selected VL CDR are shown in SEQ ID NO: 227, 228, 229, respectively. The amino acid sequences of the selected VL CDR are shown in SEQ ID NO: 230, 231, 232, respectively. The amino acid sequences of the selected VH CDR are shown in SEQ ID NO: 233, 234, 235, respectively. The amino acid sequences of the selected VL CDR are shown in SEQ ID NO: 236, 237, 238, respectively. The amino acid sequences of the selected VH CDR are shown in SEQ ID NO: 239, 240, 241, respectively. The amino acid sequences of the selected VL CDR are shown in SEQ ID NO: 242, 243, 244, respectively.(11) The selected VHCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 245, 246, and 247, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 248, 249, and 250, respectively; (12) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 251, 252, and 253, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 254, 255, and 256, respectively; (13) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 257, 258, and 259, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 260, 261, and 262, respectively; (14) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 263, 264, and 265, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 260, 261, and 262, respectively; The amino acid sequences of CDR 1, 2, and 3 are shown in SEQ ID NO: 266, 267, and 268, respectively; (15) The amino acid sequences of the selected VH CDR 1, 2, and 3 are shown in SEQ ID NO: 269, 270, and 271, respectively, and the amino acid sequences of the selected VL CDR 1, 2, and 3 are shown in SEQ ID NO: 272, 273, and 274, respectively; (16) The amino acid sequences of the selected VH CDR 1, 2, and 3 are shown in SEQ ID NO: 275, 276, and 277, respectively, and the amino acid sequences of the selected VL CDR 1, 2, and 3 are shown in SEQ ID NO: 278, 279, and 280, respectively; (17) The amino acid sequences of the selected VH CDR 1, 2, and 3 are shown in SEQ ID NO: 281, 282, and 283, respectively, and the amino acid sequences of the selected VL CDR 1, 2, and 3 are shown in SEQ ID NO: 284, 285, and 286, respectively; (18) The selected VH CDR The amino acid sequences of 1, 2, and 3 are shown in SEQ ID NO: 287, 288, and 289, respectively, and the amino acid sequences of the selected VL CDR 1, 2, and 3 are shown in SEQ ID NO: 290, 291, and 292, respectively; (19) The amino acid sequences of the selected VH CDR 1, 2, and 3 are shown in SEQ ID NO: 293, 294, and 295, respectively, and the amino acid sequences of the selected VL CDR 1, 2, and 3 are shown in SEQ ID NO: 296, 297, and 298, respectively; (20) The amino acid sequences of the selected VH CDR 1, 2, and 3 are shown in SEQ ID NO: 299, 300, and 301, respectively, and the amino acid sequences of the selected VL CDR 1, 2, and 3 are shown in SEQ ID NO: 302, 303, and 304, respectively;(21) The selected VH CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 305, 306, and 307, respectively, and the selected VL CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 308, 309, and 310, respectively; (22) The selected VH CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 311, 312, and 313, respectively, and the selected VL CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 314, 315, and 316, respectively; and (23) The selected VHCDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 317, 318, and 319, respectively, and the selected VL CDR amino acid sequences 1, 2, and 3 are shown in SEQ ID NO: 320, 321, and 322, respectively. In some embodiments, the CDR is determined by the IMGT definition.

[0010] In some embodiments, the selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) according to the Kabat definition, the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 155, 156, and 157, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 158, 159, and 160, respectively; (2) according to the Kabat definition, the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 179, 180, and 181, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 182, 183, and 184, respectively; (3) according to the IMGT definition, the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 293, 294, and 295, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 158, 159, and 160, respectively. NO: 296, 297, 298; and (4) according to the Kabat definition, the selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 317, 318, 319, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 320, 321, 322, respectively.

[0011] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to IL12p35, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the light chain variable region (VL) comprises an amino acid sequence that is at least 90% identical to a selected VL sequence. In some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 1, and the selected VL sequence is SEQ ID NO: 2; (2) the selected VH sequence is SEQ ID NO: 3, and the selected VL sequence is SEQ ID NO: 4; (3) the selected VH sequence is SEQ ID NO: 5, and the selected VL sequence is SEQ ID NO: 6; (4) the selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8; (5) the selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 9. NO: 10; (6) The selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12; (7) The selected VH sequence is SEQ ID NO: 13, and the selected VL sequence is SEQ ID NO: 14; (8) The selected VH sequence is SEQ ID NO: 15, and the selected VL sequence is SEQ ID NO: 16; (9) The selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18; (10) The selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20; (11) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (12) The selected VH sequence is SEQ ID NO: 23, and the selected VL sequence is SEQ ID NO: 24; (13) The selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 26; (14 ... NO: 27, and the selected VL sequence is SEQ ID NO: 28; (15) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 30; (16) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32; (17) the selected VH sequence is SEQ ID NO: 33, and the selected VL sequence is SEQ ID NO: 34; (18) the selected VH sequence is SEQ ID NO: 35, and the selected VL sequence is SEQ ID NO: 36;(19) The selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38; (20) The selected VH sequence is SEQ ID NO: 39, and the selected VL sequence is SEQ ID NO: 40; (21) The selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 42; (22) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 44; (23) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46; (24) The selected VH sequence is SEQ ID NO: 335, and the selected VL sequence is SEQ ID NO: 336; and (25) The selected VH sequence is SEQ ID NO: 337, and the selected VL sequence is SEQ ID NO: 338. In some embodiments, the VH and / or VL contain one or more back-to-germline (B2G) mutations. In some embodiments, the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is 38. In some embodiments, the selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46. In some embodiments, the selected VH sequence is SEQ ID NO: 335, and the selected VL sequence is SEQ ID NO: 336. In some embodiments, the selected VH sequence is SEQ ID NO: 337, and the selected VL sequence is SEQ ID NO: 338.

[0012] In some embodiments, the antibody or its antigen-binding fragment can block the binding between interleukin-12 (e.g., human interleukin-12 (IL12)) and the beta 2 subunit of the interleukin-12 receptor (e.g., human interleukin-12 receptor beta 2 subunit (IL12Rβ2)). In some embodiments, the antibody or its antigen-binding fragment can block IL12-induced intracellular signaling (e.g., JAK-STAT signaling), optionally said IL12 is human or monkey IL12. In some embodiments, the antibody or its antigen-binding fragment can inhibit IL12-induced IFN-γ production in human PBMCs. In some embodiments, the antibody or its antigen-binding fragment can prevent IFN-γ production in CD4+ T cells co-cultured with allogeneic dendritic cells. In some embodiments, the antibody or antigen-binding fragment specifically binds to human IL12p35 and / or monkey IL12p35. In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or its antigen-binding fragment. In some implementations, the antibody or antigen-binding fragment is an F(ab')2 fragment, a single-chain variable fragment (scFV), or a multispecific antibody (e.g., a bispecific antibody).

[0013] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to IL12p35, comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, a first immunoglobulin light chain, and a second immunoglobulin light chain. In some embodiments, the first immunoglobulin heavy chain and the first immunoglobulin light chain associate with each other to form a first antigen-binding site for binding to IL12p35. In some embodiments, the second immunoglobulin heavy chain and the second immunoglobulin light chain associate with each other to form a first antigen-binding site for binding to IL12p35. In some embodiments: (1) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 325, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 326; (2) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 327, and the first and second immunoglobulin light chains comprise an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 326; (3) the first and second immunoglobulin heavy chains comprise an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 326. NO: 328 has at least 90% identical amino acid sequence, and the first and second immunoglobulin light chains contain at least 90% identical amino acid sequences to SEQ ID NO: 329; (4) the first and second immunoglobulin heavy chains contain at least 90% identical amino acid sequences to SEQ ID NO: 330, and the first and second immunoglobulin light chains contain at least 90% identical amino acid sequences to SEQ ID NO: 329; (5) the first and second immunoglobulin heavy chains contain at least 90% identical amino acid sequences to SEQ ID NO: 331, and the first and second immunoglobulin light chains contain at least 90% identical amino acid sequences to SEQ ID NO: 332; and (6) the first and second immunoglobulin heavy chains contain at least 90% identical amino acid sequences to SEQ ID NO: 333, and the first and second immunoglobulin light chains contain at least 90% identical amino acid sequences to SEQ ID NO: 334. In some embodiments, the first and second immunoglobulin heavy chains are identical, and in some embodiments, the first and second immunoglobulin light chains are identical. In some embodiments, the first and second immunoglobulin heavy chains contain an Fc region (e.g., an IgG1 Fc region). In some implementations, the Fc region contains a YTE mutation (M252Y / S254T / T256E according to the EU number).

[0014] In one aspect, this disclosure relates to an antibody or an antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising VH CDR1, VHCDR2, and VH CDR3 and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, wherein in some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are identical to VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the antibody or antigen-binding fragment thereof described herein.

[0015] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof described herein.

[0016] In one aspect, this disclosure relates to an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell inhibitor.

[0017] In one aspect, this disclosure relates to a method for suppressing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein or an antibody-drug conjugate. In some embodiments, the subject suffers from an immune disorder (e.g., an autoimmune disease or an inflammatory disease).

[0018] In one aspect, this disclosure relates to a method of treating a subject with an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof or an antibody-drug conjugate as described herein. In some embodiments, the subject has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS). In some embodiments, the subject does not have psoriasis (PsO), Crohn's disease (CD), ulcerative colitis (UC), inflammatory bowel disease (IBD), or ankylosing spondylitis (AS).

[0019] In one aspect, this disclosure relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof or an antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier.

[0020] In one aspect, this disclosure relates to a method for reducing IL12-induced intracellular signaling in cells, the method comprising contacting a subject with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein or an antibody-drug conjugate.

[0021] In one aspect, this disclosure relates to a method for identifying a subject as having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS), the method comprising determining the level of IL12p35 in a sample collected from the subject using an antibody or an antigen-binding fragment thereof described herein.

[0022] In one aspect, this disclosure relates to a method for determining the risk of a subject having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS), the method comprising determining the level of IL12p35 in a sample collected from the subject using an antibody or an antigen-binding fragment thereof described herein.

[0023] In one aspect, this disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising: (1) an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2 and 3, said complementarity-determining regions (CDRs) 1, 2 and 3 comprising as shown in Figure 26 or Figure 27 The VH CDRs 1, 2, and 3, and in some embodiments, the VH binds to IL12p35 when paired with a corresponding light chain variable region (VL); or (2) an immunoglobulin light chain or a fragment thereof comprising a VL containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 contain as shown in Figure 26 or Figure 27 VL CDRs 1, 2 and 3, wherein the VL binds to IL12p35 when paired with the corresponding VH.

[0024] In some embodiments, the polypeptide comprises: (1) an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise amino acid sequences shown in SEQ ID NO: 47, 48, and 49 (or shown in SEQ ID NO: 185, 186, and 187), and in some embodiments, the VH binds to IL12p35 when paired with a light chain variable region (VL) comprising an amino acid sequence shown in SEQ ID NO: 2; (2) an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise amino acid sequences shown in SEQ ID NO: 50, 51, and 52 (or shown in SEQ ID NO: 188, 189, and 190), and in some embodiments, the VL binds to IL12p35 when paired with a light chain variable region (VL) comprising an amino acid sequence shown in SEQ ID NO: 2; (3) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 53, 54 and 55 (or shown in SEQ ID NO: 191, 192 and 193), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 4; (4) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 56, 57 and 58 (or shown in SEQ ID NO: 194, 195 and 196), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 4; (5) An immunoglobulin heavy chain or a fragment thereof comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 59, 60 and 61 (or shown in SEQ ID NO: 197, 198 and 199), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 6; (6) An immunoglobulin light chain or a fragment thereof comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 62, 63 and 64 (or shown in SEQ ID NO: 200, 201 and 202), and in some embodiments, the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 5;(7) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 contain amino acid sequences shown in SEQ ID NO: 65, 66, and 67 (or shown in SEQ ID NO: 203, 204, and 205), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 8; (8) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 contain amino acid sequences shown in SEQ ID NO: 68, 69, and 70 (or shown in SEQ ID NO: 206, 207, and 208), and in some embodiments, wherein the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 7; (9) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3; 1, 2, and 3 VHs, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 71, 72, and 73 (or shown in SEQ ID NO: 209, 210, and 211 respectively), and in some embodiments, the VHs bind to IL12p35 when paired with a VL comprising an amino acid sequence shown in SEQ ID NO: 10; (10) an immunoglobulin light chain or a fragment thereof comprising VLs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 74, 75, and 76 (or shown in SEQ ID NO: 212, 213, and 214 respectively), and in some embodiments, the VLs bind to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 9; (11) an immunoglobulin heavy chain or a fragment thereof comprising VHs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 74, 75, and 76 (or shown in SEQ ID NO: 212, 213, and 214 respectively), and in some embodiments, the VLs bind to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 9; 1, 2 and 3 respectively contain the amino acid sequences shown in SEQ ID NO: 77, 78 and 79 (or shown in SEQ ID NO: 215, 216 and 217 respectively), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing the amino acid sequence of SEQ ID NO: 12; (12) an immunoglobulin light chain or a fragment thereof comprising a VL containing CDR 1, 2 and 3, the CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 80, 81 and 82 (or shown in SEQ ID NO: 218, 219 and 220 respectively), and in some embodiments, the VL binds to IL12p35 when paired with a VH containing the amino acid sequence of SEQ ID NO: 11;(13) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 respectively contain amino acid sequences shown in SEQ ID NO: 83, 84 and 85 (or shown in SEQ ID NO: 221, 222 and 223 respectively), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 14; (14) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 respectively contain amino acid sequences shown in SEQ ID NO: 86, 87 and 88 (or shown in SEQ ID NO: 224, 225 and 226 respectively), and in some embodiments, the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 13; (15) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3; 1, 2, and 3 VHs, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 89, 90, and 91 (or shown in SEQ ID NO: 227, 228, and 229 respectively), and in some embodiments, the VHs bind to IL12p35 when paired with a VL comprising an amino acid sequence shown in SEQ ID NO: 16; (16) an immunoglobulin light chain or a fragment thereof comprising VLs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 92, 93, and 94 (or shown in SEQ ID NO: 230, 231, and 232 respectively), and in some embodiments, the VLs bind to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 15; (17) an immunoglobulin heavy chain or a fragment thereof comprising CDRs (18) VHs comprising CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 respectively contain amino acid sequences shown in SEQ ID NO: 95, 96 and 97 (or shown in SEQ ID NO: 233, 234 and 235 respectively), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 18; (18) Immunoglobulin light chains or fragments thereof comprising VLs comprising CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 respectively contain amino acid sequences shown in SEQ ID NO: 98, 99 and 100 (or shown in SEQ ID NO: 236, 237 and 238 respectively), and in some embodiments, wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 17;(19) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 101, 102, and 103 (or shown in SEQ ID NO: 239, 240, and 241 respectively), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 20; (20) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 104, 105, and 106 (or shown in SEQ ID NO: 242, 243, and 244 respectively), and in some embodiments, the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 19; (21) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3; 1, 2, and 3 VHs, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 107, 108, and 109 (or shown in SEQ ID NO: 245, 246, and 247 respectively), and in some embodiments, the VHs bind to IL12p35 when paired with a VL comprising an amino acid sequence shown in SEQ ID NO: 22; (22) an immunoglobulin light chain or a fragment thereof comprising VLs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 110, 111, and 112 (or shown in SEQ ID NO: 248, 249, and 250 respectively), and in some embodiments, the VLs bind to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 21; (23) an immunoglobulin heavy chain or a fragment thereof comprising VHs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 107, 108, and 109 (or shown in SEQ ID NO: 245, 246, and 247 respectively); 1, 2 and 3 respectively contain the amino acid sequences shown in SEQ ID NO: 113, 114 and 115 (or shown in SEQ ID NO: 251, 252 and 253 respectively), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 24; (24) an immunoglobulin light chain or a fragment thereof comprising a VL containing CDR 1, 2 and 3, the CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 116, 117 and 118 (or shown in SEQ ID NO: 254, 255 and 256 respectively), and in some embodiments, the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 23;(25) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 119, 120, and 121 (or shown in SEQ ID NO: 257, 258, and 259), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 26; (26) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 122, 123, and 124 (or shown in SEQ ID NO: 260, 261, and 262), and in some embodiments, wherein the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 25; (27) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3; 1, 2, and 3 VHs, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 125, 126, and 127 (or shown in SEQ ID NO: 263, 264, and 265), and in some embodiments, the VHs bind to IL12p35 when paired with a VL comprising an amino acid sequence shown in SEQ ID NO: 28; (28) an immunoglobulin light chain or a fragment thereof comprising VLs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 128, 129, and 130 (or shown in SEQ ID NO: 266, 267, and 268), and in some embodiments, the VLs bind to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 27; (29) an immunoglobulin heavy chain or a fragment thereof comprising VHs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 125, 126, and 127 (or shown in SEQ ID NO: 263, 264, and 268), and in some embodiments, the VLs bind to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 27; 1, 2 and 3 respectively contain the amino acid sequences shown in SEQ ID NO: 131, 132 and 133 (or shown in SEQ ID NO: 269, 270 and 271 respectively), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 30; (30) an immunoglobulin light chain or a fragment thereof comprising a VL containing CDR 1, 2 and 3, the CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 134, 135 and 136 (or shown in SEQ ID NO: 272, 273 and 274 respectively), and in some embodiments, the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 29;(31) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 137, 138, and 139 (or shown in SEQ ID NO: 275, 276, and 277 respectively), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 32; (32) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 140, 141, and 142 (or shown in SEQ ID NO: 278, 279, and 280 respectively), and in some embodiments, the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 31; (33) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 140, 141, and 142 (or shown in SEQ ID NO: 278, 279, and 280 respectively); 1, 2, and 3 VHs, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 143, 144, and 145 (or shown in SEQ ID NO: 281, 282, and 283), and in some embodiments, the VHs bind to IL12p35 when paired with a VL comprising an amino acid sequence shown in SEQ ID NO: 34; (34) an immunoglobulin light chain or a fragment thereof comprising VLs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 146, 147, and 148 (or shown in SEQ ID NO: 284, 285, and 286), and in some embodiments, the VLs bind to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 33; (35) an immunoglobulin heavy chain or a fragment thereof comprising VHs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 143, 144, and 145 (or shown in SEQ ID NO: 284, 285, and 286), and in some embodiments, the VLs bind to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 33; 1, 2 and 3 respectively contain the amino acid sequences shown in SEQ ID NO: 149, 150 and 151 (or shown in SEQ ID NO: 287, 288 and 289 respectively), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 36; (36) an immunoglobulin light chain or a fragment thereof comprising a VL containing CDR 1, 2 and 3, the CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 152, 153 and 154 (or shown in SEQ ID NO: 290, 291 and 292 respectively), and in some embodiments, the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 35;(37) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 155, 156, and 157 (or shown in SEQ ID NO: 293, 294, and 295), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 38 or 336; (38) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 158, 159, and 160 (or shown in SEQ ID NO: 296, 297, and 298), and in some embodiments, wherein the VL binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 38 or 336; (39) An immunoglobulin heavy chain or a fragment thereof comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 161, 162 and 163 (or shown in SEQ ID NO: 299, 300 and 301), and in some embodiments, the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 40; (40) An immunoglobulin light chain or a fragment thereof comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 164, 165 and 166 (or shown in SEQ ID NO: 302, 303 and 304), and in some embodiments, the VL binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 40; (41) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 167, 168 and 169 (or shown in SEQ ID NO: 305, 306 and 307), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 42; (42) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 170, 171 and 172 (or shown in SEQ ID NO: 308, 309 and 310), and in some embodiments, wherein the VL binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 39; When the amino acid sequence of NO:41 is paired with VH, it binds to IL12p35;(43) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 contain amino acid sequences shown in SEQ ID NO: 173, 174, and 175 (or shown in SEQ ID NO: 311, 312, and 313), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL containing the amino acid sequence shown in SEQ ID NO: 44; (44) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 contain amino acid sequences shown in SEQ ID NO: 176, 177, and 178 (or shown in SEQ ID NO: 314, 315, and 316), and in some embodiments, wherein the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 43; (45) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 contain amino acid sequences shown in SEQ ID NO: 176, 177, and 178 (or shown in SEQ ID NO: 314, 315, and 316), and in some embodiments, wherein the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 43; VHs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 179, 180, and 181 (or shown in SEQ ID NO: 317, 318, and 319), and in some embodiments, wherein the VH binds to IL12p35 when paired with a VL comprising an amino acid sequence shown in SEQ ID NO: 46 or 338; or (46) an immunoglobulin light chain or a fragment thereof comprising VLs comprising CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively comprise amino acid sequences shown in SEQ ID NO: 182, 183, and 184 (or shown in SEQ ID NO: 320, 321, and 322), and in some embodiments, wherein the VL binds to IL12p35 when paired with a VH comprising an amino acid sequence shown in SEQ ID NO: 45 or 337.

[0025] In some embodiments, VH specifically binds to human IL12p35 when paired with VL; or VL specifically binds to human IL12p35 when paired with VH. In some embodiments, the immunoglobulin heavy chain or a fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or a fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof. In some embodiments, the nucleic acid encodes an F(ab')2 fragment, a single-stranded variable fragment (scFv), or a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the nucleic acid is cDNA.

[0026] In one aspect, this disclosure relates to a vector comprising one or more nucleic acids described herein. In another aspect, this disclosure relates to a vector comprising two nucleic acids described herein, wherein in some embodiments, the vector encodes a VL region and a VH region that commonly bind to IL12p35. In one aspect, this disclosure relates to a pair of vectors, wherein in some embodiments, each vector comprises one nucleic acid described herein, and in some embodiments, the pair of vectors commonly encodes a VL region and a VH region that commonly bind to IL12p35.

[0027] In one aspect, this disclosure relates to a cell comprising one or more of the vectors described herein. In some embodiments, the cell is a CHO cell. In one aspect, this disclosure relates to a cell comprising one or more nucleic acids described herein. In one aspect, this disclosure relates to a cell comprising two nucleic acids described herein. In some embodiments, the two nucleic acids co-encode a VL region and a VH region that co-bind with IL12p35.

[0028] In one aspect, this disclosure relates to a method for generating an antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing the cells described herein in a manner sufficient to induce the cells to generate an antibody or an antigen-binding fragment thereof; and (b) collecting the antibody or antigen-binding fragment thereof generated by the cells.

[0029] In one aspect, this disclosure relates to a method of treating a subject with an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof that binds to IL12p35 and / or an antibody or antigen-binding fragment thereof that binds to IL12Rβ1. In some embodiments, the antibody or antigen-binding fragment thereof that binds to IL12p35 does not bind to IL12p40. In some embodiments, the antibody or antigen-binding fragment thereof that binds to IL12Rβ2 does not bind to IL12Rβ1. In some embodiments, the antibody or antigen-binding fragment thereof does not interfere with the IL23 pathway and / or the IL35 pathway. In some embodiments, the subject is a human subject. In some embodiments, the subject has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS). In some embodiments, the antibody or antigen-binding fragment thereof is a human or humanized antibody or its antigen-binding fragment. In some embodiments, the subject is a non-human mammal, such as a monkey, dog, or mouse. In some embodiments, the mammal suffers from a disease or condition similar to systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS). In some embodiments, the subject is a dog. In some embodiments, the antibody or its antigen-binding fragment is a canine or canine-derived antibody or its antigen-binding fragment.

[0030] As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (e.g., any of the three CDRs from the immunoglobulin light chain or any of the three CDRs from the immunoglobulin heavy chain) and capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may comprise the Fc region of a human antibody. The term antibody also includes derivatives formed from antibody fragments, such as bispecific antibodies, single-chain antibodies, diabody antibodies, linear antibodies, and multispecific antibodies.

[0031] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment includes at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0032] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid present in humans (e.g., a rearranged heavy or light chain human immunoglobulin locus). In some embodiments, human antibodies are collected from humans or generated in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are generated in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are generated in bacterial or yeast cells. In some embodiments, human antibodies are generated in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0033] As used herein, the term "chimeric antibody" refers to an antibody containing sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant domain of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

[0034] As used herein, the term "humanized antibody" refers to a non-human antibody containing a minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and containing a sequence derived from a human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) wherein the hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., donor antibody) (e.g., mouse, rat, or rabbit antibody) having the desired specificity, affinity, and capability. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications can be made to further improve antibody performance. In some embodiments, the humanized antibody contains at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to those of non-human (e.g., mouse) immunoglobulins, and all or substantially all of the framework regions are those of human immunoglobulins. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of human immunoglobulins. Humanized antibodies can be generated using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0035] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are capable of binding specifically to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0036] As used herein, the term "multimeric antibody" refers to an antibody containing four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is capable of crosslinking a target molecule (e.g., IL12p35) to at least one second target molecule (e.g., IL12p35) on the surface of mammalian cells.

