Antigen Binding Molecules

Abstract

The present invention provides antigen-binding molecules, specifically domain-engineered antibodies in which at least one constant region domain, CH1 / CL, is replaced with Titin T chain / Obscurin-O chain.

Description

[Technical Field]

[0001] The present disclosure relates to the field of antibody pharmaceuticals, specifically to antigen-binding molecules with substituted CH1 and CL structures and uses thereof. [Background technology]

[0002] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0003] With the continuous improvement of antibody humanization technology, monoclonal antibodies have developed rapidly in recent years. Many types of monoclonal antibodies are used to treat serious diseases such as malignant tumors and autoimmune diseases. However, tumor immune evasion always involves multiple different mechanisms, and a single monoclonal antibody can only bind to one specific target, which greatly reduces the therapeutic effect of monoclonal antibodies.

[0004] Bispecific antibodies (BsAbs) are artificial antibodies created by combining antibodies targeting two different antigens or two different antigen epitopes through genetic engineering. Unlike monoclonal antibodies, bispecific antibodies have the ability to simultaneously target two different antigens or antigen epitopes and can perform special biological functions, such as immune cell recruitment, receptor co-stimulation or co-inhibition, and polyvalent virus neutralization, which are expected to have clinical therapeutic effects superior to those of single monoclonal antibodies and even antibody combinations.

[0005] For example, various recombinant bispecific antibody formats have been developed, such as tetravalent bispecific antibodies formed by fusing IgG antibody formats and single-chain domains (see Coloma, MJ et al., Nature Biotech. 15(1997)159-163; Morrison, SL, Nature Biotech. 25(2007)1233-1234). Other bispecific formats include, for example, DVD-Ig, CrossMab, and BiTE (Spiess et al., Molecular Immunology, 67(2), pp.95-106(2015)).

[0006] Various structural models for bispecific antibodies have also been disclosed, including, for example, "knobs-into-holes" design, which involves the introduction of mutations into the Fc region (Ridgway et al., Protein Engineering, 9(7), pp. 617-21(1996)), electrostatically induced design (Gunasekaran et al., Journal of Biological Chemistry, 285(25), pp. 19637-19646(2010)), or minus-state design (Kreudenstein et al., mAbs, 5(5), pp. 646-654(2013); Leaver-Fay et al., Structure, 24(4), pp. 641-651(2016)), and swapping of CH1 and CL domains (CrossMab platform) (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187-11192(2011)), and fusion with TCR constant region (WO2019057122A1). Summary of the Invention

[0007] In the process of preparing multispecific antibodies, various products are produced by random combination of light chains and heavy chains of different antigens, but only one product is required. These by-products include antibodies lacking one light chain, incomplete antibodies, polymerized heavy chains, and mismatched light and heavy chains, which pose a significant challenge to the development of downstream processes. For this reason, the present disclosure provides dimerized polypeptides and antigen-binding molecules comprising the dimerized polypeptides.

[0008] In some embodiments, the present disclosure provides a dimerizing polypeptide comprising a Titin-T chain and an Obscurin-O chain, or a Titin-T chain and an Obscurin-Like-O chain, wherein: i) the Titin-T chain is a variant of SEQ ID NO: 32, and the variant has an amino acid residue substitution at one or more positions selected from positions 60 and 64; and / or ii) the Obscurin-O chain is a variant of SEQ ID NO: 33, wherein the variant has an amino acid residue substitution at one or more positions selected from 13, 32, 48, 66, 82, and 93; In addition, the conditions are as follows: a) if the variant does not have an amino acid residue substitution at position 13, 48, 66, 82, or 93 and has an amino acid residue substitution at position 32, the amino acid substitution at position 32 is not 32P; b) the variant does not have an amino acid residue substitution at positions 32, 48, 66, 82, or 93, and if it has an amino acid residue substitution at position 13, the amino acid substitution at position 13 is not 13Y; and c) If the above variant does not have an amino acid residue substitution at positions 48, 66, 82, or 93, and has amino acid residue substitutions at positions 13 and 32, the amino acid residue substitution at position 13 is not 13Y, and the amino acid residue substitution at position 32 is not 32P.

[0009] In some embodiments, the dimerizing polypeptide of any one of the above, wherein: i) the Titin-T chain is a variant of SEQ ID NO: 32, and the variant has an amino acid residue substitution at one or more positions selected from positions 60 and 64; and / or ii) the Obscurin-O chain is a variant of SEQ ID NO: 33, wherein the variant has one or more amino acid residue substitutions selected from 13S, 32F, an amino acid residue substitution at position 48, an amino acid residue substitution at position 66, an amino acid residue substitution at position 82, and an amino acid residue substitution at position 93; Dimerizing polypeptides are provided.

[0010] In some embodiments, the dimerizing polypeptide of any one of the preceding claims, wherein the variant of SEQ ID NO: 32 has one or more amino acid residue substitutions selected from 60S and 64T, and / or the variant of SEQ ID NO: 33 has one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H, and 93C. Dimerizing polypeptides are provided.

[0011] In some embodiments, the dimerizing polypeptide of any one of the above, wherein the variant of SEQ ID NO: 32 has a 60S and 64T amino acid residue substitution, and / or the variant of SEQ ID NO: 33 has any one of a) to c), i.e., a) 32F and 48V, b) 13S, 32F, 48V and 82H, and c) 13S, 32F, 48V, 66C, 82H and 93C, having any one amino acid residue substitution selected from Dimerizing polypeptides are provided.

[0012] In some embodiments, the present disclosure provides a dimerizing polypeptide, which consists of a Titin-T chain and an Obscurin-O chain, or a Titin-T chain and an Obscurin-Like-O chain, wherein: i) the Titin-T chain is a variant of SEQ ID NO: 32, which variant comprises at least an amino acid residue substitution at positions 60 and / or 64 compared to SEQ ID NO: 32; and / or ii) the Obscurin-O chain is a variant of SEQ ID NO: 33, which variant comprises at least one or more amino acid residue substitutions selected from the group consisting of 13, 32, 48, 66, 82, and 93 amino acid residues in SEQ ID NO: 33; In addition, the conditions are as follows: a) if the variant does not have an amino acid residue substitution at position 13, 48, 66, 82, or 93 and has an amino acid residue substitution at position 32, the amino acid substitution at position 32 is not 32P; b) the variant does not have an amino acid residue substitution at positions 32, 48, 66, 82, or 93, and if it has an amino acid residue substitution at position 13, the amino acid substitution at position 13 is not 13Y; and c) If the above variant does not have an amino acid residue substitution at positions 48, 66, 82, or 93, and has amino acid residue substitutions at positions 13 and 32, the amino acid residue substitution at position 13 is not 13Y, and the amino acid residue substitution at position 32 is not 32P.

[0013] In some embodiments, the dimerizing polypeptide of any one of the above, wherein: i) the Titin-T chain is a variant of SEQ ID NO: 32, which variant comprises at least one or more amino acid residue substitutions at positions 60 and / or 64 compared to SEQ ID NO: 32; and / or ii) The Obscurin-O chain is a variant of SEQ ID NO: 33, which variant has one or more amino acid residue substitutions compared to SEQ ID NO: 33, and the amino acid residue substitutions are selected from amino acid residue substitutions at 13S, 32F, and 48, amino acid residue substitutions at 66, amino acid residue substitutions at 82, and amino acid residue substitutions at 93.

[0014] In some embodiments, the dimerizing polypeptide of any one of the preceding claims, wherein the variant of SEQ ID NO: 32 comprises at least an amino acid residue substitution at 60S and / or 64T compared to SEQ ID NO: 32, and / or the variant of SEQ ID NO: 33 comprises at least one amino acid residue substitution selected from 13S, 32F, 48V, 66C, 82H, and 93C compared to SEQ ID NO: 33; Dimerizing polypeptides are provided.

[0015] In some embodiments, the dimerizing polypeptide of any one of the preceding claims, wherein the variant of SEQ ID NO: 32 comprises at least 60S and 64T amino acid residue substitutions compared to SEQ ID NO: 32, and / or the variant of SEQ ID NO: 33 comprises at least one of a) to c) compared to SEQ ID NO: 33, i.e., a) 32F and 48V, b) 13S, 32F, 48V and 82H, and c) 13S, 32F, 48V, 66C, 82H and 93C, containing any one amino acid residue substitution selected from Dimerizing polypeptides are provided.

[0016] In some embodiments, the dimerizing polypeptide provided herein is any one of the dimerizing polypeptides described above, wherein the variant of SEQ ID NO: 32 further has amino acid residue substitutions at one or more positions selected from 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84 compared to SEQ ID NO: 32, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residue substitutions. In some embodiments, the dimerization polypeptide provided herein is any one of the above dimerization polypeptides, wherein the variant of SEQ ID NO: 32 further comprises one or more amino acid residue substitutions selected from 3W, 8C, 11I, 13L, 20C, 22M / 22C, 25S, 26C, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 66S / 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D, and 84L compared to SEQ ID NO: 32.

[0017] In some embodiments, the dimerization polypeptide described in any one of the above is provided, wherein the variant of SEQ ID NO: 32 further comprises an amino acid residue substitution at one or more positions selected from 8, 11, 20, 25, 26, 39, 66, 79 and 81 compared to SEQ ID NO: 32; in some embodiments, the variant of SEQ ID NO: 32 further comprises one or more amino acid residue substitutions selected from 8C, 11I, 20C, 25S, 26C, 39T, 66K, 79T and 81R compared to SEQ ID NO: 32. In some embodiments, the variant of SEQ ID NO: 32 further comprises amino acid residue substitutions at positions 8, 11, 25, 39, 66, 79, and 81 compared to SEQ ID NO: 32; in some embodiments, the variant of SEQ ID NO: 32 further comprises amino acid residue substitutions of 8C, 11I, 25S, 39T, 66K, 79T, and 81R compared to SEQ ID NO: 32; in some embodiments, the variant of SEQ ID NO: 32 further comprises amino acid residue substitutions of 8C, 11I, 25S, 39T, 60S, 64T, 66K, 79T, and 81R, as well as amino acid residue substitutions of 20C and / or 26C compared to SEQ ID NO: 32, thereby providing a dimerization polypeptide.

[0018] In some embodiments, the dimerizing polypeptide of any one of the above, wherein the variant of SEQ ID NO: 32 has a) to l) a) to l) a) to l) b) a) to l) c) a) to l ... a) 8C, 25S and 39T, b) 20C, 25S and 39T, c) 25S, 26C and 39T, d) 22C, 25S and 39T, e) 8C, 25S, 39T, 66S and 77S, f) 8C, 25S, 39T, 66K, 70R, 79T and 81R, g) 3W, 8C, 11I, 13L, 22M, 25S, 39T and 82M, h) 8C, 11I, 25S, 39T, 66K, 79T and 81R, i) 8C, 25S, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 75V, 83D and 84L, j) 8C, 25S, 39T, 47E, 49G, 56S, 58E and 75V, k) 8C, 25S, 39T, 56S, 58E and 75V, and l)8C, 25S, 39T, 56S, 58E, 66S and 77S, The dimerized polypeptide further comprises any one amino acid residue substitution selected from the group consisting of:

[0019] In some embodiments, in the dimerization polypeptide described in any one of the above, the variant of SEQ ID NO: 32 has, compared to SEQ ID NO: 32, A) to C), i.e., A) 8C, 11I, 25S, 39T, 60S, 64T, 66K, 79T and 81R, B) 8C, 11I, 20C, 25S, 39T, 60S, 64T, 66K, 79T and 81R, and C) 8C, 11I, 25S, 26C, 39T, 60S, 64T, 66K, 79T and 81R The present invention provides a dimerizing polypeptide having any one amino acid residue substitution selected from the following:

[0020] In some embodiments, the dimerizing polypeptide of any one of the preceding claims, wherein the variant of SEQ ID NO: 33 further comprises an amino acid residue substitution at one or more positions selected from 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 76, 88, 89, 92, 94, and 97 compared to SEQ ID NO: 33; Variants of SEQ ID NO: 33 further comprise one or more amino acid residue substitutions selected from 2E, 3C, 7K / 7R, 9C, 11L, 12S, 13Y, 14T, 17E, 20L, 22M / 22S, 25S, 30D, 32P, 34E, 36T, 41K, 42L, 44I, 45T, 53L, 58V, 62E / 62K / 62H, 67Q / 67T, 69S, 76S, 88C, 89L, 92E, 94G and 97G relative to SEQ ID NO: 33, thereby providing dimerizing polypeptides.

[0021] In some embodiments, the dimerizing polypeptide of any one of the above, wherein the variant of SEQ ID NO: 33 has, compared to SEQ ID NO: 33, A) to R), i.e., A) 88C, B)3C, C) 9C, D) 25S, 76S and 88C, E) 25S, 76S and 3C, F) 25S, 76S and 9C, G) 7K, 25S, 62K, 76S and 88C, H) 7K, 25S, 62H, 76S and 88C, I) 7R, 25S, 62K, 76S and 88C, J)7R, 25S, 62H, 76S and 88C, K) 11L, 25S, 62K, 76S and 88C, L) 11L, 25S, 62H, 76S and 88C, M) 12S, 13Y, 14T, 22S, 25S, 62K, 76S and 88C, N)2E, 11L, 17E, 25S, 30D, 32P, 34E, 36T, 44I, 45T, 58V, 62E, 67Q, 69S, 76S, 88C and 97G, O) 11L, 20L, 22M, 25S, 53L, 62K, 76S and 88C, P) 11L, 25S, 41K, 45T, 62K, 67Q, 69S, 76S, 88C and 89L, Q) 11L, 25S, 42L, 45T, 62K, 67T, 69S, 76S, 88C, 92E and 94G, and R)11L, 12S, 13Y, 22S, 25S, 42L, 45T, 62K, 67Q, 69S, 76S, 88C, 92E and 94G The present invention provides a dimerizing polypeptide having any one amino acid residue substitution selected from the following:

[0022] In some embodiments, the dimerizing polypeptide is any one of the above, wherein the variant of SEQ ID NO: 33 further has amino acid residue substitutions at one or more positions selected from 3, 9, 25, 41, 45, 62, 67, 69, 76, 88 and 89 compared to SEQ ID NO: 33; in some embodiments, the variant of SEQ ID NO: 33 further has one or more amino acid residue substitutions selected from 3C, 9C, 25S, 41K, 45T, 62K, 67Q, 69S, 76S, 88C and 89L compared to SEQ ID NO: 33.

[0023] In some embodiments, the dimerization polypeptide is any one of the above, wherein the variant of SEQ ID NO: 33 further has amino acid residue substitutions at one or more positions selected from 41, 45, 62, 67, 69, 88 and 89 compared to SEQ ID NO: 33; in some embodiments, the variant of SEQ ID NO: 33 has one or more amino acid residue substitutions selected from 41K, 45T, 62K, 67Q, 69S, 88C and 89L compared to SEQ ID NO: 33; in some embodiments, the variant of SEQ ID NO: 33 has amino acid residue substitutions at 41K, 45T, 62K, 67Q, 69S, 88C and 89L.

[0024] In some embodiments, the dimerization polypeptide is any one of the above, wherein the variant of SEQ ID NO: 33 has, compared to SEQ ID NO: 33, amino acid residue substitutions of 13S, 32F, 48V and 82H, and one or more amino acid residue substitutions selected from 3C, 9C, 25S, 41K, 45T, 62K, 67Q, 69S, 76S, 88C and 89L. In some embodiments, the variant of SEQ ID NO: 33 further has one or more amino acid residue substitutions selected from 41K, 45T, 62K, 67Q, 69S, and 89L compared to SEQ ID NO: 33, for example, 41K, 45T, 62K, 67Q, 69S, and 89L. In some embodiments, the variant of SEQ ID NO: 33 further has one or more amino acid residue substitutions selected from 3C, 9C, 25S, 66C, 76S, 88C, and V93C compared to SEQ ID NO: 33. The amino acid sequence may have a substitution, for example, any one of amino acid residue substitutions selected from a) to j), i.e., a) 25S, 76S and 88C; b) 3C, 25S, 76S and 88C; c) 9C, 25S, 76S and 88C; d) 88C; e) 3C and 88C; f) 9C and 88C; g) 25S, 66C, 76S, 88C and 93C; h) 9C, 25S, 66C, 76S, 88C and 93C; i) 3C, 25S, 66C, 76S, 88C and 93C; and j) 3C, 9C, 25S, 66C, 76S and 93C.

[0025] In some embodiments, the dimerizing polypeptide is described in any one of the preceding paragraphs, wherein the variant of SEQ ID NO: 33 further has amino acid residue substitutions at one or more positions selected from 25, 41, 45, 62, 67, 69, 76, 88 and 89 compared to SEQ ID NO: 33; in some embodiments, the variant of SEQ ID NO: 33 further has amino acid residue substitutions at one or more positions selected from 25S, 41K, 45T, 62K, 67Q, 69S, 76S, 88C and 89L compared to SEQ ID NO: 33.

[0026] In some embodiments, the dimerizing polypeptide of any one of the above, wherein the variant of SEQ ID NO: 33 has, compared to SEQ ID NO: 33, a) to j), i.e., a) 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 88C and 89L, b) 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C and 89L, c) 3C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C and 89L, d) 9C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C and 89L, e) 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, f) 3C, 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, g) 9C, 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, h) 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C, i) 3C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C, and j) 9C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C, The present invention provides a dimerizing polypeptide having any one amino acid residue substitution selected from the following:

[0027] In some embodiments, there is provided a dimerizing polypeptide comprising any one of the above dimerizing polypeptides, wherein the Obscurin-Like-O chain is SEQ ID NO: 34 or a variant thereof, and the variant of SEQ ID NO: 34 has amino acid residue substitutions at one or more positions selected from 6, 26, 74, 77, 84, and 86 compared to SEQ ID NO: 34, and the amino acid residue substitution positions of the variant of SEQ ID NO: 34 are naturally numbered positions relative to the sequence of SEQ ID NO: 34. In some embodiments, the variant of SEQ ID NO: 34 has one or more amino acid residue substitutions selected from 6E, 26S, 74C, 77S, 84C, and 86C compared to SEQ ID NO: 33, and in some embodiments, the variant of SEQ ID NO: 34 has one or more amino acid residue substitutions selected from A) to F), i.e., A) 6E and 74C, B) 6E and 84C, C) 6E and 86C, D) 6E, 26S, 77S and 74C, E) 6E, 26S, 77S and 84C, and F) 6E, 26S, 77S and 86C, It has any one amino acid residue substitution selected from the following:

[0028] In some embodiments, the dimerizing polypeptide of any one of the preceding claims is provided, wherein the Titin-T chain is a variant of SEQ ID NO: 32, 68, or 127, the variant having one or more amino acid residue substitutions selected from 60S and 64T, and the Obscurin-O chain is a variant of SEQ ID NO: 33, 80, or 128, the variant having one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H, and 93C. The Titin-T chain residue positions are naturally numbered relative to the sequence of SEQ ID NO: 32, and the Obscurin-O chain residue positions are naturally numbered relative to the sequence of SEQ ID NO: 33.

[0029] In some embodiments, there is provided a dimerizing polypeptide according to any one of the preceding claims, wherein the Titin-T chain is a variant of SEQ ID NO: 32, 68, or 127, the variant having an amino acid residue substitution selected from 60S and / or 64T relative to SEQ ID NO: 32, 68, or 127, and the Obscurin-O chain is a variant of SEQ ID NO: 33, 80, or 128, the variant having one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H, and 93C relative to SEQ ID NO: 33, 80, or 128. The amino acid residue positions of the Titin-T chain are naturally numbered relative to the sequence of SEQ ID NO: 32, and the amino acid residue positions of the Obscurin-O chain are naturally numbered relative to the sequence of SEQ ID NO: 33.

[0030] In some embodiments, the present invention provides any one of the dimerization polypeptides described above, wherein the Titin-T chain has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 129 to 131, and the Obscurin-O chain has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 132 to 141. In some embodiments, the present invention provides any one of the dimerized polypeptides described above, wherein the amino acid sequence of the Titin-T chain is set forth in any one of SEQ ID NOs: 129 to 131, and the amino acid sequence of the Obscurin-O chain is set forth in any one of SEQ ID NOs: 132 to 141. In some embodiments, the present invention provides any one of the dimerized polypeptides described above, wherein the amino acid sequence of the Titin-T chain is set forth in SEQ ID NO: 129 and the amino acid sequence of the Obscurin-O chain is set forth in SEQ ID NO: 133, or the amino acid sequence of the Titin-T chain is set forth in SEQ ID NO: 129 and the amino acid sequence of the Obscurin-O chain is set forth in SEQ ID NO: 135, or the amino acid sequence of the Titin-T chain is set forth in SEQ ID NO: 129 and the amino acid sequence of the Obscurin-O chain is set forth in SEQ ID NO: 136.

[0031] In some embodiments, the dimerizing polypeptide of any one of the above is provided, wherein the Titin-T chain and the Obscurin-O chain are capable of associating with each other to form a dimerizing polypeptide. In some embodiments, the dimerizing polypeptide of any one of the above is provided, wherein the Titin-T chain and the Obscurin-Like-O chain are capable of associating with each other to form a dimerizing polypeptide.

[0032] In some embodiments, the present invention provides any one of the dimerized polypeptides described above, wherein one or more residues at positions 7 to 15, 19 to 24, 26, 55, 59, and 60 in the Titin-T chain are mutually bound to one or more residues at positions 3 to 6, 9, 41, 73, 75, and 80 to 90 in the Obscurin-O chain, and one or more residues at positions 1, 7 to 10, 13 to 16, 19 to 26, 59 to 60, and 96 in the Titin-T chain are mutually bound to one or more residues at positions 4 to 5, 10, 12 to 13, 74, 76, 78, and 82 to 91 in the Obscurin-Like-O chain. In some embodiments, the Titin-T chain comprises amino acids 7 to 60 of SEQ ID NO: 32, and / or further comprises an amino acid residue substitution at a corresponding site described in any one of the above sequences, and the Obscurin-O chain comprises amino acids 3 to 90 of SEQ ID NO: 33, and / or further comprises an amino acid residue substitution at a corresponding site described in any one of the above sequences. In some embodiments, the Titin-T chain comprises amino acids 1 to 96 of SEQ ID NO: 32, and / or further comprises an amino acid residue substitution at a corresponding site described in any one of the above sequences, and the Obscurin-Like-O chain comprises amino acids 4 to 91 of SEQ ID NO: 34, and / or further comprises an amino acid residue substitution at a corresponding site described in any one of the above sequences.

[0033] The present invention provides a dimerizing polypeptide described in any one of the above, wherein the Titin-T chain residue site is a natural order numbering site relative to the sequence of SEQ ID NO: 32, the Obscurin-O chain residue site is a natural order numbering site relative to the sequence of SEQ ID NO: 33, and the Obscurin-Like-O chain residue site is a natural order numbering site relative to the sequence of SEQ ID NO: 34.

[0034] In some embodiments, there is provided any one of the dimerizing polypeptides described above, wherein the Titin-T chain has one or more amino acid residue substitutions selected from positions 60S and 64T in SEQ ID NO: 32, 68, or 127, and the Obscurin-O chain has one or more amino acid residue substitutions selected from positions 13S, 32F, 48V, 66C, 82H, and 93C in SEQ ID NO: 33, 80, or 128. The Titin-T chain residue positions are naturally numbered relative to the sequence of SEQ ID NO: 32, and the Obscurin-O chain residue positions are naturally numbered relative to the sequence of SEQ ID NO: 33.

[0035] In some embodiments, the variant of SEQ ID NO:32 comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:32. In some embodiments, the variant of SEQ ID NO:32 has only one of the above amino acid residue substitutions compared to SEQ ID NO:32. In some embodiments, the variant of SEQ ID NO:33 comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:33. In some embodiments, the variant of SEQ ID NO:33 has only one of the above amino acid residue substitutions compared to SEQ ID NO:33. In some embodiments, the variant of SEQ ID NO:34 comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:34. In some embodiments, the variant of SEQ ID NO:34 has only one of the above amino acid residue substitutions compared to SEQ ID NO:34.

[0036] In some embodiments, the present disclosure provides an antigen-binding molecule comprising any one of the dimerizing polypeptides described above.

[0037] In some embodiments, the antigen-binding molecule of any one of the above is provided, comprising a first antigen-binding portion, wherein the first antigen-binding portion comprises a domain-modified Fab, wherein the domain-modified Fab comprises a heavy chain variable region VH1, a light chain variable region VL1, and the dimerization polypeptide, but does not comprise a light chain constant region CL or a heavy chain constant region CH1, and wherein the VH1 and VL1 are each linked to any one peptide chain in the dimerization polypeptide via a linker. In some embodiments, the C-terminus of the VH1 is fused to the N-terminus of the Titin-T chain via a linker, and the C-terminus of the VL1 is fused to the N-terminus of the Obscurin-O chain or Obscurin-Like-O chain via a linker. In some embodiments, the C-terminus of the VL1 is fused to the N-terminus of the Titin-T chain via a linker, and the C-terminus of the VH1 is fused to the N-terminus of the Obscurin-O chain or Obscurin-Like-O chain via a linker.

[0038] In some embodiments, the present disclosure provides an antigen-binding molecule comprising any one of the above dimerizing polypeptides, which replaces the light chain constant region CL and the heavy chain constant region CH1.

[0039] In some embodiments, the antigen-binding molecule of any one of the above is provided, comprising a first antigen-binding portion, wherein the first antigen-binding portion comprises a domain-modified Fab, wherein the domain-modified Fab comprises a heavy chain variable region VH1, a light chain variable region VL1, and the dimerization polypeptide, wherein the VH1 and VL1 are each linked to any one peptide chain in the dimerization polypeptide via a linker. In some embodiments, the C-terminus of the VH1 is fused to the N-terminus of the Titin-T chain via a linker, and the C-terminus of the VL1 is fused to the N-terminus of the Obscurin-O chain or Obscurin-Like-O chain via a linker. In some embodiments, the C-terminus of the VL1 is fused to the N-terminus of the Titin-T chain via a linker, and the C-terminus of the VH1 is fused to the N-terminus of the Obscurin-O chain or Obscurin-Like-O chain via a linker.

[0040] In some embodiments, the antigen-binding molecule of any one of the above, comprising a first antigen-binding portion, wherein the first antigen-binding portion is: a. A peptide chain consisting of [VH1]-[linker 1]-[Titin-T chain] in the order from the N-terminus to the C-terminus, and a peptide chain consisting of [VL1]-[linker 2]-[Obscurin-O chain or Obscurin-Like-O chain] in the order from the N-terminus to the C-terminus, or b. A peptide chain consisting of, from the N-terminus to the C-terminus, [VH1]-[linker 1]-[Obscurin-O chain or Obscurin-Like-O chain], and a peptide chain consisting of, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Titin-T chain], The antigen-binding molecule is provided in which the linker 1 and linker 2 are the same or different. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, A) both linker 1 and linker 2 are (G x S) y wherein x is an integer selected from 1 to 5 (e.g., 1, 2, 3, 4, or 5) and y is an integer selected from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6), and when y is 0, the linker is a bond; or B) linker 1 is a sequence obtained by truncating the C-terminus of CH1, and linker 2 is a sequence obtained by truncating the C-terminus of CL. In some other embodiments, A) linker 1 has the sequence shown in SEQ ID NO: 173, and linker 2 has the sequence shown in SEQ ID NO: 174; or B) linker 1 and linker 2 both have the sequences shown in SEQ ID NO: 175; or C) linker 1 and linker 2 both have the sequences shown in SEQ ID NO: 176.

[0041] In some embodiments, the present disclosure provides an antigen-binding molecule, which comprises a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is as defined above, the second antigen-binding portion comprises a heavy chain variable region VH2 and a light chain variable region VL2, and the first antigen-binding portion and the second antigen-binding portion bind to different antigens or bind to different epitopes on the same antigen; In some embodiments, the second antigen-binding portion comprises a Fab.

[0042] In some embodiments, the antigen-binding molecule further comprises an Fc region, the Fc region comprising a first subunit, Fc1, and a second subunit, Fc2, capable of associating with each other. In some embodiments, the Fc region is an IgG Fc region. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region has one or more amino acid substitutions that reduce homodimerization and / or one or more amino acid substitutions that can reduce binding of the Fc region to an Fc receptor. In some embodiments, the Fc region has a YTE mutation (M252Y, S254T, and T256E), an L234A mutation, an L235A mutation, and / or an S228P mutation, where the mutation numbers are numbered according to the EU index. In some embodiments, the Fc region comprises a first subunit and a second subunit that can associate with each other, and the first subunit and / or the second subunit has one or more amino acid substitutions that reduce homodimerization. In some embodiments, the first subunit has a knobs-in-hole structure and the second subunit has a hole structure, or the first subunit has a hole structure and the second subunit has a knobs-in-hole structure. In some embodiments, the first subunit has one or more amino acid substitutions at sites selected from 354, 356, 358, and 366, and the second subunit has one or more amino acid substitutions at sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the second subunit has one or more amino acid substitutions at sites selected from 354, 356, 358, and 366, and the first subunit has one or more amino acid substitutions at sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the first subunit has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the second subunit has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V.In some embodiments, the second subunit comprises one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the first subunit comprises one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the first subunit comprises amino acid substitutions 354C, 356E, 358M, and 366W, and the second subunit comprises amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the second subunit comprises amino acid substitutions 354C, 356E, 358M, and 366W, and the first subunit comprises amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the sequence of the Fc1 is set forth in SEQ ID NO: 177 and the sequence of the Fc2 is set forth in SEQ ID NO: 178, or the sequence of the Fc2 is set forth in SEQ ID NO: 177 and the sequence of the Fc1 is set forth in SEQ ID NO: 178.

