RU2847355C1 - New polypeeptide complex - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: new antigen-binding polypeptide complexes are proposed, containing the T-chain of titin and the O-chain of obscurin or the T-chain of titin and the O-chain of an obscurin-like protein, functionally linked to the variable domains of heavy and light chains. Bispecific antibodies are also proposed, in which one of the CH1 domains and one of the CL domains of the antibody are replaced, with the CH1/CL domains replaced by the titin T-chain/obscurin O-chain or the titin T-chain/obscurin-like protein O-chain.

EFFECT: effective prevention of the formation of non-homologous cross-linking molecules between light and heavy chains of IgG-like bispecific antibodies.

27 cl, 21 dwg, 20 tbl, 15 ex

Description

This application claims priority from a Chinese patent application (Application No. CN202010020878.7) filed on January 9, 2020.

AREA OF TECHNOLOGY

The present invention relates to the field of pharmaceutical preparations based on antibodies and, in particular, containing a new antibody in which the structures of CH1 (constant domain 1 of the heavy chain) and CL (constant domain of the light chain) are substituted, and its use.

PRIOR ART

The information set forth in this document is intended to provide general information relating to the present invention and may not necessarily constitute prior art.

With the constant advancement of humanized antibody technology, monoclonal antibodies have been rapidly developed in recent years. Various monoclonal antibodies have been used to treat serious conditions, such as malignant tumors and autoimmune diseases. However, tumor evasion of the immune response often involves multiple mechanisms, and a single monoclonal antibody may bind to only one specific target, which can significantly reduce its therapeutic effect.

A bispecific antibody (BsAb) is an artificial antibody created by combining antibodies targeting two different antigens or two different epitopes using genetic engineering. Unlike a monoclonal antibody, a bispecific antibody has the ability to simultaneously target two different epitopes and can perform specific biological functions, such as immune cell recruitment, receptor costimulation or coinhibition, and the neutralization of multivalent viruses. It is expected to achieve superior clinical therapeutic effects compared to single monoclonal antibodies, even when used in combination.

A wide range of recombinant bispecific antibodies have been developed, such as tetravalent bispecific antibodies created by fusing, for example, an IgG (immunoglobulin G) antibody with a single-chain domain (see Coloma, M.J., et al., Nature Biotech. 15 (1997) 159–163; Morrison, S.L., Nature Biotech. 25 (2007) 1233–1234). Other bispecific forms exist, such as DVD-Ig (DVD – dual variable domain antibody), CrossMab, and BiTE (bispecific T cell activator) (Spiess et al., Molecular Immunology, 67 (2), pp. 95–106 (2015)).

Various structural models for bispecific antibodies have also been disclosed, such as the introduction of a knob-in-hole mutation into the Fc region (Ridgway et al., Protein Engineering, 9 (7), pp. 617-21 (1996)); introduction of an electrostatic design (Gunasekaran et al., Journal of Biological Chemistry, 285, (25), pp. 19637-19646, (2010)), or introduction of a negative state design (Kreudenstein et al., mAbs, 5 (5), pp. 646-654 (2013); Leaver-Fay et al., Structure, 24 (4), pp. 641-651 (2016)), exchange 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 the constant region of TCR (T cell receptor) (WO 2019057122 A1).

BRIEF DESCRIPTION OF THE INVENTION

However, when producing a bispecific antibody, the random combination of different polypeptide chains results in the formation of multiple different products, of which only one is desired. The resulting byproducts include an antibody lacking a light chain, a half-antibody, a heavy chain polymer, mismatched light heavy chains, and the like, which poses significant challenges to the development of downstream processes. Thus, the present invention relates to a novel polypeptide complex with engineered domains.

In some embodiments, the present invention relates to a polypeptide complex that comprises a first antigen-binding fragment comprising:

A) a first polypeptide comprising a first variable heavy chain domain (VH1) from the N-terminus to the C-terminus, wherein VH1 is operably linked to a first domain containing the T-chain of titin, and

a second polypeptide comprising a first variable light chain domain (VL1) from the N-terminus to the C-terminus, wherein VL1 is operably linked to a second domain comprising a domain capable of interacting with the T-chain of titin;

or

B) a first polypeptide comprising VH1 from the N-terminus to the C-terminus, wherein VH1 is operably linked to a first domain comprising a domain capable of interacting with the T-chain of titin, and

a second polypeptide comprising VL1 from the N-terminus to the C-terminus, wherein VL1 is operably linked to a second domain containing the T-chain of titin; or

C) a first polypeptide comprising VH1 from the N-terminus to the C-terminus, wherein VH1 is operably linked to a first domain comprising an obscurin O-chain or an obscurin-like protein O-chain, and

a second polypeptide comprising VL1 from the N-terminus to the C-terminus, wherein VL1 is operably linked to a second domain comprising a domain capable of interacting with an obscurin O-chain or an obscurin-like protein O-chain;

or

D) a first polypeptide comprising VH1 from the N-terminus to the C-terminus, wherein VH1 is operably linked to a first domain comprising a domain capable of interacting with an obscurin O-chain or an obscurin-like protein O-chain, and

a second polypeptide comprising VL1 from the N-terminus to the C-terminus, wherein VL1 is operably linked to a second domain comprising an obscurin O-chain or an obscurin-like protein O-chain;

wherein the first antigen-binding fragment specifically binds to a first antigen, and the first domain and the second domain are capable of forming a dimer.