[0037] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe animals, humans, or non-humans to whom treatment is provided by the method according to the invention. The invention is contemplated for both veterinary and non-veterinary applications. Human patients can be adults or minors (e.g., persons under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, and gorillas), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), horses, dogs, felines, cattle, and other livestock, farm animals, and zoo animals.

[0038] As used herein, when referring to antibodies, the phrase "specifically binding" means that an antibody prefers to interact with its target molecule (e.g., IL12p35) compared to other molecules, because the interaction depends on the presence of a specific structure on the target molecule (i.e., an antigenic determinant or epitope); in other words, the reagent typically recognizes and binds to molecules that include a specific structure rather than all molecules. Antibodies that specifically bind to a target molecule can be called target-specific antibodies. For example, antibodies that specifically bind to the IL12p35 molecule can be called IL12p35-specific antibodies or anti-IL12p35 antibodies.

[0039] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to an amino acid polymer of any length containing at least two amino acids.

[0040] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably to refer to a nucleotide polymer of any length containing at least two nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids, and their modifications.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including the definitions, shall prevail.

[0042] Other features and advantages of the invention will be apparent from the following detailed description and drawings, as well as from the claims. Attached Figure Description

[0043] Figure 1 It is displayed via Gator ® The table shows the preliminary binding affinity of the anti-IL12p35 antibody against human IL12p70, as determined by the biolayer interferometry platform. Anti-human IL12p40 (anti-hP40) was used as a reference antibody.

[0044] Figure 2A It is displayed via Gator ® A table showing the binding affinity of selected anti-IL12p35 antibodies and their YTE variants to human, rhesus monkey, canine, or mouse IL12p70, as determined by a biolayer interferometry platform. 27H28L is an engineered antibody composed of the heavy chain of D2M008-A1A8 and the light chain of D2M008-A1A9. YTE mutations in anti-IL12p35 antibodies 27H28L and D2M008-4A4 were introduced via amino acid substitutions (M252Y / S254T / T256E) in the Fc region, generating “27H28L YTE” and “D2M-4A4YTE”, respectively. 27H28L-hblb-YTE and 4A4-hblb2-YTE have reverting germline mutations within the variable regions of “27H28L YTE” and “D2M-4A4 YTE”, respectively. OR indicates out of range. ND indicates not performed. NB means no binding was detected.

[0045] Figure 2B Displayed via BiaCore TM The binding kinetics and fitting curves of 27H28L-hblb-YTE and 4A4-hblb2-YTE with human IL12p70 were determined by the SPR platform.

[0046] Figure 2C It displays method information and is accessed through BiaCore. TMA table showing the parameters of 27H28L-hblb-YTE and 4A4-hblb2-YTE obtained from the 8K SRP platform.

[0047] Figure 3A Displayed via Gator ® The binding affinity of 27H28L and its YTE variant (“27H28L YTE”) to recombinant FcRn protein was determined by a biolayer interferometry platform at pH 6.0, pH 6.5 or pH 7.0.

[0048] Figure 3B Displayed via Gator ® The binding affinity of D2M008-4A4 and its YTE variant (“D2M-4A4 YTE”) to recombinant FcRn protein was determined by a biolayer interferometry platform at pH 6.0, pH 6.5, or pH 7.0.

[0049] Figure 4A This study demonstrates the inhibitory rates of different anti-IL12p35 antibodies against the binding of human IL12p70 to its receptor IL12Rβ2. All fixed concentrations of antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes prior to incubation with IL12Rβ2. Inhibition levels were calculated based on OD450 as measured by sandwich ELISA.

[0050] Figure 4B The inhibition rate of the selected anti-IL12p35 antibody in blocking the binding of human IL12p70 to its receptor IL12Rβ2 is shown. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes prior to incubation with IL12Rβ2. Inhibition levels were calculated based on OD450 as measured by sandwich ELISA.

[0051] Figure 4C The blocking effect of 27H28L and additional anti-IL12p35 antibody on the binding of human IL12p70 to its receptor IL12Rβ2 was demonstrated. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes prior to incubation with IL12Rβ2. The IC50 values ​​of 27H28L and the selected anti-IL12p35 antibody were calculated. 50 value.

[0052] Figure 4D The inhibitory rates of anti-IL12p35 antibodies 27H28L, D2M008-4A4, and their YTE variants in blocking the binding of human IL12p70 to its receptor IL12Rβ2 are shown. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes prior to incubation with IL12Rβ2. Inhibition levels were calculated based on OD450 as measured by sandwich ELISA.

[0053] Figure 4E The inhibition rates of anti-IL12p35 antibodies 27H28L-YTE and D2M008-4A4 YTE, and their variants with reversal germline mutations, in blocking the binding of human IL12p70 to its receptor IL12Rβ2 are shown. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes prior to incubation with IL12Rβ2. Inhibition levels were calculated based on OD450 as determined by sandwich ELISA.

[0054] Figure 5A Displayed in HEK-Blue TM The inhibition rates of different anti-IL12p35 antibodies against human IL12p70-induced signal transduction in IL12 reporter cells were investigated. All fixed concentrations of antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before incubation with reporter cells. Inhibition levels were evaluated by measuring the reporter gene signal at OD620 and performing calculations based on this signal.

[0055] Figure 5B Displayed in HEK-Blue TM In IL12 reporter cells, the selected anti-IL12p35 antibody was used to inhibit the signal transduction induced by human IL12p70. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes before incubation with reporter cells. The level of inhibition was calculated based on the reporter gene signal at OD620.

[0056] Figure 5C Displayed in HEK-Blue TM In IL12 reporter cells, the selected anti-IL12p35 antibodies (D2M008-A1A8, D2M008-A1A9, and 27H28L), anti-IL12p40 (ustekinumab, anti-hP40), and the combination of D2M008-A1A8 and anti-hP40 inhibited human IL12p70-induced signal transduction. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 min before incubation with reporter cells. Inhibition levels were calculated based on reporter gene signaling at OD620. The IC50 values ​​of the antibodies and the combination of D2M008-A1A8 and anti-hP40 were calculated.

[0057] Figure 5D Displayed in HEK-Blue TMThe inhibition rates of selected anti-IL12p35 antibody and 27H28L against human IL12p70-induced signal transduction in IL12 reporter cells were measured. Anti-human IL12p40 (ustekinumab, anti-hP40) was used as a reference antibody. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 min before incubation with reporter cells. Inhibition levels were calculated based on reporter gene signal at OD620. The IC50 values ​​of the antibodies were calculated.

[0058] Figure 5E Displayed in HEK-Blue TM In IL12 reporter cells, the inhibitory levels of anti-IL12p35 antibodies 27H28L, D2M008-4A4, and their YTE variants blocked human IL12p70-induced signal transduction. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes prior to incubation with reporter cells. Inhibition levels were evaluated by measuring reporter gene signaling at OD620.

[0059] Figure 5F Displayed in HEK-Blue TM In IL12 reporter cells, anti-IL12p35 antibodies 27H28L-YTE and D2M008-4A4YTE, along with their variants containing reversal germline mutations, blocked the inhibitory levels of human IL12p70-induced signal transduction. Serially diluted antibodies were pre-incubated with human IL12p70 at room temperature for 30 minutes prior to incubation with reporter cells. Inhibition levels were assessed by measuring reporter gene signaling at OD620.

[0060] Figure 6 Displayed in HEK-Blue TM In IL12 reporter cells, anti-IL12p35 antibodies D2M008-4A4 and 27H28L blocked the inhibitory level of IL12-induced signal transduction in rhesus monkeys. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted antibodies were pre-incubated with rhesus monkey IL12p70 at room temperature for 30 minutes before incubation with reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.

[0061] Figures 7A-7C This study demonstrates the level of IFN-γ production in vitro by human PBMCs stimulated with exogenous IL12. PBMCs from three healthy donors (#20, #22, and #23) were used. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted anti-IL12p35 antibody was pre-incubated with exogenous IL12p70 at room temperature for 30 minutes prior to incubation with PBMCs. IFN-γ production was determined by sandwich ELISA.

[0062] Figures 8A-8BThis demonstrates the level of IFN-γ produced in vitro by human CD4+ T cells co-cultured with allogeneic dendritic cells. Obtained using CD4+ T cells from donor #4. Figure 8A The data was obtained using CD4+ T cells from donor #8. Figure 8B Data from [the study / information source]. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted antibodies were mixed separately with dendritic cells (DCs) derived from monocytes from donor #18 before co-culturing with human CD4+ T cells. The ratio of dendritic cells to CD4+ T cells was 1:10. IFN-γ production was measured by sandwich ELISA.

[0063] Figures 9A-9B Displayed in HEK-Blue TM In IL23 reporter cells, anti-IL12p35 antibody 27H28L ( Figure 9A ), D2M007-4A4 ( Figure 9B The inhibitory levels of IL23-induced signal transduction were assessed by anti-human IL12p40 and its YTE variants. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. Serially diluted antibodies were pre-incubated with human IL23 at room temperature for 30 minutes before incubation with reporter cells. The level of inhibition was evaluated by measuring the reporter gene signal at OD620.

[0064] Figure 10A Melting curves of the complete IgG and F(ab')2 fragments of anti-IL12p35 antibodies 27H28L and D2M008-4A4 are shown. Anti-human IL12p40 (anti-hP40) was used as a reference antibody. qPCR was performed using a Protein ThermalShift instrument. TM The dye kit was used to determine the melting curve. The fluorescence intensity was subtracted from the lowest intensity (bottom) before the peak and normalized relative to the amplitude from bottom to peak.

[0065] Figure 10B T cells showing intact IgG and F(ab')2 fragments of anti-IL12p35 antibodies 27H28L and D2M008-4A4 m Anti-human IL12p40 (anti-hP40) was used as a reference antibody.

[0066] Figure 11A This study demonstrates the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking the binding of IL12p70 to its receptor IL12Rβ2 after exposure to stress conditions. The antibodies were subjected to stress at 40°C for two weeks in buffers at pH 5.5, pH 7.4, or pH 8.5. Their blocking kinetics were determined by ELISA.

[0067] Figure 11B This was shown in HEK-Blue after exposure to stress conditions. TM Functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70-induced transduction signaling in IL12 reporter cells. The antibodies were subjected to stress at 40°C for two weeks in buffers at pH 5.5, pH 7.4, or pH 8.5. Their blocking kinetics were evaluated by measuring reporter gene signaling at OD620.

[0068] Figure 12A This study demonstrates the functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking the binding of IL12p70 to its receptor IL12Rβ2 after exposure to human plasma or PBS. The antibodies were subjected to stress at 37°C for two weeks in fresh human plasma or PBS from different donors. Their blocking kinetics were determined by ELISA. Plasma 20 and Plasma 21 represent human plasma samples from two different donors.

[0069] Figure 12B The effect was observed in HEK-Blue after exposure to human plasma or PBS. TM Functional stability of anti-IL12p35 antibodies 27H28L and D2M008-4A4 in blocking IL12p70-induced transduction signaling in IL12 reporter cells. The antibodies were subjected to stress at 37°C for two weeks in fresh human plasma or PBS from different donors. Their blocking kinetics were evaluated by measuring the reporter gene signal at OD620. Plasma 20 and Plasma 21 represent human plasma samples from two different donors.

[0070] Figure 12C This study demonstrates the functional stability of the anti-IL12p35 antibody 27H28L-hblb-YTE in blocking the binding of IL12p70 to its receptor IL12Rβ2 after exposure to human plasma for up to 3 weeks. The antibodies were subjected to stress at 37°C for 0, 1, 2, or 3 weeks in fresh human plasma or PBS from different donors. Their blocking kinetics were determined by ELISA. Plasma samples 42, 45, 53, and 54 represent human plasma samples from four different donors.

[0071] Figure 12D This study demonstrates the functional stability of the anti-IL12p35 antibody 4A4-hblb2-YTE in blocking the binding of IL12p70 to its receptor IL12Rβ2 after exposure to human plasma for up to 3 weeks. The antibodies were subjected to stress at 37°C for 0, 1, 2, or 3 weeks in fresh human plasma or PBS from different donors. Their blocking kinetics were determined by ELISA. Plasma samples 42, 45, 53, and 54 represent human plasma samples from four different donors.

[0072] Figure 13A This displays the results of a binning competition assay performed on the selected anti-IL12p35 antibody. White indicates no competition, and black indicates competition between antibody pairs.

[0073] Figure 13B This demonstrates the competition between anti-IL12p35 antibodies 27H28L and D2M008-4A4 in binding to IL12. Anti-human Fc probes were used to immobilize one antibody (D2M008-4A4 and 27H28L, respectively), which was then incubated with human IL12p70, followed by additional incubation with a second antibody (27H28L and D2M008-4A4, respectively). Gator was used. ® The biolayer interferometry platform combines measurement and analysis capabilities.

[0074] Figure 13C This demonstrates the competition between anti-IL12p35 antibodies 27H28L and D2M008-4A4 in binding to IL12. Anti-His probes were used to immobilize human IL12p70, followed by incubation with one antibody (D2M008-4A4 and 27H28L, respectively), and then additional incubation with a second antibody (either 27H28L or D2M008-4A4, respectively). Gator was used. ® The biolayer interferometry platform combines measurement and analysis capabilities.

[0075] Figure 14 This diagram illustrates IL12-related cytokines, receptors, and signaling pathways. The illustration is adapted from Choi, J., et al., “IL-35 and autoimmunity: a comprehensive perspective.” Clinical Reviews in Allergy & Immunology 49 (2015): 327-332.

[0076] Figures 15A-15C This plot shows the correlation between genetic markers at the IL12A locus and IL12A expression (IL12A eQTL) and nine inflammatory diseases. Each point in the plot represents a genetic marker. The X-axis represents the genomic location of the genetic marker. This genetic region spans 500 KB of the genome, centered on the genomic location of the IL12A gene. The Y-axis represents the significance p-value (in log₂O₅) for each genetic marker. 10 (p-value is a metric). The horizontal line represents the significance threshold for GWAS (p-value 5 × 10⁻⁶). -8The circle size for each genetic marker was categorized by its correlation with the primary variant of IL12 eQTL (rs4680586).

[0077] Figures 16A-16C This is a region map showing the association between genetic markers at the IL12Rβ2 locus and IL12RB2 expression (IL12RB2 eQTL) and nine inflammatory diseases. Each point in the map represents a genetic marker. The X-axis represents the genomic location of the genetic marker. This genetic region spans 500 KB of the genome, centered on the genomic location of the IL12Rβ2 gene. The Y-axis represents the significance p-value (in log₂O₅) for each genetic marker. 10 (p-value is a metric). The horizontal line represents the significance threshold for GWAS (p-value 5 × 10⁻⁶). -8 The circle size for each genetic marker was categorized by its correlation with the primary variant of IL12Rβ2 eQTL (rs17129778).

[0078] Figures 17A-17C This plot shows the correlation between genetic markers at the IL12B locus and serum IL12B protein levels (IL12B pQTL) and nine inflammatory diseases. Each point in the plot represents a genetic marker. The X-axis represents the genomic location of the genetic marker. This genetic region spans 500 KB of the genome, centered on the genomic location of the IL12B gene. The Y-axis represents the significance p-value (in log₂O₅) for each genetic marker. 10 (p-value is a metric). The horizontal line represents the significance threshold for GWAS (p-value 5 × 10⁻⁶). -8 The circle size for each genetic marker was categorized by its correlation with the primary variant of IL12B pQTL (rs6556416).

[0079] Figures 18A-18C This plot shows the correlation between genetic markers at the IL23R locus and serum IL23R protein levels (IL23R pQTL) and nine inflammatory diseases. Each point in the plot represents a genetic marker. The X-axis represents the genomic location of the genetic marker. This genetic region spans 500 KB of the genome, centered on the genomic location of the IL23R gene. The Y-axis represents the significance p-value (in log₂O₅) for each genetic marker. 10 (p-value is a metric). The horizontal line represents the significance threshold for GWAS (p-value 5 × 10⁻⁶). -8 The circle size for each genetic marker was categorized by its correlation with the primary variant of IL23R pQTL (rs11581607).

[0080] Figures 19A-19CThis is a region map showing the correlation between genetic markers at the EBI3 locus and EBI3 serum protein levels (EBI3 pQTL) and nine inflammatory diseases. Each point in the map represents a genetic marker. The X-axis represents the genomic location of the genetic marker. This genetic region spans 500 KB of the genome, centered on the genomic location of the EBI3 gene. The Y-axis represents the significance p-value (in log₂O₃) for each genetic marker. 10 (p-value is a metric). The horizontal line represents the significance threshold for GWAS (p-value 5 × 10⁻⁶). -8 The circle size for each genetic marker was categorized by its correlation with the primary variant of EBI3 pQTL (rs60160662).

[0081] Figures 20A-20C This is a region map showing the correlation between genetic markers at the IL6ST locus and serum IL6ST protein levels (IL6ST pQTL) and nine inflammatory diseases. Each point in the map represents a genetic marker. The X-axis represents the genomic location of the genetic marker. This genetic region spans 500 KB of the genome, centered on the genomic location of the IL6ST gene. The Y-axis represents the significance p-value (in log₂O₅) for each genetic marker. 10 (p-value is a metric). The horizontal line represents the significance threshold for GWAS (p-value 5 × 10⁻⁶). -8 The circle size for each genetic marker was categorized by its correlation with the primary variant of EBI3 pQTL (rs11574765).

[0082] Figures 21A-21C This plot shows the correlation between genetic markers at the STAT4 locus and STAT4 gene expression levels (STAT4 eQTL) and nine inflammatory diseases. Each point in the plot represents a genetic marker. The X-axis represents the genomic location of the genetic marker. This genetic region spans 500 KB of the genome, centered on the genomic location of the STAT4 gene. The Y-axis represents the significance p-value (in log₂O₅) for each genetic marker. 10 (p-value is a metric). The horizontal line represents the significance threshold for GWAS (p-value 5 × 10⁻⁶). -8 The circle size for each genetic marker was categorized by its correlation with the primary variant of STAT4 eQTL (rs16833249).

[0083] Figures 22A-22CThis plot shows the correlation between genetic markers at the STAT3 locus and STAT3 gene expression levels (STAT3 eQTL) and nine inflammatory diseases. Each point in the plot represents a genetic marker. The X-axis represents the genomic location of the genetic marker. This genetic region spans 500 KB of the genome, centered on the genomic location of the STAT3 gene. The Y-axis represents the significance p-value (in log₂O₃) for each genetic marker. 10 (p-value is a metric). The horizontal line represents the significance threshold for GWAS (p-value 5 × 10⁻⁶). -8 The circle size for each genetic marker was categorized by its correlation with the primary variant of STAT3 pQTL (rs1053004).

[0084] Figure 23 A scatter plot comparing the genetic effects of IL12A expression relative to the risk of SSc, PBC, SjS, and SLE diseases is presented. Genetic variants significantly associated with IL12A expression were first selected. Finally, variants for each disease that overlapped with GWAS data for SSc, PBC, SjS, or SLE were selected. Each point in the plot represents a final selected genetic variant. The X-axis represents the effect of such genetic variants on IL12A gene expression. The Y-axis represents the effect of the same allele of such genetic variants on disease risk. The straight line represents the fitted regression line across all selected genetic variants. An upward line indicates that genetically increased IL12A gene expression leads to increased disease risk. R-squared and p-values ​​explain the strength of the relationship between genetically determined IL12A gene expression and genetically determined disease risk.

[0085] Figure 24 A scatter plot comparing the genetic effects of IL12Rβ2 expression with the risk of SSc, PBC, SjS, and SLE diseases is presented. Genetic variants significantly associated with IL12Rβ2 expression were first selected. Finally, variants for each disease that overlapped with GWAS data for SSc, PBC, SjS, or SLE were selected. Each point in the plot represents a final selected genetic variant. The X-axis represents the effect of such genetic variants on IL12Rβ2 gene expression. The Y-axis represents the effect of the same allele of such genetic variants on disease risk. The straight line represents the fitted regression line across all selected genetic variants. An upward line indicates that genetically increased IL12Rβ2 gene expression leads to increased disease risk. R-squared and p-values ​​explain the strength of the relationship between genetically determined IL12Rβ2 gene expression and genetically determined disease risk.

[0086] Figure 25 The heavy chain variable region (VH) and light chain variable region (VL) sequences of the anti-IL12p35 antibody are shown.

[0087] Figure 26 Showing the VH and VL CDR sequences of the anti-IL12p35 antibody according to the Kabat definition.

[0088] Figure 27 Showing the VH and VL CDR sequences of the anti-IL12p35 antibody as defined by IMGT.

[0089] Figure 28 The heavy chain (HC) and light chain (LC) sequences of the antibodies discussed in this disclosure are shown.

[0090] Figure 29 The serum concentrations of anti-IL12p35 antibodies 4A4-hblb2-YTE and 27H28L-hblb-YTE were shown in human FcRn transgenic mice after a single intravenous (iv) administration (1 mg / kg).

[0091] Figure 30 The pharmacokinetic parameters of anti-IL12p35 antibodies 4A4-hblb2-YTE and 27H28L-hblb-YTE were shown in human FcRn transgenic mice after a single intravenous administration of 1 mg / kg.

[0092] Figure 31A This demonstrates an experimental protocol for determining the in vivo effects of anti-IL12p35 antibodies on inflammatory diseases using a DNFB-induced chronic skin inflammation model.

[0093] Figure 31B The average ear thickness of mice treated with either anti-mouse IL12p35 antibody (“anti-mP35”) or anti-mouse IL12p40 antibody (“anti-mP40”) in a DNFB-induced chronic skin inflammation model is shown.

[0094] Figure 32A This demonstrates an experimental protocol for determining the in vivo effects of anti-IL12p35 antibodies on Sjögren's syndrome using an STZ-induced Sjögren's syndrome model.

[0095] Figure 32B The images show the salivation relative to body weight in mice treated with either an anti-mouse IL12p35 antibody (“anti-P35”) or an isotype antibody control (“isotype”) in an STZ-induced Sjögren syndrome model.

[0096] Figure 33A The average body weight of NZBWF1 / J mice treated with anti-mouse IL12p35 antibody (“anti-mP35”) or isotype antibody control (“isotype”) in an IMQ-induced SLE model is shown.

[0097] Figure 33BThe ratio of urinary albumin to creatinine in NZBWF1 / J mice treated with anti-mouse IL12p35 antibody (“anti-mP35”) or isotype antibody control (“isotype”) in an IMQ-induced SLE (systemic lupus erythematosus) model is shown.

[0098] Figure 33C The images show representative mouse kidney histological images of NZBWF1 / J mice treated with anti-mouse IL12p35 antibody (“anti-P35”) or isotype antibody control (“isotype”) in an IMQ-induced SLE model.

[0099] Figure 34A This shows the ratio of urinary albumin and creatinine ("Cr") in NZBWF1 / J mice treated with either anti-mouse IL12p35 antibody ("anti-mP35") or an isotype antibody control ("isotype") in a spontaneous SLE (systemic lupus erythematosus) model.

[0100] Figure 34B Showing representative mouse kidney histological images of NZBWF1 / J mice treated with anti-mouse IL12p35 antibody (sub-image eh) or allotype antibody control (sub-image ad) in a spontaneous SLE model. Scale bars: sub-images a and e: 3 mm; sub-images b and f: 100 μm; sub-images c and g: 200 μm; and sub-images d and h: 50 μm.

[0101] Figure 34C This displays a detailed score and summary of the kidney pathology score as evaluated by an independent histopathologist. Detailed Implementation

[0102] IL12 is composed of two subunits: p35 (encoded by IL12A, UniProt ID P29459) and p40 (encoded by IL12B, UniProt ID P29460). Its receptor is composed of IL12Rβ1 (which binds to p40, encoded by IL12RB1) and IL12Rβ2 (which binds to p35, encoded by IL12RB2).

[0103] The other two IL12-related cytokines are IL23 and IL35. IL23 shares p40 and its receptor IL12Rβ1 with IL12, while having a unique subunit: p19 (encoded by IL23A) and its receptor IL23R (encoded by IL23R). IL-35 shares p35 and its receptor IL12Rβ2 with IL12, and has a unique subunit: EBI3 and its receptor GP130 (encoded by IL6ST). Schematic structures of IL12, IL23, IL35, their respective receptors, and downstream signaling pathways are shown in [Figure / Image / Description]. Figure 14 .

[0104] IL23 promotes the differentiation of naive helper T cells into the Th17 phenotype, leading to the secretion of inflammatory cytokines such as IL17 and IL22. Conversely, IL12 induces Th1 polarization and the production of key cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor. Although sharing the IL12A subunit, IL12 is a pro-inflammatory heterodimer cytokine primarily produced by dendritic cells, monocytes, and macrophages, while IL35 is an inflammatory suppressor heterodimer cytokine primarily produced by regulatory T cells and regulatory B cells.