[0043] In some embodiments, the antigen-binding molecule of any one of the above, comprising a first heavy chain, a first light chain, a second heavy chain and a second light chain, a. The first heavy chain is composed of, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Titin-T chain]-[Linker 3]-[Fc1]; the first light chain is composed of, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Obscurin-O chain or Obscurin-Like-O chain], the second heavy chain is [VH2]-[CH1]-[Fc2] in order from the N-terminus to the C-terminus, the second light chain is, from N-terminus to C-terminus, [VL2]-[CL]; or b. The first heavy chain is composed of, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain or Obscurin-Like-O chain]-[Linker 3]-[Fc1]; the first light chain is composed of [VL1]-[linker 2]-[Titin-T chain] in this order from the N-terminus to the C-terminus, the second heavy chain is [VH2]-[CH1]-[Fc2] in order from the N-terminus to the C-terminus, the second light chain is [VL2]-[CL] in order from the N-terminus to the C-terminus, The linkers 1, 2, and 3 are the same or different. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization. In some embodiments, the linkers 1, 2, and 3 are all selected from the group consisting of: A) linker 1, linker 2, and linker 3; x S) y A) a linker, wherein x is an integer selected from 1 to 5 (e.g., 1, 2, 3, 4, or 5), and y is an integer selected from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6), (wherein, when y is 0, the linker is a bond); or B) linker 1 is a sequence obtained by truncating the C-terminus of CH1, linker 2 is a sequence obtained by truncating the C-terminus of CL, and linker 3 is (G x S) ywherein x is an integer selected from 1 to 5 (e.g., 1, 2, 3, 4, or 5), and y is an integer selected from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6). In some embodiments, linker 3 is a bond, and linker 1 and linker 2 are as follows: A) linker 1 has the sequence set forth in SEQ ID NO: 173, and linker 2 has the sequence set forth in SEQ ID NO: 174, or B) linker 1 and linker 2 both have the sequences set forth in SEQ ID NO: 175, or C) linker 1 and linker 2 both have the sequences set forth in SEQ ID NO: 176. In some embodiments, Fc1 has a knob structure formed by knobs-in-hole technology, and Fc2 has a hole structure formed by knobs-in-hole technology, or Fc1 has a hole structure formed by knobs-in-hole technology, and Fc2 has a knob structure formed by knobs-in-hole technology. In some embodiments, the Fc1 has one or more amino acid substitutions at sites selected from 354, 356, 358, and 366, and the Fc2 has one or more amino acid substitutions at sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the Fc2 has one or more amino acid substitutions at sites selected from 354, 356, 358, and 366, and the Fc1 has one or more amino acid substitutions at sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the Fc1 has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc2 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc2 has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc1 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc1 contains amino acid substitutions 354C, 356E, 358M, and 366W, and the Fc2 contains amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V.In some embodiments, the Fc2 comprises amino acid substitutions 354C, 356E, 358M, and 366W, and the Fc1 comprises amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc1 is represented by SEQ ID NO: 177 and the Fc2 is represented by SEQ ID NO: 178, or the Fc2 is represented by SEQ ID NO: 177 and the Fc1 is represented by SEQ ID NO: 178.

[0044] In some embodiments, the antigen-binding molecule according to any one of the above is a multispecific antibody.

[0045] In some embodiments, the antigen-binding molecule according to any one of the above is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0046] In some embodiments, the antigen-binding molecule according to any one of the above is a bispecific antibody.

[0047] In some embodiments, the antigen-binding molecule according to any one of the above, (I) The antigen-binding molecule is capable of binding to NGF and RANKL. In some embodiments, the antigen-binding molecule comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein: The first heavy chain is composed of, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[Fc1]; The first light chain is composed of, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Titin-T chain], the second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; and the second light chain is [VL2]-[CL] from the N-terminus to the C-terminus, in which VH1 and VL1 form a first antigen-binding portion that binds to NGF, and VH2 and VL2 form a second antigen-binding portion that binds to RANKL; or VH1 and VL1 form a first antigen-binding portion that binds to RANKL, and VH2 and VL2 form a second antigen-binding portion that binds to NGF.

[0048] In some embodiments, the VH1 sequence is set forth in SEQ ID NO:26, the VL1 sequence is set forth in SEQ ID NO:27, the VH2 sequence is set forth in SEQ ID NO:24, and the VL2 sequence is set forth in SEQ ID NO:25; or The sequence of the VH1 is represented by SEQ ID NO: 24, the sequence of the VL1 is represented by SEQ ID NO: 25, the sequence of the VH2 is represented by SEQ ID NO: 26, and the sequence of the VL2 is represented by SEQ ID NO: 27; and the sequence of the Obscurin-O chain is represented by any one selected from SEQ ID NOs: 132 to 141, and the sequence of the Titin-T chain is represented by any one selected from SEQ ID NOs: 129 to 131; In some embodiments, the sequence of Fc1 is set forth in SEQ ID NO: 177, the sequence of Fc2 is set forth in SEQ ID NO: 178, the sequence of CH1 is set forth in SEQ ID NO: 179, the sequence of CL is set forth in SEQ ID NO: 4, the linker 3 is a bond, and the linker 1 and linker 2 are selected from a) linker 1 and linker 2 whose sequences are both set forth in SEQ ID NO: 175, or b) linker 1 whose sequence is set forth in SEQ ID NO: 173, and linker 2 whose sequence is set forth in SEQ ID NO: 174; (II) The antigen-binding molecule is capable of binding to PDL1 and CTLA4. In some embodiments, the antigen-binding molecule comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein: The first heavy chain is composed of, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[Fc1]; The first light chain is composed of, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Titin-T chain], the second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; and the second light chain is, from N-terminus to C-terminus, [VL2]-[CL], in which VH1 and VL1 form a first antigen-binding portion that binds to PDL1, and VH2 and VL2 form a second antigen-binding portion that binds to CTLA4; or VH1 and VL1 form a first antigen-binding portion that binds to CTLA4, and VH2 and VL2 form a second antigen-binding portion that binds to PDL1.

[0049] In some embodiments, the VH1 sequence is set forth in SEQ ID NO: 156, the VL1 sequence is set forth in SEQ ID NO: 155, the VH2 sequence is set forth in SEQ ID NO: 169, and the VL2 sequence is set forth in SEQ ID NO: 170; or The sequence of the VH1 is represented by SEQ ID NO: 169, the sequence of the VL1 is represented by SEQ ID NO: 170, the sequence of the VH2 is represented by SEQ ID NO: 156, and the sequence of the VL2 is represented by SEQ ID NO: 155; The sequence of the Obscurin-O chain is represented by any one selected from SEQ ID NOs: 132 to 141, and the sequence of the Titin-T chain is represented by any one selected from SEQ ID NOs: 129 to 131; In some embodiments, the sequence of Fc1 is set forth in SEQ ID NO: 178, the sequence of Fc2 is set forth in SEQ ID NO: 177, the sequence of CH1 is set forth in SEQ ID NO: 179, the sequence of CL is set forth in SEQ ID NO: 4, linker 3 is a bond, and linker 1 and linker 2 are selected from a) linker 1 and linker 2 whose sequences are both set forth in SEQ ID NO: 175, or b) linker 1 whose sequence is set forth in SEQ ID NO: 173, and linker 2 whose sequence is set forth in SEQ ID NO: 174; or (III) The antigen-binding molecule is capable of binding to IL5 and TSLP. In some embodiments, the antigen-binding molecule comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein: The first heavy chain is composed of, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Titin-T chain]-[Linker 3]-[Fc1]; The first light chain is composed of, from the N-terminus to the C-terminus, [VL1]-[Linker 2]-[Obscurin-O chain]; the second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; and The second light chain is [VL2]-[CL] from the N-terminus to the C-terminus, VH1 and VL1 form a first antigen-binding portion that binds IL5, and VH2 and VL2 form a second antigen-binding portion that binds TSLP; or VH1 and VL1 form a first antigen-binding portion that binds TSLP, and VH2 and VL2 form a second antigen-binding portion that binds IL5.

[0050] In some embodiments, the VH1 sequence is set forth in SEQ ID NO: 16, the VL1 sequence is set forth in SEQ ID NO: 17, the VH2 sequence is set forth in SEQ ID NO: 171, and the VL2 sequence is set forth in SEQ ID NO: 172; or The sequence of the VH1 is represented by SEQ ID NO: 171, the sequence of the VL1 is represented by SEQ ID NO: 172, the sequence of the VH2 is represented by SEQ ID NO: 16, and the sequence of the VL2 is represented by SEQ ID NO: 17; The sequence of the Obscurin-O chain is represented by any one selected from SEQ ID NOs: 132 to 141, and the sequence of the Titin-T chain is represented by any one selected from SEQ ID NOs: 129 to 131; In some embodiments, the sequence of Fc1 is set forth in SEQ ID NO: 178, the sequence of Fc2 is set forth in SEQ ID NO: 177, the sequence of CH1 is set forth in SEQ ID NO: 179, the sequence of CL is set forth in SEQ ID NO: 4, linker 3 is a bond, and linker 1 and linker 2 are selected from a) linker 1 and linker 2 whose sequence is set forth in SEQ ID NO: 175, or b) linker 1 whose sequence is set forth in SEQ ID NO: 173 and linker 2 whose sequence is set forth in SEQ ID NO: 174.

[0051] In some embodiments, the antigen-binding molecule of any one of the above is a. A first heavy chain which is, from N-terminus to C-terminus, [VH1]-[Linker 1]-[Titin-T chain]-[Linker 3]-[VH2]-[CH1]-[Fc1]; a second heavy chain consisting of, from N-terminus to C-terminus, [VH1]-[Linker 1]-[Titin-T chain]-[Linker 3]-[VH2]-[CH1]-[Fc2]; a first light chain consisting of, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Obscurin-O chain or Obscurin-Like-O chain]; and a second light chain, which is [VL2]-[CL] in N- to C-terminal order; or b. A first heavy chain which is, from N-terminus to C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain or Obscurin-Like-O chain]-[Linker 3]-[VH2]-[CH1]-[Fc1]; a second heavy chain consisting of, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain or Obscurin-Like-O chain]-[Linker 3]-[VH2]-[CH1]-[Fc2]; a first light chain consisting of, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Titin-T chain]; and a second light chain, which is, from N-terminus to C-terminus, [VL2]-[CL]; Wherein, the above linker 1, linker 2 and linker 3 may be the same or different. In some embodiments, the above linker 1, linker 2 and linker 3 are A) or B): A) Linker 1, Linker 2 and Linker 3 are all (G x S) y a linker, wherein x is an integer selected from 1 to 5 and y is an integer selected from 0 to 6; and B) Linker 1 is a C-terminal truncated sequence of CH1, linker 2 is a C-terminal truncated sequence of CL, and linker 3 is a (G x S) y a linker, wherein x is an integer selected from 1 to 5 and y is an integer selected from 0 to 6; In some embodiments, the linker 1, the linker 2, and the linker 3 are selected from A) to C), i.e., A) Linker 1, whose sequence is set forth in SEQ ID NO: 173, Linker 2, whose sequence is set forth in SEQ ID NO: 174, and Linker 3, whose sequence is set forth in SEQ ID NO: 175 or SEQ ID NO: 176; B) Linker 1, linker 2 and linker 3, all of whose sequences are set forth in SEQ ID NO: 175; C) Linker 1, linker 2 and linker 3, all of whose sequences are set forth in SEQ ID NO: 176; Any one selected from In some embodiments, the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization. In some embodiments, the Fc1 and Fc2 are the same. In some embodiments, the antigen-binding molecule comprises one first heavy chain and one second heavy chain, two first light chains and two second light chains, and the amino acid sequences of the first heavy chain and the second heavy chain are the same.

[0052] In some embodiments, the antigen-binding molecule specifically binds to PDL1 and TIGIT. In some embodiments, the first antigen is PDL1 and the second antigen is TIGIT. In some embodiments, the first antigen is TIGIT and the second antigen is PDL1. In some embodiments, the antigen-binding molecule comprises a first antigen-binding portion capable of specifically binding to PDL1 and a second antigen-binding portion capable of specifically binding to TIGIT, wherein the first antigen-binding portion comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding portion comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein said VH1 comprising HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 163, SEQ ID NO: 164 and SEQ ID NO: 165, respectively; said VL1 comprising LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168, respectively; and / or The VH2 comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 157, SEQ ID NO: 158 and SEQ ID NO: 159, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 162, respectively.

[0053] In some embodiments, the sequence of the VH1 is set forth in SEQ ID NO: 156 or has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 156, and the sequence of the VL1 is set forth in SEQ ID NO: 155 or has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 155; and / or The sequence of the VH2 is set forth in SEQ ID NO: 154 or has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 154, and the sequence of the VL2 is set forth in SEQ ID NO: 153 or has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 153.

[0054] In some embodiments, the antigen binding molecule comprises: and a second light chain having a sequence set forth in SEQ ID NO: 147 or having at least 90% sequence identity to SEQ ID NO: 147; and in some embodiments, the antigen-binding molecule comprises two of the heavy chains, two of the first light chains, and two of the second light chains.

[0055] In some embodiments, the present disclosure provides an antigen-binding molecule, comprising a first antigen-binding portion capable of specifically binding to PDL1 and a second antigen-binding portion capable of specifically binding to TIGIT, wherein the first antigen-binding portion comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding portion comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein said VH1 comprising HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 163, SEQ ID NO: 164 and SEQ ID NO: 165, respectively; said VL1 comprising LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168, respectively; and / or The VH2 comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 157, SEQ ID NO: 158 and SEQ ID NO: 159, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 162, respectively.

[0056] In some embodiments, the sequence of the VH1 is set forth in SEQ ID NO: 156 or has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 156, and the sequence of the VL1 is set forth in SEQ ID NO: 155 or has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 155; and / or The sequence of the VH2 is set forth in SEQ ID NO: 154 or has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 154, and the sequence of the VL2 is set forth in SEQ ID NO: 153 or has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 153.

[0057] In some embodiments, the antigen binding molecule comprises: and a heavy chain having the sequence set forth in SEQ ID NO: 148 or having at least 90% sequence identity to SEQ ID NO: 148, a first light chain having the sequence set forth in SEQ ID NO: 146 or having at least 90% sequence identity to SEQ ID NO: 146, and a second light chain having the sequence set forth in SEQ ID NO: 147 or having at least 90% sequence identity to SEQ ID NO: 147. In some embodiments, the antigen-binding molecule has two heavy chains, two first light chains, and two second light chains.

[0058] In some embodiments, the present disclosure provides a domain-modified antibody, wherein the heavy chain constant region CH1 and the light chain constant region CL are substituted with any one of the dimerizing polypeptides described above. In some embodiments, the domain-modified antibody, wherein the heavy chain constant region CH1 is substituted with Titin-T chain and the light chain constant region CL is substituted with Obscurin-O chain. In some embodiments, the domain-modified antibody, wherein the light chain constant region CL is substituted with Titin-T chain and the heavy chain constant region CH1 is substituted with Obscurin-O chain. In some embodiments, the domain-modified antibody, wherein the heavy chain constant region CH1 is substituted with Titin-T chain and the light chain constant region CL is substituted with Obscurin-Like-O chain. In some embodiments, the domain-modified antibody, wherein the light chain constant region CL is substituted with Titin-T chain and the heavy chain constant region CH1 is substituted with Obscurin-Like-O chain.

[0059] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one of the antigen-binding molecules or domain-modified antibodies described above, and one or more pharmaceutically acceptable vectors, diluents, or excipients.

[0060] In some embodiments, the present disclosure provides the use of any one of the dimerizing polypeptides described above in reducing antibody light chain / heavy chain mismatch.

[0061] In some embodiments, the present disclosure provides the use of any one of the dimerizing polypeptides described above in reducing antibody light chain / heavy chain mismatches during the preparation of bispecific antibodies.

[0062] In some embodiments, the present disclosure provides the use of any one of the dimerizing polypeptides described above in reducing light chain / heavy chain mismatches during the preparation of multispecific antibodies.

[0063] In some embodiments, the present disclosure provides the use of any one of the dimerizing polypeptides described above in reducing light chain / heavy chain mismatches during the preparation of tetrabodies, triabodies and bispecific antibodies.

[0064] In some embodiments, the present disclosure provides a nucleic acid molecule encoding any one of the dimerizing polypeptides, antigen-binding molecules, or domain-engineered antibodies described above.

[0065] In some embodiments, the present disclosure provides an expression vector comprising any one of the above described nucleic acid molecules.

[0066] In some embodiments, the present disclosure further provides a host cell comprising any one of the above described nucleic acid molecules.

[0067] In some embodiments, the host cell described in any one of the above is obtained by transformation (or transfection) with the vector, and the host cell is selected from prokaryotic and eukaryotic cells, preferably eukaryotic cells, and more preferably mammalian cells. The host cell does not include any animal or plant cells that can develop into a complete organism, such as human embryonic stem cells, fertilized eggs, or germ cells. In some embodiments, the host cell is a eukaryotic cell, more preferably a mammalian cell, including, but not limited to, CHO, 293, NSO, and cells in which gene editing is performed in mammalian cells, such as knocking out genes such as FUT8 or GnT-III, to alter the glycosylation modification of an antibody or antigen-binding fragment thereof, thereby altering the ADCC function of the antibody or antigen-binding fragment thereof.

[0068] In some embodiments, the present disclosure provides a method for preparing any one of the dimerized polypeptides, antigen-binding molecules or domain-modified antibodies described above, comprising culturing the host cells described above and then purifying and recovering the dimerized polypeptides, antigen-binding molecules or domain-modified antibodies.

[0069] In some embodiments, the present disclosure further provides a use of any one of the antigen-binding molecules, domain-modified antibodies, or pharmaceutical compositions described above in the preparation of a medicament for treating or preventing a disease or condition.

[0070] In some embodiments, the present disclosure provides a method for treating or preventing a disease or condition, the method comprising administering to a subject in need thereof an effective amount of any one of the antigen-binding molecule, domain-modified antibody, or pharmaceutical composition described above.

[0071] In some embodiments, the present disclosure provides any one of the antigen-binding molecules, domain-modified antibodies, or pharmaceutical compositions described above for use as a medicament, and in some embodiments, the medicament is used for the treatment or prevention of a disease or condition.

[0072] In some embodiments, the disease or condition described in any one of the above is a skeletal disease, such as osteoporosis, osteopenia, or a disease or disorder of osteoarthritis, rheumatoid arthritis, periodontal disease, or multiple myeloma. In some embodiments, the tumor is selected from a carcinoma tumor, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy. In some embodiments, the tumor is selected from the group consisting of squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), T-cell / histiocyte-rich Large B-cell lymphoma, multiple myeloma, myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (digestive tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, stomach cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, pharyngeal cancer, hepatobiliary cancer In some embodiments, the inflammatory disease is selected from rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type 1 diabetes, hepatitis (e.g., hepatitis B, hepatitis A, hepatitis C), myocarditis, Sjogren's syndrome, autoimmune hemolytic anemia after transplant rejection, bullous pemphigoid, Graves' disease, Hashimoto's thyroiditis, systemic lupus erythematosus (SLE), myasthenia gravis, pemphigus, and pernicious anemia.In some embodiments, the immune-mediated disease is selected from the group consisting of rheumatoid arthritis, psoriasis, psoriatic arthritis, dermatitis, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, arteriosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, juvenile diabetes, Reiter's disease, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia (e.g., acute idiopathic thrombocytopenic purpura, chronic thrombocytopenia ... The immune-mediated disease is selected from the group consisting of chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omenn's syndrome, chronic renal failure, acute infectious mononucleosis, HIV and herpes virus-associated diseases, severe acute respiratory syndrome, choreoretinitis, and immune-mediated diseases caused by viral infection (e.g., diseases caused or mediated by infection of B cells with Ebola virus (EBV)). [Brief explanation of the drawings]

[0073] [Figure 1] Figure 1A shows the structure of the Titin-T chain and the Obscurin-O chain, and Figure 1B shows the structure of the Titin-T chain and the Obscurin-Like-O chain, forming a dimerized polypeptide complex. [Figure 2] Schematic diagram of Fab with CH1 and CL replaced. [Figure 3] Schematic of an IgG monospecific antibody structure with both CH1 and CL replaced. [Figure 4] Schematic diagram of the structure of an IgG bispecific antibody in which one CH1 and one CL are replaced. [Figure 5] FIG. 1 is a schematic diagram of the structure of the DI-1 bispecific antibody. [Figure 6]6A is a mass spectrum analysis diagram of DI-1, of which FIG. 6A is a mass spectrum analysis diagram of LC1 / LC2, FIG. 6B is a mass spectrum analysis diagram of HC1 / HC2, and FIG. 6C is a mass spectrum analysis diagram of LC1+LC2+HC1+HC2. [Figure 7] FIG. 1 is a schematic diagram of the structure of an antibody light chain / heavy chain cross-mismatch molecule. [Figure 8] FIG. 1 is a schematic diagram of the structure of the BU5 bispecific antibody. [Figure 9] 9A is a mass spectrum analysis diagram of B0, FIG. 9B is a mass spectrum analysis diagram of U0, and FIG. 9C is a mass spectrum analysis diagram of BU5. [Figure 10] FIG. 1 shows the results of an experiment on osteoclast differentiation using DI-1 bispecific antibody. [Figure 11] FIG. 1 shows the results of an experiment on TF1 cell proliferation using the DI-1 bispecific antibody. [Figure 12] FIG. 1 is a schematic diagram of the structures of bispecific antibodies HJ-1, HJ-2, HJ-3, and HJ-4. [Figure 13] 13A shows mass spectrometry results for the four-chain co-expression of HJ bispecific antibodies, where FIG. 13A shows mass spectrometry results for the four-chain co-expression of HJ-1, FIG. 13B shows mass spectrometry results for the four-chain co-expression of HJ-2, FIG. 13C shows mass spectrometry results for the four-chain co-expression of HJ-3, and FIG. 13D shows mass spectrometry results for the four-chain co-expression of HJ-4. [Figure 14] 14A and 14B show mass spectrometry analysis results of the three-chain co-expression of bispecific antibody HJ-1, consisting of HJ-1-H1, HJ-1-H2, and HJ-1-L2. FIG. 14A shows the mass spectrometry analysis results of the deconvoluted region from 120,000 to 130,000 (amu), and FIG. 14B shows the mass spectrometry analysis results of the deconvoluted region from 140,000 to 160,000 (amu). [Figure 15]15A and 15B show mass spectrometry analysis results for the co-expression of the three chains of bispecific antibody HJ-3, HJ-3-H1, HJ-3-H2, and HJ-3-L2, in which FIG. 15A shows the mass spectrometry analysis results for the deconvoluted region from 122,600 to 125,200 (amu), and FIG. 15B shows the mass spectrometry analysis results for the deconvoluted region from 120,000 to 148,000 (amu). [Figure 16] FIG. 16A is a diagram of the interaction site between Titin-T chain / Obscurin-O chain residues, and FIG. 16B is a diagram of the interaction site between Titin-T chain / Obscurin-Like-O chain residues. [Figure 17] FIG. 1 is a schematic diagram of the structure of a (FabV)2-IgG mode bispecific antibody constructed using domain-engineered Fab. [Figure 18] This figure shows the results of an experiment in which the PDL1-TIGIT bispecific antibody inhibited the binding of PD-L1 to PD-1 and the binding of TIGIT to CD155. [Figure 19] FIG. 1 shows the results of an experiment in which the PDL1-TIGIT bispecific antibody promoted IFN-γ secretion. [Figure 20] FIG. 1 shows the results of an experiment in which the PDL1-TIGIT bispecific antibody suppressed tumors transplanted into MC38-HL1 mice. DETAILED DESCRIPTION OF THE INVENTION

[0074] In order that the present disclosure may be more readily understood, the following provides an explanation of some technical and scientific terms. Unless expressly defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0075] As used in the specification and claims, the singular forms "a," "an," and "the above" include plural referents unless the context clearly dictates otherwise.

[0076] Unless the context clearly indicates otherwise, in the patent specification and claims, the words "contain," "have," "include," and the like are to be understood in the sense of "including, but not limited to," rather than in the exclusive or exhaustive sense.

[0077] The term "and / or" is intended to be inclusive. For example, the phrase "A, B and / or C" is intended to cover each of A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0078] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0079] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by genetic codons and those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon bonded to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid.

[0080] The term "amino acid mutation" includes amino acid substitution (also referred to as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be used to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy termini of the polypeptide chain. A specific amino acid mutation may be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or derivatives of the 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods for modifying amino acid side groups other than genetic engineering, such as chemical modification, may also be utilized. As used herein, the same amino acid mutation may be referred to by various names. As used herein, the amino acid residue at a particular site may be designated by the format of position + amino acid residue, for example, 366W indicates that the amino acid residue at site 366 is W. T366W indicates that the amino acid residue at site 366 has been mutated from the original T to W.

[0081] The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments or portions thereof), as long as they exhibit the desired antigen-binding activity. A "native antibody" is a naturally occurring immunoglobulin molecule. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain contains one variable heavy chain domain (VH), also called the heavy chain variable region, followed by a heavy chain constant region (CH), which generally contains three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has one variable region (VL), also called a variable light domain, light chain variable domain, followed by one constant light domain (light chain constant region, CL). The terms "full length antibody," "complete antibody," and "whole antibody" may be used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody or having heavy chains of the Fc region as defined herein. The light chain of a native intact antibody comprises a light chain variable region VL and a constant region CL, with VL at the amino terminus of the light chain, and the light chain constant region comprises a kappa chain and a lambda chain; the heavy chain comprises a variable region VH and constant regions (CH1, CH2, and CH3), with VH at the amino terminus of the heavy chain and the constant region at the carboxy terminus, of which CH3 is closest to the carboxy terminus of the polypeptide; and the heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0082] The term "variable region" or "variable domain" of an antibody refers to the domain in the heavy or light chain of the antibody that is involved in binding to an antigen. As used herein, the heavy chain variable region (VH) and light chain variable region (VL) of an antibody each contain four conserved framework regions (FR) and three complementarity-determining regions (CDR). The term "complementarity-determining region" or "CDR" refers to the region in the variable domain that primarily promotes antigen binding, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, while VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus (also referred to as the N-terminus) to the carboxy terminus (also referred to as the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0083] The boundaries of the amino acid sequences of CDRs can be determined by various known methods, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16, 9:2278). The correspondence between various numbering systems is well known to those skilled in the art and is shown, for example, in Table 1 below.

[0084] [Table 1]

[0085] Unless otherwise stated, both variable region and CDR sequences in this disclosure are governed by the "Kabat" numbering convention.

[0086] The term "antibody fragment" refers to a molecule distinct from an intact antibody and includes a portion of the intact antibody that binds to an antigen that binds to the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies composed of antibody fragments. In some embodiments, the antigen-binding fragment of the antibody is monovalent Fab (i.e., Fab), bivalent Fab (F(ab)2), trivalent Fab fragment (F(ab)3), or multivalent Fab (two or more Fab), and may be a monospecific or multispecific antigen-binding fragment comprising at least one Fab fragment.

[0087] The term "antigen-binding molecule" refers to a protein capable of specifically binding to an antigen.

[0088] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, and includes native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two subunits, which may be identical or different. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable Fc regions for use in the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may be altered, for example, by deletion of the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region, or deletion of the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system) of the Fc region. Unless otherwise specified, the numbering convention for the Fc region is the EU numbering system, also known as the EU index.

[0089] A "domain-modified antibody" as described in the present disclosure refers to an antibody formed by replacing the CH1 and / or CL of an antibody with other domains or peptide fragments, for example, by replacing CH1 / CL with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain. In some embodiments, the domain-engineered antibody is a monospecific antibody or a multispecific antibody. In some embodiments, the domain-engineered antibody is a monovalent, bivalent, or multivalent antibody. In some embodiments, the domain-engineered antibody is a whole antibody or an antigen-binding fragment thereof. In some embodiments, the domain-engineered antibody has the structure shown in FIG. 2, where the CH1 / CL of the Fab is replaced with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain, and the structure of a domain-engineered antibody has the structure shown in FIG. 3, where the CH1 / CL of a monoclonal antibody is replaced with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain, and the structure of a bispecific antibody has the structure shown in FIG. 4. Domain-engineered antibodies in which other CH1 / CLs are replaced with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain include, but are not limited to, those in which one CH1 / CL of a bivalent antibody is replaced with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain; those in which both CH1 / CLs of a bispecific antibody are replaced with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain; those in which two CH1 / CLs of F(ab)2 are replaced with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain; and those in which one, two, or three CH1 / CLs of F(ab)3 are replaced with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain.