In some embodiments of the aforementioned polypeptide complex, the domain capable of interacting with the O-chain of obscurin or the O-chain of an obscurin-like protein is the T-chain of titin, and the domain capable of interacting with the T-chain of titin is the O-chain of obscurin or the O-chain of an obscurin-like protein.

In some embodiments, for the aforementioned polypeptide complex, VH1 and VL1 of the first antigen-binding fragment form a first antigen-binding site that specifically binds to the first antigen.

In some embodiments, the C-terminus of VH1 is operably linked to the N-terminus of the first domain.

In some embodiments, the C-terminus of VL1 is operably linked to the N-terminus of the second domain.

In some embodiments of the above-mentioned polypeptide complex, the first domain and the second domain form a dimer via natural interchain linkages and/or non-natural interchain linkages.

In some embodiments, the first domain and the second domain form a dimer via natural interchain linkages; in some embodiments, one or more residues at positions selected from the group consisting of 7-15, 19-24, 26, 55, 59, and 60 on the T-chain of titin and one or more residues at positions selected from the group consisting of 3-6, 9, 41, 73, 75, and 80-90 on the O-chain of obscurin are linked to each other, or one or more residues selected from the group consisting of positions 1, 7-10, 13-16, 19-26, 59-60, and 96 on the T-chain of titin and one or more residues selected from the group consisting of positions 4-5, 10, 12-13, 74, 76, 78, and 82-91 on the O-chain of an obscurin-like protein are linked to each other; the position of the residue in the T-chain of titin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the residue in the O-chain of obscurin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the residue in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, for any of the above-mentioned polypeptide complexes, the first domain and the second domain form a dimer via at least one non-natural interchain linkage.

In some embodiments, the non-natural interchain linkage is formed between a specific mutated residue of the first domain and a specific mutated residue of the second domain. In some embodiments, at least one of the specific mutated residue of the first domain and the specific mutated residue of the second domain is a cysteine residue.

In some embodiments, the non-natural interchain bond is a disulfide bond; in some embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-natural interchain bonds.

In some embodiments, for the aforementioned polypeptide complex, the specific mutated residue of the first domain and the specific mutated residue of the second domain are located at the contact surface of the first domain and the second domain.

In some embodiments, the particular mutated residue of the aforementioned first domain and the particular mutated residue of the second domain have mutations at positions selected from the group consisting of: the T-chain of titin has one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39, and/or the O-chain of obscurin has one or more amino acid residue mutations at positions selected from the group consisting of 3, 9, 25, 76 and 88; or the T-chain of titin has one or more mutations of an amino acid residue at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39, and/or the O-chain of the obscurin-like protein has one or more mutations of an amino acid residue at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86. The position of the mutation in the T-chain of titin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some specific embodiments, said first mutated residue and said second mutated residue have mutations at positions selected from the group consisting of: the T-chain of titin has one or more amino acid residue mutations at positions selected from the group consisting of 8C, 20C, 22C, 25S, 26C and 39T, and/or the O-chain of obscurin has one or more amino acid residue mutations at positions selected from the group consisting of 3C, 9C, 25S, 76S and 88C; or the T-chain of titin has one or more amino acid residue mutations at positions selected from the group consisting of 8C, 20C, 22C, 25S, 26C and 39T, and/or the O-chain of the obscurin-like protein has one or more amino acid residue mutations at positions selected from the group consisting of 6E, 26S, 74C, 77S, 84C and 86C. In some embodiments, the T-chain of titin has the 25S, 39T and 8C mutations, and the O-chain of obscurin has the 88C mutation;

The T-chain of titin has mutations 25S, 39T and 20C, and the O-chain of obscurin has mutation 3C;

The T-chain of titin has mutations 25S, 39T and 26C, and the O-chain of obscurin has mutation 9C;

The T-chain of titin has mutations 25S, 39T and 8C, and the O-chain of obscurin has mutations 25S, 76S and 88C;

The T-chain of titin has mutations 25S, 39T and 20C, and the O-chain of obscurin has mutations 25S, 76S and 3C;

The T-chain of titin has mutations 25S, 39T and 26C, and the O-chain of obscurin has mutations 25S, 76S and 9C;

The T-chain of titin has mutations 25S, 39T and 8C, and the O-chain of obscurin-like protein has mutations 6E and 74C;

The T-chain of titin has mutations 25S, 39T and 20C, and the O-chain of obscurin-like protein has mutations 6E and 84C;

The T-chain of titin has mutations 25S, 39T and 22C, and the O-chain of obscurin-like protein has mutations 6E and 86C;

The T-chain of titin has mutations 25S, 39T and 8C, and the O-chain of obscurin-like protein has mutations 6E, 26S, 77S and 74C;

The T chain of titin has mutations 25S, 39T, and 20C, and the O chain of obscurin-like protein has mutations 6E, 26S, 77S, and 84C; or