[0105] Among the IL12 family of cytokines, several selective p40 or p19-targeting drugs have been approved for various autoimmune diseases, including psoriasis (PsO), psoriatic arthritis (PsA), Crohn's disease (CD), and ulcerative colitis (UC), which are listed below. For example, ustekinumab (Stelara), which targets p40, is approved for CD, UC, PsO, and PsA; risankizumab (Skyrizi), which targets p19, is approved for PsO, PsA, and CD; guselkumab (Tremfya), which targets p19, is approved for PsO; tildrakizumab (Ilumya), which targets p19, is approved for PsO; and mirikizumab (Omvoh), which targets p19, is approved for UC.

[0106] Meanwhile, targeting p40 has failed to demonstrate efficacy in clinical trials for primary biliary cholangitis (PBC) (Hirschfield, GM, et al. “Ustekinumab for patients with primary biliary cholangitis who have an inadequate response to ursodeoxycholic acid: a proof-of-concept study.” Hepatology 64.1 (2016): 189-199) and systemic lupus erythematosus (SLE) (van Vollenhoven, RF, et al. “Phase 3, multicentre, randomised, placebo-controlled study evaluating the efficacy and safety of ustekinumab inpatients with systemic lupus erythematosus.” Annals of the Rheumatic Diseases 81.11 (2022): 1556-1563). Furthermore, there are case reports of new onsets of SLE (Barber, C. et al. “New-onset systemic lupus erythematousin a patient receiving risankizumab for psoriasis.” JAAD Case Reports 25(2022): 104-106) or PBC (Tomše, P. et al. “Primary biliary cholangitis-cause orassociation with psoriasis: a case report.” Acta Dermatovenerologica Alpina, Pannonica, et Adriatica 32.1 (2023): 23-26). This evidence highlights that IL12p40 or IL23 pathway-specific genes are effective targets for a group of autoimmune diseases including PsO, PsA, CD, and UC, but not for other autoimmune diseases including SLE and PBC.

[0107] Despite the wide range of clinical successes and failures demonstrated in p40 and p19-targeted therapies, the role of IL12p35 targeting in autoimmune diseases has been largely overlooked. This may be driven by the following observations: In some preclinical models, IL12p35-deficient mice showed no protection or even disease exacerbation, where genetic or antibody-mediated inhibition of IL12p40 or IL12Rβ1 improved the disease (Cua, DJ, et al. “Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain.” Nature 421.6924 (2003): 744-748.; Teng, M.WL, et al. “IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases.” Nature Medicine 21.7 (2015): 719-729; and Yen, D., et al. “IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6.” The Journal of Clinical Investigation 116.5). (2006): 1310-1316). This has led to a common (erroneous) notion: that anti-IL12-p35 should be less effective than anti-IL12-p40, and that if anti-p40 is ineffective in autoimmune indications, then anti-p35 will not be effective either. To the best of our knowledge, there are no publicly disclosed ongoing activities in the development of clinical or preclinical therapies for autoimmune diseases using anti-IL12p35 or its receptor IL12Rβ2.

[0108] Subsequently, through human genetic analysis in this disclosure, we challenged the (erroneous) concept and provided evidence that targeting IL12p35 and IL12Rβ2 is effective for certain autoimmune diseases, including systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS).

[0109] This disclosure provides examples of antibodies that bind to IL12p35 (interleukin 12 subunit alpha) and their antigen-binding fragments, which may be used to treat systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE) and / or Sjogren's syndrome (SjS).

[0110] IL12 and IL12 receptor

[0111] IL-12 consists of two subunits linked by disulfide bonds. The smaller p35 monomer (35kDa α chain, IL-12A, IL-12p35, or IL-12A-p35) is encoded on chromosome 3, while the larger p40 monomer (40kDa β chain, IL-12B, IL-12p40, or IL-12B-p40) is encoded on chromosome 5. Co-expression leads to the formation of the biologically active p70 heterodimer. Within the IL-12 family of cytokines, monomers bind to different partners to produce various cytokines. While p35 can also pair with Epstein-Barr virus-induced gene 3 (EBI3) to produce IL-35, the combination of the p40 subunit with the p19 monomer results in the formation of IL-23. The fourth member of this family is IL-27, which is composed of the EBI3 and p28 subunits.

[0112] Due to the lower expression of the IL12 α chain compared to the β chain, only the free β chain, p40 homodimer, or heterodimer is secreted. Structurally, the p40 subunit shares some features with the IL6 receptor, while the p35 subunit is similar to granulocyte colony-stimulating factor (G-CSF) and IL-6. In mice, the p40 homodimer has been shown to regulate IL12 activity by counteracting IL12-induced signaling through competition with IL12p70 for receptor binding. Other functions of the p40 homodimer have been described, such as its role in dendritic cell (DC) migration, allogeneic transplant rejection, or its role in macrophage chemotactic activity.

[0113] Reflecting the heterodimeric structure of IL12 family cytokines, the corresponding receptors also consist of two subunits. The IL12 receptor β1 (IL12Rβ1) is encoded on chromosome 19 and has a molecular weight of 100 kDa. It is a transmembrane protein with an extracellular domain of 516 amino acids, responsible for interacting with IL12p40. It is also part of the IL23 receptor, pairing with IL23R. The gene for IL12Rβ2 is located on chromosome 1 and is translated into a 130 kDa transmembrane protein, with 595 amino acids forming the extracellular domain. Cellular signal transduction originates from IL12Rβ2, which interacts with IL12p35; therefore, its combination with glycoprotein 130 (gp130) is also part of the IL35 receptor. The IL27 receptor, the fourth receptor in this family, is composed of gp130 and the interleukin-27 receptor subunit alpha (WSX1). Since NK cells and T cells are the primary targets of IL12, IL12R expression is mainly confined to these cell types. Specifically, antigen contact with naive T cells induces the upregulation of IL12Rβ2, which is subsequently maintained by interferon gamma (IFN-γ) signaling but can be counteracted by IL-4. This suggests that T cell commitment to different effector T (Teff) cell lineages (such as cells with a type 1 helper T (TH1) phenotype rather than a TH2 phenotype, and where only the former consistently expresses IL12Rβ2) plays a crucial role.

[0114] IL-12 is primarily produced by professional antigen-presenting cells (APCs) such as B cells and dendritic cells (DCs), as well as phagocytes including monocytes, macrophages, and granulocytes. While the production of IL-12p35 is mainly regulated at the translational level, transcriptional regulation is responsible for the amount of IL-12p40 expressed. The initial signal that triggers IL-12 expression is exposure of these cells to bacteria, viruses, fungi, or parasites. Pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) or CpG DNA, expressed or contained in such symbionts or pathogens, are recognized by pattern recognition receptors (PRRs) of the toll-like receptor (TLR) family. This leads to the activation of several transcription factors that regulate IL-12 production, most notably NF-κB and interferon regulatory elements (IRFs).

[0115] Due to their different chromosomal locations and as described above, there are significant differences in the regulation of p40 and p35 production. The synthesis of the p40 chain far exceeds that of the p35 chain, indicating that p35 synthesis is the rate-limiting step in IL12 secretion. Furthermore, most TLRs are associated with IL12 p40 expression, while p35 expression is induced only by a limited subset of these receptors (including TLRs 3, 4, and 8). In addition to directly regulating IL12 production, TLR activation leads to the secretion of IFN-β and IFN-γ, whose signaling, in turn, induces the activation of IRF-1, IRF-7, and IRF-8. All three IRFs induce p35, and IRF-7 and IRF-8 also induce p40.

[0116] Furthermore, IL12 expression is regulated by APCs and T cells through the interaction of CD40 and its ligand CD40L. CD40 signaling via H-Ras and K-Ras enhances p38 mitogen-activated protein kinase (MAPK)-mediated pro-inflammatory IL12 production. A crucial positive feedback loop for increased IL12 secretion is so-called IFN-γ priming. Downstream IFN-γ release further promotes IL12 production via IRF-1 induction of IL12p35 and ICSBP induction of p40.

[0117] The binding of the two IL12 subunits to the two strands of the IL12 receptor, IL12Rβ1 and IL12Rβ2, activates the Janus kinase (JAK) signaling and transcription activator (STAT) signaling pathway. Specifically, IL12Rβ1 then recruits the JAK family member tyrosine kinase 2 (TYK2), and IL12Rβ2 associates with JAK2, leading to phosphorylation of JAK2. This activates the kinase activity of JAK2, which in turn phosphorylates tyrosine residues of the associated receptor subunit. The STAT molecule contains an SRC homology domain (SH2), which binds to phosphorylated IL12Rβ2 in the next step, exposing STAT to JAK and causing their phosphorylation. These activated transcription factors associate to form homodimers or heterodimers, enabling subsequent nuclear translocations. By binding to specific DNA sequences, they promote or repress gene transcription. STAT4 is the most important downstream target of IL12, while the effects of STAT1, STAT3, and STAT5 molecules play a secondary role. Furthermore, IL12R signaling activates mitogen-activated protein kinase kinase 3 / 6 (MKK) and p38 MAPK, which support IFN-γ secretion in activated T cells and TH1 cells. Importantly, this pathway is mediated by a STAT4-independent mechanism and is associated with increased STAT2 levels.

[0118] The primary function of IL12 is to induce IFN-γ production, thereby enabling cytokines to play a crucial role in adaptive and innate immunity. Additionally, it has been shown that IL12, when present early during clonal expansion, also induces CD4+ and CD8+ T cells to produce IL10. This may lead to the development of type 1 regulatory (Tr1) cells that secrete IL10 in response to IL12 and IL27, consistent with IL12-dependent Tr1 cell development observed in patients with visceral leishmaniasis. Conversely, TH2 differentiation is counteracted by IL12 because GATA-binding protein 3 (GATA3), essential for TH2 polarization, is suppressed in CD4+ and CD8+ T cell populations upon treatment with IL12 or in vivo expansion in the presence of IL12-producing DCs.

[0119] Consistent with its central role in coordinating immune responses, various studies in animal models and humans have shown that IL12 contributes to the pathogenesis of several immune-mediated inflammatory diseases, such as inflammatory bowel disease (IBD), multiple sclerosis, cancer, psoriasis, diabetes, systemic lupus erythematosus (SLE), primary biliary cholangitis (PBC), Sjögren's syndrome (SjS), and rheumatoid arthritis.

[0120] The roles of IL-12, its receptor, and their functions are described, for example, in Ullrich, KA-M., et al., “Immunology of IL-12: An update on functional activities and implications for disease.” EXCLI Journal 19 (2020): 1563; Wojno, ED, et al., “The immunobiology of the interleukin-12 family: room for discovery.” Immunity 50.4 (2019): 851-870; and Sun, L., et al., “Interleukin 12 (IL-12) family cytokines: Role in immunepathogenesis and treatment of CNS autoimmune disease.” Cytokine 75.2 (2015): 249-255; each of these is incorporated herein by reference in its entirety.

[0121] Anti-IL12p35 antibody and antigen-binding fragment

[0122] This disclosure provides antibodies that specifically bind to IL12p35 and antigen-binding fragments thereof. In some embodiments, the antibodies and antigen-binding fragments thereof described herein that specifically bind to IL12p35 are fully human antibodies or antigen-binding fragments thereof. The antibodies and antigen-binding fragments described herein are capable of binding to IL12p35 (e.g., human IL12p35), blocking the binding of IL12 to its receptor IL12Rβ2, and blocking IL12-induced intracellular signaling pathways. This disclosure provides, for example, anti-IL12p35 antibodies D2M008-A1C6, D2M008-A1A12, D2M008-A1E5, D2M008-A1A2, D2M008-A1E7, D2M008-A1G9, D2M008-A1A8, D2M008-A1A9, D2M008-A1B4, D2M008-A1E10, D2M008- B2C5, D2M008-B2B10, D2M008-B2B1, D2M008-B2B12, D2M008-B2C3, D2M008-B2B7, D2M008-3A4, D2M008-3A8, D2M008-4A4, D2M008-4A5, D2M008-3A11, D2M008-3A2, D2M008-27H28L 4A4-hblb2, 27H28L-hblb, their chimeric antibodies, and their humanized antibodies. The CDR sequences, VH, and VL of these antibodies or antibodies derived from them are shown in... Figures 25 to 27 middle.

[0123] For example, the CDR sequences of antibodies derived from “D2M008-A1C6” and “D2M008-A1C6” include CDRs of the heavy chain variable domain as defined by Kabat numbers (SEQ ID NOs: 47, 48, and 49) and CDRs of the light chain variable domain (SEQ ID NOs: 50, 51, and 52). CDRs can also be defined by IMGT numbers. According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 185, 186, and 187, and CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 188, 189, and 190.

[0124] For example, the CDR sequences of antibodies derived from “D2M008-A1A12” and “D2M008-A1A12” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NOs: 53, 54, and 55) and CDRs of the light chain variable domain (SEQ ID NOs: 56, 57, and 58). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 191, 192, and 193, and CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 194, 195, and 196.

[0125] For example, the CDR sequences of antibodies derived from “D2M008-A1E5” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 59, 60, and 61) and CDRs of the light chain variable domain (SEQ ID NO: 62, 63, and 64). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 197, 198, and 199, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 200, 201, and 202.

[0126] For example, the CDR sequences of antibodies derived from “D2M008-A1A2” and “D2M008-A1A2” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NOs: 65, 66, and 67) and CDRs of the light chain variable domain (SEQ ID NOs: 68, 69, and 70). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 203, 204, and 205, and CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 206, 207, and 208.

[0127] For example, the CDR sequences of antibodies derived from “D2M008-A1E7” and “D2M008-A1E7” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NOs: 71, 72, and 73) and CDRs of the light chain variable domain (SEQ ID NOs: 74, 75, and 76). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 209, 210, and 211, and CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 212, 213, and 214.

[0128] For example, the CDR sequences of antibodies derived from “D2M008-A1G9” and “D2M008-A1G9” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NOs: 77, 78, and 79) and CDRs of the light chain variable domain (SEQ ID NOs: 80, 81, and 82). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 215, 216, and 217, and CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 218, 219, and 220.

[0129] For example, the CDR sequences of antibodies derived from “D2M008-A1A8” and “D2M008-A1A8” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NOs: 83, 84, and 85) and CDRs of the light chain variable domain (SEQ ID NOs: 86, 87, and 88). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 221, 222, and 223, and CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 224, 225, and 226.

[0130] For example, the CDR sequences of antibodies derived from “D2M008-A1A9” and “D2M008-A1A9” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NOs: 89, 90, and 91) and CDRs of the light chain variable domain (SEQ ID NOs: 92, 93, and 94). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 227, 228, and 229, and CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 230, 231, and 232.

[0131] For example, the CDR sequences of antibodies derived from “D2M008-A1B4” and “D2M008-A1B4” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NOs: 95, 96, and 97) and CDRs of the light chain variable domain (SEQ ID NOs: 98, 99, and 100). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 233, 234, and 235, and CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 236, 237, and 238.

[0132] For example, the CDR sequences of antibodies derived from “D2M008-A1E10” and “D2M008-A1E10” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 101, 102, and 103) and CDRs of the light chain variable domain (SEQ ID NO: 104, 105, and 106). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 239, 240, and 241, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 242, 243, and 244.

[0133] For example, the CDR sequences of antibodies derived from “D2M008-B2C5” and “D2M008-B2C5” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 107, 108, and 109) and CDRs of the light chain variable domain (SEQ ID NO: 110, 111, and 112). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 245, 246, and 247, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 248, 249, and 250.

[0134] For example, the CDR sequences of antibodies derived from “D2M008-B2B10” and “D2M008-B2B10” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 113, 114, and 115) and CDRs of the light chain variable domain (SEQ ID NO: 116, 117, and 118). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 251, 252, and 253, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 254, 255, and 256.

[0135] For example, the CDR sequences of antibodies derived from “D2M008-B2B1” and “D2M008-B2B1” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 119, 120, and 121) and CDRs of the light chain variable domain (SEQ ID NO: 122, 123, and 124). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 257, 258, and 259, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 260, 261, and 262.

[0136] For example, the CDR sequences of antibodies derived from “D2M008-B2B12” and “D2M008-B2B12” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 125, 126, and 127) and CDRs of the light chain variable domain (SEQ ID NO: 128, 129, and 130). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 263, 264, and 265, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 266, 267, and 268.

[0137] For example, the CDR sequences of antibodies derived from “D2M008-B2C3” and “D2M008-B2C3” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 131, 132, and 133) and CDRs of the light chain variable domain (SEQ ID NO: 134, 135, and 136). According to IMGT definitions, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 269, 270, and 271, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 272, 273, and 274.

[0138] For example, the CDR sequences of antibodies derived from “D2M008-B2B7” and “D2M008-B2B7” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 137, 138, and 139) and CDRs of the light chain variable domain (SEQ ID NO: 140, 141, and 142). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 275, 276, and 277, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 278, 279, and 280.

[0139] For example, the CDR sequences of antibodies derived from “D2M008-3A4” and “D2M008-3A4” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 143, 144, and 145) and CDRs of the light chain variable domain (SEQ ID NO: 146, 147, and 148). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 281, 282, and 283, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 284, 285, and 286.

[0140] For example, the CDR sequences of antibodies derived from “D2M008-3A8” and “D2M008-3A8” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 149, 150, and 151) and CDRs of the light chain variable domain (SEQ ID NO: 152, 153, and 154). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 287, 288, and 289, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 290, 291, and 292.

[0141] For example, the CDR sequences of antibodies derived from “D2M008-4A4” and “D2M008-4A4” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 155, 156, and 157) and CDRs of the light chain variable domain (SEQ ID NO: 158, 159, and 160). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 293, 294, and 295, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 296, 297, and 298.

[0142] For example, the CDR sequences of antibodies derived from “4A4-hblb2” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 155, 156, and 157) and CDRs of the light chain variable domain (SEQ ID NO: 158, 159, and 160). According to IMGT definitions, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 293, 294, and 295, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 296, 297, and 298.

[0143] For example, the CDR sequences of antibodies derived from “D2M008-4A5” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 161, 162, and 163) and CDRs of the light chain variable domain (SEQ ID NO: 164, 165, and 166). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 299, 300, and 301, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 302, 303, and 304.

[0144] For example, the CDR sequences of antibodies derived from “D2M008-3A11” and “D2M008-3A11” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 167, 168, and 169) and CDRs of the light chain variable domain (SEQ ID NO: 170, 171, and 172). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 305, 306, and 307, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 308, 309, and 310.

[0145] For example, the CDR sequences of antibodies derived from “D2M008-3A2” and “D2M008-3A2” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 173, 174, and 175) and CDRs of the light chain variable domain (SEQ ID NO: 176, 177, and 178). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 311, 312, and 313, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 314, 315, and 316.

[0146] For example, the CDR sequences of antibodies derived from “D2M008-27H28L” and “D2M008-27H28L” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 179, 180, and 181) and CDRs of the light chain variable domain (SEQ ID NO: 182, 183, and 184). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 317, 318, and 319, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 320, 321, and 322.

[0147] For example, the CDR sequences of antibodies derived from “27H28L-hblb” include CDRs of the heavy chain variable domain as defined by the Kabat numbers (SEQ ID NO: 179, 180, and 181) and CDRs of the light chain variable domain (SEQ ID NO: 182, 183, and 184). According to IMGT definitions, CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 317, 318, and 319, and CDR sequences of the light chain variable domain are shown in SEQ ID NO: 320, 321, and 322.

[0148] Therefore, in one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that binds to IL12p35, said antibody or antigen-binding fragment comprising: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to the selected VL CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to the selected VL CDR1 amino acid sequence, and the VL CDR2 region comprises an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to the selected VL CDR3 amino acid sequence; ...3 The CDR2 amino acid sequence is at least 80% identical, and the VL CDR3 region contains an amino acid sequence that is at least 70%, 80%, 90%, or 100% identical to the selected VL CDR3 amino acid sequence, wherein the selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are selected from […]. Figure 26-27 The VH CDR 1, 2, 3 and VL CDR 1, 2, 3 are listed in the table.

[0149] The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-IL12p35 antibody are also provided. These VH and VL sequences are shown in... Figure 25 .

[0150] For example, VH and VL of D2M008-A1C6 are shown in SEQ ID NO: 1 and 2. For example, VH and VL of D2M008-A1A12 are shown in SEQ ID NO: 3 and 4. For example, VH and VL of D2M008-A1E5 are shown in SEQ ID NO: 5 and 6. For example, VH and VL of D2M008-A1A2 are shown in SEQ ID NO: 7 and 8. For example, VH and VL of D2M008-A1E7 are shown in SEQ ID NO: 9 and 10. For example, VH and VL of D2M008-A1G9 are shown in SEQ ID NO: 11 and 12. For example, VH and VL of D2M008-A1A8 are shown in SEQ ID NO: 13 and 14. For example, VH and VL of D2M008-A1A9 are shown in SEQ ID NO: 15 and 16. For example, VH and VL of D2M008-A1B4 are shown in SEQ ID NO: 17 and 18. For example, VH and VL of D2M008-A1E10 are shown in SEQ ID NO: 19 and 20. For example, VH and VL of D2M008-B2C5 are shown in SEQ ID NO: 21 and 22. For example, VH and VL of D2M008-B2B10 are shown in SEQ ID NO: 23 and 24. For example, VH and VL of D2M008-B2B1 are shown in SEQ ID NO: 25 and 26. For example, VH and VL of D2M008-B2B12 are shown in SEQ ID NO: 27 and 28. For example, VH and VL of D2M008-B2C3 are shown in SEQ ID NO: 29 and 30. For example, VH and VL of D2M008-B2B7 are shown in SEQ ID NO: 31 and 32. For example, VH and VL of D2M008-3A4 are shown in SEQ ID NO: 33 and 34. For example, VH and VL of D2M008-3A8 are shown in SEQ ID NO: 35 and 36. For example, VH and VL of D2M008-4A4 are shown in SEQ ID NO: 37 and 38. For example, VH and VL of D2M008-4A5 are shown in SEQ ID NO: 39 and 40. For example, VH and VL of D2M008-3A11 are shown in SEQ ID NO: 41 and 42. For example, VH and VL of D2M008-3A2 are shown in SEQ ID NO: 43 and 44. For example, VH and VL of D2M008-27H28L are shown in SEQ ID NO: 45 and 46. For example, the VH and VL of 4A4-hblb2 are shown in SEQ ID NO: 335 and 336. For example, the VH and VL of 27H28L-hblb are shown in SEQ ID NO: 337 and 338. These antibodies can be human or humanized antibodies.

[0151] In some implementations, any of these heavy chain variable region sequences (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 335, and 337) can be paired with any of these light chain variable region sequences (e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 336, and 338).

[0152] Furthermore, in some embodiments, the antibody or its antigen-binding fragment described herein may also contain a subset selected from... Figure 26-27 One, two, or three heavy chain variable regions (CDRs); and / or selected from Figure 26-27 One, two, or three light chain variable regions (CDRs). In some embodiments, the antibody or its antigen-binding fragment described herein may also contain a number according to the Kabat numbering scheme, such as... Figure 26 The document describes one, two, or three heavy chain variable region CDRs and / or one, two, or three light chain variable region CDRs. In some embodiments, the antibody or its antigen-binding fragment described herein may also contain, according to the IMGT numbering scheme, such as... Figure 27 The diagram shows one, two, or three heavy chain variable region CDRs and / or one, two, or three light chain variable region CDRs.

[0153] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain, said heavy chain variable domain containing one, two, or three of the following: having zero, one, or two amino acid insertions, deletions, or substitutions. Figure 26-27 Any of the VH CDR1s shown; having zero, one, or two amino acid insertions, deletions, or substitutions. Figure 26-27 Any of the VH CDR2 shown; having zero, one, or two amino acid insertions, deletions, or substitutions. Figure 26-27 Any of the VH CDR3 shown herein. In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain comprising one, two, or three of the following: having zero, one, or two amino acid insertions, deletions, or substitutions. Figure 26-27 Any of the VL CDR1s shown; having zero, one, or two amino acid insertions, deletions, or substitutions. Figure 26-27 Any of the VL CDR2 shown; having zero, one, or two amino acid insertions, deletions, or substitutions. Figure 26-27 Any of the VL CDR3 shown.

[0154] Insertion, deletion, and substitution can occur within the CDR sequence or at one or both ends of the CDR sequence.

[0155] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to IL12p35. The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises or is composed of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and the light chain variable region (VL) comprises or is composed of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 335, or 337. In some implementations, the selected VL sequence is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 336, or 338.

[0156] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are considered identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of vacancies introduced for optimal alignment and the length of each vacancy. For example, sequence comparison and determination of the percentage identity between two sequences can be accomplished using a Blossum 62 scoring matrix, where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5.

[0157] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chains or immunoglobulin light chains. Immunoglobulin heavy chains or immunoglobulin light chains comprise, for example... Figure 26-27 The CDR is shown (according to Kabat or IMGT number). When a polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to IL12p35.