[0090] "Titin" is a large sarcomeric protein with a complex molecular folding structure. It is known to connect thick filaments and Z-lines and maintain the integrity and stability of myofibrils. Titin is the third most abundant protein in skeletal muscle fibers, with a molecular weight of 2700 kDa (more than 25,000 amino acids) and a length of 1 μm, approximately half the length of a sarcomere.

[0091] The "Titin Ig-Like 152 domain" is an Ig-Like domain in the Titin protein, named Titin Ig-Like 152, which can bind to the Obscurin Ig-Like 1 or Obscurin-Like Ig-Like 1 domain to form a complex (available from the RCSB PDB database).

[0092] The term "Titin-T chain" or "T chain" refers to a peptide fragment or functional variant thereof containing the Titin Ig-Like 152 domain, which is 78 to 118 amino acids in length in the Titin protein. The Titin-T chain can bind to the Obscurin Ig-Like 1 or Obscurin-Like Ig-Like 1 domain to form a dimeric complex. The functional variant of the T chain is a polypeptide that can bind to the Obscurin Ig-Like 1 or Obscurin-Like Ig-Like 1 domain to form a dimeric complex, even when some amino acids of the wild-type T chain are mutated. In the present disclosure, the Titin-T chain can be used to replace the CH1 or CL domain of an antibody without affecting the antibody-antigen binding. Even when some amino acids of the Titin Ig-Like 152 domain are mutated, the Titin T chain can retain the function of associating with the Obscurin Ig-Like 1 domain or Obscurin-Like Ig-Like 1 domain to form a complex. For example, an appropriate length of amino acids can be added or truncated at the C-terminus and / or N-terminus of the Titin Ig-like 152 domain, resulting in addition or truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. For example, adding five amino acids "KAGIR" adjacent to the N-terminus of the Titin Ig-like 152 domain in wild-type Titin protein to the N-terminus of the Titin Ig-like 152 domain still retains the ability to associate with the Obscurin Ig-like 1 domain or the Obscurin-like Ig-like 1 domain to form a complex. Other amino acid mutations in the Titin Ig-like 152 domain may also be introduced, for example, to improve interchain disulfide bonds and enhance complex stability. In some embodiments, the Titin T chain is a polypeptide comprising amino acid residues 7 to 60 of SEQ ID NO: 32 or a mutant sequence thereof. In some embodiments, the Titin-T chain is a polypeptide comprising amino acid residues 1 to 96 of SEQ ID NO: 32, or a mutant sequence thereof.In some embodiments, the Titin-T chain is a variant of SEQ ID NO: 32, wherein the variant has an amino acid residue substitution at one or more positions selected from positions 60 and 64, for example, one or more amino acid residue substitutions selected from 60S and 64T. In some embodiments, the Titin-T chain is a variant of SEQ ID NO: 32, wherein the variant has an amino acid residue substitution at one or more positions selected from positions 60 and 64, and further has an amino acid residue substitution at one or more positions selected from positions 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84. The Titin-T chain may further comprise one or more amino acid residue substitutions selected from the group consisting of 3W, 8C, 11I, 13L, 20C, 22M / 22C, 25S, 26C, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 66S / 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D, and 84L. In some embodiments, the Titin-T chain is a polypeptide comprising amino acid residues 7 to 60 of SEQ ID NO: 32, or a mutant sequence thereof. In some embodiments, the Titin-T chain is a polypeptide comprising amino acid residues 1 to 96 of SEQ ID NO: 32, or a mutant sequence thereof.

[0093] Obscurin, a protein encoded by the OBSCN gene, belongs to the giant sarcosine signaling protein family. Obscurin is expressed in cardiac and skeletal muscles and plays an important role in myofibril organization during myofibril assembly. Obscurin is the major cytoplasmic ligand for the sarcoplasmic clathrin sANK1 and can prevent sANK1 degradation (Lange S et al., Molecular Biology of the Cell. 23(13): 2490-504). Obscurin also mediates signaling between sarcoplasmic reticular domains (Bagnato P et al., The Journal of Cell Biology. 160(2): 245-53). Obscurin is also involved in the formation of new sarcomeres during myofibril assembly (Borisov AB et al., Biochemical and Biophysical Research Communications. 310(3): 910-918).

[0094] The "Obscurin Ig-Like 1 domain" is an Ig-Like domain named Obscurin Ig-Like 1 in the Obscurin protein, which can bind to the Titin Ig-Like 152 domain to form a dimerization complex (available from the RCSB PDB database).

[0095] The term "Obscurin-O chain" or "O chain" refers to a peptide fragment or functional variant thereof containing the Obscurin Ig-Like 1 domain, which is 87 to 117 amino acids in length in the Obscurin protein. The Obscurin-O chain can bind to the Titin Ig-Like 152 domain to form a dimeric complex. The functional variant of the Obscurin-O chain is a polypeptide that can bind to the Titin Ig-Like 152 domain to form a dimeric complex, even when some amino acids of the wild-type O chain are mutated. In the present disclosure, the Obscurin-O chain can replace the CH1 or CL domain of an antibody without affecting the binding between the antibody and the antigen. Even when amino acids in the Obscurin Ig-Like 1 domain are mutated, the Obscurin-O chain can retain the function of associating with the Titin Ig-Like 152 domain to form a complex. For example, an appropriate length of amino acids may be added or truncated at the C-terminus and / or N-terminus of the Obscurin-O domain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be added or truncated, e.g., five amino acids "DQPQF" adjacent to the N-terminus of the Obscurin Ig-Like 1 domain in the wild-type Obscurin protein may be added to the N-terminus of the Obscurin-O domain, and the Obscurin-O domain will still retain the function of binding to the Titin Ig-Like 152 domain to form a dimerized complex. Other mutations may also be made to some amino acids in the Obscurin Ig-Like 1 domain, e.g., certain amino acids may be mutated to improve interchain disulfide bonds or increase antibody stability.In some embodiments, the Obscurin-O chain is a variant of SEQ ID NO: 33, which has an amino acid residue substitution at one or more positions selected from 13, 32, 48, 66, 82, and 93, for example, one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H, and 93C. In some embodiments, the Obscurin-O chain is a variant of SEQ ID NO: 33, which further has 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, 22, 25, 30, 32, 34, 36, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 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, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 The Obscurin-O chain has amino acid residue substitutions at one or more positions selected from positions 4, 45, 53, 58, 62, 67, 69, 76, 88, 89, 92, 94, and 97, and further has one or more amino acid residue substitutions selected from, for example, 2E, 3C, 7K / 7R, 9C, 11L, 12S, 13Y, 14T, 17E, 20L, 22M / 22S, 25S, 30D, 32P, 34E, 36T, 41K, 42L, 44I, 45T, 53L, 58V, 62E / 62K / 62H, 67Q / 67T, 69S, 76S, 88C, 89L, 92E, 94G, and 97G. In some embodiments, the Obscurin-O chain is a polypeptide comprising amino acids 3 to 90 of SEQ ID NO: 33, or a mutant sequence thereof.

[0096] "Obscurin-Like 1" is a protein encoded by the OBSL1 gene located at SPEG on human chromosome 2q35. This protein is closely related to Obscurin. Alternative splicing produces multiple isoforms with predicted molecular weights ranging from 130 to 230 kD (Geisler SB et al., (2007). Genomics. 89(4): 521-31).

[0097] The "Obscurin-Like Ig-Like 1 domain" is an Ig-Like domain named Obscurin-Like Ig-Like 1 in the Obscurin-Like 1 protein, which can form a complex with the Titin Ig-Like 152 domain through natural intermolecular interactions (available from the RCSB PDB database).

[0098] The terms "Obscurin-like O chain" and "OL chain" refer to a peptide fragment or functional variant thereof containing the Obscurin-like Ig-like 1 domain, which is 78 to 118 amino acids in length in the Obscurin-like 1 protein. The Obscurin-like O chain can bind to the Titin Ig-like 152 domain to form a dimeric complex. The functional variant of the Obscurin-like O chain is a polypeptide that can bind to the Titin Ig-like 152 domain to form a dimeric complex even when some amino acids of the wild-type OL chain are mutated. In the present disclosure, the Obscurin-like O chain can replace the CH1 or CL domain of an antibody without affecting the formation of the antigen-binding site by the antibody VH and VL and without affecting the binding of the antibody to the antigen. Mutation of some amino acids in the Obscurin-Like Ig-Like 1 domain can still retain the function of associating with the Titin Ig-Like 152 domain to form a dimerized complex. For example, an appropriate number of amino acids may be added or truncated at the C-terminus and / or N-terminus of the Obscurin-O domain, for example, by adding or truncating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Other mutations may also be made to some amino acids in the Obscurin-Like Ig-Like 1 domain; for example, certain amino acids may be mutated to improve interchain disulfide bonds or antibody stability. In some embodiments, the Obscurin-Like-O chain is a variant of SEQ ID NO: 34, which has one or more amino acid residue substitutions selected from, for example, 6E, 26S, 74C, 77S, 84C, and 86C, at one or more sites selected from positions 6, 26, 74, 77, 84, and 86. In some embodiments, the Obscurin-Like-O chain is a polypeptide comprising amino acid residues 4 to 91 of SEQ ID NO: 34, or a variant sequence thereof.

[0099] In some embodiments, even when some amino acids in the Titin Ig-Like 152 domain, the Obscurin Ig-Like 1 domain, and / or the Obscurin-Like Ig-Like 1 domain are mutated, the Titin Ig-Like 152 domain and the Obscurin Ig-Like 1 domain still retain the ability to associate with each other to form a complex, or the Titin Ig-Like 152 domain and the Obscurin-Like Ig-Like 1 domain still retain the ability to associate with each other to form a complex. In some embodiments, the present invention provides a Titin-T chain / Obscurin-O chain dimerization polypeptide and a Titin-T chain / Obscurin-Like-O chain dimerization polypeptide, in which one or more residues at positions 7 to 15, 19 to 24, 26, 55, 59, and 60 in the Titin-T chain are mutually bonded to one or more residues at positions 3 to 6, 9, 41, 73, 75, and 80 to 90 in the Obscurin-O chain, and one or more residues at positions 1, 7 to 10, 13 to 15 in the Titin-T chain are mutually bonded to one or more residues at positions 1, 7 to 10, 13 to 15 in the Obscurin-Like-O chain, and one or more residues at positions 1, 7 to 10, 13 to 15 in the Titin-T chain are mutually bonded to one or more residues at positions 1, 7 to 10, 13 to 15 in the Obscurin-Like-O chain, and one or more residues at positions 1, 7 to 10, 13 to 15 in the Obscurin-Like-O ... One or more residues at positions 16, 19-26, 59-60, and 96 are mutually bonded to one or more residues at positions 4-5, 10, 12-13, 74, 76, 78, and 82-91 in the Obscurin-Like-O chain, and the residue positions of the Titin-T chain are naturally numbered relative to sequence number 32, the residue positions of the Obscurin-O chain are naturally numbered relative to sequence number 33, and the residue positions of the Obscurin-Like-O chain are naturally numbered relative to sequence number 34.

[0100] "Fab" refers to a protein consisting of immunoglobulin VH and CH1 (Fab heavy chain) and VL and CL (Fab light chain).

[0101] "Domain-engineered Fab" or "FabV" refers to a polypeptide fragment formed by replacing CL and / or CH1 of Fab with another domain or peptide fragment. Even in a domain-engineered Fab, its VH and VL interact to form an antigen-binding site and retain the ability to bind to an antigen. In some embodiments, the domain-engineered Fab can be part of a multivalent antibody (e.g., a bivalent antibody or a trivalent antibody). In other embodiments, the domain-engineered Fab fragment forms a single antigen-binding molecule. In some embodiments, CH1 / CL of the Fab is replaced with a Titin-T chain / Obscurin-O chain or a Titin-T chain / Obscurin-Like-O chain. A schematic structure of a domain-engineered Fab is shown in Figure 2.

[0102] The terms "specific antibody" and "specifically binding antibody" refer to an antibody capable of specifically binding to a target antigen or epitope. Depending on the number of different target antigens or different antigen epitopes to which the antibody binds, antibodies can be classified as monospecific, bispecific, trispecific, tetraspecific, etc., or multispecific (binding to two or more different target antigens or different epitopes on the same antigen). For example, a "bispecific antibody" refers to an antibody capable of specifically binding to two different antigens or two different antigen epitopes on the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art. They can be classified as IgG-like bispecific antibodies and antibody fragment bispecific antibodies based on the integrity of the IgG molecule, bivalent, trivalent, tetravalent, etc., multivalent (more than two valent) bispecific antibodies based on the number of antigen-binding regions, and symmetrical bispecific antibodies and asymmetrical bispecific antibodies based on the presence or absence of bilateral symmetry in the structure.Here, bispecific antibodies based on antibody fragments are, for example, Fab fragments lacking Fc fragments. Bispecific antibodies are formed by binding two or more Fab fragments into a single molecule, which have low immunogenicity, a small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include bispecific antibodies such as F(ab)2, scFv-Fab, and (scFv)2-Fab. Antibodies such as IgG-like bispecific antibodies (e.g., antibodies with Fc fragments) have a relatively large molecular weight, and the Fc fragment contributes to the later purification of the antibody and improves its solubility and stability. The Fc portion can also bind to the receptor FcRn to increase the serum half-life of the antibody. Typical structural models of bispecific antibodies include, for example, KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, and 1Fab-IgG. Bispecific antibodies include TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, and F(ab)4-CrossMAb (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585-608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11, 2019:4516041).

[0103] The terms "monovalent," "bivalent," "trivalent," or "multivalent" antibody refer to an antibody in which a predetermined number of antigen-binding sites are present in the antibody. For example, a "monovalent antibody" refers to an antibody in which one antigen-binding site is present in the antibody, a "bivalent antibody" refers to an antibody in which two antigen-binding sites are present in the antibody, a "trivalent antibody" refers to an antibody in which three antigen-binding sites are present in the antibody, and a "multivalent antibody" refers to an antibody in which multiple (e.g., two or more) antigen-binding sites are present in the antibody.

[0104] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms are applicable to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, and to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0105] The term "antigen-binding domain" refers to a region of an antigen-binding molecule (e.g., an antibody) that specifically binds to an antigen, and the antigen-binding domain may be a portion of a ligand-binding domain capable of directly binding to an antigen, or a domain comprising an antibody variable region capable of directly binding to an antigen. The term "antigen-binding portion" refers to a portion of an antigen-binding molecule (e.g., an antibody) that comprises the antigen-binding domain.

[0106] The terms "fused" or "linked" refer to elements (e.g., two polypeptides) that are covalently linked, either directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.

[0107] The term "interaction domain" refers to a domain of a polypeptide that can promote interaction or association between two or more homo- or heteropolypeptides. For example, an interaction domain is a dimerization domain that promotes mutual association to form a dimer. A protein-protein interaction domain is a domain of a polypeptide that interacts with or associates with two or more proteins; for example, the Obscurin-Like Ig-Like 1 domain of an Obscurin-Like protein can interact with the Titin Ig-Like 152 domain of a Titin protein to form a complex. In some embodiments, the domain that interacts with the Titin-T chain is the Obscurin-O chain or the Obscurin-Like-O chain.

[0108] The term "dimeric polypeptide" refers to a dimeric polypeptide (also called a dimeric complex) formed by the association of two polypeptides with each other through covalent or non-covalent interactions. A homodimer is a dimer formed from two identical polypeptides, while a heterodimer (or heterodimer) is a dimer formed from two different polypeptides. Polypeptides can form a dimer by being linked, connected, or bonded in any suitable manner, such as by a linker, disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, hydrophobic-hydrophilic interaction, or a combination thereof. Illustratively, two molecules may form a dimer through a natural interchain bond or a non-natural interchain bond. In some embodiments of the present disclosure, the Titin-T chain and Obscurin-O chain or the Titin-T chain and Obscurin-Like-O chain of the dimeric polypeptide can form a dimer through a natural interchain bond. As is well known to those skilled in the art, when two domains associate with each other to form a dimer, the contact interface residues between the first and second domains within a distance of 6 angstroms (particularly, residues within 4.5 angstroms) play a crucial role in maintaining the association of the two domains (Yan, Changhui et al. "Characterization of protein-protein interfaces." The protein journal, vol. 27,1 (2008): 59-70).In the present disclosure, the dimerization complexes formed by the association of the Titin-T chain with the Obscurin-O chain, and the Titin-T chain with the Obscurin-Like-O chain (see Figures 1A and 1B) were analyzed using a Molecular Operating Environment (MOE) system. As a result, it was found that one or more residues at positions 7 to 15, 19 to 24, 26, 55, 59, and 60 in the Titin-T chain interact with one or more residues at positions 3 to 6, 9, 41, 73, 75, and 80 to 90 in the Obscurin-O chain, resulting in the formation of Titin-Like-O chains. One or more residues at positions 1, 7-10, 13-16, 19-26, 59-60, and 96 of the n-T chain interact with one or more residues at positions 4-5, 10, 12-13, 74, 76, 78, and 82-91 of the Obscurin-Like-O chain (see Figures 16A and 16B for binding sites), and the contact interface residue spacing between these sites in the dimerized complex is within 4.5 angstroms, which plays an important role in maintaining the association of the two domains. In some embodiments of the present disclosure, the Titin-T chain and the Obscurin-O chain or the Titin-T chain and the Obscurin-Like-O chain form a dimer via a non-natural inter-chain bond, wherein the dimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-natural inter-chain bonds. By mutating several amino acids in the Titin-T chain, Obscurin-O chain, or Obscurin-Like-O chain, the disulfide bond between the Titin-T chain and the Obscurin-O chain or the Titin-T chain and the Obscurin-Like-O chain can be made more stable, promoting the formation of a stable dimer between them.In some embodiments of the present disclosure, the interchain bonds are further stabilized by mutating one or more amino acid residues selected from positions 8, 20, 22, 25, 26, and 39 in the Titin-T chain and / or positions 3, 9, 25, 66, 76, 88, and 93 in the Obscurin-O chain, or by mutating one or more amino acid residues selected from positions 8, 20, 22, 25, 26, and 39 in the Titin-T chain and / or positions 6, 26, 74, 77, 84, and 86 in the Obscurin-Like-O chain.

[0109] In the present disclosure, "mismatching" refers to the formation of undesired dimer or multimer pairing due to interaction or association between two or more homo- or heteropolypeptides. "Less likely to cause mismatching" means, for example, that when polypeptides A1, B1, and B2 are co-expressed, production of the A1-B1 dimer is desirable, but production of the A1-B2 dimer is undesirable. If the expression level of the finally produced A1-B1 dimer is greater than that of the A1-B2 dimer, it is considered that A1 preferentially pairs with B1, i.e., A1 is less likely to mismatch with B2. In some embodiments of the present disclosure, a bispecific antibody comprising a Titin-T chain and an Obscurin-O chain or a Titin-T chain and an Obscurin-Like-O chain of a dimerizing polypeptide according to the present disclosure exhibits less mismatching between VH1 and VL2 and / or less mismatching between VL1 and VH2, and VH1 preferentially pairs with VL1 and VH2 preferentially pairs with VL2.

[0110] A "disulfide bond" is a covalent bond formed between sulfur atoms in the structure RSS-R'. The amino acid cysteine ​​contains a thiol group and may form a disulfide bond with a second thiol group, for example, with the thiol group of another cysteine ​​residue. Disulfide bonds may be formed between the thiol groups of two cysteine ​​residues located on two respective polypeptide chains, thereby forming interchain crosslinks or bonds.

[0111] Electrostatic interactions are non-covalent interactions that play an important role in protein folding, stability, flexibility, and function, and include ionic interactions, hydrogen bonds, and halogen bonds. Electrostatic interactions may form in a polypeptide, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp in a first chain and Arg, Val, or Thr in a second chain.

[0112] Salt bridges are short-range electrostatic interactions, primarily between the carboxylate anion of Asp or Glu and the ammonium cation of Lys or the guanidino group of Arg, which are pairs of oppositely charged residues in close spatial proximity in the native protein structure. Charged and polar residues at the hydrophobic interface can be hot spots for binding. Here, residues with ionizable side chains, such as His, Tyr, and Ser, can also participate in salt bridge formation.

[0113] Hydrophilic interactions are interactions in which molecules with polar groups have a strong affinity for water and can form temporary bonds with water through hydrogen bonds. Hydrophobic interactions are non-covalent interactions between non-polar molecules. These non-polar molecules (e.g., some neutral amino acid residues, also called hydrophobic residues) tend to avoid water and aggregate with each other in an aqueous environment. For example, hydrophobic interactions can form between one or more Val, Tyr, and Ala in a first chain and one or more Val, Leu, and Trp in a second chain, or between His and Ala in a first chain and Thr and Phe in a second chain (see Brinkmann et al., 2017).

[0114] The term "hydrogen bond" refers to the electrostatic attraction between two polar groups when a hydrogen atom is covalently bonded to a highly electronegative atom, such as nitrogen, oxygen, or fluorine. Hydrogen bonds can be formed between the backbone oxygen (e.g., a chalcogenide group) and the amide hydrogen (nitrogen group) of two polypeptide residues. For example, a hydrogen bond can be formed between the nitrogen group of Asn and the oxygen group of His, or between the oxygen group of Asn and the nitrogen group of Lys. Hydrogen bonds are stronger than van der Waals interactions but weaker than covalent or ionic bonds, and are crucial for maintaining secondary and tertiary structure. For example, an α-helix is ​​formed when the spacing between amino acid residues occurs regularly between positions i and i+4. A creased, twisted sheet formed by a peptide fragment 3–10 amino acids in length when two peptides are connected by at least two or three backbone hydrogen bonds is a β-pleated sheet.

[0115] A "non-natural interchain bond" is an interchain bond not found in a wild-type polypeptide polymer. For example, a non-natural interchain bond can be formed between a mutated amino acid residue in one polypeptide and a wild-type or mutated amino acid residue in another polypeptide. In one embodiment, at least one non-natural interchain bond is a "disulfide bond" formed after the amino acid is mutated.

[0116] The term "contact interface" refers to a specific region in a polypeptide that contacts or interacts with one another. A contact interface includes one or more amino acid residues, and when an interaction occurs, the amino acid residues at the contact interface in a polypeptide can interact with corresponding amino acid residues that contact it. The amino acid residues at the contact interface may be arranged contiguously or discontinuously. For example, if the interface is three-dimensional, the amino acid residues within the interface may be spaced apart at different positions in the linear sequence.

[0117] The term "linker" or "joint" refers to a linking unit that connects two polypeptide fragments. Linkers usually have a certain degree of flexibility, and the use of a joint does not impair the original function of the protein domain. Herein, linkers appearing in the same structure may be the same or different. A linker may be a peptide linker, and comprises one or more amino acids, typically about 1 to 30, 2 to 24, or 3 to 15 amino acids. Linkers as applied herein may be the same or different. In some embodiments, the linker is a (G x S) y In some embodiments, the linker is selected from the group consisting of a linker, where x is an integer selected from 1 to 5, and y is an integer selected from 0 to 6. When y is 0, the linker is a bond, which means that the two polypeptide chains are directly linked by the bond. x S) yIn the linker, x is an integer between 1 and 5 (e.g., x is 4) and y is an integer selected from 1 and 6 (e.g., 1, 2, 3, 4, 5, or 6), e.g., the linker is a "GGGGS" (SEQ ID NO: 175) or a "GGGGSGGGGS" (SEQ ID NO: 176) polypeptide. In some other embodiments, the linker is a C-terminal truncated sequence of the heavy chain constant region CH1 (i.e., a peptide sequence formed by truncating the C-terminal portion of CH1 and retaining the N-terminal portion of CH1; for example, the C-terminal truncated sequence of CH1 is a polypeptide formed from the 1st to 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th amino acid residues at the N-terminus of CH1, for example, the polypeptide "ASTKG" (SEQ ID NO: 173) consisting of the 1st to 5th residues), or the linker is a C-terminal truncated sequence of the light chain constant region CL (i.e., a peptide sequence formed by truncating the C-terminal portion of CL). and a peptide sequence formed by retaining the N-terminal portion of CL, for example, a C-terminally truncated sequence of CL is a polypeptide formed from the 1st to 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th amino acid residues of the N-terminus of CL, for example, the 1st to 5th residues "RTVAS" (SEQ ID NO: 174). In some embodiments, linker 1 is an "ASTKG" polypeptide and linker 2 is an "RTVAS" polypeptide, or both linker 1 and linker 2 are "GGGGS" polypeptide or "GGGGSGGGGS" polypeptide.

[0118] "Tm" is the melting denaturation temperature (intrinsic fluorescence). When a protein is denatured (by heating or the action of a denaturing agent), the tertiary structure opens and the microenvironment of aromatic amino acids is altered, resulting in a change in the emission fluorescence spectrum. In this disclosure, Tm1 refers to the temperature at which the fluorescence is changed to half of its maximum value.

[0119] "Tonset" is the denaturation onset temperature. It is the temperature at which the protein begins to denature, i.e., the temperature at which the fluorescence value begins to change.

[0120] "Tagg" is the aggregation onset temperature. Aggregates were detected by static light scattering at two wavelengths, 266 nm and 473 nm, to monitor the temperature at which sample aggregation began. Tagg 266 indicates that the aggregation onset temperature was monitored at 266 nm.

[0121] "SEC purity (%)" or "SEC%" refers to the percentage of SEC monomer content. SEC% = Amonomer / Atotal × 100% (Amonomer is the peak area of ​​the main peak monomer in a sample, and Atotal is the sum of all peak areas). In the present disclosure, the SEC% of an antibody can be measured by SEC size exclusion chromatography (an analytical method that separates solutes based on the correlation between the pore size of gel pores and the coil dimensions of macromolecular sample molecules). SEC measurement equipment: for example, Agilent 1260, column: water, XBrige BEH200Å SEC (300 × 7.8 mm, 3.5 μm).

[0122] "NR-CE-SDS%" or "NR-CE-SDS purity (%)" refers to the percentage of purity determined by non-reducing capillary electrophoresis. NR-CE-SDS% = A Main Peak / A Total × 100% (A Main Peak is the peak area of ​​the main light chain peak + main heavy chain peak in a sample, and A Total is the sum of all peak areas). In the present disclosure, NR-CE-SDS% of an antibody can be measured by NR-CE capillary gel electrophoresis (an electrophoresis method in which a gel is transferred to a capillary as a support medium and a sample is separated by molecular weight at a constant voltage). An example of an instrument for measuring NR-CE-SDS is a Beckman model plus800.

[0123] The antibodies of the present disclosure may be antibodies of animal origin (e.g., antibodies derived from mice, birds, rabbits, camels, monkeys, etc.), chimeric antibodies, humanized antibodies, and fully humanized antibodies.

[0124] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from another, different source or species.

[0125] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while having relatively low immunogenicity in humans, which may be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (i.e., portions of the constant and variable region framework regions).

[0126] The terms "human antibody," "humanized antibody," "fully human antibody," and "fully human antibody" are used interchangeably and refer to antibodies in which the variable and constant regions are human sequences. The term covers antibodies derived from human genes but with altered sequences, e.g., to reduce potential immunogenicity, increase affinity, or remove cysteines or glycosylation sites that may cause undesired folding. The term also covers antibodies produced recombinantly in non-human cells (which may confer glycosylation not characteristic of human cells). The term also covers antibodies that have been bred in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0127] The term "affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0128] As used herein, the terms "kassoc" or "ka" refer to the association rate of a particular antibody-antigen interaction, and the terms "kdis" or "kd" refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be measured by methods known in the art. For example, surface plasmon resonance can be measured by a biosensing system such as a surface plasmon resonance system, or affinity in solution can be measured by solution equilibrium titration (SET).

[0129] The term "effector function" refers to a biological activity that can be attributed to an antibody Fc region (a native sequence Fc region or an amino acid sequence mutated Fc region) and varies depending on the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0130] The term "monoclonal antibody" refers to a population of essentially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules within the population are the same except for possible minor natural mutations. In contrast, polyclonal antibody preparations typically contain a plurality of different antibodies with different amino acid sequences in their variable domains, which are generally specific for different epitopes. "Monoclonal" describes the character of the antibody as being obtained from an essentially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

[0131] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antigen-binding protein (including, for example, an antibody), and that can be used in an animal to raise antibodies capable of binding to the antigen. An antigen may have one or more epitopes that can interact with different antigen-binding proteins (e.g., antibodies).

[0132] The term "epitope" refers to an area or region on an antigen capable of specific binding by an antibody or antigen-binding fragment thereof. An epitope may be formed by a contiguous string of amino acids (linear epitope) or may include non-contiguous amino acids (conformational epitope) that are spatially adjacent, for example, due to antigen folding (i.e., tertiary folding of the antigen due to the nature of the protein). Conformational and linear epitopes differ in that antibody binding to conformational epitopes is lost in the presence of denaturing solvents. An epitope may include at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed by conventional methods in the art, including, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0133] The terms "capable of specifically binding," "specifically binds," or "binding" refer to an antibody's ability to bind to an antigen or an epitope within that antigen with greater affinity than to other antigens or epitopes. Typically, an antibody binds to an antigen or its epitope with an equilibrium dissociation constant (KD) of about 1×10 M or less (e.g., about 1×10 M or less). In some embodiments, the KD of an antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). KD can be measured by known methods, for example, by BIACORE® surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (e.g., humans or monkeys such as cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp)) or common marmosets (Callithrix jacchus) (marmoset).