The T-chain of titin has mutations 25S, 39T and 22C, and the O-chain of obscurin-like protein has mutations 6E, 26S, 77S and 86C;

the position of the mutation in the T-chain of titin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some specific embodiments, said first mutated residue and said second mutated residue have mutations at positions selected from the group consisting of: the T-chain of titin has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and/or the O-chain of obscurin has one or more amino acid residue mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C; or the T-chain of titin has one or more mutations of an amino acid residue at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and/or the O-chain of the obscurin-like protein has one or more mutations of an amino acid residue at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C. The position of the mutation in the T-chain of titin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, the particular mutated residue of the aforementioned first domain and the particular mutated residue of the second domain have mutations at positions selected from the group consisting of: the T-chain of titin has the mutations C25S, C39T and A8C, and the O-chain of obscurin has the mutation A88C; the T-chain of titin has the mutations C25S, C39T and V20C, and the O-chain of obscurin has the mutation A3C; the T-chain of titin has the mutations C25S, C39T and A26C, and the O-chain of obscurin has the mutation R9C; the T-chain of titin has the mutations C25S, C39T and A8C, and the O-chain of obscurin has the mutations C25S, C76S and A88C; The T chain of titin has mutations C25S, C39T, and V20C, and the O chain of obscurin has mutations C25S, C76S, and A3C; The T chain of titin has mutations C25S, C39T, and A26C, and the O chain of obscurin has mutations C25S, C76S, and R9C; The T chain of titin has mutations C25S, C39T, and A8C, and the O chain of obscurin-like protein has mutations C6E and V74C; The T chain of titin has mutations C25S, C39T, and V20C, and the O chain of obscurin-like protein has mutations C6E and G84C; The T chain of titin has mutations C25S, C39T, and T22C, and the O chain of obscurin-like protein has mutations C6E and A86C; The titin T chain has the mutations C25S, C39T, and A8C, and the obscurin-like protein O chain has the mutations C6E, C26S, C77S, and V74C; The titin T chain has the mutations C25S, C39T, and V20C, and the obscurin-like O chain has the mutations C6E, C26S, C77S, and G84C; The titin T chain has the mutations C25S, C39T, and T22C, and the obscurin-like protein O chain has the mutations C6E, C26S, C77S, and A86C. The position of the mutation in the titin T chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, said first domain and said second domain are selected from the group consisting of the following titin T-chain/obscurin O-chain or titin T-chain/obscurin-like protein O-chain: The titin T-chain is a polypeptide having one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26, and 39 based on SEQ ID NO: 32, and/or the obscurin O-chain is a polypeptide having one or more amino acid residue mutations at positions selected from the group consisting of 3, 9, 25, 76, and 88 based on SEQ ID NO: 33; or the titin T-chain is a polypeptide having one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 based on SEQ ID NO: 32, and/or the obscurin-like protein O-chain is a polypeptide having one or more amino acid residue mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34.

In some embodiments, said first domain and said second domain are selected from the group consisting of: a titin T-chain/obscurin O-chain and a titin T-chain/obscurin-like protein O-chain. The titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T based on SEQ ID NO: 32, and/or the obscurin O-chain has one or more amino acid residue mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C based on SEQ ID NO: 33; or the titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T based on SEQ ID NO: 32, and/or the obscurin-like protein O-chain has one or more amino acid residue mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C based on SEQ ID NO: 34. The position of the mutation in the titin T-chain is a position according to the natural sequence numbering of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is a position according to the natural sequence numbering of SEQ ID NO: 33; the position of the mutation in the obscurin-like protein O-chain is a position according to the natural sequence numbering of SEQ ID NO: 34.

In some embodiments, said first domain and said second domain are selected from the group consisting of: a titin T chain/obscurin O chain and a titin T chain/obscurin-like protein O chain. The titin T chain has the mutations C25S, C39T and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutation A88C based on SEQ ID NO: 33; the titin T chain has the mutations C25S, C39T and V20C based on SEQ ID NO: 32, and the obscurin O chain has the mutation A3C based on SEQ ID NO: 33; the titin T chain has the mutations C25S, C39T and A26C based on SEQ ID NO: 32, and the obscurin O chain has the mutation R9C based on SEQ ID NO: 33; The titin T chain has the mutations C25S, C39T and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S and A88C based on SEQ ID NO: 33; The titin T chain has the mutations C25S, C39T and V20C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S and A3C based on SEQ ID NO: 33; The titin T chain has the mutations C25S, C39T and A26C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S and R9C based on SEQ ID NO: SEQ ID NO: 33; The titin T chain has the mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin-like protein O chain has the mutations C6E and V74C based on SEQ ID NO: 34; the titin T chain has the mutations C25S, C39T, and V20C based on SEQ ID NO: 32, and the obscurin-like protein O chain has the mutations C6E and G84C based on SEQ ID NO: 34; the titin T chain has the mutations C25S, C39T, and T22C based on SEQ ID NO: 32, and the obscurin-like protein O chain has the mutations C6E and A86C based on SEQ ID NO: NO: 34; The titin T chain has mutations C25S, C39T and A8C based on SEQ ID NO: 32, and the obscurin-like protein O chain has mutations C6E, C26S, C77S and V74C based on SEQ ID NO: 34; the titin T chain has mutations C25S, C39T and V20C based on SEQ ID NO: 32, and the obscurin-like protein O chain has mutations C6E, C26S, C77S and G84C based on SEQ ID NO: 34; The titin T-chain has the mutations C25S, C39T and T22C based on SEQ ID NO: 32, and the obscurin-like protein O-chain has the mutations C6E, C26S, the C77S and A86C mutations based on SEQ ID NO: 34. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the obscurin-like protein O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, for the aforementioned polypeptide complex, the obscurin O chain has one or more amino acid residue mutations at positions selected from the group consisting of 7, 11, and 62. In some embodiments, the obscurin O chain has one or more amino acid residue mutations at positions selected from the group consisting of 7R and 7K, 62K and 62H, and 11L; most preferably, the titin T chain has 25S, 39T, and 8C mutations, and the obscurin O chain has 25S, 76S, 88C, 7K, and 62K mutations; the titin T chain has 25S, 39T, and 8C mutations, and the obscurin O chain has 25S, 76S, 88C, 7K, and 62H mutations; The titin T-chain has mutations at 25S, 39T and 8C, and the obscurin O-chain has mutations at 25S, 76S, 88C, 11L and 62K; or the titin T-chain has mutations at 25S, 39T and 8C, and the obscurin O-chain has mutations at 25S, 76S, 88C, 11L and 62H; the position of the mutation in the titin T-chain is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33.