[0158] Anti-IL12p35 antibodies and their antigen-binding fragments can also be antibody or antibody fragments and antibody variants (including derivatives and conjugates) of multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific (polymeric, e.g., bispecific) antibodies, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly generated antibodies (i.e., intracellular antibodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0159] Antibody fragments are suitable for the provided methods, provided they retain the desired affinity and specificity of a full-length antibody (e.g., a complete IgG antibody). Therefore, an antibody fragment binding to IL12p35 will retain the ability to bind to IL12p35. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a tightly associated heavy-chain variable domain and a light-chain variable domain, which can be covalently related, as in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Overall, six CDRs or a subset thereof confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind antigens, although typically with lower affinity than the entire binding site.

[0160] In some embodiments, the anti-IL12p35 antibody and its antigen-binding fragment described herein have an Fc region (e.g., the human IgG1 Fc region). In some embodiments, the Fc region described herein includes one or more YTE mutations (M252Y / S254T / T256E according to EU designations). The YTE mutations, located at the CH2-CH3 interface within the Fc domain, have been shown to increase the binding affinity of the antibody Fc at pH 6.0 to MHC class I neonatal FcRs (FcRn) primarily located in the acidic endosomes of endothelial cells and hematopoietic cells, thereby allowing for more efficient recycling of the administered IgG1 antibody and a longer retention time in plasma. Compared to the original IgG1 Fc structure, the increased FcRn binding at pH 6.0 by the YTE triple-mutated antibody is mediated by the creation of an additional salt bridge between the Glu 256(E) of the Fc-YTE and the Gln 2(Q) of the β2-microglobulin chain of the FcRn. Therefore, the YTE mutation can lead to higher FcRn binding and has been shown to be well tolerated in humans and to prolong the antibody half-life. Details of the YTE mutation can be found, for example, in Oganesyan, V., et al., “Structural insights intoneonatal Fc receptor-based recycling mechanisms.” Journal of Biological Chemistry 289.11 (2014): 7812-7824; and Wang, X., et al., “IgG Fc engineering tomodulate antibody effector functions.” Protein & Cell 9.1 (2018): 63-73; each of which is incorporated herein by reference in its entirety.

[0161] Single-chain Fv or (scFv) antibody fragments contain the VH and VL domains (or regions) of the antibody, where these domains are present within a single polypeptide chain. Typically, scFv polypeptides further include polypeptide linkers between the VH and VL domains, which allows scFv to form the desired structure for antigen binding.

[0162] The Fab fragment contains variable and constant domains of the light chain and a variable domain and a first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are typically covalently linked near their carboxyl termini via a hinge cysteine ​​residue. Other chemical conjugations of antibody fragments are also known in the art.

[0163] A bivalent antibody is a small antibody fragment with two antigen-binding sites, the fragment containing a VH (VH and VL) linked to a VL on the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, thus creating two antigen-binding sites.

[0164] Linear antibodies consist of a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with a complementary light chain polypeptide form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0165] The antibodies and antibody fragments disclosed herein can be modified in the Fc region to provide desired effector function or serum half-life.

[0166] Antibody polymerization can be achieved through the natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, a certain percentage of purified antibody formulations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody polymers.

[0167] Alternatively, antibody homodimers can be formed using chemical linking techniques known in the art. For example, heterobifunctional crosslinking agents, including but not limited to SMCC (succinimide-4-(maleimide-methyl)cyclohexane-1-carboxylate) and SATA (N-succinimide-S-acetylthioacetate), can be used to form antibody polymers. Exemplary schemes for forming antibody homodimers are described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94: 7509-7514, 1997). Antibody homodimers can be converted to F(ab')2 homodimers by digestion with pepsin. Another way to form antibody homodimers is by using the self-philic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0168] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant structural domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other undesirable end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0169] Bispecific antibodies include cross-linked antibodies or "heteroconjugate" antibodies. For example, one antibody in a heteroconjugate may be conjugated to avidin while the other is conjugated to biotin. Heteroconjugate antibodies can also be prepared using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0170] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stable molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stable molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation to a stable molecule may increase the half-life or prolong the biological activity of the antibody or antigen-binding fragment in vitro (e.g., in tissue cultures or when stored as a pharmaceutical composition) or in vivo (e.g., in the human body).

[0171] In some embodiments, the antibody or antigen-binding fragment described herein may be conjugated to a therapeutic agent. Antibody-drug conjugates comprising an antibody or its antigen-binding fragment may be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or cell inhibitor (e.g., cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, maytansine such as DM-1 and DM-4, ​​diketone, mitoxanthraquinone, styraxine, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogues).

[0172] In some embodiments, the antibodies or antigen-binding fragments thereof described herein include one or more reverting germline (B2G) mutations, for example by amino acid substitution, deletion, and / or insertion in the frame region (FR) of any of the VH and VL described herein. Such one or more B2G mutations may reduce potential immunogenicity to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% compared to antibodies or antigen-binding fragments thereof without one or more B2G mutations. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 B2G mutations may be introduced into any of the VH and VL described herein. In some embodiments, in any of the VH described herein, one or more B2G mutations are formed before VH CDR1, between VH CDR1 and VH CDR2, between VH CDR2 and VH CDR3, and / or after VH CDR3. In some embodiments, one or more B2G mutations are formed in any of the VLs described herein, before VL CDR1, between VL CDR1 and VLCDR2, between VL CDR2 and VL CDR3, and / or after VL CDR3. In some embodiments, one or more B2G mutations are in the frame regions (FRs) of the VH, for example, in FR1, FR2, FR3, and / or FR4 of the VH. In some embodiments, one or more B2G mutations are in the frame regions (FRs) of the VL, for example, in FR1, FR2, FR3, and / or FR4 of the VL. In some embodiments, one or more B2G mutations are not within the CDRs of the VH, for example, not within CDR1, CDR2, and / or CDR3 of the VH. In some embodiments, one or more B2G mutations are not within the CDRs of the VL, for example, not within CDR1, CDR2, and / or CDR3 of the VL.

[0173] Methods for designing B2G mutations are generally known in the art. For example, sequences of VH (e.g., any of the VHs described herein) and / or VL (e.g., any of the VLs described herein) can be aligned with the nearest phylogenetic sequence, and B2G mutations within a frame region (e.g., FR) can be identified. B2G mutations in VH are also referred to as heavy chain B2G mutations (“hb”). B2G mutations in VL are also referred to as light chain B2G mutations (“lb”). In some cases, more than one set of B2G mutations can be identified, for example when alignment is performed using different phylogenetic sequences. As a result, the first set of light chain B2G mutations can be referred to as “lb”, and the second set of light chain B2G mutations can be referred to as “lb2”. When an antibody or its antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) includes a B2G mutation in both the VH and VL sequences, the antibody or its antigen-binding fragment may be referred to as having an “hblb” or “hblb2” mutation, such as 27H28L-hblb-YTE and 4A4-hblb2-YTE.

[0174] The B2G mutation described herein can be used to expand the sequence pool of the antibodies or their antigen-binding fragments described herein, in order to identify variants with reduced immunogenicity. Details of the B2G mutation can be found, for example, Rossotti, MA, et al. “Immunogenicity and humanization of single‐domain antibodies.” The FEBS Journal 289.14 (2022): 4304-4327; and Clavero-Álvarez, A., et al. “Humanization of antibodies using a statistical inference approach.” Scientific Reports 8.1 (2018): 14820; each of which is incorporated herein by reference in its entirety.

[0175] Antibody and antigen binding fragment

[0176] This disclosure provides an anti-IL12p35 antibody and its antigen-binding fragment. Typically, antibodies (also called immunoglobulins) consist of two classes of polypeptide chains (light chains and heavy chains). The non-limiting antibody of this disclosure can be a complete four-chain immunoglobulin antibody comprising two heavy chains and two light chains (e.g., the complete IgG antibody described herein). The heavy chain of the antibody can be any isotype including IgM, IgG, IgE, IgA, or IgD, or a subisotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain. The antibody can contain two identical copies of the light chain and two identical copies of the heavy chain. Each heavy chain contains a variable domain (or variable region, V...). H The antibody's "stem" is formed by multiple constant domains (or constant regions) linked together via disulfide bonds within their constant domains. Each light chain contains a variable domain (or variable region). L Each of the light chains and a constant structural domain (or constant region) is bonded to a heavy chain via disulfide bonds. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bonded. The variable regions of both the light and heavy chains contain three highly variable regions sandwiched between more conservative framework regions (FRs).

[0177] These hypervariable regions (called complementarity-determining regions (CDRs)) form a loop containing the main antigen-binding surface of the antibody. The four framework regions primarily employ a β-sheet conformation, with CDRs forming loops within the β-sheet structure, and in some cases, CDRs forming part of the β-sheet structure. CDRs in each chain remain closely adjacent through the framework regions and, together with CDRs from other chains, contribute to the formation of the antigen-binding region.

[0178] Methods for identifying the CDR region of an antibody by analyzing its amino acid sequence are well known, and several definitions of CDR are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop region. These methods and definitions are described in, for example, Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody Engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al., “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains,” Molecular Immunology 45.14 (2008): 3832-3839; Wu, TT et al. (1970) J. Exp. Med. 132:211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., BiophysChem. 68(1-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan 1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); which are incorporated herein by reference in their entirety. Other definitions are also known in the art, including, for example, IMGT, Aho (Honneger's numbering scheme), and North. In some implementations, a combination of multiple CDR definitions is used.

[0179] CDR is important for recognizing epitopes of antigens. As used herein, an "epitaph" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The smallest size of an epitope can be about three, four, five, six, or seven amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, because the epitope can depend on the three-dimensional conformation of the antigen based on its secondary and tertiary structures.

[0180] In some implementations, antibodies are complete immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing only in their constant regions, particularly their hinges and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and described, for example, in Vidarsson et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in Immunology 5 (2014); Irani et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular Immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0181] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, camels). The antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin-binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to a portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of the antibody capable of specifically binding to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double-chain antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide comprising an antibody-binding domain or a binding domain homologous to an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, heavy chain and / or light chain CDRs of an intact antibody, variable regions of heavy chain and / or light chain of an intact antibody, full-length heavy chain or light chain of an intact antibody, or individual CDRs derived from the heavy chain or light chain of an intact antibody.

[0182] In some embodiments, the antigen-binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-stranded variable fragment (scFv) as described herein, fused with the transmembrane and inner domains of CD3-zeta. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Therefore, in one aspect, this disclosure further provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0183] F(ab) and F(ab')2 fragments are smaller antibody fragments that retain the antigen-binding specificity of the full-length antibody. The F(ab) fragment is generated by enzymatic digestion of the Ig molecule with the proteolytic enzyme papain. Papain cleaves the antibody molecule below the hinge region, resulting in two separate Fab fragments and a smaller Fc fragment. The F(ab')2 fragment is generated by enzymatic digestion of the Ig molecule with the proteolytic enzyme pepsin. Pepsin cleaves the antibody molecule below the hinge region, but leaves a small portion of the hinge region intact, resulting in two separate Fab fragments covalently linked by disulfide bonds. The F(ab')2 fragment has two antigen-binding F(ab) portions linked together by disulfide bonds, and is therefore bivalent, with a molecular weight of approximately 110 kDa. Bivalent antibody fragments (e.g., the F(ab')2 fragment) are smaller than intact IgG antibodies and can penetrate tissues more effectively, thus facilitating better antigen recognition in immunohistochemistry. The use of the F(ab')2 fragment also avoids nonspecific binding to Fc receptors on living cells or to proteins A / G. Details of the F(ab')2 fragment and its production methods can be found, for example, Rosenstein, S., et al. “Production of F(ab')2 from Monoclonal and Polyclonal Antibodies.” Current Protocols in Molecular Biology 131.1 (2020): e119, which is incorporated herein by reference in its entirety.

[0184] Antibody properties

[0185] Based on the human genetic analysis described in Example 12, the antibodies or antigen-binding fragments thereof described herein can block the binding of IL12 (e.g., IL12p70) to its receptor IL12Rβ2, thereby inhibiting the IL12p35 / IL12Rβ2-specific downstream pathway involved in a range of autoimmune diseases (e.g., SSc, PBC, SLE, and SjS). Because IL12p35 is not shared by IL23, the antibodies or antigen-binding fragments thereof described herein can specifically neutralize IL12 without interfering with the IL23 pathway. Some of the tested antibodies also exhibited excellent thermostability, tolerance to different pH stress conditions, and serum stability. Compartmentalization assays also indicated that some antibodies can target different epitopes on IL12p35.

[0186] In some embodiments, the antibody or its antigen-binding fragment described herein can reduce the binding of IL12 (e.g., IL12p70) to its receptor IL12Rβ2 to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference antibody (e.g., human IgG1). In some cases, the IC50 of the binding curve can be determined. 50 In some cases, the IC50 compared to a reference antibody (e.g., human IgG1) is... 50 In comparison, the IC50 of the antibodies or their antigen-binding fragments described in this article... 50 Less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, YTE mutations and / or B2G mutations (e.g., any of the YTE and / or B2G mutations described herein) in the antibody or its antigen-binding fragment have no significant effect on the binding between IL12 (e.g., IL12p70) and IL12Rβ2. In some embodiments, the binding between IL12 (e.g., IL12p70) and IL12Rβ2 is determined by ELISA.

[0187] In some embodiments, the antibodies described herein or their antigen-binding fragments can reduce human IL12-induced intracellular signaling in cells (e.g., human IgG1) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference antibody (e.g., human IgG1) or an anti-IL12p40 antibody (e.g., anti-hP40). In some embodiments, the IL12-induced intracellular signaling is JAK-STAT signaling. In some cases, the IC50 of the signal transduction curve can be determined. 50 In some cases, the IC50 value is higher than that of a reference antibody (e.g., human IgG1) or an anti-IL12p40 antibody (e.g., anti-hP40). 50 In comparison, the IC50 of the antibodies or their antigen-binding fragments described in this article... 50 Less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, YTE mutations and / or B2G mutations (e.g., any of the YTE and / or B2G mutations described herein) in the antibody or its antigen-binding fragment have no significant effect on IL12-induced inhibition of intracellular signaling. In some embodiments, a reporter cell system is used to evaluate the inhibition of IL12-induced intracellular signaling.

[0188] In some embodiments, the antibodies described herein or their antigen-binding fragments can reduce monkey IL12-induced intracellular signaling in cells (e.g., human reporter cells) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference antibody (e.g., human IgG1). In some embodiments, the antibodies described herein or their antigen-binding fragments can reduce monkey IL12-induced intracellular signaling in cells (e.g., human reporter cells) to levels comparable to those of an anti-IL12p40 antibody (e.g., anti-hP40). In some embodiments, the antibodies described herein or their antigen-binding fragments do not reduce monkey IL12-induced intracellular signaling in cells (e.g., human reporter cells) compared to a reference antibody (e.g., human IgG1).

[0189] In some embodiments, the antibodies described herein or their antigen-binding fragments can reduce IL12-induced IFN-γ production or secretion in human cells (e.g., human IgG1) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference antibody (e.g., human IgG1) or an anti-IL12p40 antibody (e.g., anti-hP40). In some embodiments, the IL12 is exogenous IL12 (e.g., recombinant human IL12).

[0190] In some embodiments, the antibody described herein or its antigen-binding fragment can reduce IL12-induced IFN-γ production or secretion in T cells (e.g., CD4+ T cells) co-cultured with antigen-presenting cells (e.g., allogeneic dendritic cells) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference antibody (e.g., human IgG1). In some embodiments, the IL12 is exogenous IL12 (e.g., recombinant human IL12). In some embodiments, particularly at low concentrations (e.g., less than 0.1 μg / mL, less than 0.05 μg / mL, less than 0.01 μg / mL, less than 0.005 μg / mL, or less than 0.001 μg / mL), the antibodies described herein or their antigen-binding fragments can reduce IL12-induced IFN-γ production or secretion in T cells (e.g., CD4+ T cells) co-cultured with antigen-presenting cells (e.g., allogeneic dendritic cells) to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0191] In some implementations, the antibodies described herein, their antigen-binding fragments, or their YTE and / or B2G variants do not interfere with the binding of IL23 to its receptor and / or IL23-induced signaling pathways.

[0192] In some implementations, the antibody (or its antigen-binding fragment) is delivered in less than 0.1 seconds. -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 or less than 0.0001s -1 The dissociation rate (koff or kd) specifically binds to IL12p35 (e.g., human IL12p35, monkey IL12p35, dog IL12p35, mouse IL12p35, and / or chimeric IL12p35) or IL12p70 (e.g., human IL12p70, monkey IL12p70, dog IL12p70, mouse IL12p70, and / or chimeric IL12p70). In some embodiments, the dissociation rate (koff) is greater than 0.01 s. -1 Greater than 0.001s -1 Greater than 0.0001s -1 Greater than 0.00001s -1 or greater than 0.000001s -1 .

[0193] In some implementations, the kinetic association rate (kon or ka) is greater than 1 × 10⁻⁶. 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some implementations, the kinetic association rate (kon) is less than 1 × 10⁻⁶. 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0194] The affinity can be deduced from the quotient of the kinetic rate constant (KD = koff / kon). In some implementations, KD is less than 1 × 10⁻⁶. -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, less than 1×10 -10 M, less than 1×10 -11 M or less than 1×10 -12 M. In some implementations, KD is less than 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM. In some implementations, KD is greater than 1 × 10⁻⁶. -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M or greater than 1×10 -12 M.

[0195] Common techniques for measuring antibody affinity for antigens include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some implementations, Gator is used. ® Prime BLI system or Carterra ®The SPR imaging system is used for measurement. In some embodiments, the antibody binds to human IL12p35, monkey IL12p35 (e.g., rhesus monkey or cynomolgus monkey IL12p35), dog IL12p35 (e.g., canine IL12p35), mouse IL12p35, and / or chimeric IL12p35. In some embodiments, the antibody binds to human IL12p70, monkey IL12p70 (e.g., rhesus monkey or cynomolgus monkey IL12p70), dog IL12p70 (e.g., canine IL12p70), mouse IL12p70, and / or chimeric IL12p70. In some embodiments, the human IL12p70 described herein is formed from human IL12p35 and human IL12p40. In some embodiments, the monkey IL12p70 described herein is formed from monkey IL12p35 and monkey IL12p40. In some embodiments, the canine IL12p70 described herein is formed from canine IL12p35 and canine IL12p40. In some embodiments, the mouse IL12p70 described herein is formed from mouse IL12p35 and mouse IL12p40. In some embodiments, the antibody does not bind to human IL12p40, monkey IL12p40 (e.g., rhesus monkey or cynomolgus monkey IL12p40), canine IL12p40 (e.g., canine IL12p40), mouse IL12p40, or chimeric IL12p40.

[0196] In some embodiments, a YTE mutation in the antibody or its antigen-binding fragment described herein, compared to a parental antibody (e.g., an antibody or antigen-binding fragment described herein without a YTE mutation), can increase FcRn binding affinity at acidic pH (e.g., about pH 6.0, pH 6.5, or pH 7.0) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold. As a result, compared to the half-life of the parental antibody (e.g., the antibody or antigen-binding fragment described herein without the YTE mutation), the YTE mutation can increase the half-life of the antibody or antigen-binding fragment described herein in vivo (e.g., when administered in a subject) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0197] In some implementations, thermal stability is determined. Antibody or antigen-binding fragments as described herein may have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. Because IgG can be described as a multi-domain protein, melting curves sometimes show two transitions: a first denaturation temperature Tm D1 and a second denaturation temperature Tm D2. The presence of these two peaks typically indicates denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When both peaks are present, Tm usually refers to Tm D2.

[0198] Therefore, in some embodiments, the antibody or antigen-binding fragment as described herein has a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C (e.g., for intact IgG antibodies). In some implementations, the antibody or antigen-binding fragment as described herein has a TmD2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C (e.g., for the F(ab')2 fragment). In some implementations, Tm, TmD1, and TmD2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0199] In some embodiments, the intact IgG antibody described herein (e.g., D2M008-4A4 or 27H28L) has a Tm greater than 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.1, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69, 69.5 or 70°C. In some embodiments, the F(ab')2 fragment of the antibody described herein (e.g., D2M008-4A4 or 27H28L) has a Tm greater than 71, 71.5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80, 80.5, 81, 81.5, 82, 82.5, 83, 83.5, 84, 84.5 or 85°C.

[0200] In some embodiments, the antibodies or antigen-binding fragments described herein, after being stored under stress conditions, retain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of their activity compared to unstressed control samples. In some embodiments, the antibodies or antigen-binding fragments described herein are incubated in a water bath (at approximately 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C) for approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, the antibodies or antigen-binding fragments described herein are in buffer solutions with acidic pH (e.g., about pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0) or alkaline pH (e.g., about pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, or pH 9.0). In some embodiments, the unstressed control samples described herein are in buffer solutions with neutral pH (e.g., about pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, or pH 7.9). In some embodiments, the activity of the antibody or its antigen-binding fragment described herein or an unstressed control sample is determined by measuring the blockage of binding between human IL12p70 and its receptor IL12Rβ2 (e.g., as described in Example 3 by ELISA) and / or measuring the inhibition of IL12-induced intracellular signal transduction (e.g., as described in Example 4 by cell-based assay).

[0201] In some embodiments, the antibody or antigen-binding fragment thereof described herein, after being mixed with human serum (e.g., fresh human serum collected from a healthy donor) and stored in a water bath (at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C) for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days, may retain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of its activity compared to a control sample. In some embodiments, the control sample contains the same antibody or antigen-binding fragment thereof described herein mixed with PBS. In some embodiments, the antibodies or antigen-binding fragments described herein, when mixed with human serum (e.g., fresh human serum collected from a healthy donor) and stored in a water bath (at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C) for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or longer, can retain at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of their activity compared to samples not stored under the same conditions. In some embodiments, the activity of the antibody or its antigen-binding fragment or control sample described herein is determined by measuring the blockage of binding between human IL12p70 and its receptor IL12Rβ2 (e.g., as described in Example 3 by ELISA) and / or measuring the inhibition of IL12-induced intracellular signal transduction (e.g., as described in Example 4 by cell-based assay).

[0202] In some embodiments, the antibodies or antigen-binding fragments described herein may bind to different epitopes on IL12p35. In some embodiments, the antibodies or antigen-binding fragments described herein may bind to the same epitopes on IL12p35.

[0203] In some implementations, the anti-IL12p35 antibodies described herein can reduce the immune response by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% in patients with systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS) after treatment with a therapeutically effective amount of anti-IL12p35 antibodies, compared to treatment with no anti-IL12p35 antibody or with a reference antibody (e.g., ustekinumab).

[0204] In some embodiments, the anti-IL12p35 antibody described herein is more effective than a reference antibody (e.g., ustekinumab) (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5-fold, or 10-fold more effective). The heavy and light chain sequences of ustekinumab can be found in... Figure 28 .

[0205] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may possess favorable in vivo stability and / or pharmacokinetic properties. For example, after administration at about 0.1-10 mg / kg (e.g., about 1 mg / kg), the terminal elimination half-life (T0) of the antibody or antigen-binding fragment thereof is [not specified]. 1 / 2 The time limit can be at least 100 hours, at least 150 hours, at least 200 hours, at least 250 hours, at least 300 hours, at least 350 hours, at least 375 hours, at least 400 hours, at least 425 hours, at least 450 hours, at least 475 hours, or at least 500 hours. In some implementations, T 1 / 2 Identified in subjects expressing human FcRn (e.g., human FcRn transgenic mice). In some embodiments, the antibody or antigen-binding fragment thereof described herein comprises one or more YTE mutations and / or B2G mutations.

[0206] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce the mean ear thickness in mice in a DNFB-induced chronic contact hypersensitivity mouse model. For example, the mean ear thickness can be reduced to less than 1.0 mm, less than 0.9 mm, less than 0.8 mm, less than 0.6 mm, less than 0.5 mm, less than 0.4 mm, or less than 0.3 mm. For example, compared to mice treated with PBS or anti-IL12p40 antibodies, the mean ear thickness can be reduced to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50%. In some embodiments, ear thickness is measured approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after initial DNFB sensitization. In some embodiments, the changes described herein are significant, for example, having a p-value of less than 0.01 or less than 0.005.

[0207] In some embodiments, the antibody or antigen-binding fragment thereof described herein can alleviate the reduction in salivation excretion in a mouse model of STZ-induced Sjögren syndrome. For example, the reduction in salivation excretion levels relative to body weight can be greater than 4 mg / g, greater than 3 mg / g, greater than 2 mg / g, or greater than 1 mg / g. For example, the salivation excretion level relative to body weight can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to when mice were treated with an isotype control antibody. In some embodiments, the salivation excretion level is determined using the method described herein approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after initial STZ immunization. In some embodiments, the changes described herein are significant, for example, having a p-value of less than 0.06, less than 0.05, less than 0.04, or less than 0.03.

[0208] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the body weight of mice in an IMQ-induced SLE model. For example, the weight loss following initial IMQ treatment is less than 10 g, less than 9 g, less than 8 g, less than 7 g, less than 6 g, less than 5 g, less than 4 g, less than 3 g, less than 2 g, or less than 1 g. For example, compared to mice treated with an isotype control antibody, the body weight can increase by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%. In some embodiments, body weight is measured approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after initial IMQ treatment.