[0134] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a mechanism of cell death that relies on the interaction of antibody-coated target cells with lytic effector cells (e.g., natural killer cells (NK), monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. ADCC activity of antibodies provided herein can be assessed in vitro using antigen-expressing cells as target cells and NK cells as effector cells. Cytolysis is detected by a label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein) released from the lysed cells.

[0135] The term "antibody-dependent cellular phagocytosis" ("ADCP") refers to the mechanism by which antibody-coated target cells are removed by the internalization action of phagocytes (eg, macrophages or dendritic cells).

[0136] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism for inducing cell death in which the Fc effector domain of a target-binding antibody binds and activates complement component C1q, which further activates the complement cascade, resulting in the death of the target cell. Complement activation can also lead to the deposition of complement components on the target cell surface, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0137] The term "nucleic acid" may be used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term covers nucleic acids containing known nucleoside analogs or modified backbone residues or linkers, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleoside. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphate, chiral-methyl phosphate, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule separated from a component of its natural environment. Isolated nucleic acid includes nucleic acid molecules contained in cells, as defined below, which typically contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An isolated nucleic acid encoding a polypeptide or a fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or a fusion protein, including one or more nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules present in one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence implicitly covers its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly stated sequence. Specifically, as detailed below, degenerate codon substitutions can be obtained by substituting mixed-base and / or deoxyinosine residues in the third position of one or more selected (or all) codons to produce sequences.

[0138] The term "sequence identity" refers to the degree (percentage) of amino acids / nucleic acids in two sequences that are the same at equivalent positions when optimally aligned, introducing gaps as necessary to achieve the maximum percentage of sequence identity, and not counting any conservative substitutions as part of the sequence identity. To measure the percentage of sequence identity, alignment can be achieved using techniques known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to the measured alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0139] In the present disclosure, for example, "site relative to XX sequence" means that when the sequence to be determined is optimally aligned with XX sequence and the maximum percentage identity is achieved, the site at the position corresponding to XX sequence in the sequence to be determined becomes the relative site of the two sequences. For example, in the Titin-T chain, natural order numbering site 1 in the sequence of SEQ ID NO: 32 is natural order numbering site 6 relative to SEQ ID NO: 127, and for example, in the Obscurin-O chain, natural order numbering site 3 in the sequence of SEQ ID NO: 33 is natural order numbering site 8 relative to SEQ ID NO: 128.

[0140] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop that can be ligated to an additional DNA segment. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which another DNA segment can be ligated to the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell after introduction and thereby replicate along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming a host cell and containing nucleic acid sequences that direct and / or control (with the host cell) the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, RNA splicing, which affect the coding region operably linked thereto.

[0141] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the original transformed cell and its derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. As used herein, progeny include mutants that have the same function or biological activity as screened or selected for in the initially transformed cell. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse and hamster cells, including, but not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichiaopuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, and Pichia methanolica. methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium spp., Fusarium gramineum, Fusarium venenatum venenatum, Physcomitrella patens, and Neurospora crassa.Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa.

[0142] As used herein, the expressions "cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is further understood that not all progeny will have exactly the same DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity as the initially transformed cell for which they were screened are included.

[0143] "Optionally" or "optionally" means that the subsequently described event or circumstance may occur, but need not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0144] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding fragments described herein and other chemical components, such as physiologically / pharmaceutically acceptable vectors and excipients.

[0145] The term "pharmaceutically acceptable vector" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to a subject. Pharmaceutically acceptable vectors include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0146] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" and "subject" may be used interchangeably herein. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

[0147] "Administration" or "giving," as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

[0148] The term "sample" refers to fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids, such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected from bronchial washings, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as cell and organ media (including cell or organ conditioned media), lavage fluids, tissue biopsies, fine needle aspirates, surgically resected tissues, organ cultures, or cell cultures.

[0149] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical intervention that seeks to alter the natural course of the individual being treated, and can be performed prophylactically or in the clinical pathological process. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction / reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, amelioration or alleviation of the disease state, and regression or improvement of prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the formation of disease or slow the progression of disease.

[0150] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or their underlying causes, prevent the onset of symptoms and / or their underlying causes, and / or improve or ameliorate damage caused by or associated with a disease state (e.g., lung disease). In some embodiments, the effective amount is a therapeutically or prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a condition or symptom associated with the disease state, or to otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptoms associated with the disease. A "prophylactically effective amount" is an amount that, when administered to a subject, provides a desired prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state or a reduced likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete therapeutic or prophylactic effect does not necessarily occur with a single dose, but may occur after a series of doses have been administered. Thus, a therapeutically or prophylactically effective amount can be administered in one or multiple doses. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on various factors, such as, for example, the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0151] Dimerizing polypeptide according to the present disclosure and antigen-binding molecule comprising the dimerizing polypeptide In one aspect, the present disclosure designs various dimerization polypeptides, the dimerization polypeptides comprising a Titin-T chain and an Obscurin-O chain or an Obscurin-Like-O chain, which can be used to replace antibody CH1 / CL to improve mismatch between the heavy chain and light chain of a multispecific antibody (e.g., a bispecific antibody) without affecting antibody-antigen binding.

[0152] In some embodiments, the present disclosure provides a dimerizing polypeptide comprising a Titin-T chain and an Obscurin-O chain or an Obscurin-Like-O chain.

[0153] In some embodiments, the present disclosure provides a dimerizing polypeptide, wherein the Titin-T chain has one or more amino acid residue substitutions selected from positions 8, 20, 22, 25, 26, and 39, and / or the Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76, and 88, or the Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26, and 39, and / or the Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74, 77, 84, and 86, wherein the Titin-T chain mutation sites are naturally numbered relative to the sequence of SEQ ID NO: 32, the Obscurin-O chain mutation sites are naturally numbered relative to the sequence of SEQ ID NO: 33, and the Obscurin-Like-O chain mutation sites are naturally numbered relative to the sequence of SEQ ID NO: 34.

[0154] In some specific embodiments, the Titin-T chain has one or more amino acid residue substitutions selected from 8C, 20C, 22C, 25S, 26C, and 39T, and / or the Obscurin-O chain has one or more amino acid residue substitutions selected from 3C, 9C, 25S, 76S, and 88C, or the Titin-T chain has one or more amino acid residue substitutions selected from 8C, 20C, 22C, 25S, 26C, and 39T, and / or the Obscurin-Like-O chain has one or more amino acid residue mutations selected from 6E, 26S, 74C, 77S, 84C, and 86C.In some embodiments, the Titin-T chain and the Obscurin-O chain, or the Titin-T chain and the Obscurin-Like-O chain, have the following amino acid residue substitutions: the Titin-T chain has 25S, 39T, and 8C substitutions, and the Obscurin-O chain has 88C substitutions; the Titin-T chain has 25S, 39T, and 20C substitutions, and the Obscurin-O chain has 3C substitutions; and the Titin-T chain has 25S, 39T, and 20C substitutions. The Titin-T chain has 25S, 39T, and 26C substitutions, and the Obscurin-O chain has 9C substitutions; the Titin-T chain has 25S, 39T, and 8C substitutions, and the Obscurin-O chain has 25S, 76S, and 88C substitutions; the Titin-T chain has 25S, 39T, and 20C substitutions, and the Obscurin-O chain has 25S, 76S, and 3C substitutions; the Titin-T chain has 25S, 39T, and 26C substitutions, and the Obscurin-O chain has 25 The Titin-T chain has 25S, 76S, and 9C substitutions, the Titin-T chain has 25S, 39T, and 8C substitutions, and the Obscurin-Like-O chain has 6E and 74C substitutions, the Titin-T chain has 25S, 39T, and 20C substitutions, and the Obscurin-Like-O chain has 6E and 84C substitutions, the Titin-T chain has 25S, 39T, and 22C substitutions, and the Obscurin-Like-O chain has 6E and 86C substitutions, and the Titin-T the Titin-T chain has 25S, 39T, and 8C substitutions, and the Obscurin-Like-O chain has 6E, 26S, 77S, and 74C substitutions, the Titin-T chain has 25S, 39T, and 20C substitutions, and the Obscurin-Like-O chain has 6E, 26S, 77S, and 84C substitutions, or the Titin-T chain has 25S, 39T, and 22C substitutions, and the Obscurin-Like-O chain has 6E, 26S, 77S, and 86C substitutions. In some embodiments, the Titin-T chain has the site substitutions based on SEQ ID NO: 32 or 127, the Obscurin-O chain has the site substitutions based on SEQ ID NO: 33 or 128, and the Obscurin-Like-O chain has the site substitutions based on SEQ ID NO: 34.

[0155] In some embodiments, in the dimerized polypeptide, the Obscurin-O chain has one or more amino acid residue mutations selected from positions 7, 11, and 62. In some embodiments, the Obscurin-O chain has one or more amino acid residue substitutions selected from 7R or 7K, 62K or 62H, and 11L; in some embodiments, the Titin-T chain has a 25S, 39T, and 8C substitution, and the Obscurin-O chain has a 25S, 76S, 88C, 7K, and 62K substitution; the Titin-T chain has a 25S, 39T, and 8C substitution, and the Obscurin-O chain has a 25S, 76S, 88C, 7K, and 62H substitution; the Titin-T chain has a 25S, 39T, and 8C substitution, and the Obscurin-O chain has a 25S, 76S, 88C, 11L, and 62K substitution; or the Titin-T chain has a 25S, 39T, and 8C substitution, and the Obscurin-O chain has a 25S, 76S, 88C, 11L, and 62H substitution. In some embodiments, the Obscurin-O chain has the above-mentioned site substitution based on SEQ ID NO: 33 or 45. The Titin-T chain substitution site is a naturally numbered site relative to the sequence of SEQ ID NO: 32, and the Obscurin-O chain substitution site is a naturally numbered site relative to the sequence of SEQ ID NO: 33.

[0156] In some embodiments, in the dimerization polypeptide, the Titin-T chain has one or more amino acid mutations selected from positions 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84, and / or the Obscurin-O chain has one or more amino acid mutations selected from positions 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 89, 92, 94, and 97. The Titin-T chain mutation sites are naturally numbered relative to the sequence of SEQ ID NO: 35, and the Obscurin-O chain mutation sites are naturally numbered relative to the sequence of SEQ ID NO: 50. In some embodiments, the Titin-T chain has one or more amino acid substitutions selected from 3W, 11I, 13L, 22M, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 66S or 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D, and 84L, and / or the Obscurin-O chain has one or more amino acid substitutions selected from 2E, 11K, 12S, 13Y, 14T, 17E, 20L, 22M or 22S, 30D, 32P, 34E, 36T, 41K, 42L, 44I, 45T, 53L, 58V, 62E, 67Q or 67T, 69S, 89L, 92E, 94G, and 97G.In some embodiments, the Titin-T chain has 66S and 77S amino acid substitutions, and / or the Obscurin-O chain has 11K, 12S, 13Y, 14T, and 22S amino acid substitutions, the Titin-T chain has 66K, 70R, 79T, and 81R amino acid substitutions, and / or the Obscurin-O chain has 2E, 17E, 30D, 32P, 34E, 36T, 44I, 45T, 58V, 62E, 67Q, 69S, and 97G amino acid substitutions, and the Titin The in-T chain has 3W, 11I, 13L, 22M, and 82M amino acid substitutions, and / or the Obscurin-O chain has 20L, 22M, and 53L amino acid substitutions, the Titin-T chain has 11I, 66K, 79T, and 81R amino acid substitutions, and / or the Obscurin-O chain has 41K, 45T, 67Q, 69S, and 89L amino acid substitutions, and the Titin-T chain has 40S, 42K, 45S, 47E, 49G, 56S, 58E, 75V, 83D, and 84L amino acid substitutions. and / or the Obscurin-O chain has 42L, 45T, 67T, 69S, 92E, and 94G amino acid substitutions, and the Titin-T chain has 47E, 49G, 56S, 58E, and 75V amino acid substitutions; and / or the Obscurin-O chain has 42L, 45T, 67T, 69S, 92E, and 94G amino acid substitutions, and the Titin-T chain has .... The Titin-T chain has 45T, 67T, 69S, 92E, and 94G amino acid substitutions, or the Titin-T chain has 56S, 58E, 66S, and 77S amino acid substitutions, and / or the Obscurin-O chain has 12S, 13Y, 22S, 42L, 45T, 67Q, 69S, 92E, and 94G amino acid substitutions, wherein the Titin-T chain substitution sites are naturally numbered relative to the sequence of SEQ ID NO: 35, and the Obscurin-O chain substitution sites are naturally numbered relative to the sequence of SEQ ID NO: 50. In some embodiments, the Titin-T chain has the amino acid substitutions based on SEQ ID NO: 35, and the Obscurin-O chain has the amino acid substitutions based on SEQ ID NO: 50.

[0157] In some embodiments, the dimerized polypeptide described above, wherein the Titin-T chain further has amino acid residue mutations at one or more sites selected from positions 60 and 64, and / or the Obscurin-O chain has amino acid residue mutations at one or more sites selected from positions 13, 32, 48, 66, 82, and 93, the amino acid residue sites of the Titin-T chain are naturally numbered sites relative to the sequence of SEQ ID NO: 32, and the amino acid residue sites of the Obscurin-O chain are naturally numbered sites relative to the sequence of SEQ ID NO: 33. In some embodiments, the dimerized polypeptide has the Titin-T chain having one or more, for example, one or two, amino acid residue substitutions selected from 60S and 64T, and / or the Obscurin-O chain having one or more, for example, one, two, three, four, five, six or more, amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H and 93C, wherein the amino acid residue positions of the Titin-T chain are naturally numbered positions relative to the sequence of SEQ ID NO: 32, and the amino acid residue positions of the Obscurin-O chain are naturally numbered positions relative to the sequence of SEQ ID NO: 33. In some embodiments, in the dimerized polypeptide, the Titin-T chain has an amino acid residue substitution at positions 60S and 64T, and / or the Obscurin-O chain has any one amino acid residue substitution selected from a) to c), i.e., a) an amino acid residue substitution at positions 13S and 48V, b) an amino acid residue substitution at positions 13S, 32F, 48V, and 82H, and c) an amino acid residue substitution at positions 13S, 32F, 48V, 66C, 82H, and 93C, wherein the amino acid residue positions of the Titin-T chain are naturally numbered relative to the sequence of SEQ ID NO: 32, and the amino acid residue positions of the Obscurin-O chain are naturally numbered relative to the sequence of SEQ ID NO: 33.In some embodiments, in the dimerized polypeptide, the Titin-T chain has one or more amino acid residue substitutions selected from positions 60S and 64T in SEQ ID NO: 32, 68, or 127, and the Obscurin-O chain has one or more amino acid residue substitutions selected from positions 13S, 32F, 48V, 66C, 82H, and 93C in SEQ ID NO: 33, 80, or 128. The amino acid residue positions of the Titin-T chain are naturally numbered relative to the sequence of SEQ ID NO: 32, and the amino acid residue positions of the Obscurin-O chain are naturally numbered relative to the sequence of SEQ ID NO: 33.

[0158] In some embodiments, in the dimerized polypeptide, one or more residues selected from positions 7 to 15, 19 to 24, 26, 55, 59, and 60 in the Titin-T chain bind to one or more residues selected from positions 3 to 6, 9, 41, 73, 75, and 80 to 90 in the Obscurin-O chain to form a dimerized complex, or one or more residues selected from positions 1, 7 to 10, 13 to 16, 19 to 26, 59 to 60, and 96 in the Titin-T chain bind to one or more residues selected from positions 1, 7 to 10, 13 to 16, 19 to 26, 59 to 60, and 96 in the Titin-T chain to form a dimerized complex. or a plurality of residues bond with one or more residues selected from residues 4-5, 10, 12-13, 74, 76, 78, and 82-91 in the Obscurin-Like-O chain to form a dimeric complex, wherein the Titin-T chain residue site is a naturally numbered site relative to the sequence of SEQ ID NO: 32, the Obscurin-O chain residue site is a naturally numbered site relative to the sequence of SEQ ID NO: 33, and the Obscurin-Like-O chain residue site is a naturally numbered site relative to the sequence of SEQ ID NO: 34. In some embodiments, the Titin-T chain comprises amino acids 7-60 of SEQ ID NO: 32 or a variant thereof, and the Obscurin-O chain comprises amino acids 3-90 of SEQ ID NO: 33 or a variant thereof, or the Titin-T chain comprises amino acids 1-96 of SEQ ID NO: 32 or a variant thereof, and the Obscurin-Like-O chain comprises amino acids 4-91 of SEQ ID NO: 34 or a variant thereof.

[0159] In some embodiments, the dimerizing polypeptide comprises a Titin-T chain and an Obscurin-O chain, or a Titin-T chain and an Obscurin-Like-O chain, wherein i) the Titin-T chain is a variant of SEQ ID NO: 32, wherein the variant has amino acid residue substitutions at one or more positions selected from positions 60 and 64 relative to SEQ ID NO: 32, and / or ii) the Obscurin-O chain is a variant of SEQ ID NO: 33, wherein the variant has amino acid residue substitutions at one or more positions selected from positions 13, 32, 48, 66, 82, and 93 relative to SEQ ID NO: 33, and a) the variant a) if the variant does not have an amino acid residue substitution at position 13, 48, 66, 82 or 93, but has an amino acid residue substitution at position 32, the amino acid substitution at position 32 is not 32P; b) if the variant does not have an amino acid residue substitution at position 32, 48, 66, 82 or 93, but has an amino acid residue substitution at position 13, the amino acid substitution at position 13 is not 13Y; and c) if the variant does not have an amino acid residue substitution at position 48, 66, 82 or 93, but has amino acid residue substitutions at positions 13 and 32, the amino acid substitution at position 13 is not 13Y, and the amino acid substitution at position 32 is not 32P.

[0160] In some embodiments, the dimerizing polypeptide has the variant of SEQ ID NO: 32 having one or more amino acid residue substitutions selected from the group consisting of 60S and 64T compared to SEQ ID NO: 32, and / or the variant of SEQ ID NO: 33 having one or more amino acid residue substitutions selected from the group consisting of 13S, 32F, 48V, 66C, 82H, and 93C compared to SEQ ID NO: 33.

[0161] In some embodiments, the variant of SEQ ID NO: 32 has an amino acid residue substitution of 60S and 64T compared to SEQ ID NO: 32, and / or the variant of SEQ ID NO: 33 has an amino acid residue substitution of any one selected from a) to c) compared to SEQ ID NO: 32, i.e., a) 32F and 48V, b) 13S, 32F, 48V and 82H, and c) 13S, 32F, 48V, 66C, 82H and 93C.

[0162] In some embodiments, the dimerization polypeptide further comprises a variant of SEQ ID NO: 32 having an amino acid substitution at one or more positions selected from 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84 compared to SEQ ID NO: 32; in some embodiments, the variant of SEQ ID NO: 32 further comprises an amino acid substitution at one or more positions selected from 3W, 8C, 11I, 13L, 20C, 22M / 22L, 23M / 23L, 24M / 24L, 25M / 25L, 26M / 26L, 27M / 27L, 28M / 28L, 29M / 29L, 30M / 30L, 31M / 31L, 32M / 32L, 33M / 33L, 34M / 34L, 35M / 35L, 36M / 36L, 37M / 37L, 38M / 38L, 39M / 39L, 40M / 39L, 41M / 39L, 42M / 39L, 43M / 39L, 44M / 39L, 45M / 39L, 46M / 39L, 47M / 39L, 48M / 39L, 49M / 49L, 56M / 58L, 58M / 59L, 60M / 59L, 61M / 59L, 62M / 59L, 63M / 59L, 64M / 59L, 6 and 84L. In some embodiments, the variant of SEQ ID NO: 32 has one or more amino acid residue substitutions selected from the group consisting of C, 25S, 26C, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 66S / 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D, and 84L. In some embodiments, the variant of SEQ ID NO: 32 has one or more amino acid residue substitutions selected from the group consisting of: ... a) 8C, 25S, and 39T; b) 20C, 25S, and 39T; c) 25S, 26C, and 39T; d) 22C, 25S, and 39T; e) 8C, 25S, 39T, 66 S and 77S, f) 8C, 25S, 39T, 66K, 70R, 79T and 81R, g) 3W, 8C, 11I, 13L, 22M, 25S, 39T and 82M, h) 8C, 11I, 25S, 39T, 66K, 79T and 81R, i) 8C, 25S, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 75V, 83D and 84L, j) 8C, 25S, 39T, 47E, 49G, 56S, 58E and 75V, k) 8C, 25S, 39T, 56S, 58E and 75V, and l) 8C, 25S, 39T, 56S, 58E and 75V. The variant of SEQ ID NO: 32 further comprises any one amino acid residue substitution selected from 8E, 66S, and 77S. In some embodiments, the variant of SEQ ID NO: 32 has any one amino acid residue substitution selected from A) to C) compared to SEQ ID NO: 32, i.e., A) 8C, 11I, 25S, 39T, 60S, 64T, 66K, 79T, and 81R; B) 8C, 11I, 20C, 25S, 39T, 60S, 64T, 66K, 79T, and 81R; and C) 8C, 11I, 25S, 26C, 39T, 60S, 64T, 66K, 79T, and 81R.

[0163] In some embodiments, the dimerization polypeptide has a variant of SEQ ID NO: 33 that further has amino acid residue substitutions at one or more positions selected from positions 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 76, 88, 89, 92, 94, and 97 compared to SEQ ID NO: 33. In some embodiments, the variant of SEQ ID NO: 33 further comprises one or more amino acid residue substitutions selected from the group consisting of 2E, 3C, 7K / 7R, 9C, 11L, 12S, 13Y, 14T, 17E, 20L, 22M / 22S, 25S, 30D, 32P, 34E, 36T, 41K, 42L, 44I, 45T, 53L, 58V, 62E / 62K / 62H, 67Q / 67T, 69S, 76S, 88C, 89L, 92E, 94G, and 97G compared to SEQ ID NO: 33. In some embodiments, the variant of SEQ ID NO: 33 further comprises one of A) to R), i.e., A) 88C, B) 3C, C) 9C, D) 25S, 76S and 88C, E) 25S, 76S and 3C, F) 25S, 76S and 9C, G) 7K, 25S, 62K, 76S and 88C, H) 7K, 25S, 6 2H, 76S and 88C, I) 7R, 25S, 62K, 76S and 88C, J) 7R, 25S, 62H, 76S and 88C, K) 11L, 25S, 62K, 76S and 88C, L) 11L, 25S, 62H, 76S and 88C, M) 12S, 13Y, 14T, 22S, 25S, 62K, 76S and 88C, N) 2E , 11L, 17E, 25S, 30D, 32P, 34E, 36T, 44I, 45T, 58V, 62E, 67Q, 69S, 76S, 88C and 97G, O) 11L, 20L, 22M, 25S, 53L, 62K, 76S and 88C, P)11L, 25S, 41K, 45T, 62K, 67Q, 69S, 76S, 88 C and 89L, Q) 11L, 25S, 42L, 45T, 62K, 67T, 69S, 76S, 88C, 92E and 94G, and R) 11L, 12S, 13Y, 22S, 25S, 42L, 45T, 62K, 67Q, 69S, 76S, 88C, 92E and 94G.In some embodiments, the variants of SEQ ID NO: 33 include any of a) to j) of the following variants relative to SEQ ID NO: 33: a) 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 88C, and 89L; b) 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C, and 89L; c) 3C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C and 89L, d) 9C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C and 89L, e) 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, f) 3C, 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, g) 9C, 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, h) 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C, i) has any one amino acid residue substitution selected from 3C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C, and j) 9C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C.

[0164] In some embodiments, in the dimerized polypeptide, the Obscurin-Like-O chain is SEQ ID NO: 34 or a variant thereof, and the variant of SEQ ID NO: 34 has amino acid residue substitutions at one or more positions selected from positions 6, 26, 74, 77, 84, and 86 compared to SEQ ID NO: 34. In some embodiments, the variant of SEQ ID NO: 34 has one or more amino acid residue substitutions selected from the group consisting of 6E, 26S, 74C, 77S, 84C, and 86C compared to SEQ ID NO: 34. In some embodiments, the variant of SEQ ID NO: 34 has any one amino acid residue substitution selected from A) to F), i.e., A) 6E and 74C, B) 6E and 84C, C) 6E and 86C, D) 6E, 26S, 77S, and 74C, E) 6E, 26S, 77S, and 84C, and F) 6E, 26S, 77S, and 86C compared to SEQ ID NO: 34.

[0165] In some embodiments, in the dimerization polypeptide, the Titin-T chain is a variant of SEQ ID NO: 32, 68, or 127, wherein the variant of SEQ ID NO: 32 has one or more amino acid residue substitutions selected from 60S and 64T compared to SEQ ID NO: 32, the variant of SEQ ID NO: 68 has one or more amino acid residue substitutions selected from 60S and 64T compared to SEQ ID NO: 68, the variant of SEQ ID NO: 127 has one or more amino acid residue substitutions selected from 60S and 64T compared to SEQ ID NO: 127, and the Obscurin- The O chain is a variant of SEQ ID NO: 33, 80 or 128, wherein the variant of SEQ ID NO: 33 has one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H and 93C compared to SEQ ID NO: 33, the variant of SEQ ID NO: 80 has one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H and 93C compared to SEQ ID NO: 80, and the variant of SEQ ID NO: 128 has one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H and 93C compared to SEQ ID NO: 128.

[0166] In some embodiments, the antigen-binding molecule of any one of the above, comprising a first heavy chain, a first light chain, a second heavy chain and a second light chain, a. The first heavy chain is, from N-terminus to C-terminus, [VH1]-[Linker1]-[Titin-T chain]-[Fc1]; The first light chain is composed of, from the N-terminus to the C-terminus, [VL1]-[Linker 2]-[Obscurin-O chain]; The second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; the second light chain is, from N-terminus to C-terminus, [VL2]-[CL]; or b. The first heavy chain is, from N-terminus to C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain]-[Fc1]; The first light chain is composed of, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Titin-T chain], The second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; The second light chain is [VL2]-[CL] from the N-terminus to the C-terminus, The linker 1 and the linker 2 are the same or different. In some embodiments, the Fc1 and the Fc2 each independently have one or more amino acid substitutions that reduce homodimerization. In some embodiments, the linker 1 and the linker 2 each have one or more amino acid substitutions that reduce homodimerization. In some embodiments, the linker 1 and the linker 2 each have one or more amino acid substitutions that reduce homodimerization. x S) yA) linker, wherein x is an integer selected from 1 to 5 (e.g., 1, 2, 3, 4, or 5) and y is an integer selected from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6), (wherein, when y is 0, the linker is a bond); or B) linker 1 is a sequence in which the C-terminus of CH1 is truncated, and linker 2 is a sequence in which the C-terminus of CL is truncated, and in some embodiments, linker 1 and linker 2 are any one selected from A) to C), i.e., A) linker 1 has the sequence shown in SEQ ID NO: 173, and linker 2 has the sequence shown in SEQ ID NO: 174; B) linker 1 and linker 2 both have the sequences shown in SEQ ID NO: 175; and C) linker 1 and linker 2 both have the sequences shown in SEQ ID NO: 176. In some embodiments, the Fc1 has knobs configured using knobs-in-hole technology and the Fc2 has holes configured using knobs-in-hole technology, or the Fc1 has holes configured using knobs-in-hole technology and the Fc2 has knobs configured using knobs-in-hole technology. In some embodiments, the Fc1 has one or more amino acid substitutions at sites selected from 354, 356, 358, and 366, and the Fc2 has one or more amino acid substitutions at sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the Fc2 has one or more amino acid substitutions at sites selected from 354, 356, 358, and 366, and the Fc1 has one or more amino acid substitutions at sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the Fc1 has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc2 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc2 has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc1 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V.In some embodiments, the Fc1 comprises amino acid substitutions 354C, 356E, 358M, and 366W, and the Fc2 comprises amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc2 comprises amino acid substitutions 354C, 356E, 358M, and 366W, and the Fc1 comprises amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc1 is represented by SEQ ID NO: 177 and the Fc2 is represented by SEQ ID NO: 178, or the Fc2 is represented by SEQ ID NO: 177 and the Fc1 is represented by SEQ ID NO: 178.