In some embodiments, the obscurin O chain has one or more amino acid residue mutations at positions selected from the group consisting of L7K and L7R, K11L, T62K and T62H; in some embodiments, the obscurin O chain has the mutations C25S, C39T and A8C, and the obscurin O chain has the mutations C25S, C76S, A88C, L7K and T62K; the titin T chain has the mutations C25S, C39T and A8C, and the obscurin O chain has the mutations C25S, C76S, A88C, L7K and T62H; the titin T chain has the mutations C25S, C39T and A8C, and the obscurin O chain has the mutations C25S, C76S, A88C, K11L and T62K; The titin T-chain has the mutations C25S, C39T, and A8C, and the obscurin O-chain has the mutations C25S, C76S, A88C, K11L, and T62H. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33.

In some embodiments, with respect to the aforementioned polypeptide complex, the obscurin O chain is an obscurin O chain having one or more amino acid residue mutations at positions selected from the group consisting of 7, 11, and 62 based on SEQ ID NO: 45; in some embodiments, the obscurin O chain has one or more amino acid residue mutations at positions selected from the group consisting of L7K and L7R, K11L and T62K and T62H based on SEQ ID NO: 45; in some embodiments, the titin T chain has the mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S, A88C, L7K, and T62K based on SEQ ID NO: 45; The titin T chain has the mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S, A88C, L7K, and T62H based on SEQ ID NO: 45; The titin T chain has the mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S, A88C, K11L, and T62K based on SEQ ID NO: 45; The titin T-chain has the mutations C25S, C39T and the mutation A8C based on SEQ ID NO: 32, and the obscurin O-chain has the mutations C25S, C76S, A88C, K11L and T62H based on SEQ ID NO: 45. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33.

In some embodiments, for the aforementioned polypeptide complex, the first domain and the second domain have one or more amino acid residue mutations at positions selected from the group consisting of: the T-chain of titin has one or more amino acid mutations at positions selected from the group consisting of 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or the O-chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of 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 position of the mutation in the T-chain of titin is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 35; the position of the mutation in the O-chain of obscurin is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 50.

In some embodiments, for the above-mentioned polypeptide complex, the first domain and the second domain are selected from the group consisting of the following titin T chain/obscurin O chain: The titin T chain is a polypeptide having one or more amino acid mutations at positions selected from the group consisting of 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84 based on SEQ ID NO: 35, and the obscurin O chain is a polypeptide having one or more amino acid mutations at positions selected from the group consisting of 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 based on SEQ ID NO: 50. The position of the mutation in the T-chain of titin is the numbering position of the natural sequence relative to the sequence of SEQ ID NO: 35; the position of the mutation in the O-chain of obscurin is the numbering position of the natural sequence relative to the sequence of SEQ ID NO: 50.

In some embodiments, said first domain and said second domain have one or more residue mutations at positions selected from the group consisting of: the T chain of titin has one or more amino acid mutations at positions selected from the group consisting of 3W, 11I, 13L, 22M, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 66S and 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D and 84L, and/or the O chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of 2E, 11K, 12S, 13Y, 14T, 17E, 20L, 22M and 22S, 30D, 32P, 34E, 36T, 41K, 42L, 44I, 45T, 53L, 58V, 62E, 67Q and 67T, 69S, 89L, 92E, 94G and 97G. In some embodiments, the titin T chain has amino acid mutations 66S and 77S, and/or the obscurin O chain has amino acid mutations 11K, 12S, 13Y, 14T and 22S; The T chain of titin has amino acid mutations 66K, 70R, 79T, and 81R, and/or the O chain of obscurin has amino acid mutations 2E, 17E, 30D, 32P, 34E, 36T, 441, 45T, 58V, 62E, 67Q, 69S, and 97G; The T chain of titin has amino acid mutations 3W, 11I, 13L, 22M, and 82M, and/or the O chain of obscurin has amino acid mutations 20L, 22M, and 53L; The titin T chain has amino acid mutations 11I, 66K, 79T, and 81R, and/or the obscurin O chain has amino acid mutations 41K, 45T, 67Q, 69S, and 89L; the titin T chain has amino acid mutations 40S, 42K, 45S, 47E, 49G, 56S, 58E, 75V, 83D, and 84L, and/or the obscurin O chain has amino acid mutations 42L, 45T, 67T, 69S, 92E, and 94G; The T chain of titin has amino acid mutations 47E, 49G, 56S, 58E and 75V, and/or the O chain of obscurin has amino acid mutations 42L, 45T, 67T, 69S, 92E and 94G; The T chain of titin has amino acid mutations 56S, 58E and 75V, and/or the O chain of obscurin has amino acid mutations 42L, 45T, 67T, 69S, 92E and 94G amino acid mutations; or the titin T-chain has 56S, 58E, 66S and 77S amino acid mutations, and/or the obscurin O-chain has 12S, 13Y, 22S, 42L, 45T, 67Q, 69S, 92E and 94G amino acid mutations; the position of the mutation in the titin T-chain is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 35; the position of the mutation in the obscurin O-chain is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 50.