[0209] In some embodiments, the antibody or its antigen-binding fragment described herein can increase urinary albumin levels in mice in an IMQ-induced SLE model. For example, the urinary albumin / creatinine ratio can be higher than 100 μg / mg, higher than 1000 μg / mg, higher than 10000 μg / mg, or higher than 100000 μg / mg. In some embodiments, the urinary albumin / creatinine ratio can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, or at least 120% compared to when mice are treated with an isotype control antibody. In some embodiments, the ratio described herein is determined approximately 1, 2, 3, 4, 5, 6, 7, or 8 weeks after initial IMQ treatment.

[0210] In some embodiments, the antibody or its antigen-binding fragment described herein can increase urinary albumin levels in mice in a spontaneous SLE model. For example, the urinary albumin / creatinine ratio can be higher than 1 mg / mg, higher than 10 mg / mg, higher than 20 mg / mg, higher than 30 mg / mg, higher than 40 mg / mg, higher than 50 mg / mg, higher than 60 mg / mg, higher than 70 mg / mg, higher than 80 mg / mg, higher than 90 mg / mg, or higher than 100 mg / mg. In some embodiments, the urinary albumin / creatinine ratio can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, or at least 120% compared to when mice are treated with an isotype control antibody. In some implementations, the proportions described herein are determined approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the first day of the experiment using the spontaneous SLE model.

[0211] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can significantly reduce SLE-related renal pathology symptoms compared to treatment of mice with an allotype control antibody. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can slow disease progression in spontaneous SLE models. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase activity scores by at least 50%, at least 75%, or at least 100% compared to treatment of mice with an allotype control antibody in a spontaneous SLE model. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce chronicity scores by less than 50%, less than 40%, less than 30%, or less than 20% compared to treatment of mice with an allotype control antibody in a spontaneous SLE model. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce independent lesion scores by less than 80%, less than 60%, less than 40%, or less than 20% compared to treatment of mice with an allotype control antibody in a spontaneous SLE model.

[0212] Methods for preparing anti-IL12p35 antibodies

[0213] Fragments of isolated human IL12p35 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody formulations. Polyclonal antibodies can be generated in animals through multiple injections of antigenic peptides or proteins (e.g., subcutaneous or intraperitoneal injections). In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an agent that is immunogenic in the species to be immunized. Animals may be injected with the antigenic peptide or protein more than once (e.g., twice, three times, or four times).

[0214] Full-length peptides or proteins can be used, or alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of the protein contains at least eight (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of IL12p35 and covers the epitope of the protein, such that antibodies generated against the peptide form specific immune complexes with the protein. As described above, the full-length sequence of human IL12p35 is known in the art. In some embodiments, Fc-labeled or His-labeled human IL12p35 protein is used as an immunogen.

[0215] Immunogens are commonly used to prepare antibodies by immunizing a suitable subject (e.g., a human or a transgenic animal expressing at least one human immunoglobulin locus). Appropriate immunogenic formulations may contain, for example, recombinantly expressed or chemically synthesized peptides (e.g., recombinant chimeric IL12 as described herein, or fragments of human IL12p35). Formulations may further include adjuvants, such as Freund's complete or incomplete adjuvants or similar immunostimulants.

[0216] Polyclonal antibodies can be prepared as described above by immunizing suitable subjects (e.g., RenMab™ mice) with the recombinant chimeric IL12 described herein as an immunogen. Optionally, an IL12p35 polypeptide or an antigenic peptide thereof (e.g., a fraction of IL12p35) can be used as an immunogen. Antibody titers in immunized subjects can be monitored over time using standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized human IL12p35 or a fragment thereof. If desired, antibody molecules can be isolated from mammals (e.g., from blood) and further purified by known techniques (such as protein A or protein G chromatography) to obtain IgG fractions. At an appropriate time following immunization, such as when the specific antibody titer is at its highest, antibody-producing cells can be obtained from the subject, and monoclonal antibodies can be prepared using said cells via standard techniques such as hybridoma techniques originally described by Kohler et al. (Nature 256:495-497, 1975), human B-cell hybridoma techniques (Kozbor et al., Immunol. Today 4:72, 1983), EBV hybridoma techniques (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma techniques. Techniques for generating hybridomas are well-known (see generally Current Protocols in Immunology, 1994, Coligan et al. (ed.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells that produce monoclonal antibodies can be detected by screening the hybridoma culture supernatant for the presence of antibodies that bind to peptides or epitopes of interest, for example, using a standard ELISA assay.

[0217] Variants of the antibody or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding human, humanized, or chimeric antibodies or their antigen-binding fragments, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence constituting the antigen-binding site or antigen-binding domain of the antibody. Within this group of variants, some antibody or antigen-binding fragments will have increased affinity for target proteins such as IL12p35. Any combination of deletions, insertions, and / or combinations can be made to obtain antibodies or their antigen-binding fragments with increased binding affinity to the target. Introducing amino acid changes into the antibody or antigen-binding fragment can also alter the antibody or antigen-binding fragment or introduce new post-translational modifications, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are linked by enzymes present in the cell), or introducing new glycosylation sites.

[0218] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include those derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camels (e.g., camels and alpacas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits) (including genetically engineered rodents to produce human antibodies).

[0219] In some implementations, antibodies are generated using mice (e.g., RenMab™ mice) that possess both humanized heavy chain immunoglobulin loci and humanized kappa chain immunoglobulin loci. Heavy chain immunoglobulin loci are regions of chromosomes containing genes encoding antibody heavy chains. Loci may include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (linkage) genes, and mouse heavy chain constant domain genes. Kappa chain immunoglobulin loci are regions of chromosomes containing genes encoding antibody light chains (kappa chains). Kappa chain immunoglobulin loci may include, for example, human IGKV (variable) genes, human IGKJ (linkage) genes, and mouse light chain constant domain genes. A detailed description of RenMab™ mice can be found in WO / 2020 / 169022, which is incorporated herein by reference in its entirety.

[0220] In some implementations, a recombinant chimeric IL12 comprising mouse IL12p40 and human IL12p35 is used as an immunogen to immunize mice (e.g., RenMab™ mice). As a result, the resulting antibody can specifically target human IL12p35 but not mouse IL12p40.

[0221] Human antibodies and humanized antibodies include antibodies having variable and constant regions derived from human immunoglobulin sequences (or having the same amino acid sequence as human immunoglobulin sequences). Human antibodies may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR.

[0222] Humanized antibodies typically possess a human framework (FR) grafted with a non-human CDR. Therefore, humanized antibodies have one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are generally referred to as “import” residues, which are typically derived from the “import” variable domain. Humanization can be performed, for example, by replacing the corresponding sequence of a human antibody with rodent CDRs or CDR sequences. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); each of which is incorporated herein by reference in its entirety. Thus, a “humanized” antibody is a chimeric antibody in which a portion far less than the complete human V domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies, in which some CDR residues and some FR residues are replaced by residues from similar sites in human antibodies.

[0223] The selection of human VH and VL domains for the preparation of humanized antibodies is crucial for reducing immunogenicity. Following a so-called "best-fit" approach, sequences of the V domains of mouse antibodies were screened across an entire library of known human domain sequences. The human sequence that best approximates the mouse sequence was then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).

[0224] Further important is the humanization of antibodies, in which high specificity and affinity for antigens, as well as other advantageous biological properties, are retained. To achieve this, humanized antibodies can be prepared by analyzing parental sequences and various conceptual humanization products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly used and well-known to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. These displays allow for the analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues affecting the ability of the candidate immunoglobulin to bind its antigens. In this way, FR residues can be selected and combined from the accepting and input sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0225] Typically, amino acid sequence variants of human, humanized, or chimeric anti-IL12p35 antibodies will contain an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence present in the light or heavy chain of the original antibody.

[0226] The antibody generated from mice has a complete human VH, a complete human VL, and a mouse constant region. In some embodiments, the human VH and human VL are linked to human IgG constant regions (e.g., IgG1, IgG2, IgG3, and IgG4).

[0227] Identity or homology relative to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to those present in the human, humanized, or chimeric anti-IL12p35 antibody or fragment. If necessary, vacancies are introduced after the alignment sequence to achieve the maximum percentage of sequence identity, and any conserved substitutions are not considered as part of the sequence identity.

[0228] Antibodies against IL12p35 or antigen-binding fragments can be further modified. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in that region. The resulting homodimeric antibodies can have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-lives can also be prepared using heterobifunctional crosslinking agents as described, for example, by Wolff et al. (Cancer Res. 53:2560-2565, 1993). Optionally, antibodies with dual Fc regions can be engineered (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0229] In some embodiments, antibodies against IL12p35 or their antigen-binding fragments can be covalently modified. These covalent modifications can be performed by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications to antibodies or antibody fragments can be introduced into the molecule by reacting the target amino acid residues of the antibody or fragment with an organic derivatizer capable of reacting with selected side chains or N- or C-terminal residues.

[0230] Recombinant vector

[0231] This disclosure also provides recombinant vectors (e.g., expression vectors) comprising isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), wherein a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains polynucleotides and / or a vector containing polynucleotides), and recombinant antibody polypeptides or fragments thereof are generated by recombinant technology.

[0232] As used herein, “vector” refers to any construct capable of delivering one or more polynucleotides of interest to a host cell when introduced into the host cell. An “expression vector” is capable of delivering one or more polynucleotides of interest and expressing them as encoded polypeptides in a host cell that has already been introduced with an expression vector. Therefore, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operatively linked to regulatory elements such as promoters, enhancers, and / or poly-A tails at or near the integration site of the polynucleotide of interest within the vector or in the host cell genome, such that the polynucleotide of interest will be translated in the host cell in which the expression vector has been introduced.

[0233] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection, and infection and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0234] In some implementations, a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses) is used to introduce the polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein). This may involve using a non-pathogenic (deficient) replicative virus, or a replication-deficient virus may be used. In the latter case, viral transmission will typically occur only in complementary viral packaging cells. Suitable systems are disclosed in, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NYAcad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; US ​​Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89 / 01973; US Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner et al., Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA can also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating DNA onto biodegradable beads that are efficiently transported into cells.

[0235] For expression, a DNA insert containing a polynucleotide encoding an antibody or a polynucleotide encoding a polypeptide disclosed herein can be operatively linked to a suitable promoter (e.g., a heterologous promoter) such as the bacteriophage λPL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, to name just a few. Other suitable promoters are known to those skilled in the art. The expression construct may further contain sites for transcription initiation and termination, and ribosome-binding sites for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct may include translation initiated at initiation and a stop codon (UAA, UGA, or UAG) appropriately located at the end of the polypeptide to be translated.

[0236] As shown, the expression vector may include at least one selective biomarker. Such biomarkers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in *E. coli* and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as *E. coli*, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Noctuidae Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0237] Non-limiting vectors for use with bacteria include pQE70, pQE60, and pQE-9, available from Qiagen; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5, available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL, available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0238] Suitable non-restrictive bacterial promoters include the *E. coli* lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, lambda PR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs such as the Raúl's sarcoma virus (RSV) promoter, and metallothionein promoters such as the mouse metallothionein-I promoter.

[0239] In the yeast *Saccharomyces cerevisiae*, many vectors containing constitutive or inducible promoters such as alpha factors, alcohol oxidases, and PGH can be used. For related reviews, see Ausubel et al. (1989) *Current Protocols in Molecular Biology*, John Wiley & Sons, New York, NY, and Grant et al., *Methods Enzymol.*, 153: 516-544 (1997).

[0240] Introducing constructs into host cells can be achieved through calcium phosphate transfection, DEAE-glucan-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0241] Transcription of DNA encoding the disclosed antibodies in higher eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically about 10 to 300 bp, that enhance the transcriptional activity of promoters in a given host cell type. Examples of enhancers include the SV40 enhancer located on the late side of the origin of replication at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polytumor enhancer on the late side of the origin of replication, and the adenovirus enhancer.

[0242] To facilitate the secretion of translated proteins into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. These signals can be endogenous to the polypeptide or they can be heterologous signals.

[0243] Peptides (e.g., antibodies) can be expressed in modified forms such as fusion proteins (e.g., GST-fusions) or with histidine tags, and can include not only secretion signals but also additional heterologous functional regions. For example, regions of additional amino acids (especially charged amino acids) can be added to the N-terminus of a peptide to improve its stability and persistence in host cells during purification or during subsequent processing and storage. Similarly, peptide moieties can be added to peptides to facilitate purification. Such regions can be removed prior to the final preparation of the peptide. Adding peptide moieties to peptides to induce secretion or excretion, improve stability, and facilitate purification are well-known and conventional techniques in the art.

[0244] Treatment

[0245] The antibodies or antigen-binding fragments disclosed herein can be used for a variety of therapeutic purposes.

[0246] In one aspect, this disclosure provides methods for treating conditions associated with abnormal or undesirable immune responses, such as autoimmune diseases, preventing conditions associated with abnormal or undesirable immune responses, such as autoimmune diseases, or reducing the risk of developing conditions associated with abnormal or undesirable immune responses, such as autoimmune diseases, for example, by inhibiting the IL12p35 / IL12Rβ2 specific downstream pathway. In some embodiments, the methods described herein can inhibit the IL12 signaling pathway without interfering with the IL23 signaling pathway. In some embodiments, the methods described herein can neutralize IL12 in vivo but preserve IL23.

[0247] Systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS) represent a group of inflammatory diseases that are often associated with each other, implying the existence of potentially universally shared pathological mechanisms (Conrad, N., et al. “Incidence, prevalence, and co-occurrence of autoimmune disorders over time and by age, sex, and socioeconomic status: a population-based cohort study of 22 million individuals in the UK.” The Lancet 401.10391 (2023): 1878–1890; and Selmi, C. et al. “Chronic autoimmunepithelitis in Sjögren's syndrome and primary biliary cholangitis: a comprehensive review.” Rheumatology and Therapy 4 (2017): 263–279). Most widely used immunotherapies, such as anti-TNF, JAK inhibitors, and anti-p40, are ineffective against them, highlighting the significant unmet need for developing novel therapies for this group of autoimmune diseases.

[0248] Systemic sclerosis (SSc), or scleroderma, exhibits complex autoimmune characteristics, characterized by vascular damage and fibrosis in various organs. It has the highest case-specific mortality rate among systemic autoimmune diseases (Scherlinger, M., et al. “Worldwide trends in all-cause mortality of auto-immune systemic diseases between 2001 and 2014.” Autoimmunity reviews 19.6 (2020): 102531). Treatment options for fibrotic lesions, sclerosis, and SSc-associated interstitial lung disease (SSc-ILD) are limited. Primary biliary cholangitis (PBC) is a chronic autoimmune cholestatic liver disease characterized by progressive intrahepatic cholestasis and end-stage liver disease requiring transplantation (Onofrio, FQ, et al. “Apractical review of primary biliary cholangitis for the gastroenterologist.” Gastroenterology & Hepatology 15.3 (2019): 145). Systemic lupus erythematosus (SLE) is a challenging autoimmune disease with diverse clinical manifestations and an unpredictable course. Despite advances in pathophysiological understanding and some newly developed treatments, SLE patients still face a significant risk of mortality and progressive organ damage (Piga, M., et al. “The main challenges in systemic lupus erythematosus: wheredo we stand?.” Journal of Clinical Medicine 10.2 (2021): 243). Sjogren's syndrome (SjS) is an epidemic chronic autoimmune rheumatic disease characterized by lymphocytic infiltration of exocrine glands and various extra-glandular organs. Treatment focuses primarily on symptom management and complication prevention, with no cure yet (Zhan, Q., et al. “Pathogenesis and treatment of Sjogren's syndrome: Review and update.” Frontiers in Immunology 14 (2023): 1127417).

[0249] In some embodiments, the anti-IL12p35 antibody or its antigen-binding fragment described herein can block the binding of IL12 to its receptor IL12Rβ2, thereby inhibiting IL12-induced intracellular pathways (e.g., the IL12p35 / IL12Rβ2 specific pathway). Based on the human genetic analysis discussed in Example 12, these anti-IL12p35 antibodies or their antigen-binding fragments can be used to treat a group of immune disorders (e.g., SSc, PBC, SLE, and SjS). In some embodiments, the subject described herein has systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS). In some embodiments, the subject is a human subject. In some embodiments, the subject is a non-human mammal, such as a monkey, dog, mouse, or any commonly used model animal known in the art. In some embodiments, the subject is a dog with a disease or condition similar to SSc, PBC, SLE, and / or SjS. In some implementations, the dog has immune-mediated diseases (e.g., autoimmune diseases), such as hypothyroidism, lupus, immune-mediated polyarthritis (IMPA), inflammatory bowel disease (IBD), immune-mediated hemolytic anemia (IMHA), immune-mediated thrombocytopenia (IMT), diabetes, myasthenia gravis, rheumatoid arthritis, Addison's disease (adrenocortical insufficiency), bullous autoimmune skin disease, and periodontal disease. For details, see, for example, Pedersen, NC, “A review of immunologic diseases of the dog.” Veterinary Immunology and Immunopathology 69.2-4 (1999): 251-342, which is incorporated herein by reference in its entirety.

[0250] In some embodiments, the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein do not block the binding of IL12p40 to its receptor IL12Rβ1. Therefore, these anti-IL12p35 antibodies or antigen-binding fragments thereof do not interfere with the binding of IL23 to its receptor or transduction signals (e.g., the IL12p40 / IL12Rβ1 specific pathway). Based on the human genetic analysis discussed in Example 12, because IL12p40 and IL23R are associated with PsO, CD, UC, IBD, and AS, but not with SSc, PBC, SLE, or SjS, the anti-IL12p35 antibodies or antigen-binding fragments thereof described herein may not be suitable for treating a group of immune disorders (e.g., PsO, CD, UC, IBD, and AS). In some embodiments, the subjects described herein did not have psoriasis (PsO), Crohn's disease (CD), ulcerative colitis (UC), inflammatory bowel disease (IBD), or ankylosing spondylitis (AS).

[0251] In some implementations, the anti-IL12p35 antibody or its antigen-binding fragment described herein can inhibit the IL12 signaling pathway without interfering with the IL23 signaling pathway.

[0252] As used herein, “effective amount” means an amount or dose sufficient to achieve a beneficial or desired outcome, including stopping, slowing, delaying, or inhibiting the progression of a disease (e.g., any of the autoimmune diseases described herein). Effective amounts will vary depending on the age and weight of the subject, the severity of symptoms, and the route of administration, such as the antibody to be administered, the antigen-binding fragment, the polynucleotide encoding the antibody, the carrier containing the polynucleotide, and / or a combination thereof, and therefore can be determined on an individual basis.

[0253] An effective amount can be administered in one or more doses. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to reduce, stop, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to reduce, stop, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As understood in the art, the effective amount of an antibody or antigen-binding fragment can vary depending on (among other things) the patient's medical history and other factors such as the type (and / or dosage) of the antibody used.

[0254] The effective amount and schedule for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and such determination is within the scope of the art. Those skilled in the art will understand that the dosage that must be administered will vary, for example, depending on the mammal to which the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein will be received, the route of administration, the specific type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed herein used, and other drugs administered to the mammal. Guidance on selecting appropriate dosages of antibodies or antigen-binding fragments can be found in literature concerning the therapeutic use of antibodies and antigen-binding fragments, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0255] In any of the methods described herein, at least one antibody, its antigen-binding fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and optionally at least one additional therapeutic agent may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered as a sustained-release oral formulation.

[0256] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that the biological activity periods of one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) overlap.

[0257] In some implementations, subjects may be administered at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). Skilled medical professionals may determine the length of treatment using any of the methods described herein for diagnosing or tracking treatment effectiveness (e.g., observing at least one symptom of cancer). As described herein, skilled medical professionals may also vary the identity and quantity (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and may also adjust (e.g., increase or decrease) the dose or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to the subject based on an assessment of treatment effectiveness (e.g., using any of the methods described herein and known in the art).

[0258] Pharmaceutical Compositions and Routes of Administration

[0259] This document also provides pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0260] The pharmaceutical composition is formulated to suit its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixative oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, an antibacterial or antifungal agent (such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, and thimerosal), an antioxidant (such as ascorbic acid or sodium bisulfite), a chelating agent (such as ethylenediaminetetraacetic acid), a buffer (such as acetate, citrate, or phosphate), and an isotonic agent (such as a sugar (e.g., dextran)), a polyol (e.g., mannitol or sorbitol), or a salt (e.g., sodium chloride) or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent No. 4,522,811). The composition may be formulated and packaged in ampoules, disposable syringes, or multi-dose vials. Where necessary (e.g., in injectable formulations), adequate fluidity can be maintained by using coating agents such as lecithin or surfactants. Absorption of the antibody or its antigen-binding fragment can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Optionally, controlled release can be achieved via implants and microencapsulated delivery systems that may include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0261] Compositions containing one or more of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dose units (i.e., physically discrete units containing a predetermined amount of the active compound for ease of administration and dose uniformity).

[0262] Pharmaceutical compositions intended for parenteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., a single-dose dose). The pharmaceutical composition may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, antibodies may be formulated in an aqueous solution, preferably in a physiologically compatible buffer to reduce injection site discomfort. The solution may contain a formulation agent, such as a suspending agent, stabilizer, and / or dispersant. Optionally, the antibody may be in lyophilized form for preparation with a suitable solvent (e.g., sterile, pyrogen-free water) prior to use.

[0263] The toxicity and therapeutic efficacy of the composition can be determined in cell cultures or laboratory animals (e.g., monkeys) using standard pharmaceutical procedures. For example, the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population) can be determined: the therapeutic index is the ratio of LD50 to ED50. Agents exhibiting a high therapeutic index are preferred. In cases where the agent exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce unnecessary side effects). Toxicity and therapeutic efficacy can be determined using other standard pharmaceutical procedures.

[0264] Data obtained from cell culture assays and animal studies can be used to formulate an appropriate dose of any given agent for a subject (e.g., human, dog, monkey, or mouse). The therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or their antigen-binding fragments (e.g., any of the antibodies or antibody fragments described herein) will be the amount by which the disease is treated (e.g., by killing cancer cells) in a subject (e.g., a human subject identified as having cancer) or a subject identified as being at risk of developing the disease (e.g., a subject who previously had cancer but has now been cured), and the amount by which the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., human, dog, monkey, or mouse) is reduced. The effectiveness and dosage of any of the antibodies or antigen-binding fragments described herein can be determined by a healthcare professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in a subject (e.g., human, dog, monkey, or mouse). Certain factors may influence the dosage and timing required for effective treatment of a subject (e.g., the severity of the disease or condition, prior treatment, the subject's general health and / or age, and the presence of other diseases).

[0265] Exemplary doses include milligrams or micrograms per kilogram of the antibody or antigen-binding fragment described herein (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg). While these doses cover a wide range, those skilled in the art will understand that therapeutic agents, including antibodies and their antigen-binding fragments, vary in potency and that effective amounts can be determined by methods known in the art. Typically, a relatively low dose is administered initially, which may then be gradually increased by a primary healthcare professional or veterinary professional (in the case of therapeutic applications) or an investigator (when still in the development phase) until an appropriate response is obtained. Furthermore, it should be understood that the specific dose level used for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health condition, sex and diet, the time of administration, the route of administration, the rate of release, and the half-life of the antibody or antibody fragment in the body.

[0266] The pharmaceutical composition may be included in a container, package, or dispenser along with instructions for use. This disclosure also provides methods for manufacturing antibodies or antigen-binding fragments thereof for the various uses described herein.

[0267] Example

[0268] The invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0269] Example 1: Generation of anti-IL12p35 antibody

[0270] This example describes how to generate anti-IL12p35 antibodies. A set of antibodies that selectively bind to the human cynomolgus monkey (IL12p35) antigen were generated in RenMab™ mice (Biocytogen) by immunization with recombinant chimeric IL12.

[0271] immunityRecombinant chimeric IL12 is a heterodimer composed of mouse IL12p40 and human IL12p35, engineered to promote immunogenicity against the human IL12p35 subunit of IL12p70. A total of seven mice were initially immunized subcutaneously via neck and ankle injections with a recombinant chimeric IL12 emulsion prepared in complete Freund's adjuvant (CFA). Subsequently, all mice were boosted three times with a recombinant chimeric IL12 emulsion prepared in incomplete Freund's adjuvant (CFA). Five days after the final booster immunization, four mice with higher serum titers were sacrificed for Beacon binding selection. The remaining three mice were further boosted three times. Four days after the final booster immunization, two mice with higher serum titers were sacrificed for Beacon binding selection.

[0272] Preparation of B cells Lymphatic organs, including lymph nodes, bone marrow, and spleen, were harvested from euthanized mice. The harvested organs were homogenized to break down tissue structures. Single-cell suspensions were obtained by passing the homogenized material through a 40 μm cell filter. Cell number and viability were assessed. Plasma B cells were enriched using a mouse CD138+ plasma cell separation kit (StemCell Technologies, Cat#: 18957).