[0167] In some embodiments, the antigen-binding molecule of any one of the above, comprising a first heavy chain, a first light chain, a second heavy chain and a second light chain, a. The first heavy chain is, from N-terminus to C-terminus, [VH1]-[Linker1]-[Titin-T chain]-[Fc1]; The first light chain is composed of, from the N-terminus to the C-terminus, [VL1]-[Linker 2]-[Obscurin-Like-O chain]; The second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; the second light chain is, from N-terminus to C-terminus, [VL2]-[CL]; or b. The first heavy chain is, from N-terminus to C-terminus, [VH1]-[Linker1]-[Obscurin-Like-O chain]-[Fc1]; The first light chain is composed of, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Titin-T chain], The second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; The second light chain is [VL2]-[CL] from the N-terminus to the C-terminus, The linker 1 and the linker 2 are the same or different. In some embodiments, the Fc1 and the Fc2 each independently have one or more amino acid substitutions that reduce homodimerization. In some embodiments, the linker 1 and the linker 2 each have one or more amino acid substitutions that reduce homodimerization. In some embodiments, the linker 1 and the linker 2 each have one or more amino acid substitutions that reduce homodimerization. x S)yA) linker, wherein x is an integer selected from 1 to 5 (e.g., 1, 2, 3, 4, or 5) and y is an integer selected from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6), (wherein, when y is 0, the linker is a bond); or B) linker 1 is a sequence in which the C-terminus of CH1 is truncated, and linker 2 is a sequence in which the C-terminus of CL is truncated, and in some embodiments, linker 1 and linker 2 are any one selected from A) to C), i.e., A) linker 1 has the sequence shown in SEQ ID NO: 173, and linker 2 has the sequence shown in SEQ ID NO: 174; B) linker 1 and linker 2 both have the sequences shown in SEQ ID NO: 175; and C) linker 1 and linker 2 both have the sequences shown in SEQ ID NO: 176. In some embodiments, the Fc1 has knobs configured using knobs-in-hole technology and the Fc2 has holes configured using knobs-in-hole technology, or the Fc1 has holes configured using knobs-in-hole technology and the Fc2 has knobs configured using knobs-in-hole technology. In some embodiments, the Fc1 has one or more amino acid substitutions at sites selected from 354, 356, 358, and 366, and the Fc2 has one or more amino acid substitutions at sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the Fc2 has one or more amino acid substitutions at sites selected from 354, 356, 358, and 366, and the Fc1 has one or more amino acid substitutions at sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the Fc1 has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc2 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc2 has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc1 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V.In some embodiments, the Fc1 comprises amino acid substitutions 354C, 356E, 358M, and 366W, and the Fc2 comprises amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc2 comprises amino acid substitutions 354C, 356E, 358M, and 366W, and the Fc1 comprises amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc1 is represented by SEQ ID NO: 177 and the Fc2 is represented by SEQ ID NO: 178, or the Fc2 is represented by SEQ ID NO: 177 and the Fc1 is represented by SEQ ID NO: 178.

[0168] Fc region modifications In one aspect, the Fc region of the present disclosure comprises one or more amino acid substitutions that reduce binding to its Fc receptor, e.g., binding to its Fcγ receptor, and reduce or eliminate effector function. Native IgG Fc regions are specifically IgG1 Fc regions or IgG4 Fc regions, potentially allowing fusion proteins of the present disclosure to target cells expressing the Fc receptor rather than cells expressing the antigen. In some embodiments, modified Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and / or reduced effector function. In some embodiments, the modified Fc region exhibits 50%, 80%, 90%, or 95% or more reduced binding affinity to an Fc receptor compared to a native Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIB, or FcγRIIIa. In some embodiments, the altered Fc region also has reduced binding affinity to complement, such as C1q, compared to the native Fc region. In some embodiments, the altered Fc region does not have reduced binding affinity to the neonatal Fc receptor (FcRn) compared to the native Fc region. In some examples, the altered Fc region has reduced effector functions, which may include, but are not limited to, one or more of: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen-presenting cell uptake, reduced NK cell binding, reduced macrophage cell binding, reduced monocyte binding, reduced polymorphonuclear cell binding, reduced direct signaling-induced cell apoptosis, reduced dendritic cell maturation, or reduced T cell triggering. For the IgG1 Fc region, substitution of amino acid residues at positions such as 238, 265, 269, 270, 297, 327, and 329 can reduce effector function.In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For IgG4 Fc regions, substitution of amino acid residues at positions such as 228 can reduce effector function.

[0169] Antigen-binding molecules may contain different antigen-binding domains fused to two subunits in the Fc region, potentially resulting in undesired homodimerization. To improve yield and purity, it is advantageous to introduce modifications that promote heterodimerization into the Fc region of the antigen-binding molecules of the present disclosure. In some embodiments, the Fc region of the present disclosure is modified using the knob-into-hole (KIH) technique, which involves introducing a knob structure at the interface of the first subunit and a hole structure at the interface of the second subunit, or introducing a knob structure at the interface of the first subunit and a hole structure at the interface of the second subunit. This positions the knob structure at the hole structure, promoting heterodimer formation and inhibiting homodimer formation. The knob structure is constructed by replacing a small amino acid side chain from the interface of the first subunit with a relatively large side chain (e.g., tyrosine or tryptophan). Alternatively, hole structures may be constructed at the interface of the second subunit by substituting relatively small amino acid side chains (e.g., alanine or threonine) for larger amino acid side chains. Knob and hole structures are prepared by altering the nucleic acid encoding the polypeptide; illustratively, selectable amino acid substitutions are shown in Table 2 below:

[0170] [Table 2]

[0171] In addition to the knobs-in-holes technique, other techniques for modifying the CH3 domain of the heavy chain of a multispecific antibody so as to achieve heterodimerization are also known in the art, e.g., WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO 007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954 and WO 013 / 096291.

[0172] The C-terminus of the Fc region may be a complete C-terminus ending with the amino acid residue PGK, or may be a truncated C-terminus, e.g., one or two C-terminal amino acid residues have been removed. In one preferred embodiment, the C-terminus of the heavy chain is a truncated C-terminus ending with PG. Thus, in some embodiments, a composition of complete antibodies may include antibodies in which any of the K447 and / or G446+K447 residues have been removed. In some embodiments, a composition of complete antibodies may include antibodies in which the K447 and / or G446+K447 residues have not been removed. In some embodiments, a composition of complete antibodies includes a mixture of antibodies with and without the K447 and / or G446+K447 residues.

[0173] Recombinant methods Antigen-binding molecules or polypeptides can be produced by recombinant methods. For these methods, one or more isolated nucleic acids encoding the polypeptide or antigen-binding molecule are provided.

[0174] In one embodiment, the present disclosure provides isolated nucleic acids encoding the above-described polypeptides or antigen-binding molecules. Such nucleic acids can each independently encode any one of the above-described polypeptide chains. In another aspect, the present disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In another aspect, the present disclosure provides host cells comprising such nucleic acids. In one embodiment, a method for preparing a polypeptide or antigen-binding molecule is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the polypeptide or fusion protein under conditions suitable for expression, as described above, and optionally recovering the protein from the host cell (or host cell culture medium).

[0175] To produce a recombinant polypeptide or antigen-binding molecule, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using routine procedures, or produced by recombinant methods, or obtained by chemical synthesis.

[0176] Suitable host cells for cloning or expressing vectors encoding the polypeptides or antigen-binding proteins include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. After expression, they can be isolated from bacterial cell paste in a soluble fraction and further purified.

[0177] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeast are also suitable cloning or expression hosts for vectors encoding antigen-binding molecules, including fungal and yeast strains. Suitable host cells for expressing antigen-binding molecules may be derived from multicellular organisms (invertebrates and vertebrates), and examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have already been identified and can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures may also be used as hosts, as described in, for example, US Pat. No. 5,959,177, US Pat. No. 6,040,498, US Pat. No. 6,420,548, US Pat. No. 7,125,978, and US Pat. No. 6,417,429, or vertebrate cells, such as mammalian cell lines suitable for growth in suspension, may also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7), human embryonic kidney (293 or 293T) cells, baby hamster kidney (BHK) cells, mouse Sertoli (TM4) cells, monkey kidney (CV1) cells, African green monkey kidney (VERO-76) cells, human cervical carcinoma (HELA) cells, canine kidney (MDCK) cells, buffalo rat hepatocytes (BRL3A), human lung (W138) cells, human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, MRC 5 cells, and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of several mammalian host cell lines suitable for the production of antibodies, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0178] measurement The physical / chemical characteristics and / or biological activity of the polypeptides or antigen-binding molecules provided herein can be identified, screened, or expressed by various assay methods known in the art. In one embodiment, the activity of the polypeptides or antigen-binding molecules of the present disclosure is tested by known methods, such as ELISA and Western blotting.

[0179] Treatment methods and routes of administration Any antigen-binding molecule provided herein can be applied to a method of treatment. In a further aspect, the present disclosure provides a use of an antigen-binding molecule in the manufacture or preparation of a medicament. In some embodiments, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). The "subject" according to any of the above embodiments may be a human.

[0180] In a further aspect, pharmaceutical compositions of the antigen-binding molecules described above are provided, for example, for use in any of the pharmaceutical applications or therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises any of the antigen-binding molecules provided herein and a pharmaceutically acceptable vector. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0181] The antigen-binding molecules of the present disclosure can be used alone or in combination with other reagents for treatment. For example, antibodies of the present disclosure can be administered together with at least one additional therapeutic agent.

[0182] The antigen-binding molecules of the present disclosure (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, when localized treatment is required, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration, and pulse infusion.

[0183] The antigen-binding molecules of the present disclosure are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the reagent, the method of administration, the administration schedule, and other factors known to medical professionals. The polypeptide or fusion protein may or may not be formulated with one or more other reagents currently used to prevent or treat the condition. The effective amount of such other reagents will depend on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These will generally be used in the same dosages and by the same routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at different dosages and via any route determined empirically / clinically appropriate.

[0184] The appropriate dose of an antigen-binding molecule of the present disclosure (used alone or in combination with one or more additional therapeutic agents) for preventing or treating a disease will depend on the type of disease being treated, the type of therapeutic molecule, the severity and course of the disease, whether the administration is prophylactic or therapeutic, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is administered to the patient at one time or over a series of treatments, as appropriate.

[0185] product Another aspect of the present disclosure provides an article of manufacture containing materials applicable to the treatment, prevention, and / or diagnosis of the above-described medical conditions. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition that effectively treats, prevents, and / or diagnoses a disease, alone or in combination with another composition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a plug pierceable by a hypodermic injection needle). At least one active agent in the composition is an antigen-binding molecule of the present disclosure. The label or package insert indicates use of the composition to treat a selected medical condition. The article of manufacture may also include (a) a first container containing a composition comprising an antigen-binding molecule of the present disclosure and (b) a second container containing a composition comprising another cytotoxic or other therapeutic agent. The product in this embodiment of the present disclosure may further include a package insert indicating that the composition is applicable to the treatment of a particular medical condition. Optionally, or in addition, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer. From a business and user perspective, it may further include other necessary materials, including other buffers, diluents, filters, needles, and syringes.

[0186] Although the antibodies used in the examples target specific antigens, those skilled in the art can understand the following under the teachings of the present disclosure: the realization of the technical effect is not dependent on a specific CDR sequence or a specific antigen sequence, but rather on improving the mismatch between the heavy chain and the light chain by replacing CH1 / CL with Titin T chain / Obscurin-O chain or Titin T chain / Obscurin-Like-O chain. [Example]

[0187] Examples and Test Examples The present disclosure will be further explained below with reference to examples and test examples, but these examples and test examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples and test examples of the present disclosure generally follow standard conditions, such as those in the Reisenko Antibody Technology Experiment Manual and Molecular Cloning Manual, or conditions recommended by the manufacturers of raw materials or products. Reagents for which specific sources are not specified are standard commercially available reagents.

[0188] Example 1: Method for preparing antibodies or polypeptide proteins Primer PCR is designed to assemble gene fragments (e.g., antibody VH / VK gene fragments), which are then homologously recombined with an expression vector (e.g., pHr (a gene with a signal peptide and constant region (e.g., CH1-Fc / CL) fragment)) to construct an expression vector (e.g., VH-CH1-Fc-pHr / VK-CL-pHr). The constructed expression vector is then introduced into prokaryotes or eukaryotes for expression, and the product is finally purified to obtain the desired antibody or polypeptide protein. Non-limiting examples of antibody constant regions include those selected from human kappa and lambda light chain constant regions and heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4. Non-limiting examples further include optimizing the human antibody constant region, for example, by mutations at sites such as L234A / L235A or L234F / L235E in the heavy chain constant region. Exemplary antibody light / heavy chain constant region sequences are as follows: > IgG1 heavy chain constant region (abbreviated as hIgG1) [ka] Note: In the sequences, the single underlined part is CH1, the dotted part is CH2, and the italicized part is CH3.

[0189] >knob-IgG1 heavy chain constant region (abbreviated as knob -IgG1): [ka] Note: In the sequences, the single underlined part is CH1, the dotted part is CH2, and the italicized part is CH3.

[0190] >hole-IgG1 heavy chain constant region (abbreviated as hole-IgG1): [ka] Note: In the sequences, the single underlined part is CH1, the dotted part is CH2, and the italicized part is CH3.

[0191] >kappa light chain constant region (abbreviated as kappa or hκ) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).

[0192] Lambda light chain constant region (abbreviated as lambda or hλ) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 5).

[0193] >hole-Fc partial amino acid sequence of IgG1 heavy chain constant region: [ka] >Fc partial amino acid sequence of knob-IgG1 heavy chain constant region: [ka] >Partial amino acid sequence of CH1 of IgG1 heavy chain constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 179); The amino acid sequences of the light and heavy chain variable regions of antibodies against antigens such as B7H3 and CD3 according to the examples or test examples of the present disclosure are as follows: >F0 antibody VH amino acid sequence: QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVAVISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSARLYASFDYWGQGALVTVSS (SEQ ID NO: 6); >F0 antibody VL amino acid sequence DTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKLTVL (SEQ ID NO: 7); >N0 antibody VH amino acid sequence DVQLVQSGGGLVQPGGSLRLSCAGSGFTSSLSWMSWLRQAPGKGLEWVANINQDGSEKNYVDSVKGRFTISRDNAQNSLYLQMSSLRAEDTAVYYCARGLWTFDSWGQGTLLTVSP (SEQ ID NO: 8); >N0 antibody VL amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQNINNRFLAWYQQRPGQAPRLLIYAASSRATGVPDRFSGSGSGTDFTLTISRLEAEDFAMYHCQQYGSFPRTFGQGTMVDIK (SEQ ID NO: 9); >S0 antibody VH amino acid sequence EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS (SEQ ID NO: 10); >S0 antibody VL amino acid sequence DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK (SEQ ID NO: 11); >V0 antibody VH amino acid sequence QVQLQQSAAELARPGASVKMSCKASGYSFTSYTIHWVKQRPGQGLEWIGYINPNSRNTDYNQKFKDETTLTADRSSSTAYMQLISLTSEDSAVYYCARYSGSTPYWYFDVWGAGTTVTVSS (SEQ ID NO: 12); >V0 antibody VL amino acid sequence QIVLTQSPAILSASPGEKVTMTCRASSSVSYMNWYQQKPGSSPKPWIYATSNLASGVPARFSVSVSGTSHSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 13); >J0 antibody VH amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYWMHWVRQAPGKGLEWVGHIKSKTDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAREIYYYAFDSWGQGTLVTVSS (SEQ ID NO: 14); >J0 antibody VL amino acid sequence SYELTQPLSVSVALGQTARITCSGDNIGSKYVHWYQQKPGQAPVLVIYGDNERPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQAADWVDFYVFGGGTKLTVL (SEQ ID NO: 15); >H0 antibody VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVTSISYEGDITYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCASQTLRESFDYWGQGTLVTVSS (SEQ ID NO: 16); >H0 antibody VL amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQDIANYLSWYQQKPGKSPKLLIYGTSNLEVGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQDKEFPRTFGGGTKVEIK (SEQ ID NO: 17); >R0 antibody VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGLSLTSNSVNWIRQAPGKGLEWVGLIWSNGDTDYNSAIKSRFTISRDTSKSTVYLQMNSLRAEDTAVYYCAREYYGYFDYWGQGTLVTVSS (SEQ ID NO: 18); >R0 antibody VL amino acid sequence DIQMTQSPSSLSASVGDRVTITCLASEGISSYLAWYQQKPGKAPKLLIYGANSLQTGVPSRFSGSGSATDYTLTISSLQPEDFATYYCQQSYKFPNTFGQGTKVEVK (SEQ ID NO: 19); >Bmab (abbreviated as B0) antibody VH amino acid sequence QVQLQQSGAEVKKPGSSVRVSCKASGGTFNNNAINWVRQAPGQGLEWMGGIIPMFGTAKYSQNFQGRVAITADESTGTASMELSSLRSEDTAVYYCARSRDLLLFPHHALSPWGRGTMVTVSS (SEQ ID NO: 20); >Bmab (abbreviated as B0) antibody VL amino acid sequence SSELTQDPAVSVALGQTVRVTCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCSSRDSSGNHWVFGGGTELTVL (SEQ ID NO: 21); >Umab (abbreviated as U0) antibody VH amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIMSPVDSDIRYSPSFQGQVTMSVDKSITTAYLQWNSLKASDTAMYYCARRRPGQGYFDFWGQGTLVTVSS (SEQ ID NO: 22); >Umab (abbreviated as U0) antibody VL amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPYTFGQGTKLEIK (SEQ ID NO: 23); >Dmab (abbreviated as D0) antibody VH amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPGTTVIMSWFDPWGQGTLVTVSS (SEQ ID NO: 24); >Dmab (abbreviated as D0) antibody VL amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVRGRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGSSPRTFGQGTKVEIK (SEQ ID NO: 25); >Tmab (abbreviated as I0) antibody VH amino acid sequence QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 26); >Tmab (abbreviated as I0) antibody VL amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIK (SEQ ID NO: 27); >C0 antibody VH amino acid sequence EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYHMNWVRQAPGQRLEWMGDINPDIGGTSYNQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARWDFDSFANWGQGTLVTVSS (SEQ ID NO: 28); >C0 antibody VL amino acid sequence DIVMTQSPDSLAVSLGERATINCRASESVSIIGTNLIHWYQQKPGQPPKLLIYHASNLETGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCLQSRKIPYTFGGGTKVEIK (SEQ ID NO: 29); >A0 antibody VH amino acid sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSS (SEQ ID NO: 105); >A0 antibody VL amino acid sequence QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL (SEQ ID NO: 106); The heavy chain constant region sequences of the above-mentioned antibodies F0, N0, S0, V0, J0, H0, R0, B0, U0, D0, I0, C0, and A0 antibodies are IgG1 heavy chain constant regions (sequence number 1), the light chain constant region sequences of the N0, S0, H0, R0, U0, I0, and C0 antibodies are kappa light chain constant regions (sequence number 4), and the light chain constant region sequences of the F0, V0, J0, B0, D0, and A0 antibodies are lambda light chain constant regions (sequence number 5).

[0194] The sequence of the hB7H3 antigen protein is as follows: LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSL QVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPT GAVEVQVPEDPVVALVGTDATLRCSSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAV SLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMT DYKDDDDKHHHHHH (SEQ ID NO: 111); The hCD3 antigen protein is a heterodimer consisting of the δ and ε subunits of the hCD3 antigen, where the δ subunit sequence of the hCD3 antigen is as follows: FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVASAENAQCEKELQALEKENAQLEWELQALEKELAQDYKDDDDK (SEQ ID NO: 112); The sequence of the epsilon subunit of the hCD3 antigen is as follows: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDAKNAQCKKKLQALKKKNAQLKWKLQALKKKLAQHHHHHH (SEQ ID NO: 113).

[0195] Example 2: Replacement of antibody CH1 / CL domains The Ig-like domains, which have natural intermolecular interactions in the natural Titin / Obscurin complex, Titin / Obscurin-Like O complex, and IL6Ra / IL6Rb complex, were used to replace the CH1 / CL domains of antibodies, yielding antibodies with modified CH1 / CL domains.

[0196] Here, in the IL6Ra / IL6Rb complex, the Ig-like domain in the IL6Ra protein for replacing antibody CH1 or CL is the IL6Ra.0 chain, and the Ig-like domain in the IL6Rb protein for replacing antibody CL or CH1 is the IL6Rb.0 chain; in the Titin / Obscurin complex, the Ig-like domain (Titin Ig-Like 152 domain) in the Titin protein for replacing antibody CH1 or CL is the T.0 chain, and the Ig-like domain (Obscurin Ig-Like-1 domain) in the Obscurin protein for replacing antibody CL or CH1 is the O.0 chain; in the Titin / Obscurin-Like O complex, the Ig-like domain (Obscurin-Like-Ig-Like-1 domain) in the Obscurin-like protein for replacing antibody CH1 or CL is the O.0 chain, and the Ig-like domain (Titin Ig-Like 152 domain) is the T.0 chain. The sequence of the IL6Ra.0 chain is as follows: GILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPW (SEQ ID NO: 30); The sequence of the IL6Rb.0 chain is as follows: FDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGIT (SEQ ID NO: 31); The sequence of the T.0 strand is as follows: GIPPKIEALPSDISIDEGKVLTVACAFTGEPTPEVTWSCGGRKIHSQEQGRFHIENTDDLTTLIIMDVQKQDGGLYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 32); The sequence of the O.0 strand is: SGAPRFLTRPKAFVVSVGKDATLSCQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVCRARNAIGEAFAAVGLQVDAEA (SEQ ID NO: 33); The sequence of the OL.0 strand is: QGSPPCFLRFPRPVRVVSGAEAELKCVVLGEPPPVVVWEKGGQQLAASERLSFPADGAEHGLLLTAALPTDAGVYVCRARNAAGEAYAAAAVTVLEPP (SEQ ID NO: 34); Several types of antibodies with CH1 / CL replacement were constructed, and the antibody purity and binding activity to their corresponding antigens were detected (see Test Examples 2 and 4 of the present disclosure for detection methods). The experimental results are shown in Table 3 below. The experimental results show that antibodies that bind to different antigens, such as F0, V0, S0, N0, J0, H0, R0, and C0, retain good binding activity and high antibody purity even when their CH1 / CL are simultaneously replaced with T.0 chain / O.0 chain or T.0 chain / O.0 chain.

[0197] [Table 3-1] [Table 3-2] Note: For example, F1 represents an antibody obtained by replacing the heavy chain CH1 of the F0 antibody with the IL6Rb.0 chain (SEQ ID NO: 31) and the light chain CL with the IL6Ra.0 chain (SEQ ID NO: 30), while maintaining the other portions the same as F0. The others are inferred from this. In the table, "-" indicates not detected, and SEC is the result of detecting antibody purity by size exclusion chromatography (NA indicates not detected due to too low expression level).

[0198] Example 3: Optimized design of CH1 / CL domain-modified antibodies First, individual amino acid residues in the Titin Ig-Like 152 domain (T.0 chain), Obscurin Ig-Like 1 domain (O.0 chain), and Obscurin-Like Ig-Like 1 domain (OL.0 chain) were mutated. For example, first, amino acid residues in the T.0, O.0, and OL.0 chains were mutated to increase interchain disulfide bonds, and then other individual amino acids in the domains were mutated. For example, amino acid mutations were performed at positions 7, 62, and 11 of the Obscurin Ig-Like 1 domain. The five amino acids "KAGIR (SEQ ID NO: 180)" adjacent to the N-terminus of the Titin Ig-Like 152 domain in five wild-type Titin proteins were added to the N-terminus of the Titin Ig-Like 152 domain. The five amino acids "DQPQF (SEQ ID NO: 181)" adjacent to the N-terminus of the Obscurin Ig-Like 1 domain in five wild-type Obscurin proteins were added to the N-terminus of the Obscurin Ig-Like 1 domain. Specific domain optimization designs are shown in Tables 4-1 to 4-3.

[0199] [Table 4-1] Note: In the table, for example, the mutation pattern of T.1, "C25S, C39T, A8C," indicates that the 25th amino acid residue of the T.0 (sequence number 32) sequence is mutated from C to S, the 39th amino acid residue is mutated from C to T, and the 8th amino acid residue is mutated from C to A. The mutation pattern of T.6, "N-terminus + Titin_KAGIR," indicates that five amino acids, "KAGIR," are added to the N-terminus of the T.0 (sequence number 32) sequence. The others are inferred accordingly.

[0200] [Table 4-2] Note: In the table, for example, the mutation pattern "A88C" for O.1 means that the 88th amino acid residue in the O.0 (sequence number 33) sequence is mutated from A to C, and the mutation pattern for O.11 means that the C25S, C76S, A88C amino acid mutations are made to the O.0 (sequence number 33) sequence, and the five amino acids "DQPQF" are added to the N-terminus of O.0; the others are inferred accordingly.

[0201] [Table 4-3] Note: In the table, for example, the mutation pattern for OL.1, "C6E, V74C," indicates that the 6th amino acid residue in the OL.0 (sequence number 34) sequence is mutated from C to E, and the 74th amino acid residue is mutated from V to C; the others are inferred accordingly.

[0202] The sequences of the Titin-T chain, Obscurin-O chain, and Obscurin-Like-O chain after amino acid mutation are as follows: >T.1 (T.0 has C25S, C39T, and A8C mutations) GIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIIMDVQKQDGGLYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 35); >T.2 (T.0 has C25S, C39T, and V20C mutations) GIPPKIEALPSDISIDEGKCLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIIMDVQKQDGGLYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 36); >T.3 (T.0 has mutations C25S, C39T, and A26C) GIPPKIEALPSDISIDEGKVLTVASCFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIIMDVQKQDGGLYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 37); >T.4 (T.0 has C25S, C39T, and T22C mutations) GIPPKIEALPSDISIDEGKVLCVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIIMDVQKQDGGLYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 38); >T.5 (T.0 has C25S, C39T, and A8C mutations, and KAGIR is added to the N-terminus) KAGIRGIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIIMDVQKQDGGLYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 39); >T.6 (KAGIR added to the N-terminus of T.0) KAGIRGIPPKIEALPSDISIDEGKVLTVACAFTGEPTPEVTWSCGGRKIHSQEQGRFHIENTDDLTTLIIMDVQKQDGGLYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 127); >T.1-L1 (T.0 has C25S, C39T, and A8C mutations, and a (G4S)1 linker was added to the N-terminus) [ka] >T.1-L2 (T.0 has C25S, C39T, and A8C mutations, and a (G4S)2 linker was added to the N-terminus) [ka] >O.1 (O.0 has the A88C mutation) SGAPRFLTRPKAFVVSVGKDATLSCQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVCRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 42); >O.2 (O.0 has A3C mutation) SGCPRFLTRPKAFVVSVGKDATLSCQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVCRARNAIGEAFAAVGLQVDAEA (SEQ ID NO: 43); >O.3 (O.0 has the R9C mutation) SGAPRFLTCPKAFVVSVGKDATLSCQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVCRARNAIGEAFAAVGLQVDAEA (SEQ ID NO: 44); >O.4 (O.0 has mutations C25S, C76S, and A88C) SGAPRFLTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 45); >O.5 (O.0 has C25S, C76S, and A3C mutations) SGCPRFLTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVSRARNAIGEAFAAVGLQVDAEA (SEQ ID NO: 46); >O.6 (O.0 has C25S, C76S, and R9C mutations) SGAPRFLTCPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVSRARNAIGEAFAAVGLQVDAEA (SEQ ID NO: 47); >O.7 (O.0 has mutations C25S, C76S, A88C, L7K, and T62K) SGAPRFKTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLKILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 48); >O.8 (O.0 has mutations C25S, C76S, A88C, L7K, and T62H) SGAPRFKTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLHILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 49); >O.9 (O.0 has mutations C25S, C76S, A88C, K11L, and T62K) SGAPRFLTRPLAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLKILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 50); >O.10 (O.0 has mutations C25S, C76S, A88C, K11L, and T62H) SGAPRFLTRPLAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLHILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 51); >O.11 (O.0 has C25S, C76S, and A88C mutations, and DQPQF is added to the N-terminus) DQPQFSGAPRFLTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 52); >O.12 (O.0 has mutations C25S, C76S, A88C, L7K, and T62K, and DQPQF is added to the N-terminus) DQPQFSGAPRFKTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLKILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 53); >O.13 (O.0 has C25S, C76S, A88C, L7K, and T62H mutations, and DQPQF is added to the N-terminus) DQPQFSGAPRFKTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLHILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 54); >O.14 (O.0 has C25S, C76S, A88C, L7R, and T62K mutations, with DQPQF added to the N-terminus) DQPQFSGAPRFRTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLKILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 55); >O.15 (O.0 has C25S, C76S, A88C, L7R, and T62H mutations, and DQPQF is added to the N-terminus) DQPQFSGAPRFRTRPKAFVVSVGKDATLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLHILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 56); >O.16 (DQPQF added to the N-terminus of O.0) DQPQFSGAPRFLTRPKAFVVSVGKDATLSCQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLTILDLALGDSGQYVCRARNAIGEAFAAVGLQVDAEA (SEQ ID NO: 128); >O.4-L1 (O.0 has C25S, C76S, and A88C mutations, and a (G4S)1 linker was added to the N-terminus) [ka] >O.4-L2 (O.0 has the mutations C25S, C76S, and A88C, and a (G4S)2 linker was added to the N-terminus) [ka] >OL.1 (OL.0 has C6E and V74C mutations) QGSPPEFLRFPRPVRVVSGAEAELKCVVLGEPPPVVVWEKGGQQLAASERLSFPADGAEHGLLLTAALPTDAGCYVCRARNAAGEAYAAAAVTVLEPP (SEQ ID NO: 59); >OL.2 (OL.0 has C6E and G84C mutations) QGSPPEFLRFPRPVRVVSGAEAELKCVVLGEPPPVVVWEKGGQQLAASERLSFPADGAEHGLLLTAALPTDAGVYVCRARNAACEAYAAAAVTVLEPP (SEQ ID NO: 60); >OL.3 (OL.0 has mutations C6E and A86C) QGSPPEFLRFPRPVRVVSGAEAELKCVVLGEPPPVVVWEKGGQQLAASERLSFPADGAEHGLLLTAALPTDAGVYVCRARNAAGECYAAAAVTVLEPP (SEQ ID NO: 61); >OL.4 (OL.0 has mutations C6E, C26S, C77S, and V74C) QGSPPEFLRFPRPVRVVSGAEAELKSVVLGEPPPVVVWEKGGQQLAASERLSFPADGAEHGLLLTAALPTDAGCYVSRARNAAGEAYAAAAVTVLEPP (SEQ ID NO: 62); >OL.5 (OL.0 has mutations C6E, C26S, C77S, and G84C) QGSPPEFLRFPRPVRVVSGAEAELKSVVLGEPPPVVVWEKGGQQLAASERLSFPADGAEHGLLLTAALPTDAGVYVSRARNAACEAYAAAAVTVLEPP (SEQ ID NO: 63); >OL.6 (OL.0 has mutations C6E, C26S, C77S, and A86C) QGSPPEFLRFPRPVRVVSGAEAELKSVVLGEPPPVVVWEKGGQQLAASERLSFPADGAEHGLLLTAALPTDAGVYVSRARNAAGECYAAAAVTVLEPP (SEQ ID NO: 64).