In some embodiments, said first domain and said second domain have one or more mutations of residues at positions selected from the group consisting of: the T chain of titin has one or more amino acid mutations at positions selected from the group consisting of P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or the O chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 35; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 50.

In some embodiments, said first domain and said second domain are selected from the group consisting of the following titin T chain/obscurin O chain: the titin T chain has one or more amino acid mutations at positions selected from the group consisting of P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L based on SEQ ID NO: 35, and/or the obscurin O chain has one or more amino acid mutations at positions selected from the group consisting of G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G based on SEQ ID NO: 35. The position of the mutation in the T-chain of titin is the position according to the numbering of the natural sequence of SEQ ID NO: 35; the position of the mutation in the O-chain of obscurin is the position according to the numbering of the natural sequence of SEQ ID NO: 50.

In some embodiments, the aforementioned titin T chain has the amino acid mutations M66S and T77S, and/or the obscurin O chain has the amino acid mutations L11K, A12S, F13Y, V14T and T22S; the titin T chain has the amino acid mutations M66K, K70R, S79T and G81R, and/or the obscurin O chain has the amino acid mutations G2E, V17E, N30D, T32P, Q34E, S36T, V44I, A45T, L58V, K62E, A67Q, G69S and A97G; The titin T chain has the amino acid mutations P3W, S11I, I13L, T22M, and N82M, and/or the obscurin O chain has the amino acid mutations D20L, T22M, and A53L; The titin T chain has the amino acid mutations S11I, M66K, S79T, and G81R, and/or the obscurin O chain has the amino acid mutations Q41K, A45T, A67Q, G69S, and V89L; The titin T chain has the amino acid mutations G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D, and F84L; and/or the obscurin O chain has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E, and D94G; The T-chain of titin has amino acid mutations Q47E, Q49G, N56S, D58E and L75V, and/or the O-chain of obscurin has amino acid mutations Q42L, A45T, A67T, G69S, Q92E and D94G; the T-chain of titin has amino acid mutations N56S, D58E and L75V, and/or the O-chain of obscurin has amino acid mutations Q42L, A45T, A67T, G69S, amino acid mutations Q92E and D94G; or the titin T-chain has the amino acid mutations N56S, D58E, M66S and T77S, and/or the obscurin O-chain has the amino acid mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E and D94G. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 35; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 50.

In some embodiments, said first domain and said second domain are selected from the group consisting of the following titin T-chain/obscurin O-chain: titin T-chain has amino acid mutations M66S and T77S based on SEQ ID NO: 35, and/or obscurin O-chain has amino acid mutations L11K, A12S, F13Y, V14T and T22S based on SEQ ID NO: 50; The titin T chain has the amino acid mutations M66K, K70R, S79T and G81R based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations G2E, V17E, N30D, T32P, Q34E, S36T, V44I, A45T, L58V, K62E, A67Q, G69S and A97G based on SEQ ID NO: 50; the titin T chain has the amino acid mutations P3W, S11I, I13L, T22M and N82M based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations D20L, T22M and A53L based on SEQ ID NO: 50; The titin T chain has the amino acid mutations S11I, M66K, S79T and G81R based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations Q41K, A45T, A67Q, G69S and V89L based on SEQ ID NO: 50; the titin T chain has the amino acid mutations G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D and F84L based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E and D94G based on SEQ ID NO: 50; The titin T chain has the amino acid mutations Q47E, Q49G, N56S, D58E and L75V based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E and D94G based on SEQ ID NO: 50; the titin T chain has the amino acid mutations N56S, D58E and L75V based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E and D94G based on SEQ ID NO: 50; or the titin T-chain has the amino acid mutations N56S, D58E, M66S and T77S based on SEQ ID NO: 35, and/or the obscurin O-chain has the amino acid mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E and D94G based on SEQ ID NO: 50. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence of SEQ ID NO: 35; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence of SEQ ID NO: 50.

In some embodiments, for the above-mentioned polypeptide complex, VH1 is operably linked to the first domain via a first linker domain, and VL1 is operably linked to the second domain via a second linker domain.

In some embodiments, said first and/or second linker domain is selected from the group consisting of: an N-terminal fragment of the T-chain of titin, an N-terminal fragment of the O-chain of obscurin, and an N-terminal fragment of the O-chain of an obscurin-like protein, and a linker (G _x S) _y , where X is selected from the group consisting of integers from 1 to 5 (e.g., 1, 2, 3, 4, or 5), and Y is selected from the group consisting of integers from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6).

In some embodiments, said first and/or second linker domain is selected from the group consisting of: "KAGIR", "DQPQF", (G ₄ S) ₁ and (G ₄ S) ₂ .

In some embodiments for the above-mentioned polypeptide complex, when the first domain is the titin T-chain, the first linker domain is the KAGIR polypeptide; when the second domain is the obscurin O-chain, the second linker domain is the DQPQF polypeptide. In other embodiments, when the second domain is the titin T-chain, the second linker domain is the KAGIR polypeptide, and when the first domain is the obscurin O-chain, the first linker domain is the DQPQF polypeptide.