[0273] Beacon filtering Using Beacon ® Optical flow control system (model: 110-08023) and OptoSelect ®11K microarrays (Berkeley Lights, Cat#: 500-12012) were used for Beacon screening of plasma B cells. The Beacon bloom screening assay used streptavidin beads (Berkeley Lights, Cat#: 520-00053) conjugated with biotinylated chimeric IL12 or biotinylated chimeric IL12 to capture secreted binding antibodies, and simultaneously quantified the binding antibodies using fluorescently labeled anti-mouse Fc. A total of 128 cells were successfully unloaded from the blooming Beacon culture pin, and RNA was isolated and then RT-PCR was performed to amplify the nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL). The PCR amplicon was sent to Genewiz / Azenta for sequencing. The sequences were analyzed using the IMGT program. The unique clones were converted into the complete human IgG1 form and synthesized for further characterization. Some clones are engineered to improve pharmacokinetics by introducing Fc mutations with enhanced FcRn affinity via amino acid substitution (e.g., M252Y / S254T / T256E or YTE mutations) and to reduce potential immunogenicity by introducing reverting germline mutations via amino acid substitution within the framework of the variable region.

[0274] Example 2: Anti-IL12p35 antibody with high affinity binding to human and monkey IL12p70

[0275] The binding kinetics of the antibodies identified in Example 1 were determined. Gator was used. ®Successfully generated antibodies were measured using the Prime BLI (Biolayer Interferometry) system (Gator Bio, USA), and selected variants were measured using the BiaCore™ SPR platform. Specifically, anti-IL12p35 antibody was loaded onto an HFC probe (anti-hIgG Fc, Gator Bio, Cat#: 160003). Subsequently, the probe was incubated with the corresponding monomeric antigens—human IL12p70-His (Sino Bio, Cat#: CT011-H08H), rhesus monkey IL12-His (Sino Bio, Cat#: CT045-C08H), canine IL12p70 (Kingfisher Biotech, Cat#: RP2228D-005), or mouse IL12p70 (BioLegend, Cat#: 577002)—in 250 µl K buffer (Gator Bio, Cat#: 120011) at 25°C for 3–4 minutes. Dissociation was monitored for approximately 10 minutes. The probes were regenerated with regeneration buffer (Gator Bio, Cat#: 120012) between binding cycles. Binding kinetics were analyzed using software provided by the manufacturer. Specifically, the following settings were used: antibody for all tests: 4 μg / ml; canine IL12p70: 2 μg / ml; and mouse IL12p70: 1 μg / ml.

[0276] To measure binding kinetics more accurately, the selected antibody was analyzed using the BiaCore™ 8K SPR platform. Specifically, the anti-IL12p35 antibody was loaded onto a Protein A chip (Cytiva, Cat#:29127556). The chip was then incubated with serially diluted monomeric antigen human IL12p70-His (Sino Bio, Cat#: CT011-H08H) to allow association for 90 seconds and dissociation for 210 seconds, and kinetics were monitored. The chip was regenerated with glycine hydrochloride buffer at pH 1.5 between cycles. Binding kinetics were analyzed using the BiaCore™ Insight evaluation software provided by the manufacturer.

[0277] To compare the binding kinetics of YTE variants with their corresponding parental antibodies, anti-IL12p35 antibody was loaded with HFC (anti-hIgG Fc, Gator Bio, Cat#: 160003), and the probe was incubated with recombinant FcRn protein (Sino Bio, Cat#: CT009-H08H) in internally prepared K buffer at pH 6.0, pH 6.5, or pH 7.0, followed by a dissociation step in the corresponding K buffer.

[0278] via Gator® The Prime system measures the preliminary binding affinity of the antibody to human IL12p70, and the results are shown in... Figure 1 In the middle. For some antibodies, because their binding affinity is too high (e.g., having less than 1 × 10⁻⁶),... -11 M or less than 1 × 10 -12 The estimated KD values ​​of M are beyond the detection capabilities of the Gator BLI technology (“OR”), so their KD values ​​cannot be calculated.

[0279] like Figure 2A As shown, further analysis revealed that some antibodies, such as D2M008-A1A8, D2M008-A1A9, and D2M008-4A4, exhibited high binding affinity for human and rhesus monkey IL12p70. D2M008-A1A8 and D2M008-A1A9 showed approximately a 100-fold difference in binding affinity for human and monkey IL12. A recombinant antibody (named 27H28L) composed of the heavy chain of D2M008-A1A8 and the light chain of D2M008-A1A9 showed a similar magnitude of difference when binding to human and rhesus monkey IL12p70. Antibody D2M008-4A4 exhibited very high binding affinity for human and rhesus monkey IL12p70. Because the IL12 protein sequences of cynomolgus monkeys and rhesus monkeys are identical, the high binding affinity to rhesus monkey IL12p70 indicates that the antibody can also bind to cynomolgus monkey IL12p70, suggesting that cynomolgus monkeys (as a related species) could be used for future non-clinical studies. Figure 2B-2C As shown, the YTE mutation and reverted germline mutation showed no effect on the binding affinity for 27H28L and D2M008-4A4. Notably, the 27H28L variant 27H28L-hblb-YTE showed a very high affinity for canine IL-12, comparable to its affinity for human IL-12, as both exceed the range on Gator. Figures 3A-3B As shown, the YTE variant exhibits improved FcRn binding affinity at acidic pH compared to the parent antibody, suggesting that the YTE variant may theoretically improve pharmacokinetics in humans compared to the parent antibody.

[0280] Example 3: Anti-IL12p35 antibody blocks the binding of IL12 to its receptor IL12Rβ2.

[0281] The ability of the anti-IL12p35 antibody to block the interaction between human IL12p70 and recombinant IL12Rβ2 (interleukin 12 receptor, β2 subunit) protein was determined by an ELISA-based assay. Specifically, 1 μg / mL biotinylated human IL12p70 (SinoBio, Cat#: CT011-H08H-B) was pre-incubated with a fixed concentration (or serially diluted) of anti-IL12p35 or the control antibody anti-IL12p40 (ustekinumab, Biointron, Cat#: B779149; or "anti-hP40") at room temperature (RT) for 30 min, and then added to a 96-well plate coated with 1 μg / mL IL12Rβ2 (Biointron, Cat#: B21709001), followed by incubation at RT with gentle shaking for 2 h. Bound IL12p70 was detected using HRP-conjugated streptavidin (BioLegend, Cat#: 405210) and TMB substrate (Surmodics, Cat#: TMBS-1000-01). OD450 was measured using a Varioskan™ Lux microplate reader (Thermo Scientific).

[0282] like Figure 4A As shown, in Example 2, it was identified and Figure 1 The 27 binding agents listed showed differences in their ability to block the interaction between human IL12p70 and recombinant IL12Rβ2. Further analysis of eight selected anti-IL12p35 antibodies showed that they blocked the interaction between human IL12p70 and recombinant IL12Rβ2 in a dose-dependent manner. Figure 4B Titrated 27H28L and several additional anti-IL12p35 antibodies were tested. Specifically, 27H28L, D2M008-4A4, and D2M008-4A5 exhibited dose-dependent blockade of the interaction between human IL12p70 and recombinant IL12Rβ2. Figure 4C Furthermore, the YTE mutation showed no significant effect on the blocking function of 27H28L and D2M008-4A4. Figure 4D Furthermore, reversing germline mutations showed no effect on blocking function. Figure 4E ).

[0283] Example 4: Anti-IL12p35 antibody blocks IL12-induced intracellular signaling in IL12 reporter cells

[0284] The ability of anti-IL12p35 antibody to inhibit IL12p70-induced signaling was evaluated in HEK-Blue™ IL12 reporter cells (InvivoGen, Cat#: hkb-il12). Specifically, 20 ng / ml recombinant human IL12 (BioLegend, Cat#: 573004) or rhesus monkey IL12 (Sino Bio, Cat#: CT045-C08H) was pre-incubated with different concentrations of IgG1, anti-hP40, or anti-IL12p35 antibody at room temperature for 30 minutes, and then added to cells seeded with 5 × 10⁶ cells. 4 HEK-Blue™ IL12 reporter cells were placed in flat-bottomed 96-well plates and then incubated at 37°C for 24 hours. 20 μl of supernatant was collected from each well and mixed with 180 μl of QUANTI-Blue™ solution (Invivogen, Cat#: rep-qbs) in a new flat-bottomed 96-well plate, and incubated at 37°C for 50 minutes. OD620 was measured using a Varioskan™ Lux microplate reader (Thermo Scientific, SN#3020-80467).

[0285] like Figure 5A As shown, in Example 2, it was identified and Figure 1 The 27 binding agents listed showed varying abilities to inhibit IL12p70-induced intracellular signaling in HEK-Blue™ IL12 reporter cells. D2M008-A1A8 and D2M008-A1A9 exhibited the best blocking ability among these IL12 binding agents. D2M008-A1B4 and D2M008-A1E10 also showed blocking ability. Further analysis of these four selected anti-IL12p35 antibodies was conducted. Figure 5B As shown, D2M008-A1A8 and D2M008-A1A9 strongly inhibit IL12p70-induced intracellular signaling in HEK-Blue™ IL12 reporter cells in a dose-dependent manner.

[0286] like Figure 5C As shown, the engineered variant 27H28L, composed of the heavy chain of D2M008-A1A8 and the light chain of D2M008-A1A9, exhibited significantly stronger potency in a dose-dependent manner than its parental antibodies D2M008-A1A8 and D2M008-A1A9. Interestingly, 27H28L was also more potent than anti-IL12p40 (ustekinumab), and its activity was comparable to the combination of anti-IL12p40 (ustekinumab) and D2M008-A1A8. Therefore, as Figure 5B and Figure 5CAs shown, D2M008-A1A8 and D2M008-A1A9 are slightly superior, or at least comparable, to anti-IL12p40 (ustekinumab) in inhibiting IL12p70 activity. These results indicate that the engineered anti-IL12p35 variant 27H28L can block the interaction between IL12p70 and its receptor via a different mode of action than D2M008-A1A8 and D2M008-A1A9 (superior to the anti-IL12p40 (ustekinumab)).

[0287] Although D2M008-4A4 is less effective than 27H28L in inhibiting IL12 activity, it is significantly superior to anti-IL12p40 (ustekinumab) in a dose-dependent manner. Figure 5D YTE and reverted germline mutations showed no significant effect on the ability of 27H28L and D2M008-4A4 to inhibit IL12 activity. Figure 5E-5F While both 27H28L and D2M008-4A4 inhibited human IL12p70 more effectively than anti-IL12p40 (ustekinumab), only D2M008-4A4 inhibited IL12p70-induced signaling in rhesus monkeys in HEK-Blue™ IL12 reporter cells (similar to ustekinumab), while 27H28L did not. Figure 6 ).

[0288] Example 5: Anti-IL12p35 antibody inhibits IL12-induced IFN-γ production in human PBMCs

[0289] The primary function of IL12 is to activate T cells and NK cells, leading to increased IFN-γ production, proliferation, and cytotoxic potential. The ability of identified anti-IL12p35 antibodies to inhibit human IFN-γ production in response to exogenous IL12p70 was evaluated using human PBMCs. Specifically, 10 ng / ml recombinant human IL12 (BioLegend, Cat#: 573004) was pre-incubated with different concentrations of IgG1, anti-IL12p40, or anti-IL12p35 antibodies at room temperature for 30 minutes, and then added to each well seeded with 1–2 × 10⁶ antibodies. 5 Individual PBMCs were placed in U-bottom 96-well plates and then incubated at 37°C for 24 hours. Supernatant was collected from each well and human IFN-γ was measured using the MAX™ Deluxe Set Human IFN-γ ELISA Kit (BioLegend, Cat#: 430116).

[0290] like Figures 7A-7CAs shown, the anti-IL12p35 antibody inhibited exogenous IL12-induced IFN-γ production in human PBMCs in a dose-dependent manner. Significantly, consistent with results observed in IL12 reporter cells, D2M008-4A4 and 27H28L were more effective than anti-IL12p40 (ustekinumab) in inhibiting IL12-induced IFN-γ production in human PBMCs.

[0291] Example 6: Anti-IL12p35 antibody inhibits IFN-γ production in CD4+ T cells co-cultured with allogeneic dendritic cells.

[0292] The ability to inhibit endogenous IL12p70 activity was evaluated using primary human CD4+ T cells and allogeneic dendritic cells in an MLR assay. Specifically, human dendritic cells were generated from monocytes by incubation for 6 days with 50 ng / ml human GM-CSF (BioLegend, Cat#: 766106) and 20 ng / ml human IL-4 (BioLegend, Cat#: 766206), followed by maturation for 1 day with 1 mg / ml LPS (Sigma, Cat#: L2630-10MG). 1×10⁶ cells were added to 50 ml of complete 1640 RPMI medium. 4 Dendritic cells were seeded into each well of a flat-bottomed 96-well plate. Then, different concentrations of IgG1, anti-IL12p40, 27H28L, or D2M008-4A4 (starting at 60 μg / ml, then 3-fold dilution) in 100 ml of complete 1640 RPMI medium were added to the designated wells. Next, 50 μl of 1×10⁻⁶ DMG PLC in complete 1640 RPMI medium was added. 5 Allogeneic CD4+ T cells were added to the designated wells. The cells were mixed and incubated at 37°C for 24 hours. The supernatant was collected from each well and human IFN-γ was measured using the MAX™ Deluxe Set Human IFN-γ ELISA kit (BioLegend, Cat#: 430116).

[0293] like Figures 8A-8BAs shown, in an MLR assay using allogeneic dendritic cells to stimulate primary CD4 T cells, anti-IL12p35 antibodies (engineered variants 27H28L and D2M008-4A4) and anti-IL12p40 (ustekinumab) inhibited IFN-γ production in a dose-dependent manner. Interestingly, D2M008-4A4 and 27H28L, especially 27H28L, were more effective than anti-IL12p40 (ustekinumab) in inhibiting IFN-γ production at low concentrations. However, at high concentrations, anti-IL12p40 (ustekinumab) showed greater efficacy, indicating that anti-IL12p40 not only prevents IL12-induced IFN-γ production but also inhibits IFN-γ production via other mechanisms (e.g., through other factors). Combined with observations related to exogenous IL12, the results indicate that specific inhibition of the IL12p35 / IL12Rβ2 interaction is a more effective strategy for inhibiting the biological activity of IL12 compared to blocking the IL12p40 / IL12Rβ1 interaction. This enhanced efficacy can be attributed to the different roles of IL12Rβ1 and IL12Rβ2 in the heterodimeric IL12 receptor complex. Furthermore, inhibiting the IL12p35 / IL12Rβ2 interaction provides an effective strategy for specifically inhibiting the biological activity of IL12 without interfering with IL23, whereas blocking the IL12p40 / IL12Rβ1 interaction does not achieve the same specificity.

[0294] Example 7: Anti-IL12p35 antibody does not interfere with IL23 binding to its receptor and transduction signaling.

[0295] The effect of anti-IL12p35 antibody on the downstream JAK-STAT signaling pathway activated by IL23 was examined in HEK-Blue™ IL23 reporter cells (InvivoGen, Cat#: hkb-il23). Specifically, 10 ng / ml recombinant human IL23 (BioLegend, Cat#: 574104) was pre-incubated with different concentrations of IgG1, anti-hP40, or anti-IL12p35 antibody at room temperature for 30 minutes, and then added to each well seeded with 5 × 10⁶ antibodies. 4 HEK-Blue™ IL23 reporter cells were placed in flat-bottomed 96-well plates and incubated at 37°C for 24 hours. 20 μl of supernatant was collected from each well and mixed with 180 μl of QUANTI-Blue™ solution (Invivogen, Cat#: rep-qbs) in a new flat-bottomed 96-well plate, followed by incubation at 37°C for 30–60 minutes. OD620 was measured using a Varioskan™ Lux microplate reader (Thermo Scientific).

[0296] like Figures 9A-9B As shown, although anti-IL12p40 (ustekinumab) effectively neutralized IL23, 27H28L, D2M008-4A4, or their YTE variants did not inhibit IL23 biological activity. The results confirm that anti-IL12p35 antibody can specifically neutralize IL12, unlike anti-IL12p40, which can neutralize both IL12 and IL23.

[0297] Example 8: Anti-IL12p35 antibody exhibits excellent thermal stability

[0298] The melting points of intact IgG and F(ab')2 of humanized anti-IL12p35 antibody were measured using a Protein Thermal Shift™ dye kit (ThermoFisher, Cat#: 4461146) and qPCR. To prepare F(ab')2, 17.25 μL (1 mg / mL) of intact IgG antibody was digested with 0.25 μL of IdeZ protease (IgG specific) (NEB, Cat#: P0770S) along with 2 μL of 10× GlycoBuffer 2 (NEB, Cat#: P0770S) in a 37°C water bath for 2 hours. 10 μL of CaptureSelect™ IgG-Fc magnetic agarose beads (ThermoFisher, Cat#: 2882852005) were washed with PBS and added to the digestion reaction. The mixture was incubated at room temperature for 15 minutes. Undigested antibody and Fc fragments were removed by centrifugation at 300 g for 2 minutes. Add 12.5 μL aliquots of supernatant containing F(ab')2 or intact IgG antibodies to the wells of a 0.2 mL low-profile 96-well PCR plate (Thermo Scientific, Cat#: AB-0700 / W). Add 5 μL of buffer and 2.5 μL of diluted dye (8×) from a Protein Thermal Shift™ dye kit (ThermoFisher, Cat#: 4461146) to each well and mix thoroughly. Heat the plate from 25 °C to 99 °C at a rate of 0.05 °C / s and detect fluorescence at each temperature using QuantStudio™ 6 software (ThermoFisher, Cat#: A43180). Subtract the lowest intensity before the peak (baseline value) from the fluorescence intensity and normalize to the peak-to-baseline amplitude. Calculate the Tg for each antibody based on the temperature at which the amplitude from baseline to peak is 50% of the peak value using an internally developed Perl script. m .

[0299] like Figures 10A-10BAs shown, both anti-IL12p35 antibodies 27H28L and D2M008-4A4 exhibited excellent thermal stability, and their F(ab')2 forms showed T values ​​of 73.99°C and 79.53°C, respectively. m .

[0300] Example 9: Anti-IL12p35 antibody exhibits stability under different pH stress conditions

[0301] Common chemical modifications of antibodies include deamidation and isomerization. Asparagine deamidation and aspartic acid isomerization can be induced in vitro under high and low pH conditions, respectively. To investigate the stability of anti-IL12p35 antibodies under stress conditions, 100 μL of antibody (1 mg / mL) was exchanged with pH 5.5 buffer (50 mM sodium acetate) or pH 8.5 buffer (20 mM Tris and 10 mM EDTA) using a Zebra™ spin desalting column (40K MWCO, ThermoFisher, Cat#: 87767). The antibody was then incubated in a 40°C water bath for 2 weeks. The function of anti-IL12p35 antibodies under different stress conditions was measured using the methods described in Examples 3-4, which detected the ability of anti-IL12p35 antibodies to block the binding of human IL12p70 to its receptor IL12Rβ2 and to inhibit IL12-induced signal transduction. The control sample (in pH 7.4 buffer) was not subjected to a 2-week 40°C water bath.

[0302] like Figure 11A-11B As shown, the stressed anti-IL12p35 antibodies (27H28L and D2M008-4A4) exhibited similar blocking ability compared to antibodies incubated at normal pH, indicating that the anti-IL12p35 antibodies are stable under high or low pH stress conditions.

[0303] Example 10: Anti-IL12p35 antibody exhibits serum stability

[0304] To further investigate the stability of the anti-IL12p35 antibodies, 50 μg of anti-IL12p35 (approximately 1 mg / mL) was mixed with an equal volume of fresh human serum collected from healthy volunteers and incubated in a 37°C water bath for 2 weeks. Then, using the ELISA and cell-based assays described in Examples 3 and 4, the anti-IL12p35 antibodies exposed to fresh plasma serum were compared with their untreated stock samples (in PBS, without 2 weeks of incubation in a 37°C water bath), measuring their ability to block IL12 binding to IL12Rβ2 and transduce intracellular signaling.

[0305] like Figure 12A-12BAs shown, anti-IL12p35 antibodies incubated with human serum for up to 2 weeks exhibited similar activity compared to those incubated with PBS, indicating their serum stability.

[0306] Similarly, the stability of 27H28L-hblb-YTE and 4A4-hblb2-YTE was determined by mixing 50 μg of anti-IL12p35 (approximately 1 mg / mL) with an equal volume of fresh human serum collected from healthy volunteers and incubating at 37°C for 1, 2, or 3 weeks. Subsequently, 1 μg / mL of biotinylated human IL12p70 (Sino Bio, Cat#: CT011-H08H-B) was pre-incubated with serially diluted anti-IL12p35 antibody at room temperature (RT) for 30 minutes. Next, 100 μl of the pre-incubated solution was added to the designated wells and incubated at RT for 2 hours. Next, 100 μl of HRP-conjugated streptavidin was added to each well and incubated for 30 minutes. Next, 100 μl of HPR substrate was added to each well and incubated at RT for 3 minutes. Finally, 100 μl of stop solution was added to each well. Then, OD450 was measured using the Varioskan™ Lux microplate reader (ThermoScientific).

[0307] like Figure 12C-12D As shown, 27H28L-hblb-YTE and 4A4-hblb2-YTE, incubated with human serum for up to 3 weeks, exhibited similar activity compared to the control sample (“week 0”), indicating their serum stability.

[0308] Example 11: Anti-IL12p35 antibodies 27H28L and D2M008-4A4 do not compete with each other when binding to IL12.

[0309] Using Gator ® The Prime BLI (Biolayer Interferometry) system (Gator Bio, USA) uses a matrix to analyze samples selected from... Figure 1 The anti-IL12p35 antibody was subjected to a binning-competitive assay. Specifically, the anti-IL12p35 antibody was immobilized separately onto an anti-human Fc probe (anti-hIgG Fc, Gator Bio, Cat#: 160003). The probe was then incubated with the monomeric antigen human IL12p70-His (Sino Bio, Cat#: CT011-H08H) in K buffer (Gator Bio, Cat#: 120011) at 25°C for 3–4 minutes. The probe was then incubated with the anti-IL12p35 antibody separately in K buffer at 25°C for 3–4 minutes. Binding kinetics were analyzed using software provided by the manufacturer.

[0310] Further verification was conducted to determine whether anti-IL12p35 antibodies 27H28L and D2M008-4A4 competed for binding. Specifically, anti-IL12p35 antibodies 27H28L and D2M008-4A4 were individually immobilized on a probe by binding to an anti-human Fc probe (anti-hIgG Fc, Gator Bio, Cat#: 160003). Subsequently, the probe was incubated with the monomeric antigen human IL12p70-His (Sino Bio, Cat#: CT011-H08H) in K buffer (Gator Bio, Cat#: 120011) at 25°C for 3–4 minutes. The probe was then incubated with either 27H28L or D2M008-4A4 in K buffer at 25°C for 3–4 minutes. In separate experiments, human IL12p70-His was immobilized by binding to an anti-His probe (GatorBio, Cat#: 160009). The probe was then incubated with 27H28L and D2M008-4A4 for 3–4 minutes, followed by an additional 3–4 minutes of incubation with either 27H28L or D2M008-4A4. Binding kinetics were analyzed using software provided by the manufacturer.

[0311] like Figure 13AAs shown, there are two large boxes, one consisting of antibodies that compete with almost every anti-IL12p35 antibody except for antibodies D2M008-4A4, D2M008-A1E7, D2M008-A1C6, D2M008-B2B1, and D2M008-B2C5; the other consisting of antibodies that compete with almost every anti-IL12p35 antibody except for antibodies D2M008-A1B4 and D2M008-A1E10. Both large boxes overlap with D2M008-A1A8, D2M008-A1A9, D2M008-B2B7, D2M008-A1E5, D2M008-B2C3, D2M008-B2B10, D2M008-A1A2, D2M008-A1G9, and D2M008-B2B12. Therefore, all anti-IL12p35 samples can be further divided into three smaller boxes: the first box includes D2M008-4A4, A1E7, D2M008-A1C6, D2M008-B2B1, and D2M008-B2C5; the second box includes D2M008-A1A8, D2M008-A1A9, D2M008-B2B7, D2M008-A1E5, D2M008-B2C3, D2M008-B2B10, D2M008-A1A2, D2M008-A1G9, and D2M008-B2B12; and the third box includes D2M008-A1B4 and D2M008-A1E10. D2M008-A1A8 and D2M008-A1A9 originate from the same IGHV and IGKV alleles. D2M008-A1B4 and D2M008-A1E10 originate from the same IGHV and IGKV alleles.