[0203] Note: In the above sequence, the double underlined part is the linker sequence. Antibodies with CH1 / CL replacements (see Table 5 for specific replacements) were constructed using the Titin-T chain, Obscurin-O chain, and Obscurin-Like-O chain described above, and the binding activity of each optimized mutant antibody to its corresponding antigen was detected using the method described in Test Example 4 of the present disclosure. The experimental results are shown in Table 5. The results show that the modified antibodies retain good antigen binding activity.

[0204] [Table 5] Note: In the table, an antibody, for example, "F5", represents an antibody in which the heavy chain CH1 of the F0 antibody is replaced with the T.1 chain (sequence number 35), and the light chain CL is replaced with the O.1 chain (sequence number 42), and the other parts are obtained in the same manner as F0, and the rest are inferred thereby.

[0205] In addition, antibodies with CH1 / CL replaced were constructed using the above-mentioned mutated or domain-modified Titin-T chain and Obscurin-O chain, and the functional activity of other antibodies with CH1 / CL replaced was detected using the methods of Test Examples 2 and 4 of the present disclosure. The experimental results are shown in Table 6. The experimental results show that even with CH1 / CL replaced, the antibodies have good antigen-binding activity and high antibody purity (SEC) (%).

[0206] [Table 6-1] [Table 6-2] Note: For example, in the table, antibody "F16" represents an antibody obtained by replacing the heavy chain CH1 of the F0 antibody with the T.5 chain (sequence number 39), replacing the light chain CL with the O.4 chain (sequence number 45), and maintaining other parts the same as F0; the rest are inferred therefrom.

[0207] Second, other amino acids in the Titin Ig-Like 152 domain and the Obscurin Ig-Like 1 domain were mutated.

[0208] The specific amino acid mutation designs for the Titin Ig-Like 152 domain and the Obscurin Ig-Like 1 domain are shown in Table 7.

[0209] [Table 7] Note: In the table, for example, T.7 represents the Titin-T chain obtained by performing amino acid mutations M66S and T77S on the T.1 (sequence number 35) sequence, O.23 represents the Obscurin-O chain obtained by first performing amino acid mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E, and D94G on O.9 (sequence number 50), and then adding the "DQPQF" sequence to the N-terminus, and the others are inferred therefrom.

[0210] The variant sequences of the T.1 and O.9 chains are as follows: >T.7 (T.1 has the mutations M66S and T77S) GIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIISDVQKQDGGLYSLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 65); >T.8 (T.1 has the mutations M66K, K70R, S79T, and G81R) GIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIIKDVQRQDGGLYTLTLRNEFGSDSATVNIHIRSI (SEQ ID NO: 66); >T.9 (T.1 has mutations P3W, S11I, I13L, T22M, and N82M) GIWPKIECLPIDLSIDEGKVLMVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIIMDVQKQDGGLYTLSLGMEFGSDSATVNIHIRSI (SEQ ID NO: 67); >T.10 (T.1 has S11I, M66K, S79T, and G81R mutations) GIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDLTTLIIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI (SEQ ID NO: 68); >T.11 (T.1 has the following mutations: G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D, and F84L) GIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTSGKKISSEEGGRFHIESTEDLTTLIIMDVQKQDGGVYTLSLGNDLGSDSATVNIHIRSI (SEQ ID NO: 69); >T.12 (T.1 has mutations Q47E, Q49G, N56S, D58E, and L75V) GIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSEEGGRFHIESTEDLTTLIIMDVQKQDGGVYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 70); >T.13 (T.1 has the mutations N56S, D58E, and L75V) GIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIESTEDLTTLIIMDVQKQDGGVYTLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 71); >T.14 (T.1 has N56S, D58E, M66S, and T77S mutations) GIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIESTEDLTTLIISDVQKQDGGLYSLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 72); >T.15 (T.1 has N56S, D58E, M66S, and T77S mutations, N-terminal + KAGIR) KAGIRGIPPKIECLPSDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIESTEDLTTLIISDVQKQDGGLYSLSLGNEFGSDSATVNIHIRSI (SEQ ID NO: 74); >T.10-L1 (T.1 has S11I, M66K, S79T, and G81R mutations, and +(G4S)1 ligation sequence) [ka] >T.15-L1 (T.1 has N56S, D58E, M66S, and T77S mutations, and the N-terminal + KAGIR + (G4S)1 ligation sequence) [ka] >T.14-L1 (T.1 has N56S, D58E, M66S, and T77S mutations, and +(G4S)1 ligation sequence) [ka] >O.17 (O.9 has L11K, A12S, F13Y, V14T, and T22S mutations) SGAPRFLTRPKSYTVSVGKDASLSSQIVGNPTPQVSWEKDQQPVAAGARFRLAQDGDLYRLKILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 77); >O.18 (O.9 has mutations G2E, V17E, N30D, T32P, Q34E, S36T, V44I, A45T, L58V, K62E, A67Q, G69S, and A97G) SEAPRFLTRPLAFVVSEGKDATLSSQIVGDPPPEVTWEKDQQPITAGARFRLAQDGDVYRLEILDLQLSDSGQYVSRARNAIGEAFACVGLQVDAEG (SEQ ID NO: 78); >O.19 (O.9 has D20L, T22M, and A53L mutations) SGAPRFLTRPLAFVVSVGKLAMLSSQIVGNPTPQVSWEKDQQPVAAGARFRLLQDGDLYRLKILDLALGDSGQYVSRARNAIGEAFACVGLQVDAEA (SEQ ID NO: 79); >O.20 (O.9 has mutations Q41K, A45T, A67Q, G69S, and V89L) SGAPRFLTRPLAFVVSVGKDATLSSQIVGNPTPQVSWEKDKQPVTAGARFRLAQDGDLYRLKILDLQLSDSGQYVSRARNAIGEAFACLGLQVDAEA (SEQ ID NO: 80); >O.21 (O.9 has mutations Q42L, A45T, A67T, G69S, Q92E, and D94G) SGAPRFLTRPLAFVVSVGKDATLSSQIVGNPTPQVSWEKDQLPVTAGARFRLAQDGDLYRLKILDLTLSDSGQYVSRARNAIGEAFACVGLEVGAEA (SEQ ID NO: 81); >O.22 (O.9 has mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E, and D94G) SGAPRFLTRPLSYVVSVGKDASLSSQIVGNPTPQVSWEKDQLPVTAGARFRLAQDGDLYRLKILDLQLSDSGQYVSRARNAIGEAFACVGLEVGAEA (SEQ ID NO: 82); >O.23 (O.9 has mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E, D94G, and N-terminal +DQPQF) DQPQFSGAPRFLTRPLSYVVSVGKDASLSSQIVGNPTPQVSWEKDQLPVTAGARFRLAQDGDLYRLKILDLQLSDSGQYVSRARNAIGEAFACVGLEVGAEA (SEQ ID NO: 85); >O.20-L1 (O.9 has mutations Q41K, A45T, A67Q, G69S, and V89L, and has a +(G4S)1 sequence) [ka] >O.22-L1 (O.9 has mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E, and D94G, and has a +(G4S)1 sequence) [ka] >O.23-L1 (O.9 has mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E, and D94G, and has an N-terminal sequence of +DQPQF and +(G4S)1) [ka] .

[0211] Note: In the above sequence, the double underlined portion is the linker sequence.

[0212] The above-mentioned mutated or domain-modified Titin-T chain and Obscurin-O chain were used to construct antibodies with replaced CH1 / CL, and the antigen-binding activity of the antibodies with replaced CH1 / CL was detected using the method of Test Example 4 of the present disclosure. The experimental results are shown in Table 8. The results show that even when CH1 / CL was replaced with Titin-T chain / Obscurin-O chain, the antibodies retained good antigen-binding activity.

[0213] [Table 8] Note: In the table, for example, antibody "B1" represents an antibody in which the heavy chain CH1 of the B0 antibody is replaced with the T.1 chain (sequence number 35), the light chain CL is replaced with the O.9 chain (sequence number 50), and other parts are obtained in the same manner as B0, and the rest are inferred thereby.

[0214] In addition, the protein expression levels of the domain-modified antibodies were detected using the method of Test Example 1 of the present disclosure, and the experimental results are shown in Table 9. The results show that mutating some amino acids in the Titin-T chain / Obscurin-O chain significantly improved the antibody expression levels.

[0215] [Table 9] Note: In the table, for example, antibody "B1" represents an antibody in which the heavy chain CH1 of the B0 antibody is replaced with the T.1 chain (sequence number 35), the light chain CL is replaced with the O.9 chain (sequence number 50), and other parts are obtained in the same manner as B0, and the rest are inferred thereby.

[0216] Third, a new Titin-T chain / Obscurin-O chain. 1. Design of amino acid mutations in Titin-T chain / Obscurin-O chain Based on T.10 / O.20, the Titin-T chain / Obscurin-O chain was mutated as follows, and the specific amino acid mutations are shown in Table 10.

[0217] [Table 10] Note: In the table, for example, T.16 represents a Titin-T chain formed by substituting L60S and I64T amino acid residues in T.10 (SEQ ID NO: 68), and the others are inferred therefrom.

[0218] The sequence of Titin-T chain / Obscurin-O chain after amino acid residue substitution is as follows: >T.16 (T.10 has the L60S and I64T mutations) GIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI (SEQ ID NO: 129) >T.17 (T.10 has the mutations V20C, L60S, and I64T) GIPPKIECLPIDISIDEGKCLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI (SEQ ID NO: 130) >T.18 (T.10 has A26C, L60S, and I64T mutations) GIPPKIECLPIDISIDEGKVLTVASCFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI (SEQ ID NO: 131) >O.24 (O.20 has L11K, T32F and A48V mutations) SGAPRFLTRPKAFVVSVGKDATLSSQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVSRARNAIGEAFACLGLQVDAEA (SEQ ID NO: 132) >O.25 (O.20 has the mutations L11K, F13S, T32F, A48V, and I82H) SGAPRFLTRPKASVVSVGKDATLSSQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVSRARNAHGEAFACLGLQVDAEA (SEQ ID NO: 133) >O.26 (O.20 has mutations A3C, L11K, F13S, T32F, A48V, and I82H) SGCPRFLTRPKASVVSVGKDATLSSQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVSRARNAHGEAFACLGLQVDAEA (SEQ ID NO: 134) >O.27 (O.20 has the mutations R9C, L11K, F13S, T32F, A48V, and I82H) SGAPRFLTCPKASVVSVGKDATLSSQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVSRARNAHGEAFACLGLQVDAEA (SEQ ID NO: 135) >O.28 (O.20 has mutations S25C, S76C, L11K, F13S, T32F, A48V, and I82H) SGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA (SEQ ID NO: 136) >O.29 (O.20 has mutations A3C, S25C, S76C, L11K, F13S, T32F, A48V, and I82H) SGCPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA (SEQ ID NO: 137) >O.30 (O.20 has the mutations R9C, S25C, S76C, L11K, F13S, T32F, A48V, and I82H) SGAPRFLTCPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA (SEQ ID NO: 138) >O.31 (O.20 has the mutations L66C, V93C, L11K, F13S, T32F, A48V, and I82H) SGAPRFLTRPKASVVSVGKDATLSSQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDCQLSDSGQYVSRARNAHGEAFACLGLQCDAEA (SEQ ID NO: 139) >O.32 (O.20 has mutations A3C, L66C, V93C, L11K, F13S, T32F, A48V, and I82H) SGCPRFLTRPKASVVSVGKDATLSSQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDCQLSDSGQYVSRARNAHGEAFACLGLQCDAEA (SEQ ID NO: 140) >O.33 (O.20 has the mutations R9C, L66C, V93C, L11K, F13S, T32F, A48V, and I82H) SGAPRFLTCPKASVVSVGKDATLSSQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDCQLSDSGQYVSRARNAHGEAFACLGLQCDAEA (SEQ ID NO: 141)

[0219] 2. DI bispecific antibody constructed with Titin-T chain / Obscurin-O chain and its detection The anti-hNGF and hRANKL DI bispecific antibodies DI-2 to DI-20 were constructed, which consisted of the following first heavy chain, second heavy chain, first light chain and second light chain: A first heavy chain consisting of, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[Fc1]; A first light chain consisting of [VL1]-[linker 2]-[Titin-T chain] in order from the N-terminus to the C-terminus; a second heavy chain consisting of [VH2]-[CH1]-[Fc2] in order from N-terminus to C-terminus; and a second light chain, which is [VL2]-[CL] from the N-terminus to the C-terminus.

[0220] wherein VH1 and VL1 are the heavy chain variable region and light chain variable region of I0, VH2 and VL2 are the heavy chain variable region and light chain variable region of D0, the amino acid sequence of VH1 is shown in SEQ ID NO: 26, the amino acid sequence of VL1 is shown in SEQ ID NO: 27, the amino acid sequence of VH2 is shown in SEQ ID NO: 24, and the amino acid sequence of VL2 is shown in SEQ ID NO: 25, Fc1 is the first subunit of Fc of IgG1 containing a hole mutation (amino acid sequence shown in SEQ ID NO: 177), Fc2 is the second subunit of Fc of IgG1 containing a knob mutation (amino acid sequence shown in SEQ ID NO: 178). The amino acid sequence of CH1 is shown in SEQ ID NO: 179, the amino acid sequence of CL is shown in SEQ ID NO: 4, the N-terminus of the Obscurin-O chain is linked to VH1 via linker 1, the C-terminus of the Obscurin-O chain is linked to Fc1 via a bond (i.e., linker 3 is a bond), and the N-terminus of the Titin-T chain is linked to VL1 via linker 2. The structures of the Obscurin-O chain / Titin-T chain, linker 1, and linker 2 in the different DI bispecific antibodies in this example are shown in Table 11.

[0221] [Table 11]

[0222] The antigen-binding activity of bispecific antibodies DI-2 to DI-20 was determined using the method described in Test Example 4 of the present disclosure. The thermal stability of DI-2, DI-4 to DI-8, DI-10 to DI-16, and DI-20 was also studied. The antibodies were diluted to 5 mg / mL with PBS and their thermal stability was measured using Units (loading volume: 9 μL; parameter settings: Start Temp 20°C, Incubation 0 s, Rate 0.3°C / min, Plate Hold 5 s, End Temp 95°C). The experimental results are shown in Tables 12 and 13. The modified bispecific antibodies showed no significant change in antigen-binding activity, and DI-4 to DI-8, DI-10 to DI-16, and DI-20 showed significantly improved Tm1 (°C) and Tonset (°C) compared to DI-2, demonstrating superior thermal stability of the bispecific antibodies.

[0223] [Table 12]

[0224] [Table 13]

[0225] Furthermore, a DI bispecific antibody solution was prepared using a buffer containing 10 mM acetic acid, pH 5.5, and 9% sucrose (see Table 14 for details). The solution was incubated in an incubator at 40°C for 4 weeks. After the fourth week of incubation, the bispecific antibody concentration was concentrated to the same concentration as at the start of incubation, and the solution was observed for precipitation. The experimental results are shown in Table 14 below. The experimental results show that precipitation occurred in the solution of the DI-2 bispecific antibody group, while DI-3 to DI-7 have better stability than DI-2.

[0226] [Table 14]

[0227] 3. PL bispecific antibody constructed with Titin-T chain / Obscurin-O chain and its detection The anti-hPDL1 and hCTLA4 PL bispecific antibodies PL-1 to PL-19 were constructed, which comprise the following first heavy chain, second heavy chain, first light chain, and second light chain: A first heavy chain consisting of, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[Fc1]; A first light chain consisting of [VL1]-[linker 2]-[Titin-T chain] in order from the N-terminus to the C-terminus; a second heavy chain consisting of [VH2]-[CH1]-[Fc2] in order from N-terminus to C-terminus; and a second light chain, which is [VL2]-[CL] from the N-terminus to the C-terminus.

[0228] wherein VH1 and VL1 are the heavy chain variable region and light chain variable region of the P0 antibody (i.e., the h1831K antibody in WO2020177733A1), VH2 and VL2 are the heavy chain variable region and light chain variable region of the L0 antibody, the amino acid sequence of VH1 is shown in SEQ ID NO: 156, the amino acid sequence of VL1 is shown in SEQ ID NO: 155, the amino acid sequence of VH2 is shown in SEQ ID NO: 169, and the amino acid sequence of VL2 is shown in SEQ ID NO: 170, Fc1 is the first subunit of the Fc of IgG1 containing a knob mutation (the amino acid sequence is shown in SEQ ID NO: 178), and Fc2 is the first subunit of the Fc of IgG1 containing a hole mutation. The second Fc subunit (amino acid sequence shown in SEQ ID NO: 177) has the amino acid sequence of CH1 shown in SEQ ID NO: 179 and the amino acid sequence of CL shown in SEQ ID NO: 4. The N-terminus of the Obscurin-O chain is linked to VH1 via linker 1, the C-terminus of the Obscurin-O chain is directly linked to Fc1 via a bond (i.e., linker 3 is a bond), and the N-terminus of the Titin-T chain is linked to VL1 via linker 2. The structures of the Obscurin-O chain / Titin-T chain, linker 1, and linker 2 in different PL bispecific antibodies are shown in Table 15.

[0229] [Table 15]

[0230] The heavy chain variable region and light chain variable region of the anti-CTLA-4 antibody ipilimumab (abbreviated as L0, trade name Yervoy) are as follows: Heavy chain variable region of L0: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 169) L0 light chain variable region: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 170) Illustratively, the four-stranded sequence of PL-1 is as follows: The first heavy chain amino acid sequence of PL-1: [ka] The first light chain amino acid sequence of PL-1: [ka] The second heavy chain amino acid sequence of PL-1: [ka] The amino acid sequence of the second light chain of PL-1: [ka] Note: In the above sequences, the bolded parts are variable regions, the dotted parts are Fc regions, the wavy parts are CH1, the dashed underlined parts are CL, the double underlined parts are linkers, and the single underlined parts are Obscurin-O chain / Titin-T chain.

[0231] The binding activity of the PL bispecific antibody was detected using the ELISA assay described in Test Example 4 of the present disclosure (hPDL1 and hCTLA4 antigens were purchased from Sinobiology). The thermal stability of the PL bispecific antibody was also investigated using a high-throughput differential scanning fluorometer (UNCHAINED, specifications: Unit). The method was as follows: 9 μL of sample diluted with PBS (phosphate buffer solution) (PL bispecific antibody concentration: 1.4-3 mg / mL) was added to the sample cell. The instrument was run under the following parameters: Start Temp 25°C, Incubation 180 s, Rate 0.3°C / min, Plate Hold 3 s, End Temp 95°C. The experimental results were analyzed using Uncle Analysis software.

[0232] The experimental results are shown in Tables 16 and 17. The PL bispecific antibodies constructed with the new Titin-T chain / Obscurin-O chain still had good binding activity to the antigen, and PL-2 to PL-19 had significantly improved Tm1 (°C), Tagg 266 (°C), and Tonset (°C) compared to PL-1, indicating superior thermal stability of the bispecific antibodies.

[0233] [Table 16]

[0234] [Table 17]

[0235] 4. HJ bispecific antibody constructed with Titin-T chain / Obscurin-O chain and its detection Anti-hIL5 and hTSLP HJ bispecific antibodies HJ-3 to HJ-11 were constructed, which consisted of the following first heavy chain, second heavy chain, first light chain, and second light chain: A first heavy chain consisting of, from the N-terminus to the C-terminus, [VH1]-[linker 1]-[Titin-T chain]-[linker 3]-[Fc1]; A first light chain consisting of [VL1]-[linker 2]-[Obscurin-O chain] in order from the N-terminus to the C-terminus; a second heavy chain consisting of [VH2]-[CH1]-[Fc2] in order from N-terminus to C-terminus; and a second light chain which is, from N-terminus to C-terminus, [VL2]-[CL], Among them, VH1 and VL1 are the heavy chain variable region and light chain variable region of the H0 antibody, VH2 and VL2 are the heavy chain variable region and light chain variable region of the J1 antibody, the amino acid sequence of VH1 is shown in SEQ ID NO: 16, the amino acid sequence of VL1 is shown in SEQ ID NO: 17, the amino acid sequence of VH2 is shown in SEQ ID NO: 171, and the amino acid sequence of VL2 is shown in SEQ ID NO: 172, Fc1 is the first subunit of Fc of IgG1 containing a knob mutation (amino acid sequence shown in SEQ ID NO: 178), and Fc2 is the second subunit of Fc of IgG1 containing a hole mutation (amino acid sequence shown in SEQ ID NO: 179). The amino acid sequence of CH1 is shown in SEQ ID NO: 179, the amino acid sequence of CL is shown in SEQ ID NO: 4, the N-terminus of the Titin-T chain is linked to VH1 via linker 1, the C-terminus of the Titin-T chain is directly linked to Fc1 via a bond (i.e., linker 3 is a bond), and the N-terminus of the Obscurin-O chain is linked to VL1 via linker 2. The structures of the Titin-T chain / Obscurin-O chain, linker 1, and linker 2 in the different HJ bispecific antibodies constructed are shown in Table 18.

[0236] [Table 18]

[0237] The binding activity of the HJ-3 and HJ-5 to HJ-11 bispecific antibodies to hIL5 and hTSLP antigens was detected by the method in Test Example 4 of the present disclosure. The thermal stability of the bispecific antibody was also investigated as follows: a diluted solution of the HJ bispecific antibody was prepared in a 10 mM acetic acid, pH 5.5, 9% sucrose buffer solution, and then the bispecific antibody was concentrated by ultrafiltration to obtain HJ bispecific antibody solutions of various concentrations (see Table 19-2 for the HJ bispecific antibody concentrations). The concentrated solutions were then incubated in a 40°C incubator, and the SEC purity of the samples was determined on day 0 (i.e., before the start of incubation at 40°C, D0), day 7 (day 7 of incubation at 40°C, D7), day 14 (day 14 of incubation at 40°C, D14), day 21 (day 21 of incubation at 40°C, D21), and day 28 (day 28 of incubation at 40°C, D28). After 28 days of incubation at 40°C, the samples were immediately sampled for non-reducing CE-SDS purity determination.

[0238] The experimental results are shown in Tables 19-1 and 19-2 below. The experimental results show that the HJ bispecific antibodies constructed in the present disclosure have no significant changes in antigen-binding activity, and HJ-5 to HJ-11 have better thermal stability than HJ-3.

[0239] [Table 19-1]

[0240] [Table 19-2] Note: D28△SEC purity (%) = D0 SEC purity (%) - D28 SEC purity (%)

[0241] Example 4: Half-life of CH1 / CL domain-modified antibodies To evaluate whether the antibody's in vivo stability was affected after its CH1 / CL was replaced with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain, the half-life of the antibody in rats was measured before and after the CH1 / CL replacement. As an example, the half-lives of antibody F20, in which the CH1 / CL of the F0 antibody was replaced with T.1 / O.9, and antibody C3, in which the CH1 / CL of the C0 antibody was replaced with T.1 / O.9, were measured in rats. The specific method is as follows: Three mpk of antibody was administered intravenously to rats, and 0.3 mL of whole blood was collected 5 min, 8 h, 1 d, 2 d, 4 d, 7 d, 10 d, 14 d, 21 d, and 28 d after administration. No anticoagulant was added. After collection, the blood was left to stand at 4°C for 30 min and then centrifuged at 1000 g for 15 min. The supernatant (serum) was collected, placed in an EP tube, and stored at -80°C. The antibody drug concentration at each time point was detected by sandwich ELISA. The corresponding antigen was coated onto an ELISA plate, incubated overnight at 4°C, washed with washing buffer, and then added with blocking buffer and incubated at room temperature for 1-3 hours. Washed with washing buffer, 100 μL of standard or serum sample to be measured was added and incubated at 37°C for 3 hours. Washed with washing buffer, secondary antibody Anti-Human IgG FC(HRP) mouse preadsorbed (Abcam, product number ab98624, diluted 1:10000) was added and incubated at 37°C for 1-1.5 hours. Washed with buffer, TMB was added and incubated at room temperature in the dark for 10 minutes. Stopping solution was added, and the OD450 value was read.

[0242] The experimental results are shown in Table 20. The results show that even when the antibody's CH1 / CL was replaced with Titin-T chain / Obscurin-O chain, the antibody maintained the same half-life as the original antibody in the rat body, suggesting that the antibody with the replaced CH1 / CL has good stability in the body, similar to the original antibody.

[0243] [Table 20]

[0244] Example 5. Construction and detection of bispecific antibodies Bispecific antibodies were constructed by bridge PCR using antibodies that bind to the first antigen and antibodies that bind to the second antigen. For example, multiple types of bispecific antibodies were constructed.

[0245] 1. Construction of bispecific antibodies First, a DI-1 bispecific antibody was constructed. The DI-1 bispecific antibody contained one heavy chain of the D0 antibody (with a knob modification in the Fc region), one light chain of the D0 antibody, and one heavy chain and one light chain of the domain-engineered I0 antibody (with CH1 / CL replaced with Titin-T chain / Obscurin-O chain and with a hole modification in the Fc region). The DI-1 bispecific antibody is an IgG-like bispecific antibody, and its structural diagram is shown in Figure 5. The four-chain sequence of DI-1 is as follows: Amino acid sequence of DI-1 chain 1 (DI-1-H1): [ka] Note: In the sequences, the bolded part is VH of D0, the single underlined part is CH1, the dotted part is CH2, and the italicized part is CH3.

[0246] Amino acid sequence of DI-1 chain 2 (DI-1-L1): [ka] Note: In the sequences, the bolded part is the VL of D0, and the single underlined part is the D0 light chain constant region CL.

[0247] Amino acid sequence of DI-1 chain 3 (DI-1-H2): [ka] Note: In the sequences, the bolded part is VH of I0, the single underlined part is T.10, the double underlined part is the linker sequence, the dotted part is CH2, the italicized part is CH3, and the double underlined part is the linker sequence.

[0248] Amino acid sequence of DI-1 chain 4 (DI-1-L2): [ka] Note: In the sequences, the bolded part is the VL of I0, the single underlined part is O.20, and the double underlined part is the linker sequence.

[0249] Furthermore, an IgG-like BU bispecific antibody was constructed using B0 or ​​a B0 domain-engineered antibody and U0 or a U0 domain-engineered antibody (see, for example, Figure 8 for a schematic diagram of the BU5 structure). The four-chain sequence of the resulting bispecific antibody is shown in Table 21:

[0250] [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4]

[0251] Note: In the sequences, the bolded parts are variable region sequences, the single underlined parts are CH1 or Titin-T chain, the dotted parts are CH2, the italicized parts are CH3, the double underlined parts are CL or Obscurin-O chain, and the dashed underlined parts are linker L1.

[0252] Furthermore, the FA-1 bispecific antibody was constructed. This FA-1 bispecific antibody is an IgG-like bispecific antibody consisting of one heavy chain (knob-modified in the Fc region) and one light chain of the A0 antibody, and one heavy chain (hole-modified in the Fc region, with CH1 replaced with T.10-L1) and one light chain (CL replaced with O.20-L1) of the F0 domain-modified antibody. The four-chain sequence of FA-1 is shown in Table 22.

[0253] Chain 1 of FA-1 (heavy chain CH1 of F0 was replaced with T.10-L1 and the Fc region contained knob modifications): [ka] Chain 2 of FA-1 (light chain CL of F0 replaced by O.20-L1): [ka] Chain 3 of FA-1 (A0 heavy chain contained knob modification): [ka] Chain 4 of FA-1 (light chain of A0): [ka] Note: In the above sequences, the bolded parts are variable region sequences, the single underlined parts are CH1 or Titin-T chain, the dotted parts are CH2, the italicized parts are CH3, the double underlined parts are CL or Obscurin-O chain, and the dashed underlined parts are linker L1.