In some embodiments of the polypeptide complex, wherein

(A) The titin T chain has the sequence shown in SEQ ID NO: 32, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 8, 20, 22, 25, 26, 39, 3, 11, 13, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or 5 KAGIR amino acid residues added at the N-terminus based on SEQ ID NO: 32; preferably, the titin T chain has a sequence having one or more amino acid mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S and M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or the amino acid residue KAGIR added at the N-terminus; and/or

(B) The O-chain of obscurin has the sequence shown in SEQ ID NO: 33, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 3, 9, 25, 76, 88, 7, 11, 62, 2, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 67, 69, 89, 92, 94 and 97, and/or a KAGIR amino acid residue added at the N-terminus based on SEQ ID NO: 33; preferably, the O-chain of obscurin has a sequence having one or more amino acid mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S, A88C, L7K and L7R, T62K and T62H, G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G, and/or the amino acid residue DQPQF added at the N-terminus; or

The O-chain of the obscurin-like protein has a sequence as presented in SEQ ID NO: 34, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34; preferably, the O-chain of the obscurin-like protein has a sequence having one or more amino acid mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C;

the position of the mutation in the T-chain of titin is the position according to the numbering of the natural sequence of SEQ ID NO: 32; the position of the mutation in the O-chain of obscurin is the position according to the numbering of the natural sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is the position according to the numbering of the natural sequence of SEQ ID NO: 34.

In some embodiments of the polypeptide complex, wherein (A) the titin T chain has one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 based on SEQ ID NO: 32, and preferably has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and the position of the mutation in the titin T chain is a position according to the numbering of the natural sequence of SEQ ID NO: 32; more preferably, the titin T chain has one or more amino acid mutations at positions selected from the group consisting of 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84 based on SEQ ID NO: 35, and preferably has one or more amino acid mutations at positions selected from the group consisting of P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L; the position of the mutation in the titin T-chain is a position according to the numbering of the natural sequence SEQ ID NO: 35, and/or

(B) The O-chain of obscurin has one or more amino acid residue mutations at positions selected from the group consisting of 3, 9, 25, 76 and 88 based on SEQ ID NO: 33, and preferably has one or more amino acid residue mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C, and the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence of SEQ ID NO: 33; preferably, the O-chain of obscurin has one or more mutations of an amino acid residue at positions selected from the group consisting of 7, 11 and 62 based on SEQ ID NO: 45, and preferably has one or more mutations of an amino acid residue at positions selected from the group consisting of L7K and L7R, T62K and T62H and K11L, and the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence of SEQ ID NO: 45; more preferably, the O-chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of 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 based on SEQ ID NO: 50, and preferably has one or more amino acid mutations at positions selected from the group consisting of G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G, and the position of the mutation in the O-chain of obscurin is the position according to the numbering of the natural sequence of SEQ ID NO: 50; or

The O-chain of the obscurin-like protein has one or more mutations of the amino acid residue at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34, and preferably has one or more mutations of the amino acid residue at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C, and the position of the mutation in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence of SEQ ID NO: 34.

In some embodiments of the polypeptide complex, wherein the titin T chain comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 32, 35-41, 65-76, or a polypeptide having a sequence at least 80% identical to any one of SEQ ID NOs: 32, 35-41, 65-76, and/or the obscurin O chain comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 33, 42-58, 77-86, or a polypeptide having a sequence at least 80% identical to any one of SEQ ID NOs: 33, 42-58, 77-86; or the titin T-chain comprises a polypeptide having a sequence as presented in any of SEQ ID NOs: 32, 35-41, 65-76, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76, and/or the obscurin-like protein O-chain comprises a polypeptide having a sequence as presented in any of SEQ ID NOs: 34, 59-64, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 34, 59-64. Moreover, a sequence that is at least 80% identical to a sequence refers, for example, to a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence.

In some embodiments, the aforementioned titin T-chain comprises a polypeptide having the sequence presented in SEQ ID NO: 68, and the obscurin O-chain comprises a polypeptide having the sequence presented in SEQ ID NO: 80.

In other embodiments, the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 73, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 83.

In other embodiments, the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 76, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 84.

In other embodiments, the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 76, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 86.

In other embodiments, the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 76, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 86.

In other embodiments, the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 75, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 86.

In some embodiments, the titin T chain is a polypeptide having the sequence presented in SEQ ID NO: 68, and the obscurin O chain is a polypeptide having the sequence presented in SEQ ID NO: 80; the titin T chain is a polypeptide having the sequence presented in SEQ ID NO: 73, and the obscurin O chain is a polypeptide having the sequence presented in SEQ ID NO: 83; the titin T chain is a polypeptide having the sequence presented in SEQ ID NO: 76, and the obscurin O chain is a polypeptide having the sequence presented in SEQ ID NO: 84; the titin T chain is a polypeptide having the sequence presented in SEQ ID NO: 76, and the obscurin O chain is a polypeptide having the sequence presented in SEQ ID NO: 86; The titin T-chain is a polypeptide having the sequence presented in SEQ ID NO: 76, and the obscurin O-chain is a polypeptide having the sequence presented in SEQ ID NO: 86; or the titin T-chain is a polypeptide having the sequence presented in SEQ ID NO: 75, and the obscurin O-chain is a polypeptide having the sequence presented in bSEQ ID NO: 86.

In some embodiments of the above-mentioned polypeptide complex, the first antigen is selected from the group consisting of: a foreign antigen, an endogenous antigen, an autoantigen, a neoantigen, a viral antigen, and a tumor antigen.

In some embodiments, the disclosed is a multispecific polypeptide complex comprising:

a first polypeptide complex that is any of the above-mentioned polypeptide complexes and specifically binds to the first antigen, and

a second polypeptide complex containing a second antigen-binding fragment and specifically binding to a second antigen.

In some embodiments, the first polypeptide complex and the second polypeptide complex bind to two different antigens.