[0312] like Figure 13B-13C As shown, the results were obtained through two separate experiments: first, anti-IL12 antibody was initially immobilized with an anti-human Fc probe; then, the antigen IL12 was added; and finally, another antibody was allowed to bind to IL12. Figure 13B Alternatively, antigen IL12 can be initially immobilized using an anti-His probe, followed by allowing 27H28L and D2M008-4A4 to bind to IL12 sequentially. Figure 13C The results revealed that 27H28L and D2M008-4A4 did not exhibit competition for binding to IL12, and competition was observed between 27H28L or D2M008-4A4 and anti-hP40. In summary, based on these results, 27H28L and D2M008-4A4 represent two primary performers in their respective functional families, possessing completely different binding epitopes on IL12.

[0313] Example 12. Human genetic analysis

[0314] The human immune system is complex. Different autoimmune diseases share similar or different mechanisms. Animal models help us understand some autoimmune diseases; however, they have limitations and cannot fully replicate the complexity of the human immune system. Many human autoimmune diseases lack suitable animal models. In such cases, relying on human data, especially evidence of human genetic associations related to immune diseases, can provide more relevant insights.

[0315] Here, we integrated data from a wide range of genome-wide association studies (GWAS) of autoimmune diseases, GWAS of gene expression (eQTL: quantitative trait loci), and GWAS of protein expression (pQTL: quantitative trait loci), and used colocalization tests and comparisons of the genetic effects of eQTL / pQTL on disease risk to conduct a comprehensive human genetic analysis of the components of the IL12 / IL23 / IL35 pathway.

[0316] Our extensive and in-depth genetic analysis revealed that while IL12A (p35) and IL12B (p40) are heterodimers of the same IL12 cytokine, their genetic associations with different human diseases differ. IL12B (p40) and IL23R exhibit specific causal effects on autoimmune diseases such as psoriasis (PsO), Crohn's disease (CD), ulcerative colitis (UC), inflammatory bowel disease (IBD), and ankylosing spondylitis (AS), but not on other autoimmune diseases. Conversely, IL12A (p35) and IL12Rβ2 show specific causal effects on autoimmune diseases such as systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and Sjogren's syndrome (SjS), but there is no evidence of association with PsO, CD, UC, IBD, and AS.

[0317] Based on our in-depth genetic analysis of a broad range of autoimmune diseases, we concluded that targeting IL12p35 / IL12Rβ2 will specifically benefit patients with SSc, PBC, SLE, and SjS. Based on this insight, we discovered and developed specific anti-IL12Ap35 antibodies (as discussed in the foregoing examples) for the treatment of this class of autoimmune diseases, including SSc, PBC, SLE, and SjS.

[0318] Example 12.1: Method

[0319] (1) Data collection

[0320] To fully utilize findings in immune diseases and human genetics, detailed association information from various genetic variations is required. Complete disease GWAS data can be collected from the GWAS catalog database:

[0321] Table 1.

[0322]

[0323] Genetic determinants of the molecular phenotypes of IL12 / IL23 / IL35 pathway ligands and receptors, such as gene expression GWAS in blood cells (eQTLs) and protein levels in serum (pQTLs), were collected from various resources. Data resources are as follows:

[0324] Table 2.

[0325]

[0326] eQTL: quantitative trait locus; # pQTL: Quantitative protein trait locus

[0327] (2) Data cleaning and harmonization

[0328] To ensure that all GWAS datasets are encoded in a consistent manner with genomic location, variant name, and effector allele type, data preprocessing is a necessary step. Dataset preprocessing is performed as follows:

[0329] First, use the LiftOver tool in the UCSC Genome Browser (genome.ucsc.edu / cgi-bin / hgLiftOver?hgsid=1770184142_UzmzHb9sIFwTgvO2zwt0sLLOTsM7) to convert the dataset, which is aligned based on the human genome version hg38, to hg37.

[0330] In datasets that only include effect alleles, the corresponding other alleles are derived from dbSNP. Furthermore, for datasets lacking effect allele frequencies (EAF) data, we utilize minor allele frequencies (MAF) details from dbSNP to infer the necessary EAF information. Finally, the datasets are reconciled using the IEU GWAS catalog reconciliation process (https: / / www.ebi.ac.uk / gwas / docs / methods / summary-statistics).

[0331] (3) Genetic association region map

[0332] To effectively visualize association results centered on specific gene regions, region maps were generated using LocusZoom v1.4, which is accessible at github.com / statgen / locuszoom-standalone. For these maps, build="hg19", pop="EUR", and source="1000G_Nov2014".

[0333] (4) Co-localization of disease sites, gene expression eQTLs, and protein expression pQTLs

[0334] Colocation is a statistical method for determining whether two distinct traits (such as gene expression levels and disease risk) share a common genetic cause. This process typically involves Bayesian statistical methods to estimate the probabilities of shared and distinct causal variations in overlapping genomic regions. To further evaluate the causal relationship between selected genes and the disease of interest, colocation analysis was performed using the coloc Rpackage v5.2.2 described in chr1swallace.github.io / coloc / articles / a01_intro.html.

[0335] We first calculate the gene center by averaging the start and end positions, then perform co-location analysis on all SNPs falling within 500kb on either side of the gene center. SNPs with MAF = 0 are removed. Assuming each trait has at most one causal variant within the considered region, the function "coloc.abf(..)" is used for co-location analysis.

[0336] (5) Comparing the genetic effects of gene / protein expression on disease risk

[0337] To explore the impact of changes in gene expression or protein concentration on disease outcomes, β-β plots were created. The β value represents the magnitude of the effect of a genetic variant (typically a single nucleotide polymorphism, SNP) on outcomes such as gene expression levels, protein levels, or disease risk. In the β-β scatter plot, the X-axis represents the genetic effect of a gene or protein quantitative trait locus (QTL), while the Y-axis shows the genetic effect of the disease outcome. Each point on the plot corresponds to a genetic variant. Only genetic variants common to both gene / protein and disease outcomes were included in the β-β plots. In our analysis, we restricted genetic variants significantly associated with gene / protein levels (e.g., eQTL / pQTL) to reduce noise in the data representation.

[0338] Example 12.2: Results

[0339] (1) IL12A (IL12p35) and its receptor IL12Rβ2 are associated with SSc, PBC, SLE and SjS, but not with PsO, CD, UC, IBD or AS.

[0340] like Figures 15A-15C As shown, IL12A eQTL analysis revealed that SNP rs4680536, located near the IL12A gene, was the primary variant associated with IL12A gene expression levels (Figure 4). Genetic markers in this region also showed significant associations with SSc, SLE, and PBC (p < 5 × 10⁻⁶). -8 The primary association marker was highly associated with the primary variant of IL12A eQTL (rs4680536). Although no genetic variant reached GWAS significance for SjS in this dataset, newer literature reports rs485497 (p=1.17×10⁻⁶) in the IL12A region. -10 ) is associated with Sjögren's syndrome (Lessard, Christopher J., et al. "Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjögren's syndrome." Nature Genetics 45.11 (2013): 1284-1292). Furthermore, rs4680536 and rs485497 are associated (in a 1000-genome European population, R...). 2 =0.27). The significant association pattern between genotype and IL12A gene expression level is highly consistent with the significant association pattern of SSc, PBC, SLE, and SjS risk. The posterior probabilities of co-localization of IL12 eQTL with genetic markers associated with SSc, PBC, SLE, and SjS are 0.948, 0.975, 0.985, and 0.744, respectively. The results strongly suggest that IL12A gene expression level shares common causal genomic variation with SSc, PBC, SLE, and SjS risk. Conversely, in the data presented here or in other published GWAS studies, no genetic variation in this IL12A region is significantly associated with PsO, CD, UC, IBD, or AS (p < 5 × 10⁻⁶). -8 ).

[0341] like Figures 16A-16CAs shown, IL12Rβ2 eQTL analysis revealed that the SNP rs17129778, located near the IL12Rβ2 gene, was associated with IL12Rβ2 gene expression levels (Figure 4). Genetic markers in this region also showed significant associations with SSc, PBC, and SLE (p < 5 × 10⁻⁶). -8 The primary association marker was highly correlated with the primary variant of IL12RB2 eQTL (rs17129778). The significant association pattern between genotype and IL12Rβ2 gene expression level was highly consistent with the significant association pattern of SSc, PBC, SLE, and SjS risk. The results indicate that IL12Rβ2 gene expression level shares common causal genomic variants with SSc, PBC, and SLE risk. Conversely, different groups of genetic variants in this genetic region not associated with IL12Rβ2 eQTLs were significantly associated with PsO, CD, UC, IBD, or AS (p < 5 × 10⁻⁶). -8 IL23R is a neighboring gene upstream of IL12Rβ2. (See later...) Figures 18A-18C As shown, a group of variants associated with PsO, CD, UC, IBD, or AS are actually associated with those variants that affect the expression of the protein (pQTL) of IL23R.

[0342] (2) IL12B (IL12p40) and IL23R are associated with PsO, CD, UC, IBD, and AS, but not with SSc, PBC, SLE, or SjS.

[0343] like Figures 17A-17C As shown, IL12B pQTL analysis revealed an association between the SNP rs6556416, located near the IL12B gene, and serum IL12B protein levels (Figure 4). Genetic markers in this region also showed significant associations with PsO, CD, UC, IBD, and AS (p < 5 × 10⁻⁶). -8 The primary association marker was highly correlated with the primary variant of IL12B pQTL (rs6556416). The significant association pattern between genotype and IL12B protein levels closely matched the significant association patterns with the risks of PsO, CD, UC, IBD, and AS. These results indicate that IL12B protein levels share common causal genomic variants with the risks of PsO, CD, UC, IBD, and AS. Conversely, in the data presented here or in other published GWAS studies, no genetic variant in this IL12B genetic region was significantly associated with SSc, PBC, SLE, or SjS (p < 5 × 10⁻⁶). -8 ).

[0344] like Figures 18A-18CAs shown, IL23R pQTL analysis revealed an association between the SNP rs11581607, located near the IL23R gene, and serum IL23R protein levels (Figure 4). Genetic markers in this region also showed significant associations with PsO, CD, UC, IBD, and AS (p < 5 × 10⁻⁶). -8 The primary association marker was highly associated with the primary variant of IL23R pQTL (rs11581607). The significant association pattern between genotype and IL23R protein levels closely matched the significant association patterns with PsO, CD, UC, IBD, and AS risks. These results indicate that IL23R protein levels share common causal genomic variants with PsO, CD, UC, IBD, and AS risks. Conversely, in the data presented here or in other published GWAS studies, no genetic variant in this IL23R genetic region was significantly associated with SjS (p < 5 × 10⁻⁶). -8 Furthermore, genetic variations in different groups that were not associated with pQTLs of IL23R in this genetic region were significantly associated with SSc, PBC, and SLE (p < 5 × 10⁻⁶). -8 ) is associated. IL12Rβ2 is a neighboring gene downstream of IL23R. As previously mentioned in Figures 16A-16C As shown, a group of variants associated with SSc, PBC, and SLE are actually associated with those variants that affect the expression of gene IL12Rβ2 (eQTL).

[0345] (3) EBI3 and IL6ST are not associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS.

[0346] like Figures 19A-19C As shown, EBI3 pQTL analysis revealed an association between the SNP rs60160662, located near the EBI3 gene, and EBI3 serum protein levels (Figure 4). However, in the data presented here or in other published GWAS studies, no genetic variation in this EBI3 genetic region was significantly associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS (p < 5 × 10⁻⁶). -8 Since functional genetic variations regulating EBI3 protein levels exist in this region but have no effect on disease risk, EBI3 protein levels are unlikely to be a causal effect on these diseases.

[0347] like Figures 20A-20CAs shown, IL6ST pQTL analysis revealed an association between the SNP rs11574765, located near the IL6ST gene, and serum IL6ST protein levels (Figure 4). However, in the data presented here or in other published GWAS studies, no genetic variants in this IL6ST genetic region were significantly associated with PsO, CD, UC, IBD, AS, SSc, PBC, SLE, or SjS (p < 5 × 10⁻⁶). -8 Since functional genetic variations regulating IL6ST protein levels exist in this region but have no effect on disease risk, IL6ST protein levels are unlikely to be a causal effect of these diseases.

[0348] (4) STAT4 is associated with SSc, PBC, SLE, SjS, IBD, CD, and UC, while STAT3 is associated with CD, UC, and IBD.

[0349] like Figures 21A-21C As shown, STAT4 eQTL analysis revealed an association between the SNP rs16833249, located near the STAT4 gene, and STAT4 gene expression levels (Figure 4). Genetic markers in this region also showed significant associations with SSc, PBC, SLE, SjS, IBD, CD, and UC (p < 5 × 10⁻⁶). -8 The data showed that a significant association marker was associated with the primary STAT4 eQTL variant (rs16833249). The significant association pattern between genotype and STAT4 gene expression level was highly consistent with the significant association pattern of SSc, PBC, SLE, SjS, IBD, CD, and UC risks. These results indicate that STAT4 gene expression level shares a common causal genomic variant with the risks of SSc, PBC, SLE, SjS, IBD, CD, and UC. Conversely, in the data presented here or in other published GWAS studies, no genetic variant in this STAT4 genetic region was significantly associated with PsO or AS (p < 5 × 10⁻⁶). -8 ).

[0350] like Figures 22A-22C As shown, STAT3 eQTL analysis revealed an association between the SNP rs1053004, located near the STAT3 gene, and STAT3 gene expression levels (Figure 4). Genetic markers in this region also showed significant associations with IBD, CD, and UC (p < 5 × 10⁻⁶). -8The data showed that a significant association marker was associated with the primary STAT3 eQTL variant (rs1053004). The pattern of significant association between genotype and STAT3 gene expression levels closely matched the pattern of significant association between IBD, CD, and UC risk. These results indicate that STAT3 gene expression levels and IBD, CD, and UC risk share a common causal genomic variant. Conversely, in the data presented here or in other published GWAS studies, no genetic variant in this STAT3 genetic region was significantly associated with SSc, PBC, SLE, SjS, PsO, or AS (p < 5 × 10⁻⁶). -8 ).

[0351] (5) Genetically determined higher expression of IL12A and IL12Rβ2 is associated with a higher risk of SSc, PBC, SLE, and SjS.

[0352] Based on significant expression of IL12A (p < 5 × 10⁻⁶), -8 Genetic variations associated with IL12A expression were fitted with positive regression lines to a scatter plot comparing the genetic effects of IL12A expression with the genetic effects on the risk of SSc, PBC, SjS, and SLE diseases. Figure 23 This indicates that increased IL12A expression through genetic perturbation is positively associated with an increased risk of SSc, PBC, SLE, and SjS.

[0353] Based on significant expression of IL12Rβ2 (p < 5 × 10⁻⁶), -8 Genetic variations associated with IL12Rβ2 expression were fitted with positive regression lines to a scatter plot comparing the genetic effects of IL12Rβ2 expression with the genetic effects on the risk of SSc, PBC, SjS, and SLE diseases. Figure 24 This indicates that increased IL12Rβ2 expression through genetic perturbation is positively associated with an increased risk of SSc, PBC, SLE, and SjS.

[0354] Example 12.3: Conclusion

[0355] IL12A-p35 / IL12Rβ2 is specifically associated with immune diseases such as SSc, PBC, SLE, and SjS. While IL12B forms IL12 along with IL12A, it exhibits a different pattern of association with immune diseases. IL12B is associated with PsO, CD, UC, IBD, and AS, but not with SSc, PBC, SLE, and SjS. IL12B-p40 is also a component of IL23. The disease association pattern of IL12B is the same as that of IL23R. Pharmacological targeting of IL12B or IL23A-p19 is successful in IBD and PsO, but unsuccessful in PBC and SLE, consistent with the genetic association patterns of IL12B and IL23R. These genetic association characteristics suggest that pharmacological targeting of IL12B will have effects similar to targeting the IL23 pathway, but potentially different from targeting IL12A.

[0356] Furthermore, STAT4 is a major downstream signaling molecule of the IL12 cytokine pathway. STAT4 is associated with all diseases related to IL12A-p35 / IL12Rβ2, including SSc, PBC, SLE, and SjS. STAT3 is a downstream signaling molecule of the IL23 cytokine pathway, and STAT3 is associated only with IBD, UC, and CD. The different immune disease associations derived from STAT4 and STAT3 also support different indications for targeting IL12 and IL23.

[0357] Meanwhile, IL12A-p35 / IL12Rβ2 is also a component of IL35's cytokines and receptors. One explanation for the different genetic associations of IL12A and IL12B could be the different effects of IL35 and IL12 cytokines. However, although genetic variations significantly affect the protein levels of EBI3 / IL6ST (another component of IL35 and its receptor), they have not been associated with any of the autoimmune diseases evaluated above. Therefore, IL12A-p35 / IL12Rβ2 is unlikely to influence immune diseases through the immunomodulatory effects of IL35.

[0358] Furthermore, a positive correlation was identified between the genetic effects of IL12A and IL12Rβ2 expression and the genetic effects on the risk of SSc, PBC, SLE, and SjS. Since higher IL12A / IL12Rβ2 expression is associated with a higher disease risk, we propose that the development of IL12A-p35 / IL12Rβ2 antagonists would be particularly beneficial for patients with SSc, PBC, SLE, and / or SjS.

[0359] Example 13. 27H28L-hblb-YTE and 4A4-hblb2-YTE exhibited excellent pharmacokinetic properties in human FcRn transgenic mice.

[0360] To investigate the pharmacokinetic properties of anti-IL12p35 antibodies in humans, hFcRN transgenic C57BL / 6 mice (Shanghai Model Organisms Center, Inc.) were administered intravenously (iv) 1 mg / kg of either anti-IL12p35 antibody 4A4-hblb2-YTE or 27H28L-hblb-YTE. Pharmacokinetic (PK) blood samples were collected at 2 hours (“hr”), 6 hours, 24 hours, 48 ​​hours, 72 hours, 7 days (“D”), 10 days, 14 days, 17 days, 21 days, 24 days, and 28 days post-administration. Serum concentrations of anti-IL12p35 antibodies were analyzed by ELISA. For example, human IL-12 was used to capture anti-IL12p35 antibodies in serum, and anti-human Fc antibodies were used to detect anti-IL12p35 antibodies bound to human IL-12.

[0361] The serum concentrations of 4A4-hblb2-YTE and 27H28L-hblb-YTE at the corresponding time points are shown in... Figure 29 The pharmacokinetic parameters of 27H28L-hblb-YTE and 4A4-hblb2-YTE are shown in the figure. Figure 30 .like Figure 30 As shown, the anti-IL12p35 antibodies with TYE and reverting germline mutations exhibited good in vivo stability and excellent PK characteristics in human FcRn transgenic mice. Specifically, after a single dose of 1 mg / kg in human FcRn transgenic mice, 4A4-hblb2-YTE and 27H28L-hblb-YTE showed T values ​​of 375 hours and 500 hours, respectively. 1 / 2 .

[0362] Example 14. Anti-IL12p35 antibody instead of anti-IL12p40 antibody reduces DNFB-induced skin inflammation.

[0363] To investigate the in vivo effects of anti-IL12p35 antibodies on inflammatory diseases, a mouse model of chronic contact hypersensitivity induced by 1-fluoro-2,4-dinitrophenyl (DNFB) was established, and anti-mouse IL12p35 was used as an alternative to antibody treatment. Specifically, twenty-four 8-week-old CB57BL / 6 mice were sensitized on day 0 with 50 μL of 0.5% DNFB (%v / v) on shaved right dorsal skin. Prior to administration, DNFB was freshly dissolved in acetone and olive oil (4:1, v / v). Then, all animals received 10 μL of 0.3% DNFB (%v / v) on the back of both ears on days 5–7. Subsequently, on days 8, 9, 10, 11, and 12, 10 μL of 0.1% DNFB was administered to the back of both ears. On day 0, mice were randomly divided into three groups (n=8 per group) and administered 100 μl PBS (control), 10 mg / kg rat anti-mouse IL12p35 antibody (anti-mP35) (BioXcell, Cat#: BE0371), or 10 mg / kg rat anti-mouse IL12p40 antibody (anti-mP40) (BioXcell, Cat#: BE0051) intraperitoneally (ip) on days 0, 4, 7, and 11, respectively. Ear thickness was determined using a digital portable pachymeter from day 5 to day 12 prior to DNFB administration. Each ear was measured three times to determine the mean thickness at each data point.

[0364] The experimental protocol is shown below Figure 31A .like Figure 31B As shown, mean ear thickness was increased in all DNFB-induced skin inflammation groups. Anti-mouse IL12p35 antibody treatment significantly reduced ear thickness compared to the control group (treated with PBS), while anti-mouse IL12p40 antibody treatment did not significantly affect ear thickness compared to the control group.

[0365] Example 15. Anti-IL12p35 antibody alleviates Sjögren's syndrome in a mouse model

[0366] Sjögren's syndrome (SjS) is an autoimmune disease mediated by lymphocyte infiltration into exocrine glands, leading to progressive destruction of the lacrimal and salivary glands and glandular dysfunction. To investigate the in vivo effects of anti-IL12-p35 antibody on Sjögren's syndrome, NOD mice (NOD / MrkTac, Taconic US) were treated with anti-mouse IL12p35 antibody. In NOD / ShiLtJ mice, type 1 diabetes accelerates the progression of Sjögren's syndrome. Briefly, 8-week-old NOD / ShiLtJ mice were fasted for 24 hours and then administered 3.6 mg of streptozotocin (STZ; dissolved in 0.1 M sodium citrate, pH 4.5) intraperitoneally (ip). On day 0, mice were randomly divided into two groups and treated with either 10 mg / kg rat IgG2a isotype antibody (BioXcell, Cat#: BE0089) or rat anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371) on days 0, 3, 6, and 9, respectively. Mice were anesthetized using an isoflurane machine on days 0, 3, 7, and 10. Pre-weighed absorbent material was inserted into the mice's mouths. Each mouse received 100 μg pilucarpine intraperitoneally (ip). After 12 minutes, the absorbent material was removed and weighed to determine the amount of saliva released.

[0367] The experimental protocol is shown below Figure 32A .like Figure 32B As shown, during the development of STZ-induced Sjogren's syndrome in NOD / ShiLtJ mice, salivation was significantly reduced in response to pilucarpine stimulation. Treatment with anti-mouse IL12-p35 antibody significantly alleviated the reduction in salivation in mice with Sjogren's syndrome.

[0368] Example 16. Anti-IL12p35 antibody significantly inhibited weight loss and reduced urinary albumin levels and renal pathology in an IMQ-induced SLE model.

[0369] Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease. NZBWF1 / J mice are used as a classic model of SLE because they spontaneously generate an immune response similar to that in human SLE. Studies have shown that mutations and activation of Toll-like receptor (TLR) 7 are associated with the pathogenesis of various autoimmune diseases, including SLE. To investigate the therapeutic effect of anti-IL-12p35 antibody on SLE in mice, an NZBWF1 / J (Jackson Laboratory, Cat#100008) mouse model induced by the TLR7 agonist imiquimod (IMQ) was used. Specifically, female NZBWF1 / J mice were received and acclimatized for one week at 18 weeks of age. On day 0, all mice received a topical administration of 2.5 mg of 2.5% IMQ cream per 25 g of mouse body weight to the right inner ear. IMQ was administered twice weekly for a total of 5 doses. IMQ cream was prepared by mixing 100 mg (~100 µL) of Vanish cream with 5 mg of IMQ and an additional 100 μL of pure H2O. On day 0 / week 0, mice were randomly assigned to two groups based on body weight, receiving either 10 mg / kg of rat IgG2a isotype antibody (BioXcell, Cat#: BE0089; as the control group) or anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371; as the treatment group). All mice were administered the medication twice weekly until the experimental endpoint.

[0370] Urine samples were collected at weeks 0, 1, 2, 3, 4, and 5 to measure urinary albumin and creatinine levels using an in-house developed ELISA method and a commercially available creatinine assay kit, respectively. The albumin / creatinine ratio was calculated by dividing the albumin concentration by the creatinine concentration. To measure urinary albumin concentration, 100 μL of anti-mouse albumin (2 µg / mL, Exalpha, Cat#: 3 RAM / Alb / 7S) was spread in a 96-well plate and incubated overnight. The plate was then washed and blocked with 300 μL of blocking buffer (containing 1% BSA) at room temperature for 1 hour. The plate was then washed again, and 100 μL of diluted urine sample and standard (Sigma, Cat#: 126674-25MG) was added to the wells and incubated at room temperature for 2 hours. After incubation, the plate was washed and 100 μL of anti-mouse albumin polyclonal antibody HRP (diluted 1% BSA 1:25000, Bethyl Laboratories, Cat#: A90-134P) was added and incubated at room temperature for 1 hour. The plate was then washed and developed with TMB to determine albumin concentration based on standards. Urinary creatinine concentration was measured using a creatinine assay kit (Bioassay Systems, Cat#: DICT500) according to the manufacturer's instructions. Mice were euthanized at week 5. Their kidneys were collected. The left kidney was frozen, while the right kidney was fixed with 10% formalin and sent to iHisto (Salem, MA) for histological analysis.