[0254] 2. Detection of bispecific antibodies DI-1 was detected using the method of Test Example 3 of the present disclosure. The four chains of DI-1 were co-transfected into cells and expressed, followed by mass spectrometry analysis. The experimental results are shown in Table 22 and Figures 6A to 6C. The results of mass spectrometry analysis showed that no homodimers or mismatched molecules were detected, indicating that DI-1 was assembled correctly.

[0255] [Table 22]

[0256] The expression of BU5 was also detected using the methods of Test Examples 3 and 2 of the present disclosure. First, the four chains of BU5 were co-transfected into cells to express the bispecific antibody, and B0 and U0 were used as controls. The expression product was then subjected to purity and mass spectrometry analysis. The experimental results are shown in Table 23 and Figures 9A to 9C. The experimental results show that the bispecific antibody BU5 was successfully expressed, the four chains of BU5 were assembled into the target molecule, and no mismatches were observed. The bispecific antibody BU5 also had high purity, with an SEC% of 88%.

[0257] [Table 23] Note: In the table, "○" indicates correct pairing.

[0258] In addition, the purity and antigen-binding activity of the constructed BU1-BU4 bispecific antibody were determined using the methods of Test Examples 2 and 4 of the present disclosure, and the experimental results are shown in Table 24. The experimental results show that the bispecific antibody with CH1 / CL replacement still retains good antigen-binding activity and has high antibody purity.

[0259] [Table 24]

[0260] Test Example Test Example 1: Test method for antibody expression level HEK293E cells were transfected with monoclonal or bispecific antibody expression plasmids. After 6 days, the expression supernatant was collected and centrifuged at high speed to remove impurities. The supernatant was purified using a Protein A column (GE Healthcare). The column was washed with PBS until the A280 readings were reduced to baseline, followed by further washing with 100 mM acetate buffer (pH 3.5) and neutralization with 1 M Tris-HCl (pH 8.0). The amount of antibody expression was quantified by dividing the final purified antibody amount by the expression volume.

[0261] Test Example 2: Method for detecting antibody purity Antibody purity was monitored by SEC-HPLC. Detection was performed according to the instrument's instructions. The Waters e2695 chromatography column was a Waters Xbridge BEH 200A SEC column, and the mobile phase was PBS (pH adjusted to 6.8 with dilute hydrochloric acid). 100 μg of protein was injected, and isocratic elution was performed at a flow rate of 0.5 mL / min. Antibody purity was calculated as the percentage of the main peak area relative to the total peak area (SEC (%)).

[0262] Test Example 3: Mass Spectral Detection Method for Antibodies (1) Sample preparation: 1. Measurement of deglycosylated complete molecular weight: 30 μg of expressed antibody was taken and freeze-dried, then 10 μL of 8 M Gua-HCl was added and denatured in a 70°C water bath for 10 minutes. 90 μL of distilled water was added, and 30 μL of the antibody was taken and 0.8 μL of PNG enzyme F (peptide N-glycosidase F) was added. The antibody was then placed in a 37°C water bath and incubated for 2 hours. 0.5 μg of the antibody was taken and the deglycosylated complete molecular weight was measured.

[0263] 2. Measurement of deglycosylated reduced molecular weight: 30 μg of expressed antibody was lyophilized, then added with 10 μL of 8 M Gua-HCl and denatured in a 70°C water bath for 10 minutes. 90 μL of distilled water was added, and 30 μL of the antibody was added with 0.8 μL of PNG enzyme F (peptide N-glycosidase F) and incubated in a 37°C water bath for 2 hours. 2 μL of 0.025 M DTT was added, and the antibody was reduced in a 70°C water bath for 10 minutes. 0.5 μg of the antibody was then measured for deglycosylated reduced molecular weight.

[0264] (2) Chromatography-mass spectrometry conditions: Chromatography conditions: Chromatography column: Poroshell 300SB-C8 5 μm 2.1 x 75 mm Mobile phase: A: 0.1%HCOOH / H2O B: 0.1%HCOOH / CAN Column temperature: 75℃ Gradient: See Table 25.

[0265] [Table 25]

[0266] Mass spectrum conditions: Mass spectrum: Agilent 6530 Q-TOF LC-MS Ionization mode ESI Acquisition mode: 1 GHz (mass range 500-5000 m / z) Drying gas temperature: 325℃ Drying gas flow rate: 10 L / min Atomizer: 40 psi Sheath gas temperature: 350℃ Sheath gas flow rate: 12 L / min Spray voltage: 500 V Capillary voltage: 3,500 V Decomposition voltage: 200 V Skimmer voltage: 65V RF voltage: 7500 V Mass spectrometry analysis was performed on the antibody awaiting measurement, and the experimental results showed that after CH1 / CL was replaced with Titin-T / Obscurin-O, the antibody retained its correct assembly.

[0267] Test Example 4: Method for detecting antibody binding activity Antibodies targeting membrane proteins can be detected by FACS to detect their antigen binding activity. For example, C0, N0, F0, V0, S0, and their CH1 / CL domain-modified antibodies can be detected by FACS. Cells (2 x 106 cells / mL, 90 µL) resuspended in FACS buffer (98% PBS, 2% FBS) were added to a 96-well U-bottom plate (Corning, 3795). 10 µL of gradient-diluted antibodies were added and incubated at 4°C for 1 hour. After washing twice with FACS buffer, Alexa Fluor 488 sheep anti-human IgG (H+L) (Invitrogen, Cat # 2015982, diluted 1:1000) was added to each well. The cells were incubated at 4°C for 1 hour and washed twice. The cells were resuspended in FACS buffer, and the fluorescence signal was read using a FACS Canto II (BD). Finally, data were processed and analyzed with FlowJo 7.6 and Graphpad Prism 5.

[0268] For antibodies targeting soluble proteins, the binding activity of the antibody to the soluble protein was detected using ELISA. For example, DO, I, B, U, H, R, J, and their CH1 / CL domain-modified antibodies can be detected using ELISA. The method is as follows: Protein was diluted to 1 μg / mL in PBS (Genbyou Bioscience, B320) buffer at pH 7.4, placed in a 96-well microplate (Corning, 9018) at a volume of 100 μL per well, and incubated overnight at 4°C. After discarding the liquid, 300 μL of 5% nonfat milk (BD, 232100) diluted in PBS was added to each well to block the binding, and the plate was incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20). Then, 100 μL of the gradient diluted antibody solution was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST, and 100 μL of mouse anti-human IgG (H+L) (Jackson ImmunoResearch, 209-035-088, diluted 1:8000) was added to each well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB chromogenic substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes. The reaction was stopped by adding 450 μL of 1 M H2SO4 to each well, and the absorbance value at 450 nm was read using a microplate reader. The antibody-antigen binding curve was fitted using software to calculate the EC50 value.

[0269] In this test example, the antigens corresponding to the antibodies are as follows: the antigen bound by D0 and its CH1 / CL domain-modified antibody is hRANKL (purchased from Sinobiological, 11682-HNCH), the antigen bound by I0 and its CH1 / CL domain-modified antibody is hNGF (purchased from Sinobiological, 11050-HNAC), the antigen bound by B0 and its CH1 / CL domain-modified antibody is hBAFF (purchased from Sinobiological, 10056-HNCH), the antigen bound by U0 and its CH1 / CL domain-modified antibody is hP40 (purchased from Sinobiological, 10052-H08H), and the antigen bound by H0, R0, and their CH1 / CL domain-modified antibodies is hIL-5 (R&D The antigen bound by J0 and its CH1 / CL domain-modified antibodies was hTSLP (purchased from Sinobiological Systems, 16135-H08H). The antigen bound by C0, N0, and its CH1 / CL domain-modified antibodies was CEA (CEA-overexpressing MKN45 cells (purchased from Nanjing Kebai, CBP60488)). The antigen bound by F0, V0, S0, and its CH1 / CL domain-modified antibodies was B7H3 (B7H3-overexpressing recombinant cell line CT26-B7H3 (CT26 was derived from the Cell Bank of the Chinese Academy of Sciences; the B7H3 sequence is ID: XP_005254757.1)).

[0270] Test Example 5: Experimental detection of mismatches in CH1 / CL domain modified antibodies 1. Method for detecting the expression of mismatched light / heavy chain molecules in different antibodies This experiment analyzed the pairing expression of different light / heavy chain antibodies to verify that the occurrence of heavy / light chain mismatches can be effectively reduced after replacing CH1 / CL with Titin-T chain / Obscurin-O chain or Titin-T chain / Obscurin-Like-O chain. Specifically, when expressing antibodies in cells, we examined whether wild-type CH1 and CL could form cross-mismatched molecules by exchanging the heavy / light chains of D0 and I0 (i.e., I / D-1 (D0 heavy chain + I0 light chain) and I / D-2 (I0 heavy chain + D0 light chain)). At the same time, we also examined the pairing expression of domain-engineered antibody D3, in which the CH1 / CL of D0 and I0 were replaced with Titin-T chain / Obscurin-O chain (the heavy chain CH1 of D0 is T). The heavy and light chains of I / D-3 (D0 heavy chain + I3 light chain), I / D-4 (D3 heavy chain + I0 light chain), I / D-5 (I0 heavy chain + I3 light chain), and I / D-6 (I3 heavy chain + D0 light chain) were exchanged, respectively, to construct mismatched antibodies. The structural diagrams of the constructed antibodies are shown in Figure 7.

[0271] The antibody expression level was detected using the method of Test Example 1, and mass spectrometry analysis of the expression product was performed using the method of Test Example 3 to detect whether mismatched molecules were formed. The experimental results are shown in Table 26. The experimental results showed that when the D0 heavy chain + I0 light chain or the I0 heavy chain + D0 light chain were co-expressed, they were able to form intact IgG molecules (150 kDa), with very high expression levels of 74.4 mg / L and 73.8 mg / L, respectively. LC-MS results confirmed that the resulting proteins were mismatched IgG molecules. Therefore, unless CH1-CL is modified or replaced, the co-expression of the four different heavy and light chains of the bispecific antibody will result in the formation of a large number of light / heavy chain mismatched molecules. On the other hand, after replacing CH1 / CL with Titin-T chain / Obscurin-O chain, the four cross-mismatched forms I / D-3 (D0 heavy chain + I3 light chain), I / D-4 (D3 heavy chain + I0 light chain), I / D-5 (I0 heavy chain + I3 light chain), and I / D-6 (I3 heavy chain + D0 light chain) did not generate target molecules (150 kDa). The D3 heavy chain + I0 light chain combination formed a small number of mismatched heavy chains (100 kDa), but the other three forms did not generate mismatched molecules. This indicates that nonhomologous pairing after modification does not form mismatched molecules in the IgG structural form. Therefore, replacing CH1 / CL with Titin-T chain / Obscurin-O chain effectively prevents or reduces the formation of nonhomologous cross-paired molecules between the light and heavy chains of IgG-like bispecific antibodies.

[0272] [Table 26] Note: In the table, "○" indicates that the mismatched molecule was detected, and "×" indicates that the mismatched molecule was not detected.

[0273] 2. Method for detecting light / heavy chain mismatch in bispecific antibodies To verify that mismatches do not occur or are reduced when CH1 / CL is replaced with Titin-T chain / Obscurin-O chain, this example was designed to express three forms of light / heavy chain cross-pairing: expressing antibodies by co-transfecting cells with two different heavy chains and one identical VL-CL light chain; expressing antibodies by co-transfecting cells with two different heavy chains and one identical light chain in which CL is replaced with Obscurin-O chain; or expressing antibodies by co-transfecting cells with two different heavy chains and two different light chains (see Table 27 for specific pairings).

[0274] The expression product was then subjected to mass spectrometry analysis (see Test Example 3 of the present disclosure for the method) to detect the resulting antibody purity (SEC) (see Test Example 2 of the present disclosure for the method). The experimental results are shown in Table 27, and the results show that the correct molecule was formed only when a light chain replaced with the Obscurin-O chain and a light chain without CL replacement were added together, but the correct target molecule was not observed in cross-pairings where only one light chain without CL replacement or only one light chain with CL replaced by the Obscurin-O chain was added. This indicates that light chains in which CL has been replaced by Obscurin-O chains more easily pair and bind to heavy chains in which CH1 has been replaced by Titin-T chains, without cross-matching with heavy chains in which CH1 has not been replaced by Titin-T chains, to form molecules, and that light chains in which CL has not been replaced by Obscurin-O chains more easily pair and bind to heavy chains in which CH1 has not been replaced by Titin-T chains, without binding to heavy chains in which CH1 has been replaced by Titin-T chains.

[0275] [Table 27] Notes (in the table, "○" indicates that the four-stranded paired molecule was detected, and "--" indicates that the four-stranded paired molecule was not detected).

[0276] Test Example 6: Detection of affinity between bispecific antibodies and antigens The affinity of the target antibody to the antigen protein was measured using a Biacore T200 (GE) instrument. Following the instructions in the Human Capture Prevention Reagent Kit (GE, Cat. # BR-1008-39) manual, the target antibody was affinity-captured on a Protein A biosensor chip (Cat. # 29127556, GE). A gradient of soluble antigen was then applied across the chip surface, and the Biacore T200 instrument detected the response signal in real time to obtain binding and dissociation curves. After the dissociation cycle for each experiment was completed, the biosensor chip was regenerated by washing with glycine-HCl regeneration solution (pH 1.5, Cat. # BR-1003-54, GE). The data obtained from the experiment were fitted with a (1:1) Langmuir model using BIAevaluation version 4.1, GE software, to obtain affinity values. The experimental results are shown in Table 28.

[0277] Experimental results have shown that the domain-engineered bispecific antibodies of the present disclosure fully retain the affinity for their antigens of the two parent monoclonal antibodies. In this experiment, the antigens used were hRANKL (purchased from Sino biological, 11682-HNCH), hNGF (purchased from Sino biological, 11050-HNAC), hBAFF (purchased from Sino biological, 10056-HNCH), hP40 (purchased from Sino biological, 10052-H08H), hB7H3 (prepared in the laboratory, the sequence of which is shown in SEQ ID NO: 111), and hCD3 antigen (prepared in the laboratory, a heterodimer consisting of the δ subunit (sequence of which is shown in SEQ ID NO: 112) and the ε subunit (sequence of which is shown in SEQ ID NO: 113) of hCD3).

[0278] [Table 28]

[0279] Test Example 7: Antigen binding experiment of CH1 / CL domain modified double antibody The ability of the disclosed CH1-CL domain engineered bispecific antibody to simultaneously bind two target antigens was verified by measuring the affinity of the antibody to the antigen protein using a Biacore T200 (GE) instrument. The specific experimental procedure was as follows: After affinity capturing of antibody BU5 using a Protein A biosensor chip (Cat. # 29127556, GE), the first antigen molecule of the bispecific antibody, hP40 (purchased from Sino biological, 10052-H08H), was injected onto the chip surface until saturation was reached, and then a fixed concentration of the second antigen molecule, hBAFF (purchased from Sino biological, 10056-HNCH), was injected. The Biacore T200 instrument detected the reaction signal in real time, and binding and dissociation curves were obtained. After the cycle dissociation for each experiment was completed, the biosensor chip was washed and regenerated with glycine-HCl regeneration solution (pH 1.5 Cat. # BR-1003-54, GE), and the data fitting model was fitted with a (1:1) Langmuir model using BIAevaluation version 4.1, GE software. The antibody BU5 was then affinity-captured using a Protein A biosensor chip (Cat. # 29127556, GE). The first antigen molecule of the bispecific antibody, hP40 (purchased from Sino biological, 10052-H08H), was then injected onto the chip surface until saturation was reached. A fixed concentration of the second antigen molecule, hBAFF (purchased from Sino biological, 10056-HNCH), was then injected. The reaction signals were detected in real time using a Biacore T200 instrument to obtain binding and dissociation curves. After the cycle dissociation for each experiment was completed, the biosensor chip was regenerated by washing with glycine-HCl regeneration solution (pH 1.5, Cat. # BR-1003-54, GE). The data were fitted using the (1:1) Langmuir model using BIAevaluation version 4.1, GE software.

[0280] The experimental results are shown in Table 29. The experimental results show that when the binding of bispecific antibody BU5 to the antigen hP40 becomes saturated, it can continue to bind to the antigen hBAFF, and its ability to further bind to hBaFF is equivalent to that of monoclonal antibody B0. Similarly, when the binding of bispecific antibody BU5 to the antigen hBAFF becomes saturated, it can continue to bind to the antigen hP40, and its ability to further bind to hP40 is equivalent to that of monoclonal antibody B0. This demonstrates that the bispecific antibody of the present disclosure in which CH1 / CL is replaced with Titin-T chain / Obscurin-O chain can simultaneously bind to two target antigens.

[0281] [Table 29]

[0282] Test Example 8: Osteoclast differentiation experiment using DI-1 bispecific antibody Raw264.7 cells (Chinese Academy of Sciences Cell Bank, SCSP-5036) were digested, resuspended, and counted. They were then seeded into 24-well cell culture plates (Corning, 3524) and cultured overnight in a 37°C cell incubator. The next day, the antibody solution was diluted to different concentrations, homogenously mixed with RANKL (Sino biological, 11682-HNCH), and added to the cell culture plates to a final RANKL concentration of 50 ng / mL. The cells were then cultured at 37°C. After 96 h, the solution in the 24-well plate was removed, and 100 μL of cell lysis solution (Beyotime, P0013J) was added to each well. The cells were mixed homogenously by pipetting. The lysates were transferred to EP tubes and centrifuged at 12,000 g for 5 min. The supernatant was then detected using a tartrate-resistant acid phosphatase detection reagent kit (Beyotime, P0332) according to the manufacturer's instructions. The absorbance at 405 nm was read using a microplate reader. The detected values ​​were fitted to a curve using software to calculate the IC50 value. The experimental results are shown in Figure 10. The experimental results show that the CH1 / CL-substituted bispecific antibody DI-1 retains good activity and effectively inhibits osteoclast differentiation.

[0283] Test Example 9: TF1 cell proliferation experiment with DI-1 bispecific antibody The cellular activity of the antibody was evaluated using an NGF-induced proliferation experiment using TF-1 cells (ATCC, CRL-2003). The experimental procedure was as follows: TF1 cells were digested, harvested, resuspended, and counted. They were then seeded into a 96-well plate (Corning, 3903) and cultured overnight in a 37°C incubator. The following day, the antibody solution was diluted to different concentrations, homogenously mixed with NGF (Sino biological, 11050-HNAC), and added to the cell culture plate. The final NGF concentration was 10 ng / mL, and the cells were placed in the incubator and continued to grow. After 72 h, the cell culture plate was removed, and 50 μL of Cell-titer Glo (Promega, G755B) detection solution was added to each well. The plate was then incubated on a shaker for 10 min and then left at room temperature for 10 min. Luminescence signals were detected using a microplate reader (PerkinElmer, Victor3). The detected signal values ​​were fitted to a curve using software to calculate the IC50 value. The experimental results are shown in FIG. 11, which show that the CH1 / CL replaced bispecific antibody DI-1 has good activity.

[0284] Test Example 10: Detection of light-heavy chain mismatch after CH1 / CL replacement of bispecific antibodies The following experiments were conducted to analyze the light and heavy chain mismatch of bispecific antibodies when CH1 / CL was replaced with Titin-T chain / Obscurin-O chain or TCRα / TCRβ. The specific experiments are as follows: 1. Light-heavy chain mismatch experiment when the four chains of a bispecific antibody are co-expressed The four-chain bispecific antibodies HJ-1 and HJ-2 were constructed by replacing the CH1 / CL of H0 or J1 with TCRα / TCRβ, and the four-chain bispecific antibodies HJ-3 and HJ-4 were constructed by replacing the CH1 / CL of H0 or J1 with Titin-T chain / Obscurin-O chain. The structural diagrams of the bispecific antibodies are shown in Figure 12, and the full-length sequences of the bispecific antibodies are shown in Table 30:

[0285] [Table 30-1] [Table 30-2] [Table 30-3] Note: In the sequences, the bolded parts are the Titin-T chain / Obscurin-O chain parts in the TCR sequence, the dotted parts are the Fc or CL constant region parts, the single underlined parts are the variable region parts, and the dashed underlined parts are the linker L1.

[0286] J1 heavy chain: [ka] J1 light chain: [ka] J1 heavy chain variable region: [ka] J1 light chain variable region: [ka] The four chains, HJ-1, HJ-2, HJ-3, and HJ-4, were co-transfected into cells and expressed, and the expression products were subjected to mass spectrometry (see Example 3 of the present disclosure for the method) to detect the presence of mismatched light-heavy chain molecules. The experimental results are shown in Figures 13A-13D and Table 31.

[0287] [Table 31] Note: In the table, "○" indicates that the molecule in question was detected, "×" indicates that the molecule in question was not detected, "HJ(1+2+3+4)" indicates a bispecific antibody consisting of four chains whose corresponding numbers to the left are 1, 2, 3, and 4, "mismatch molecule 1(1+2+3+3)" indicates mismatch molecule 1 consisting of four chains whose corresponding numbers to the left are 1, 2, 3, and 3, and "mismatch molecule 2(1+2+4+4)" indicates mismatch molecule 2 consisting of four chains whose corresponding numbers to the left are 1, 2, 4, and 4.

[0288] Experimental results showed that when the four chains of a bispecific antibody in which CH1 / CL are replaced with TCRβ / TCRα are co-transfected and expressed, mismatches occur in the light and heavy chains. In contrast, no mismatches occur in the light and heavy chains of bispecific antibodies in which CH1 / CL are replaced with Titin-T chain / Obscurin-O chain. This demonstrates that the bispecific antibody disclosed herein in which CH1 / CL is replaced with Titin-T chain / Obscurin-O chain has an excellent ability to reduce light and heavy chain mismatches.

[0289] Experiments with two- and three-chain co-expressed bispecific antibodies In this test, three chains (one heavy chain in which CH1 was replaced with TCRβ or Titin-T chain, one heavy chain in which CH1 was not replaced, and one wild-type light chain (VL-CL) in which CL was not replaced; specific sequences are shown in Table 30) were co-transfected into cells and expressed. The expression products were then subjected to mass spectrometry analysis (see Test Example 3 of the present disclosure for the method) and antibody purity (SEC) was detected (see Test Example 2 of the present disclosure for the method) to verify whether the wild-type light chain (VL-CL) combined with the heavy chain in which CH1 was replaced with TCRβ or Titin-T chain to form a mismatch molecule. The experimental results are shown in Figures 14A, 14B, 15A, 15B, and Table 32.

[0290] [Table 32] Note: In the table, "○" indicates that the molecule was detected, "×" indicates that the molecule was not detected, "molecule (1+2+3)" indicates a molecule consisting of three chains with the corresponding numbers 1, 2, and 3 on the left, and "mismatch molecule (1+2+3+3)" indicates a mismatch molecule consisting of four chains with the corresponding numbers 1, 2, 3, and 3 on the left.

[0291] Experimental results showed that VL-CL easily forms mismatched molecules with two identical light chains by binding to a heavy chain in which CH1 has been replaced with TCRβ, whereas VL-CL light chains do not form mismatched molecules with two identical light chains by binding to a heavy chain in which CH1 has been replaced with Titin-T chain. This indirectly demonstrates that the bispecific antibody disclosed herein in which the heavy chain CH1 has been replaced with Titin-T chain has a better ability to reduce light-heavy chain mismatches than a bispecific antibody in which the heavy chain CH1 has been replaced with TCRβ.

[0292] Test Example 11: CH1 / CL-replaced (FabV)2-IgG bispecific antibody 1. Construction of PDL1-TIGIT bispecific antibody with CH1 / CL replacement Using the anti-TIGIT antibody (antibody name: h1708-04) disclosed in International Patent Application WO2019062832A1 and the anti-PDL1 antibody (antibody name: h1831K) disclosed in International Patent Application WO2020177733A1, a pair of (FabV)2-IgG anti-PDL1-TIGIT bispecific antibodies POT (see FIG. 17 for its structural diagram) was constructed in which CH1 / CL was replaced with Titin-T chain / Obscurin-O chain (T.10 / O.24). POT comprises two identical light chains 1, two identical light chains 2, and two identical heavy chains. The amino acid sequences of the polypeptide chains of POT are as follows: POT light chain 1: [ka] (Note: The single underlined portion is the light chain variable region VL1 portion (similar to VL of h1831K), the dashed underlined portion is the linker portion, and the dotted line portion is the T.10 portion.) POT light chain 2: [ka] (Note: The single underlined portion is the light chain variable region VL2 portion (similar to VL in h1708-04), and the wavy line portion is the light chain constant region (similar to CL in h1708-04).) POT heavy chain: [ka] (Note: The single-underlined portion is the heavy chain variable region VH1 portion (similar to the VH of h1831K), the double-underlined portion is the heavy chain variable region VH2 portion (similar to the VH of h1708-04), the dotted portion is the O.24 portion, the dashed underlined portion is the linker portion, and the wavy portion is the heavy chain constant region portion (similar to the heavy chain constant region of h1708-04)) The sequences of h1831K (abbreviated as P-IgG1) and h1708-04 (abbreviated as T-IgG1) are as follows: Light chain of h1708-04: [ka] Heavy chain of h1708-04: [ka] h1831K light chain: [ka] h1831K heavy chain: [ka] Note: In the above sequences, the portions indicated by single underlines are variable regions, and the portions indicated by wavy lines are constant regions.

[0293] Additionally, the sequences of the variable regions and CDRs (see Table 33) of h1708-04 and h1831K are as follows: h1708-04 light chain variable region: [ka] Heavy chain variable region of h1708-04: [ka] h1831K light chain variable region: [ka] Heavy chain variable region of h1831K: [ka]

[0294] [Table 33] Note: The sequences of the CDR regions are determined according to the Kabat numbering convention.

[0295] 2. Detection of the binding ability of PDL1-TIGIT bispecific antibodies with CH1 / CL replacement to PDL1 and TIGIT The affinity of the presently disclosed dual antibody molecule POT and human PDL1-his (hPDL1, Cat. No. 10084-H08H, SB) and human TIGIT-his (hTIGIT, Cat. No. 10917-H08H, SB) was measured using a Biacore T200 instrument. IgG was affinity-captured using a Protein A biosensor chip (Cat. No. 29127556, GE). After affinity capture of IgG, a high concentration of antigen 1 (100 nM hPDL1 or 100 nM hTIGIT) was injected over the chip surface for 180 s to saturate the antibody binding site for antigen 1. Antigen 2 (hTIGIT or hPDL1) was then injected, and the reaction signal was detected in real time using the Biacore T200 instrument to obtain binding and dissociation curves. After each experimental cycle, the biosensor chip was regenerated by washing with 10 mM Gly-HCl, pH 1.5. The data fitting model was a 1:1 model. The results are shown in Table 34 below:

[0296] [Table 34] The detection results showed that the (FabV)2-IgG double antibody POT, in which CH1 / CL was replaced with Titin-T chain / Obscurin-O chain, had excellent binding ability to hPDL1 and hTIGIT, and the binding of POT to one antigen did not affect the binding to another antigen.

[0297] 3. Detection of the inhibitory effect of CH1 / CL-substituted PDL1-TIGIT bispecific antibody on the binding of PD-L1 to PD-1 and the binding of TIGIT to CD155 The following in vitro cell experiments can measure the inhibitory effect of the test antibody on the binding of PD-L1 to PD-1 and the binding of TIGIT to CD155, and the activity can be measured using EC 50On the first day of the experiment, CHOK1 / PD-L1 cells (Promega, CS187108) stably expressing human CD155 were seeded into a 96-well plate at a density of 4000 cells / well in complete F-12 Nutrient Mixture (Gibco, 11765-054) medium containing 10% FBS (Gibco, 10099-141) and antibiotics, and 100 μL of cell suspension was added to each well and cultured overnight in a 37°C, 5% CO2 cell incubator. The next day, the medium in the culture plate was aspirated, and 40 μL of gradient-diluted antibody to be tested, prepared in RPMI (Gibco, 11875119) containing 2% FBS (Gibco, 10099-141), and 40 μL of Jurkat / PD-1 / NFAT-luc2 cells (Promega, CS187102) stably expressing human TIGIT and human CD226, resuspended in the same medium, were added to each well at 50,000 cells / well. The final antibody concentration was 100 nM, with nine concentration points diluted 3-fold. No-antibody control wells and no-cell control wells were also included, and the plate was placed in a cell incubator at 37°C and 5% CO2 for 6 hours. After 6 hours, the 96-well cell culture plate was removed, and each well was filled with Bio-Glo TM After adding 40 μL of substrate from Luciferase Assay (Promega, G7573) and allowing to stand at room temperature for 10 minutes, the luminescence signal was read using a microplate reader (PerkinElmer, VICTOR 3). The relative activation level of each antibody concentration was calculated from the control wells, and curve fitting was performed using GraphPad Prism based on the logarithmic concentration of the antibody and the relative activation level to obtain the EC 50 The value was calculated.

[0298] The results are shown in Figure 18. The experimental results show that the PDL1-TIGIT bispecific antibody with CH1 / CL replacement has additive blocking activity compared to the PDL1 monoclonal antibody and TIGIT monoclonal antibody.