In some embodiments, the first polypeptide complex and the second polypeptide complex bind to two different epitopes on the same antigen.

In some embodiments, the multispecific polypeptide complex is a multispecific polypeptide complex, and more preferably, the multispecific polypeptide complex is a bispecific antibody.

In some embodiments, the second polypeptide complex of the multispecific polypeptide complex comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2). Mispairing between VH1 of the first polypeptide complex and VL2 of the second polypeptide complex and/or between VH2 of the second polypeptide complex and VL1 of the first polypeptide complex is less likely to occur. In some embodiments, VH2 and VL2 of the second antigen-binding fragment form a second antigen-binding site that specifically binds to a second antigen.

In some embodiments for the above-mentioned multispecific polypeptide complex, the second antigen-binding fragment of the second polypeptide complex comprises:

a third polypeptide comprising VH2 from the N-terminus to the C-terminus, wherein VH2 is operably linked to a third CH1-containing domain, and

a fourth polypeptide comprising VL2 from the N-terminus to the C-terminus, wherein VL2 is operably linked to the fourth CL-containing domain.

In some embodiments, the C-terminus of VH2 is linked to the N-terminus of CH1 and the C-terminus of VL2 is linked to the N-terminus of CL.

In some embodiments, for the aforementioned multispecific polypeptide complex, the first polypeptide complex further comprises an operably linked first dimerization domain, and the second polypeptide complex further comprises an operably linked second dimerization domain, wherein the first dimerization domain and the second dimerization domain are linked together.

Preferably, the C-terminus of the first domain of the first polypeptide complex is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the third domain of the second polypeptide complex is operably linked to the N-terminus of the second dimerization domain.

In some embodiments, said first dimerization domain and said second dimerization domain are linked to form a dimer via a hinge region of the antibody or a portion thereof, a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, or a combination thereof.

In some embodiments, the aforementioned first dimerization domain and the aforementioned second dimerization domain are linked to form a dimer via the hinge region of the antibody or a portion thereof, and preferably the hinge region of IgG1, IgG2, or IgG4 or a portion thereof. In some embodiments, the hinge region of IgG1, IgG2, IgG3, or IgG4 or a portion thereof is preferred for dimer formation.

In some embodiments, for the aforementioned multispecific polypeptide complex, the first dimerization domain and/or the second dimerization domain further comprises an antibody CH2 domain and/or an antibody CH3 domain.

In some embodiments of the aforementioned multispecific polypeptide complex, the CH2 domain of the antibody and/or the CH3 domain of the antibody are derived from IgG1, IgG2, or IgG4. Preferably, the CH2 domain of the antibody and/or the CH3 domain of the antibody are derived from IgG1, IgG2, IgG3, or IgG4.

In some embodiments, for the aforementioned multispecific polypeptide complex, the first polypeptide sequentially comprises VH1-L1-T-chain-L2-CH2-CH3 (T-chain - T-chain of titin) from the N-terminus to the C-terminus, the second polypeptide sequentially comprises VL1-L3-O-chain (O-chain - O-chain of obscurin) or VL1-L4-OL-chain (OL-chain - O-chain of obscurin-like protein) from the N-terminus to the C-terminus, the third polypeptide sequentially comprises VH2-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the fourth polypeptide sequentially comprises VL1-CL from the N-terminus to the C-terminus; or the first polypeptide sequentially comprises VH1-L2-chain O-L3-CH2-CH3 or VH1-L4-OL chain-L5-CH2-CH3 from the N-terminus to the C-terminus, the second polypeptide sequentially comprises VL1-L1-chain T from the N-terminus to the C-terminus, the third polypeptide sequentially comprises VH2-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the fourth polypeptide sequentially comprises VL2-CL from the N-terminus to the C-terminus; L1-L5 are spacers (also known as linker domains), which may or may not be present. Preferably, the spacer is selected from the group consisting of: an N-terminal fragment of the T-chain of titin, an N-terminal fragment of the O-chain of obscurin, an N-terminal fragment of the O-chain of an obscurin-like protein and a linker (i.e., a (G _x S) _y linker), where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6. Most preferably, the spacer is selected from the group consisting of: "KAGIR", "DQPQF", (G ₄ S) ₁ and (G ₄ S) ₂ .

In some embodiments, for the aforementioned multispecific polypeptide complex, the first dimerization domain and the second dimerization domain are not identical and are associated in a manner that prevents homodimerization and/or promotes heterodimerization.

In some embodiments, said first dimerization domain and said second dimerization domain are associated to form a dimer via knob-into-hole technology, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility.

In some embodiments, the aforementioned first dimerization domain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 2, and the second dimerization domain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 3; or the first dimerization domain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 3, and the second dimerization domain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 2.

In some embodiments, the present invention provides a domain engineered antibody in which at least one CH1 constant region domain and at least one CL constant region domain are substituted, wherein

the CH1 domain is replaced by the T-chain of titin, and the CL domain is replaced by a domain capable of forming protein-protein interactions with the T-chain of titin;

the CL domain is replaced by the T-chain of titin, and the CH1 domain is replaced by a domain capable of forming an interprotein interaction with the T-chain of titin;

the CH1 domain is replaced by the O-chain of obscurin, and the CL domain is replaced by a domain capable of forming an interprotein interaction with the O-chain of obscurin;

the CL domain is replaced by the O-chain of obscurin, and the CH1 domain is replaced by a domain capable of forming an interprotein interaction with the O-chain of obscurin;

the CH1 domain is replaced by the O-chain of an obscurin-like protein, and the CL domain is replaced by a domain capable of forming a protein-protein interaction with the O-chain of an obscurin-like protein; or

The CL domain is replaced by the O-chain of an obscurin-like protein, and the CH1 domain is replaced by a domain capable of forming protein-protein interactions with the O-chain of an obscurin-like protein.