[0371] like Figures 33A-33C As shown, anti-IL12p35 antibody treatment significantly inhibited weight loss and reduced urinary albumin levels. The most significant difference in urinary albumin levels between the treatment and control groups occurred at week 3. After three weeks, possibly due to a decrease in the effect of IMQ, some mice began to show improvement in SLE symptoms, leading to a reduction in the albumin / creatinine ratio in these mice. Figure 33CAs shown, the images present representative mouse renal histological images from each group (all with high levels of urinary albumin). In the isotype-treated group, multifocal glomerulonephritis and multifocal glomerular atrophy (as indicated by black arrows) were observed, accompanied by partial replacement by fibrous connective tissue (as indicated by black rhombuses), multifocal tubular dilatation and hyaline casts (as indicated by hollow stars), and significant interstitial infiltration by mixed cell populations (as indicated by black triangles). In contrast, the anti-IL12p35 antibody-treated group showed largely intact glomerular structure with only a few instances of mild glomerulonephritis (as indicated by black arrows); and no fibrosis or interstitial infiltration was observed. These results indicate that local administration of IMQ accelerates the development of SLE in young NZBWF1 / J mice, enabling rapid evaluation of the effects of anti-IL12p35 antibody treatment on SLE. Anti-IL12p35 antibody treatment significantly inhibited weight loss, reduced urinary albumin levels, and significantly reduced SLE-related renal pathological changes.

[0372] Example 17. Anti-IL12p35 antibody significantly reduced renal pathology in a spontaneous SLE model.

[0373] To further investigate the therapeutic effect of anti-mouse IL12-p35 antibody on SLE in mice, a spontaneously occurring SLE model was established using NZBWF1 / J mice (Jackson Laboratory, Cat#100008). Specifically, female NZBWF1 / J mice were received and acclimatized for one week at 18 weeks of age. At week 0, all mice were randomly divided into two groups according to body weight, and treated weekly via intraperitoneal (ip) with either 15 mg / kg rat IgG2a isotype antibody (BioXcell, Cat#: BE0089; as control group) or anti-mouse IL12p35 antibody (BioXcell, Cat#: BE0371; as treatment group) until the experimental endpoint. Urine samples were collected at weeks 0, 2, 4, 6, 8, 10, 12, 14, and 15 to measure albumin and creatinine levels using an internally developed ELISA method and a creatinine assay kit, respectively (as described in Example 16). The albumin / creatinine ratio was calculated by dividing albumin concentration by creatinine concentration. Mice were euthanized on the second day of week 15. Their kidneys were collected. The left kidney was frozen, while the right kidney was fixed with 10% formalin and sent to iHisto (Salem, MA) for histological analysis. Histological pathological analysis was performed internally by an independent histopathologist from Alliance Bio (Hangzhou, China).

[0374] like Figure 34AAs shown, treatment with anti-IL12p35 antibody significantly reduced SLE renal symptoms. For example, when SLE occurred spontaneously in NZBWF1 / J mice, urinary albumin levels showed a significant reduction at 14 weeks post-treatment. It is important to note that the onset time of SLE symptoms varied significantly among mice. Notably, some mice in the control group did not show an increase in albumin / creatinine ratio throughout the experiment, while some mice in both the treatment and control groups had very early onset. Furthermore, the development of the anti-drug antibody (mouse anti-rat) resulted in adverse pharmacokinetic (PK) exposure to the anti-IL12p35 antibody, impairing its therapeutic efficacy. This was particularly evident in mice with elevated albumin / creatinine ratios, which were also associated with adverse PK.

[0375] like Figure 34B As shown in the images, histological analysis of the kidneys revealed that, in the isotype control group, the kidney tissues primarily exhibited end-stage disease (subfigure ad; #42): 1) Five of the seven samples showed glomerular sclerosis (as indicated by black arrows), and some were accompanied by fibrous crescent formation (as indicated by white arrows); 2) Tubular degeneration or atrophy was observed (as indicated by black triangles), as well as protein casts in the tubules (as indicated by black rhombuses); and 3) Interstitial inflammation of varying degrees was observed, with some samples showing interstitial fibrosis. However, in the anti-mouse IL12p35 antibody treatment group (subfigure eh; #51), the pathological changes in the kidney tissue primarily reflected early damage: 1) common glomerular mesangial proliferation with inflammatory cell infiltration; 2) 3 out of 7 samples showed mild to moderate-to-severe glomerulosclerosis, but no fibrous crescent formation was observed; 3) renal tubular degeneration ranged from moderate to moderate-to-severe, with 3 out of 7 samples showing protein casts; and 4) inflammatory cell infiltration was noted in the interstitial region, with one sample showing mild interstitial fibrosis. Overall, in the treatment group, compared with the isotype control group, the WTS (whole tissue score) was significantly reduced, while the AS (activity score) was increased, and the CS (chronicity score) was reduced. Detailed scoring and summaries of the pathological scores evaluated by independent histopathologists are shown in [reference needed]. Figure 34C .

[0376] In summary, although the development of anti-drug antibodies has weakened their therapeutic efficacy, in spontaneous SLE models, treatment with anti-IL12p35 antibodies reduced urinary albumin levels, significantly reduced SLE-associated renal pathological changes, and slowed disease progression.

[0377] Other implementation plans

[0378] It should be understood that although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to the interleukin 12 subunit alpha (IL12p35), comprising: The heavy chain variable region (VH) contains complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH CDR1 contains an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, VH CDR2 contains an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and VH CDR3 contains an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and The light chain variable regions (VLs) include CDRs 1, 2, and 3, wherein the VL CDR1 region contains an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region contains an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region contains an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence. The selected VH CDR 1, 2 and 3 amino acid sequences and the selected VL CDR 1, 2 and 3 amino acid sequences are selected from the VH CDR 1, 2, 3 and VL CDR 1, 2, 3 listed in Figure 26 or Figure 27.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 47, 48, and 49, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 50, 51, and 52, respectively; (2) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 53, 54, and 55, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 56, 57, and 58, respectively; (3) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 59, 60, and 61, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 62, 63, and 64, respectively; (4) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 65, 66, and 67, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 68, 69, and 70, respectively; (5) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 71, 72, and 73, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 74, 75, and 76, respectively; (6) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 77, 78, and 79, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 80, 81, and 82, respectively; (7) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 83, 84, and 85, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 86, 87, and 88, respectively; (8) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 89, 90, and 91, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 92, 93, and 94, respectively; (9) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 95, 96, and 97, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 98, 99, and 100, respectively; (10) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 101, 102, and 103, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 104, 105, and 106, respectively; (11) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 107, 108, and 109, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 110, 111, and 112, respectively; (12) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 113, 114, and 115, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 116, 117, and 118, respectively; (13) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 119, 120, and 121, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 122, 123, and 124, respectively; (14) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 125, 126, and 127, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 128, 129, and 130, respectively; (15) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 131, 132, and 133, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 134, 135, and 136, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 137, 138, and 139, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 140, 141, and 142, respectively; (17) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 143, 144, and 145, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 146, 147, and 148, respectively; (18) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 149, 150, and 151, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 152, 153, and 154, respectively; (19) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 155, 156, and 157, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 158, 159, and 160, respectively; (20) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 161, 162, and 163, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 164, 165, and 166, respectively; (21) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 167, 168, and 169, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 170, 171, and 172, respectively; (22) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 173, 174, and 175, respectively; and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 176, 177, and 178, respectively; and (23) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 179, 180, and 181, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 182, 183, and 184, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the CDR is determined by the Kabat definition.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 185, 186, 187, respectively, and the selected VLCDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 188, 189, 190, respectively; (2) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 191, 192, and 193, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 194, 195, and 196, respectively; (3) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 197, 198, and 199, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 200, 201, and 202, respectively; (4) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 203, 204, and 205, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 206, 207, and 208, respectively; (5) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 209, 210, and 211, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 212, 213, and 214, respectively; (6) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 215, 216, and 217, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 218, 219, and 220, respectively; (7) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 221, 222, and 223, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 224, 225, and 226, respectively; (8) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 227, 228, and 229, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 230, 231, and 232, respectively; (9) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 233, 234, and 235, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 236, 237, and 238, respectively; (10) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 239, 240, and 241, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 242, 243, and 244, respectively; (11) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 245, 246, and 247, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 248, 249, and 250, respectively; (12) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 251, 252, and 253, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 254, 255, and 256, respectively; (13) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 257, 258, and 259, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 260, 261, and 262, respectively; (14) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 263, 264, and 265, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 266, 267, and 268, respectively; (15) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 269, 270, and 271, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 272, 273, and 274, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 275, 276, and 277, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 278, 279, and 280, respectively; (17) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 281, 282, and 283, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 284, 285, and 286, respectively; (18) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 287, 288, and 289, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 290, 291, and 292, respectively; (19) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 293, 294, and 295, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 296, 297, and 298, respectively; (20) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 299, 300, and 301, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 302, 303, and 304, respectively; (21) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 305, 306, and 307, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 308, 309, and 310, respectively; (22) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 311, 312, and 313, respectively; and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 314, 315, and 316, respectively; and (23) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 317, 318, and 319, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 320, 321, and 322, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 4, wherein the CDR is determined by the IMGT definition.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) According to Kabat’s definition, the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 155, 156, and 157, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 158, 159, and 160, respectively. (2) According to Kabat’s definition, the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 179, 180, and 181, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 182, 183, and 184, respectively. (3) According to the IMGT definition, the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 293, 294, and 295, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 296, 297, and 298, respectively; and (4) According to the definition of Kabat, the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 317, 318, and 319, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 320, 321, and 322, respectively.

7. An antibody or antigen-binding fragment thereof that binds to IL12p35, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the light chain variable region (VL) comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 1, and the selected VL sequence is SEQ ID NO: 2; (2) The selected VH sequence is SEQ ID NO: 3, and the selected VL sequence is SEQ ID NO: 4; (3) The selected VH sequence is SEQ ID NO: 5, and the selected VL sequence is SEQ ID NO: 6; (4) The selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8; (5) The selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 10; (6) The selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12; (7) The selected VH sequence is SEQ ID NO: 13, and the selected VL sequence is SEQ ID NO: 14; (8) The selected VH sequence is SEQ ID NO: 15, and the selected VL sequence is SEQ ID NO: 16; (9) The selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18; (10) The selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20; (11) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (12) The selected VH sequence is SEQ ID NO: 23, and the selected VL sequence is SEQ ID NO: 24; (13) The selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 26; (14) The selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 28; (15) The selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 30; (16) The selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32; (17) The selected VH sequence is SEQ ID NO: 33, and the selected VL sequence is SEQ ID NO: 34; (18) The selected VH sequence is SEQ ID NO: 35, and the selected VL sequence is SEQ ID NO: 36; (19) The selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38; (20) The selected VH sequence is SEQ ID NO: 39, and the selected VL sequence is SEQ ID NO: 40; (21) The selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 42; (22) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 44; (23) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46; (24) The selected VH sequence is SEQ ID NO: 335, and the selected VL sequence is SEQ ID NO: 336; and (25) The selected VH sequence is SEQ ID NO: 337, and the selected VL sequence is SEQ ID NO: 338; Optionally, VH and / or VL contain one or more reverting germline (B2G) mutations.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the selected VH sequence is SEQ ID NO: 37 and the selected VL sequence is 38; or wherein the selected VH sequence is SEQ ID NO: 335 and the selected VL sequence is SEQ ID NO:

336.

9. The antibody or antigen-binding fragment thereof according to claim 7, wherein the selected VH sequence is SEQ ID NO: 45 and the selected VL sequence is SEQ ID NO: 46; or wherein the selected VH sequence is SEQ ID NO: 337 and the selected VL sequence is SEQ ID NO:

338.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof is capable of blocking the binding between interleukin 12 (e.g., human interleukin 12 (IL12)) and the beta 2 subunit of the interleukin 12 receptor (e.g., human interleukin 12 receptor beta 2 subunit (IL12Rβ2)).

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof is capable of blocking IL12-induced intracellular signaling (e.g., JAK-STAT signaling), optionally wherein the IL12 is human or monkey IL12.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof is capable of inhibiting IL12-induced IFN-γ production in human PBMCs.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is capable of preventing the production of IFN-γ in CD4+ T cells co-cultured with allogeneic dendritic cells.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody or antigen-binding fragment specifically binds to human IL12p35 and / or monkey IL12p35.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody or antigen-binding fragment is an F(ab')2 fragment, a single-chain variable fragment (scFV), or a multispecific antibody (e.g., a bispecific antibody).

17. An antibody or antigen-binding fragment thereof that binds to IL12p35, comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, a first immunoglobulin light chain, and a second immunoglobulin light chain, wherein the first immunoglobulin heavy chain and the first immunoglobulin light chain are associated with each other to form a first antigen-binding site for binding to IL12p35, wherein the second immunoglobulin heavy chain and the second immunoglobulin light chain are associated with each other to form a first antigen-binding site for binding to IL12p35, wherein: (1) The first and second immunoglobulin heavy chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 325, and the first and second immunoglobulin light chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 326; (2) The first and second immunoglobulin heavy chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 327, and the first and second immunoglobulin light chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 326; (3) The first and second immunoglobulin heavy chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 328, and the first and second immunoglobulin light chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 329; (4) The first and second immunoglobulin heavy chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 330, and the first and second immunoglobulin light chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 329; (5) The first and second immunoglobulin heavy chains contain at least 90% identical amino acid sequences to SEQ ID NO: 331, and the first and second immunoglobulin light chains contain at least 90% identical amino acid sequences to SEQ ID NO: 332; and (6) The first and second immunoglobulin heavy chains contain at least 90% of the same amino acid sequence as SEQ ID NO: 333, and the first and second immunoglobulin light chains contain at least 90% of the same amino acid sequence as SEQ ID NO:

324.

18. The antibody or antigen-binding fragment thereof according to claim 17, wherein the first and second immunoglobulin heavy chains are identical, and wherein the first and second immunoglobulin light chains are identical.

19. The antibody or antigen-binding fragment thereof according to claim 17 or 18, wherein the first and second immunoglobulin heavy chains comprise an Fc region (e.g., an IgG1 Fc region).

20. The antibody or antigen-binding fragment thereof according to claim 19, wherein the Fc region comprises a YTE mutation (M252Y / S254T / T256E according to EU designation).

21. An antibody or an antigen-binding fragment thereof, comprising: The heavy chain variable region (VH) contains VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region (VL) contains VL CDR1, VL CDR2, and VLCDR3. The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 thereon are the same as the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20.

22. An antibody or an antigen-binding fragment thereof, which cross-competes with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21.

23. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22 covalently bound to a therapeutic agent.

24. The antibody-drug conjugate of claim 23, wherein the therapeutic agent is a cytotoxic agent or a cell inhibitor.

25. A method for suppressing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 22 or an antibody-drug conjugate according to claim 23 or 24.

26. The method of claim 25, wherein the subject has an immune disorder (e.g., an autoimmune disease or an inflammatory disease).

27. A method of treating a subject suffering from an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 22 or an antibody-drug conjugate according to claim 23 or 24.

28. The method of claim 27, wherein the subject suffers from systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS).

29. The method of claim 27 or 28, wherein the subject does not suffer from psoriasis (PsO), Crohn's disease (CD), ulcerative colitis (UC), inflammatory bowel disease (IBD), or ankylosing spondylitis (AS).

30. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 22, or an antibody-drug conjugate according to claim 23 or 24, and a pharmaceutically acceptable carrier.

31. A method for reducing IL12-induced intracellular signaling in cells, the method comprising: The subject is given contact with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22 or an antibody-drug conjugate according to claim 23 or 24.

32. A method for identifying a subject as having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS), the method comprising: The level of IL12p35 in samples collected from the subject was determined using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 22.

33. A method for determining the risk of a subject having systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS), the method comprising: The level of IL12p35 in samples collected from the subject was determined using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 22.

34. A nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising: (1) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, wherein the complementarity-determining regions (CDRs) 1, 2, and 3 contain VH CDRs 1, 2, and 3 as shown in Figure 26 or Figure 27, and wherein the VH binds to IL12p35 when paired with a corresponding light chain variable region (VL); or (2) Immunoglobulin light chains or fragments thereof, comprising VLs containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain VLs CDRs 1, 2 and 3 as shown in Figure 26 or Figure 27, wherein the VLs bind to IL12p35 when paired with the corresponding VH.

35. The nucleic acid of claim 31, wherein the polypeptide comprises: (1) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2 and 3, wherein the complementarity-determining regions (CDRs) 1, 2 and 3 respectively contain amino acid sequences shown in SEQ ID NO: 47, 48 and 49 (or shown in SEQ ID NO: 185, 186 and 187 respectively), and wherein the VH binds to IL12p35 when paired with a light chain variable region (VL) containing an amino acid sequence shown in SEQ ID NO: 2; (2) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 50, 51 and 52 (or shown in SEQ ID NO: 188, 189 and 190, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 1; (3) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 53, 54 and 55 (or shown in SEQ ID NO: 191, 192 and 193), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 4; (4) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 56, 57 and 58 (or shown in SEQ ID NO: 194, 195 and 196, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 3; (5) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 59, 60 and 61 (or shown in SEQ ID NO: 197, 198 and 199, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 6; (6) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 62, 63 and 64 (or shown in SEQ ID NO: 200, 201 and 202, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 5; (7) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 65, 66 and 67 (or shown in SEQ ID NO: 203, 204 and 205, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 8; (8) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 68, 69 and 70 (or shown in SEQ ID NO: 206, 207 and 208, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 7; (9) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 71, 72 and 73 (or shown in SEQ ID NO: 209, 210 and 211, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 10; (10) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 74, 75 and 76 (or shown in SEQ ID NO: 212, 213 and 214, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 9; (11) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 77, 78 and 79 (or shown in SEQ ID NO: 215, 216 and 217, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 12; (12) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 80, 81 and 82 (or shown in SEQ ID NO: 218, 219 and 220, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 11; (13) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 83, 84 and 85 (or shown in SEQ ID NO: 221, 222 and 223, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 14; (14) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 86, 87 and 88 (or shown in SEQ ID NO: 224, 225 and 226, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 13; (15) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 89, 90 and 91 (or shown in SEQ ID NO: 227, 228 and 229, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 16; (16) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 92, 93 and 94 (or shown in SEQ ID NO: 230, 231 and 232, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 15; (17) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 95, 96 and 97 (or shown in SEQ ID NO: 233, 234 and 235, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 18; (18) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 98, 99 and 100 (or shown in SEQ ID NO: 236, 237 and 238, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 17; (19) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 101, 102 and 103 (or shown in SEQ ID NO: 239, 240 and 241, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 20; (20) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 104, 105 and 106 (or shown in SEQ ID NO: 242, 243 and 244, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 19; (21) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 107, 108 and 109 (or shown in SEQ ID NO: 245, 246 and 247, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 22; (22) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 110, 111 and 112 (or shown in SEQ ID NO: 248, 249 and 250, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 21; (23) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 113, 114 and 115 (or shown in SEQ ID NO: 251, 252 and 253), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 24; (24) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 116, 117 and 118 (or shown in SEQ ID NO: 254, 255 and 256, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 23; (25) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 respectively contain amino acid sequences shown in SEQ ID NO: 119, 120, 121 (or shown in SEQ ID NO: 257, 258 and 259 respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 26; (26) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 122, 123 and 124 (or shown in SEQ ID NO: 260, 261 and 262), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 25; (27) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 125, 126 and 127 (or shown in SEQ ID NO: 263, 264 and 265), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 28; (28) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 128, 129 and 130 (or shown in SEQ ID NO: 266, 267 and 268, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 27; (29) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 131, 132 and 133 (or shown in SEQ ID NO: 269, 270 and 271, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 30; (30) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 134, 135 and 136 (or shown in SEQ ID NO: 272, 273 and 274), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 29; (31) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 137, 138 and 139 (or shown in SEQ ID NO: 275, 276 and 277), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 32; (32) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 140, 141 and 142 (or shown in SEQ ID NO: 278, 279 and 280, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 31; (33) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 143, 144 and 145 (or shown in SEQ ID NO: 281, 282 and 283), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 34; (34) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 146, 147 and 148 (or shown in SEQ ID NO: 284, 285 and 286, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 33; (35) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 149, 150 and 151 (or shown in SEQ ID NO: 287, 288 and 289, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 36; (36) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 152, 153 and 154 (or shown in SEQ ID NO: 290, 291 and 292, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 35; (37) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 155, 156 and 157 (or shown in SEQ ID NO: 293, 294 and 295, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 38 or 336; (38) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 158, 159 and 160 (or shown in SEQ ID NO: 296, 297 and 298, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 37 or 335; (39) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 161, 162 and 163 (or shown in SEQ ID NO: 299, 300 and 301, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 40; (40) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 164, 165 and 166 (or shown in SEQ ID NO: 302, 303 and 304, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 39; (41) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 167, 168 and 169 (or shown in SEQ ID NO: 305, 306 and 307, respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 42; (42) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 170, 171 and 172 (or shown in SEQ ID NO: 308, 309 and 310, respectively), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 41; (43) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 173, 174 and 175 (or shown in SEQ ID NO: 311, 312 and 313), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 44; (44) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 176, 177 and 178 (or shown in SEQ ID NO: 314, 315 and 316), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 43; (45) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3, wherein CDRs 1, 2, and 3 respectively contain amino acid sequences shown in SEQ ID NO: 179, 180, and 181 (or shown in SEQ ID NO: 317, 318, and 319 respectively), and wherein the VH binds to IL12p35 when paired with a VL containing an amino acid sequence shown in SEQ ID NO: 46 or 338; or (46) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3, wherein the CDRs 1, 2 and 3 contain amino acid sequences shown in SEQ ID NO: 182, 183 and 184 (or shown in SEQ ID NO: 320, 321 and 322), and wherein the VL binds to IL12p35 when paired with a VH containing an amino acid sequence shown in SEQ ID NO: 45 or 337.

36. The nucleic acid according to claim 34 or 35, wherein the VH specifically binds to human IL12p35 when paired with VL; or the VL specifically binds to human IL12p35 when paired with VH.

37. The nucleic acid according to any one of claims 34 to 36, wherein the immunoglobulin heavy chain or a fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or a fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof.

38. The nucleic acid according to any one of claims 34 to 37, wherein the nucleic acid encodes an F(ab')2 fragment, a single-stranded variable fragment (scFv), or a multispecific antibody (e.g., a bispecific antibody).

39. The nucleic acid according to any one of claims 34 to 38, wherein the nucleic acid is cDNA.

40. A vector comprising one or more nucleic acids according to any one of claims 34 to 39.

41. A vector comprising two nucleic acids according to any one of claims 34 to 39, wherein the vector encodes a VL region and a VH region that commonly bind to IL12p35.

42. A pair of vectors, wherein each vector comprises a nucleic acid according to any one of claims 34 to 39, wherein the pair of vectors co-encode a VL region and a VH region that co-bind with IL12p35.

43. A cell comprising the vector according to claim 40 or 41 or a pair of vectors according to claim 42.

44. The cell of claim 43, wherein the cell is a CHO cell.

45. A cell comprising one or more nucleic acids according to any one of claims 34 to 39.

46. ​​A cell comprising two nucleic acids according to any one of claims 34 to 39.

47. The cell of claim 46, wherein the two nucleic acids co-encode the VL region and VH region that co-bind with IL12p35.

48. A method for generating an antibody or an antigen-binding fragment thereof, the method comprising: (a) Culturing cells according to any one of claims 43 to 47 under conditions sufficient to induce the cells to produce antibodies or antigen-binding fragments thereof; and (b) Collect the antibody or its antigen-binding fragment produced by the cells.

49. A method of treating a subject suffering from an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof that binds to IL12p35 and / or an antibody or antigen-binding fragment thereof that binds to IL12Rβ1.

50. The method of claim 49, wherein the antibody or antigen-binding fragment thereof that binds to IL12p35 does not bind to IL12p40.

51. The method according to claim 49 or 50, wherein the antibody or its antigen-binding fragment that binds to IL12Rβ2 does not bind to IL12Rβ1.

52. The method according to any one of claims 49 to 51, wherein the antibody or its antigen-binding fragment does not interfere with the IL23 pathway and / or IL35 pathway.

53. The method according to any one of claims 49 to 52, wherein the subject is a human subject.

54. The method of claim 53, wherein the subject suffers from systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS).

55. The method according to any one of claims 49 to 54, wherein the antibody or antigen-binding fragment thereof is a human or humanized antibody or antigen-binding fragment thereof.

56. The method according to any one of claims 49 to 52, wherein the subject is a non-human mammal, such as a monkey, dog, or mouse, optionally said mammal suffering from a disease or condition similar to systemic sclerosis (SSc), primary biliary cholangitis (PBC), systemic lupus erythematosus (SLE), and / or Sjogren's syndrome (SjS).

57. The method of claim 56, wherein the subject is a dog, and optionally the antibody or its antigen-binding fragment is a canine or canine-derived antibody or its antigen-binding fragment.

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