[0299] 4. Activation of antibodies against tuberculin-stimulated peripheral blood mononuclear cells The following in vitro cell experiment can measure the activating effect of the test antibody on IFN-γ secretion by peripheral blood mononuclear cells (PBMC) stimulated with tuberculin, and the activity is shown to be EC 50 On the first day of the experiment, PBMCs were isolated from fresh healthy human blood using SepMate™-50 separation tubes (STEMCELL, 86450) according to the manufacturer's instructions, and the cells were diluted to 4 × 10 in RPMI medium (Gibco, 11875119) containing 10% FBS (Gibco, 10099-141) and 800-fold diluted tuberculin (Synbiotics, 97-8800). 6 Cells were seeded into 6-well plates at a density of 1 x 10 cells / well, and 2 mL of cell suspension was added to each well and cultured at 37°C in a 5% CO2 cell incubator for 5 days. On day 4, human CD155 / PVR protein (Acro, CD5-H5223) was diluted to 2.5 μg / mL in PBS and added at 100 μL / well to a 96-well cell culture plate. The plate was then placed in a 4°C refrigerator overnight. On day 5, the protein-coated 96-well cell culture plate was washed twice with PBS, and tuberculin-stimulated PBMCs were harvested and cultured at 1 x 10 cells in RPMI medium containing 10% FBS. 5Cells were seeded into a 96-well plate at a density of 10 cells / well, with 90 μL of cell suspension per well. Then, 10 μL of the antibody to be tested, diluted in PBS, was added to each well, resulting in nine concentration points, each diluted 3-fold starting from 200 nM. A control well without antibody was included. The well plate was then placed in a cell incubator at 37°C and 5% CO2 for 3 days. On day 8, the 96-well cell culture plate was centrifuged at 250 g for 5 minutes, and the supernatant was transferred to a new 96-well plate. After diluting the supernatant 20-fold, the IFN-γ content in the supernatant was assayed using a Human IFN-γ ELISA kit (Xinbosheng, EHC102g.96.10). The relative activation levels of the antibody-treated groups were calculated from the control wells, and curve fitting was performed using Graphpad Prism 5 software based on the antibody concentration and the corresponding relative activation levels to obtain the EC. 50 The value was calculated.

[0300] The experimental results are shown in Figure 19, which show that the PDL1-TIGIT bispecific antibody with CH1 / CL replacement can block immunosuppression and promote IFN-γ secretion better than the monoclonal antibody.

[0301] V. In vivo efficacy of PDL1-TIGIT bispecific antibody with CH1 / CL replacement In this experiment, MC38-HL1 cells stably transfected with human PD-L1 were inoculated into human PD-1-TIGIT double transgenic mice, and after tumor formation, the mice were divided into groups to compare the efficacy of combined administration of PD-L1 and TIGIT with that of the PDL1-TIGIT bispecific antibody.

[0302] PD-1-TIGIT double transgenic mice were purchased from Biocytogen. They were 6-8 weeks old, 82 female mice, weighing approximately 16-18 g. They were housed in cages of 5 mice in an SPF-grade environment at a temperature of 20-25°C and humidity of 40-60%. License number: SCXK (Kyoto) 2015-0008. They were allowed to acclimate to the environment for approximately 10 days. MC38-HL1 cells (2.0 × 10 5100 μL of the cells was inoculated subcutaneously into the right flank of 82 hPD-1 / TIGIT double transgenic mice. After tumor formation (~110 mm 3 Mice with tumors that were too large or too small were excluded, and the mice were randomly divided into five groups based on tumor volume: the negative control C25-IgG1 (unrelated to the target IgG1 protein) group, the P-IgG1 + T-IgG1 group, the POT group, the P-IgG1 group, and the T-IgG1 group, with eight mice per group. Details are shown in Table 35. On the first day of the experiment, the antibodies were injected intraperitoneally once a week for three weeks. Tumor volume was measured twice a week, and mice were weighed and recorded.

[0303] [Table 35] Note: In the table, ip means intraperitoneal injection, and qw means once-weekly administration. The data were recorded using Excel statistical software, plotted using GraphPad Prism software, and statistically analyzed by two-way ANOVA, one-way ANOVA, and t-test, so that the mean value was calculated as avg, the SD value was calculated as STDEV, and the SEM value was calculated as STDEV / SQRT (number of animals in each group).

[0304] Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 The relative tumor growth rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment and control groups at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0305] The experimental results are shown in Table 36 and FIG.

[0306] [Table 36]

[0307] According to the experimental results, on day 22, compared with the negative control C25-IgG1 group, the tumor inhibition rate in the 9 mg / kg P-IgG1 and 9 mg / kg T-IgG1 co-administration group was 43%, and the tumor inhibition rate in the 5 mg / kg POT administration group reached 55%, demonstrating significant inhibition of MC38-HL1 tumor growth and demonstrably superior tumor inhibition effects to those in the P-IgG1 or T-IgG1 single-administration groups.

Claims

1. A dimerized polypeptide comprising a Titin-T chain and an Obscurin-O chain, or a Titin-T chain and an Obscurin-Like-O chain, i) the Titin-T chain is a variant of SEQ ID NO: 32, which variant has an amino acid residue substitution at one or more positions selected from positions 60 and 64 compared to SEQ ID NO: 32; and / or ii) the Obscurin O chain is a variant of SEQ ID NO: 33, which variant has amino acid residue substitutions at one or more positions selected from 13, 32, 48, 66, 82, and 93 compared to SEQ ID NO: 33; In addition, the conditions are as follows: a) if the variant does not have an amino acid residue substitution at position 13, 48, 66, 82, or 93 and has an amino acid residue substitution at position 32, the amino acid substitution at position 32 is not 32P; b) the variant does not have an amino acid residue substitution at positions 32, 48, 66, 82, or 93, and if it has an amino acid residue substitution at position 13, the amino acid substitution at position 13 is not 13Y; and c) if the variant does not have an amino acid residue substitution at positions 48, 66, 82, or 93, and has amino acid residue substitutions at positions 13 and 32, the amino acid residue substitution at position 13 is not 13Y and the amino acid residue substitution at position 32 is not 32P; Dimerizing polypeptides.

2. the variant of SEQ ID NO: 32 has one or more amino acid residue substitutions selected from 60S and 64T, and / or the variant of SEQ ID NO: 33 has one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H and 93C; Preferably, the variant of SEQ ID NO: 32 has 60S and 64T amino acid residue substitutions, and / or the variant of SEQ ID NO: 33 has any of a) to c), i.e. a) 32F and 48V, b) 13S, 32F, 48V and 82H, and c) 13S, 32F, 48V, 66C, 82H and 93C, having any one amino acid residue substitution selected from The dimerized polypeptide of claim 1.

3. The variant of SEQ ID NO: 32 further has an amino acid residue substitution at one or more positions selected from 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84, compared to SEQ ID NO: 32; Preferably, the variant of SEQ ID NO: 32 further comprises one or more amino acid residue substitutions compared to SEQ ID NO: 32 selected from 3W, 8C, 11I, 13L, 20C, 22M / 22C, 25S, 26C, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 66S / 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D and 84L; More preferably, the variant of SEQ ID NO: 32 has the following characteristics compared to SEQ ID NO: 32: a) 8C, 25S and 39T, b) 20C, 25S and 39T; c) 25S, 26C and 39T, d) 22C, 25S and 39T, e) 8C, 25S, 39T, 66S and 77S, f) 8C, 25S, 39T, 66K, 70R, 79T and 81R, g) 3W, 8C, 11I, 13L, 22M, 25S, 39T and 82M, h) 8C, 11I, 25S, 39T, 66K, 79T and 81R, i) 8C, 25S, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 75V, 83D and 84L, j) 8C, 25S, 39T, 47E, 49G, 56S, 58E and 75V; k) 8C, 25S, 39T, 56S, 58E and 75V, and l) 8C, 25S, 39T, 56S, 58E, 66S and 77S, Further comprising any one amino acid residue substitution selected from Most preferably, the variant of SEQ ID NO: 32 has the following structure compared to SEQ ID NO: 32: A) to C), i.e. A) 8C, 11I, 25S, 39T, 60S, 64T, 66K, 79T and 81R, B) 8C, 11I, 20C, 25S, 39T, 60S, 64T, 66K, 79T and 81R, C) 8C, 11I, 25S, 26C, 39T, 60S, 64T, 66K, 79T and 81R having any one amino acid residue substitution selected from The dimerized polypeptide of claim 1.

4. The variant of SEQ ID NO: 33 further has an amino acid residue substitution at one or more positions selected from 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 76, 88, 89, 92, 94, and 97, compared to SEQ ID NO: 33; Preferably, The variant of SEQ ID NO: 33 further has one or more amino acid residue substitutions selected from 2E, 3C, 7K / 7R, 9C, 11L, 12S, 13Y, 14T, 17E, 20L, 22M / 22S, 25S, 30D, 32P, 34E, 36T, 41K, 42L, 44I, 45T, 53L, 58V, 62E / 62K / 62H, 67Q / 67T, 69S, 76S, 88C, 89L, 92E, 94G and 97G compared to SEQ ID NO: 33; More preferably, the variant of SEQ ID NO: 33 has the following amino acids A) to R) compared to SEQ ID NO: 33, i.e. A) 88C, B) 3C, C) 9C, D) 25S, 76S and 88C, E) 25S, 76S and 3C, F) 25S, 76S and 9C, G) 7K, 25S, 62K, 76S and 88C, H) 7K, 25S, 62H, 76S and 88C, I) 7R, 25S, 62K, 76S and 88C, J) 7R, 25S, 62H, 76S and 88C, K) 11L, 25S, 62K, 76S and 88C, L) 11L, 25S, 62H, 76S and 88C, M) 12S, 13Y, 14T, 22S, 25S, 62K, 76S and 88C, N) 2E, 11L, 17E, 25S, 30D, 32P, 34E, 36T, 44I, 45T, 58V, 62E, 67Q, 69S, 76S, 88C and 97G, O) 11L, 20L, 22M, 25S, 53L, 62K, 76S and 88C, P) 11L, 25S, 41K, 45T, 62K, 67Q, 69S, 76S, 88C and 89L, Q) 11L, 25S, 42L, 45T, 62K, 67T, 69S, 76S, 88C, 92E and 94G, R) 11L, 12S, 13Y, 22S, 25S, 42L, 45T, 62K, 67Q, 69S, 76S, 88C, 92E and 94G, Further having any one amino acid residue substitution selected from Most preferably, the variant of SEQ ID NO: 33 has the following structure compared to SEQ ID NO: 33: a) 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 88C and 89L, b) 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C and 89L, c) 3C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C and 89L, d) 9C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 76S, 82H, 88C and 89L, e) 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, f) 3C, 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, g) 9C, 13S, 32F, 41K, 45T, 48V, 62K, 67Q, 69S, 82H, 88C and 89L, h) 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C, i) 3C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C, and j) 9C, 13S, 25S, 32F, 41K, 45T, 48V, 62K, 66C, 67Q, 69S, 76S, 82H, 88C, 89L and 93C, having any one amino acid residue substitution selected from The dimerized polypeptide of claim 1.

5. the Obscurin-Like-O chain is SEQ ID NO: 34 or a variant thereof, and the variant of SEQ ID NO: 34 has amino acid residue substitutions at one or more sites selected from positions 6, 26, 74, 77, 84, and 86; Preferably, the variant of SEQ ID NO: 34 has one or more amino acid residue substitutions selected from 6E, 26S, 74C, 77S, 84C and 86C; More preferably, the variant of SEQ ID NO: 34 is any of A) to F), i.e. A) 6E and 74C, B) 6E and 84C, C) 6E and 86C, D) 6E, 26S, 77S and 74C, E) 6E, 26S, 77S and 84C, and F) 6E, 26S, 77S and 86C, having any one amino acid residue substitution selected from The dimerized polypeptide of claim 1.

6. the Titin-T chain is a variant of SEQ ID NO: 32, 68 or 127, which has one or more amino acid residue substitutions selected from 60S and 64T; the Obscurin-O chain is a variant of SEQ ID NO: 33, 80 or 128, which has one or more amino acid residue substitutions selected from 13S, 32F, 48V, 66C, 82H and 93C; Preferably, the Titin-T chain has at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 129 to 131, and the Obscurin-O chain has at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 132 to 141; More preferably, the amino acid sequence of the Titin-T chain is represented by any one selected from SEQ ID NOs: 129 to 131, and the amino acid sequence of the Obscurin-O chain is represented by any one of SEQ ID NOs: 132 to 141. The dimerized polypeptide of claim 1.

7. An antigen-binding molecule comprising the dimerized polypeptide of claim 1.

8. a first antigen-binding portion, the first antigen-binding portion comprising a domain-modified Fab, the domain-modified Fab comprising a heavy chain variable region VH1, a light chain variable region VL1 and the dimerizing polypeptide, but not comprising a light chain constant region CL or a heavy chain constant region CH1, and the VH1 and VL1 are each linked to any one of the peptide chains in the dimerizing polypeptide via a linker; Preferably, the C-terminus of the VH1 is fused via a linker to the N-terminus of the Titin-T chain of the dimerizing polypeptide of claim 1, and the C-terminus of the VL1 is fused via a linker to the N-terminus of the Obscurin-O chain or Obscurin-Like-O chain of the dimerizing polypeptide of claim 1, or The C-terminus of the VL1 is fused via a linker to the N-terminus of the Titin-T chain of the dimerized polypeptide of claim 1, and the C-terminus of the VH1 is fused via a linker to the N-terminus of the Obscurin-O chain or Obscurin-Like-O chain of the dimerized polypeptide of claim 1. The antigen-binding molecule of claim 7.

9. a first antigen-binding portion, said first antigen-binding portion comprising: a. A peptide chain consisting of [VH1]-[Linker 1]-[Titin-T chain] in the order from the N-terminus to the C-terminus, and a peptide chain consisting of [VL1]-[Linker 2]-[Obscurin-O chain or Obscurin-Like-O chain] in the order from the N-terminus to the C-terminus, or b. A peptide chain consisting of [VH1]-[Linker 1]-[Obscurin-O chain or Obscurin-Like-O chain] in the order from the N-terminus to the C-terminus, and a peptide chain consisting of [VL1]-[Linker 2]-[Titin-T chain] in the order from the N-terminus to the C-terminus, The linker 1 and the linker 2 are the same or different, The Titin-T chain and the Obscurin-O chain or the Obscurin-Like-O chain are as defined in claim 1; Preferably, A) Linker 1 and Linker 2 are both (G ｘ S) ｙ a linker, wherein x is an integer selected from 1 to 5 and y is an integer selected from 0 to 6; or B) Linker 1 is a C-terminal truncated sequence of CH1, and Linker 2 is a C-terminal truncated sequence of CL; More preferably, A) Linker 1 is represented by SEQ ID NO: 173 and Linker 2 is represented by SEQ ID NO: 174, or B) Linker 1 and Linker 2 are both set forth in SEQ ID NO: 175; or C) Linker 1 and linker 2 are both represented by SEQ ID NO: 176; The antigen-binding molecule of claim 7.

10. The antigen-binding molecule further comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other; Preferably, the Fc region comprises one or more amino acid substitutions that reduce homodimerization, and / or the Fc region comprises one or more amino acid substitutions that can reduce binding of the Fc region to an Fc receptor; More preferably, the Fc1 has a knob structure based on knob-in-hole technology, and the Fc2 has a hole structure based on knob-in-hole technology, or the Fc2 has a knob structure based on knob-in-hole technology, and the Fc1 has a hole structure based on knob-in-hole technology; Most preferably, the sequence of Fc1 is set forth in SEQ ID NO: 177 and the sequence of Fc2 is set forth in SEQ ID NO: 178, or the sequence of Fc1 is set forth in SEQ ID NO: 178 and the sequence of Fc2 is set forth in SEQ ID NO:

177. The antigen-binding molecule of claim 7.

11. The antibody comprises a first antigen-binding portion and a second antigen-binding portion, wherein the second antigen-binding portion comprises a heavy chain variable region VH2 and a light chain variable region VL2, and the first antigen-binding portion and the second antigen-binding portion bind to different antigens or different epitopes on the same antigen; Preferably, the second antigen-binding portion comprises a Fab. The antigen-binding molecule of claim 7.

12. a first heavy chain, a first light chain, a second heavy chain, and a second light chain, a. the first heavy chain is, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Titin-T chain]-[Linker 3]-[Fc1]; the first light chain is [VL1]-[linker 2]-[Obscurin-O chain or Obscurin-Like-O chain] in this order from the N-terminus to the C-terminus, the second heavy chain is [VH2]-[CH1]-[Fc2] in order from the N-terminus to the C-terminus; the second light chain is, from N-terminus to C-terminus, [VL2]-[CL]; or b. the first heavy chain is, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain or Obscurin-Like-O chain]-[Linker 3]-[Fc1]; the first light chain is [VL1]-[linker 2]-[Titin-T chain] in this order from the N-terminus to the C-terminus, the second heavy chain is [VH2]-[CH1]-[Fc2] in order from the N-terminus to the C-terminus; the second light chain is [VL2]-[CL] in order from the N-terminus to the C-terminus, The linker 1, the linker 2 and the linker 3 are the same or different, Preferably, the Fc1 and Fc2 each independently comprise one or more amino acid substitutions that reduce homodimerization; More preferably, A) Linker 1, Linker 2 and Linker 3 are all (G ｘ S) ｙ wherein x is an integer selected from 1 to 5 and y is an integer selected from 0 to 6; or B) Linker 1 is a C-terminal truncated sequence of CH1, linker 2 is a C-terminal truncated sequence of CL, and linker 3 is (G ｘ S) ｙ wherein x is an integer selected from 1 to 5, and y is an integer selected from 0 to 6; Most preferably, A) Linker 1 is represented by SEQ ID NO: 173, Linker 2 is represented by SEQ ID NO: 174, and Linker 3 is a bond; or B) Linker 1 and Linker 2 are both set forth in SEQ ID NO: 175, and Linker 3 is a bond; or C) Linker 1 and Linker 2 are both represented by SEQ ID NO: 176, and Linker 3 is a bond; The antigen-binding molecule of claim 11.

13. (I) the antigen-binding molecule is capable of binding to NGF and RANKL; Preferably, The antigen-binding molecule comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, the first heavy chain is, from N-terminus to C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[Fc1]; the first light chain is, from the N-terminus to the C-terminus, [VL1]-[Linker 2]-[Titin-T chain]; the second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; and the second light chain is, from N-terminus to C-terminus, [VL2]-[CL]; wherein VH1 and VL1 form a first antigen-binding portion that binds to NGF, and VH2 and VL2 form a second antigen-binding portion that binds to RANKL; or VH1 and VL1 form a first antigen-binding portion that binds to RANKL, and VH2 and VL2 form a second antigen-binding portion that binds to NGF; More preferably, the sequence of the VH1 is set forth in SEQ ID NO:26, the sequence of the VL1 is set forth in SEQ ID NO:27, the sequence of the VH2 is set forth in SEQ ID NO:24, and the sequence of the VL2 is set forth in SEQ ID NO:25; or The sequence of the VH1 is represented by SEQ ID NO: 24, the sequence of the VL1 is represented by SEQ ID NO: 25, the sequence of the VH2 is represented by SEQ ID NO: 26, and the sequence of the VL2 is represented by SEQ ID NO: 27; The sequence of the Obscurin O chain is represented by any one selected from SEQ ID NOs: 132 to 141, and the sequence of the Titin T chain is represented by any one selected from SEQ ID NOs: 129 to 131; Most preferably, the sequence of Fc1 is represented by SEQ ID NO: 177, the sequence of Fc2 is represented by SEQ ID NO: 178, the sequence of CH1 is represented by SEQ ID NO: 179, the sequence of CL is represented by SEQ ID NO: 4, the linker 3 is a bond, and the linker 1 and linker 2 are selected from a) linker 1 and linker 2 are both represented by SEQ ID NO: 175, or b) linker 1 is represented by SEQ ID NO: 173, and linker 2 is represented by SEQ ID NO: 174; (II) the antigen-binding molecule is capable of binding to PDL1 and CTLA4; Preferably, The antigen-binding molecule comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, the first heavy chain is, from N-terminus to C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[Fc1]; the first light chain is, from the N-terminus to the C-terminus, [VL1]-[Linker 2]-[Titin-T chain]; the second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; and The second light chain is [VL2]-[CL] from the N-terminus to the C-terminus, VH1 and VL1 form a first antigen-binding portion that binds to PDL1, and VH2 and VL2 form a second antigen-binding portion that binds to CTLA4; or VH1 and VL1 form a first antigen-binding portion that binds to CTLA4, and VH2 and VL2 form a second antigen-binding portion that binds to PDL1; More preferably, the sequence of the VH1 is set forth in SEQ ID NO: 156, the sequence of the VL1 is set forth in SEQ ID NO: 155, the sequence of the VH2 is set forth in SEQ ID NO: 169, and the sequence of the VL2 is set forth in SEQ ID NO: 170; or The sequence of the VH1 is represented by SEQ ID NO: 169, the sequence of the VL1 is represented by SEQ ID NO: 170, the sequence of the VH2 is represented by SEQ ID NO: 156, and the sequence of the VL2 is represented by SEQ ID NO: 155; The sequence of the Obscurin-O chain is represented by any one selected from SEQ ID NOs: 132 to 141, and the sequence of the Titin-T chain is represented by any one selected from SEQ ID NOs: 129 to 131; Most preferably, the sequence of Fc1 is represented by SEQ ID NO: 178, the sequence of Fc2 is represented by SEQ ID NO: 177, the sequence of CH1 is represented by SEQ ID NO: 179, the sequence of CL is represented by SEQ ID NO: 4, the linker 3 is a bond, and the linker 1 and linker 2 are selected from a) linker 1 and linker 2 are both represented by SEQ ID NO: 175, or b) linker 1 is represented by SEQ ID NO: 173 and linker 2 is represented by SEQ ID NO: 174, or (III) the antigen-binding molecule is capable of binding to IL5 and TSLP; Preferably, The antigen-binding molecule comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, the first heavy chain is, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Titin-T chain]-[Linker 3]-[Fc1]; the first light chain is, from the N-terminus to the C-terminus, [VL1]-[linker 2]-[Obscurin-O chain]; the second heavy chain is, from N-terminus to C-terminus, [VH2]-[CH1]-[Fc2]; and The second light chain is [VL2]-[CL] from the N-terminus to the C-terminus, VH1 and VL1 form a first antigen-binding portion that binds IL5, and VH2 and VL2 form a second antigen-binding portion that binds TSLP; or VH1 and VL1 form a first antigen-binding portion that binds TSLP, and VH2 and VL2 form a second antigen-binding portion that binds IL5; More preferably, the sequence of the VH1 is set forth in SEQ ID NO: 16, the sequence of the VL1 is set forth in SEQ ID NO: 17, the sequence of the VH2 is set forth in SEQ ID NO: 171, and the sequence of the VL2 is set forth in SEQ ID NO: 172; or The sequence of the VH1 is represented by SEQ ID NO: 171, the sequence of the VL1 is represented by SEQ ID NO: 172, the sequence of the VH2 is represented by SEQ ID NO: 16, and the sequence of the VL2 is represented by SEQ ID NO: 17; The sequence of the Obscurin O chain is represented by any one selected from SEQ ID NOs: 132 to 141, and the sequence of the Titin T chain is represented by any one selected from SEQ ID NOs: 129 to 131; Most preferably, The sequence of the Fc1 is represented by SEQ ID NO: 178, the sequence of the Fc2 is represented by SEQ ID NO: 177, the sequence of the CH1 is represented by SEQ ID NO: 179, the sequence of the CL is represented by SEQ ID NO: 4, the linker 3 is a bond, and the linker 1 and linker 2 are selected from a) linker 1 and linker 2 whose sequences are represented by SEQ ID NO: 175, or b) linker 1 whose sequence is represented by SEQ ID NO: 173 and linker 2 whose sequence is represented by SEQ ID NO: 174; The antigen-binding molecule of claim 12.

14. a. a first heavy chain which is, from N-terminus to C-terminus, [VH1]-[Linker 1]-[Titin-T chain]-[Linker 3]-[VH2]-[CH1]-[Fc1]; a second heavy chain which is, from N-terminus to C-terminus, [VH1]-[Linker1]-[Titin-T chain]-[Linker3]-[VH2]-[CH1]-[Fc2]; A first light chain consisting of, from the N-terminus to the C-terminus, [VL1]-[Linker 2]-[Obscurin-O chain or Obscurin-Like-O chain]; and a second light chain that is, from N-terminus to C-terminus, [VL2]-[CL]; or b. A first heavy chain which is, from N-terminus to C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain or Obscurin-Like-O chain]-[Linker 3]-[VH2]-[CH1]-[Fc1]; a second heavy chain which is, from the N-terminus to the C-terminus, [VH1]-[Linker 1]-[Obscurin-O chain or Obscurin-Like-O chain]-[Linker 3]-[VH2]-[CH1]-[Fc2]; A first light chain consisting of, from the N-terminus to the C-terminus, [VL1]-[Linker 2]-[Titin-T chain]; and a second light chain which is, in N-terminal to C-terminal order, [VL2]-[CL]; The linker 1, the linker 2 and the linker 3 are the same or different, Preferably, the Fc1 and Fc2 are the same, or the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization; More preferably, A) Linker 1, Linker 2 and Linker 3 are all (G ｘ S) ｙ a linker, wherein x is an integer selected from 1 to 5 and y is an integer selected from 0 to 6, preferably both of which are represented by SEQ ID NO: 175 or SEQ ID NO: 176; or B) Linker 1 is a C-terminal truncated sequence of CH1, preferably represented by SEQ ID NO: 173; Linker 2 is a C-terminal truncated sequence of CL, preferably represented by SEQ ID NO: 174; Linker 3 is (G ｘ S) ｙ A linker, wherein x is an integer selected from 1 to 5 and y is an integer selected from 0 to 6, preferably represented by SEQ ID NO: 175 or SEQ ID NO: 176; Most preferably, The antigen-binding molecule is capable of binding to PDL1 and TIGIT. The antigen-binding molecule of claim 11.

15. The antibody comprises a first antigen-binding portion capable of specifically binding to PDL1 and a second antigen-binding portion capable of specifically binding to TIGIT, wherein the first antigen-binding portion comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding portion comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein: said VH1 comprises an HCDR1, an HCDR2 and an HCDR3 set forth in SEQ ID NO: 163, SEQ ID NO: 164 and SEQ ID NO: 165, respectively; said VL1 comprises an LCDR1, an LCDR2 and an LCDR3 set forth in SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168, respectively; and / or The VH2 comprises HCDR1, HCDR2 and HCDR3 represented by SEQ ID NOs: 157, 158 and 159, respectively; the VL2 comprises LCDR1, LCDR2 and LCDR3 represented by SEQ ID NOs: 160, 161 and 162, respectively; Preferably, the sequence of said VH1 is set forth in SEQ ID NO: 156 or has at least 90% sequence identity to SEQ ID NO: 156, and the sequence of said VL1 is set forth in SEQ ID NO: 155 or has at least 90% sequence identity to SEQ ID NO: 155; and / or the sequence of said VH2 is set forth in SEQ ID NO: 154 or has at least 90% sequence identity to SEQ ID NO: 154, and the sequence of said VL2 is set forth in SEQ ID NO: 153 or has at least 90% sequence identity to SEQ ID NO: 153; More preferably, the antigen-binding molecule is a heavy chain whose sequence is set forth in SEQ ID NO: 148 or has at least 90% sequence identity to SEQ ID NO: 148; a first light chain whose sequence is set forth in SEQ ID NO: 146 or has at least 90% sequence identity to SEQ ID NO: 146; a second light chain having a sequence set forth in SEQ ID NO: 147 or having at least 90% sequence identity to SEQ ID NO: 147; Antigen-binding molecules.

16. An antibody in which the heavy chain constant region CH1 and the light chain constant region CL are substituted with the dimerizing polypeptide of claim 1, Preferably, the heavy chain constant region CH1 is substituted with Titin-T chain and the light chain constant region CL is substituted with Obscurin-O chain, or the light chain constant region CL is substituted with Titin-T chain and the heavy chain constant region CH1 is substituted with Obscurin-O chain. Domain-engineered antibodies.

17. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 7 to 15 or the domain-modified antibody of claim 16, and one or more pharmaceutically acceptable vectors, diluents or excipients.

18. Reducing light chain / heavy chain mismatches during the preparation of multispecific antibodies; Preferably, use of a dimerizing polypeptide according to any one of claims 1 to 6 in reducing light chain / heavy chain mismatches during the preparation of bispecific antibodies.

19. A nucleic acid molecule encoding the dimerized polypeptide of any one of claims 1 to 6, the antigen-binding molecule of any one of claims 7 to 15, or the domain-modified antibody of claim 16.

20. 20. A host cell comprising the nucleic acid molecule of claim 19.

21. A method for preparing the dimerized polypeptide of any one of claims 1 to 6, the antigen-binding molecule of any one of claims 7 to 15, or the domain-modified antibody of claim 16, the method comprising the steps of culturing the host cell of claim 20, and then purifying and recovering the dimerized polypeptide, the antigen-binding molecule, or the domain-modified antibody.

22. Use of the antigen-binding molecule of any one of claims 7 to 15 or the domain-modified antibody of claim 16 in the preparation of a medicament for treating or preventing a disease or condition.

23. The pharmaceutical composition of claim 17 for treating or preventing a disease or condition.

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