In some embodiments, for the above-mentioned domain-engineered antibody, wherein the domain capable of interacting with the O-chain of obscurin or the O-chain of an obscurin-like protein is the T-chain of titin, and the domain capable of interacting with the T-chain of titin is the O-chain of obscurin or the O-chain of an obscurin-like protein.

In some embodiments, the antibody comprises a heavy chain variable region VH1 and a light chain variable region VL1, wherein VH1 and VL1 form a primary binding site that specifically binds to its antigen; the C-terminal region of VH1 is operably linked to the N-terminus of the T-chain of titin, the C-terminal region of VL1 is operably linked to the N-terminus of the O-chain of obscurin or the O-chain of an obscurin-like protein, or the C-terminal region of VL1 is operably linked to the N-terminus of the T-chain of titin, and the C-terminal region of VH1 is operably linked to the N-terminus of the O-chain of obscurin or the O-chain of an obscurin-like protein.

In some embodiments, the present invention relates to a domain engineered antibody Fab fragment in which the CH1 constant region domain and the CL constant region domain are substituted, wherein

the CH1 domain is replaced by the T-chain of titin, and the CL domain is replaced by a domain capable of forming a protein-protein interaction with the T-chain of titin; or

the CL domain is replaced by the T-chain of titin, and the CH1 domain is replaced by a domain capable of forming a protein-protein interaction with the T-chain of titin; or

the CH1 domain is replaced by an obscurin O-chain, and the CL domain is replaced by a domain capable of forming a protein-protein interaction with the obscurin O-chain; or

the CL domain is replaced by an obscurin O chain, and the CH1 domain is replaced by a domain capable of forming a protein-protein interaction with the obscurin O chain; or

the CH1 domain is replaced by the O-chain of an obscurin-like protein, and the CL domain is replaced by a domain capable of forming a protein-protein interaction with the O-chain of an obscurin-like protein; or

The CL domain is replaced by the O-chain of an obscurin-like protein, and the CH1 domain is replaced by a domain capable of forming an interprotein interaction with the O-chain of an obscurin-like protein.

In some embodiments of the aforementioned Fab fragment, the domain having a protein-protein interaction with the T-chain of titin is the O-chain of obscurin or the O-chain of an obscurin-like protein; the domain having a protein-protein interaction with the O-chain of obscurin or the obscurin-like O-chain is the T-chain of titin.

In some embodiments, the Fab comprises a heavy chain variable region VH1 and a light chain variable region VL1, wherein VH1 and VL1 form a primary binding site that specifically binds to its antigen; the C-terminal region of VH1 is operably linked to the N-terminus of the T-chain of titin, the C-terminal region of VL1 is operably linked to the N-terminus of the O-chain of obscurin or the O-chain of an obscurin-like protein, or the C-terminal region of VL1 is operably linked to the N-terminus of the T-chain of titin, and the C-terminal region of VH1 is operably linked to the N-terminus of the O-chain of obscurin or the O-chain of an obscurin-like protein.

In some embodiments, the present invention relates to an engineered domain antibody comprising the aforementioned Fab fragment.

In some embodiments, the present invention relates to a bispecific antibody comprising a first heavy chain and a first light chain specifically binding to a first antigen, and further comprising a second heavy chain and a second light chain specifically binding to a second antigen, wherein

the CH1 domain of the first heavy chain is replaced by the T-chain of titin, and the CL domain of the first light chain is replaced by a domain capable of forming a protein-protein interaction with the T-chain of titin; or

the CL domain of the first light chain is replaced by the T chain of titin, and the CH1 domain of the first heavy chain is replaced by a domain capable of forming a protein-protein interaction with the T chain of titin; or

the CH1 domain of the first heavy chain is replaced by the O-chain of obscurin, and the CL domain of the first light chain is replaced by a domain capable of forming an interprotein interaction with the O-chain of obscurin;

the CL domain of the first light chain is replaced by an O-chain of obscurin, and the CH1 domain of the first heavy chain is replaced by a domain capable of forming a protein-protein interaction with the O-chain of obscurin; or

the CH1 domain of the first heavy chain is replaced by the O-chain of an obscurin-like protein, and the CL domain of the first light chain is replaced by a domain capable of forming a protein-protein interaction with the O-chain of an obscurin-like protein; or

The CL domain of the first light chain is replaced by the O-chain of an obscurin-like protein, and the CH1 domain of the first heavy chain is replaced by a domain capable of forming an interprotein interaction with the O-chain of an obscurin-like protein.

A bispecific antibody binds to a first antigen and a second antigen that are not identical, or binds to two different epitopes on the same antigen.

In particular embodiments, mispairing between the first heavy chain and the second light chain, and between the first light chain and the second heavy chain, is less likely to occur.

In some embodiments of the aforementioned bispecific antibody, the domain having a protein-protein interaction with the T-chain of titin is the O-chain of obscurin or the O-chain of an obscurin-like protein; the domain having a protein-protein interaction with the O-chain of obscurin or the O-chain of an obscurin-like protein is the T-chain of titin.

In some embodiments, the first heavy chain comprises a first heavy chain variable region (VH1), and the first light chain comprises a first light chain variable region (VL1), wherein VH1 and VL1 form an antigen-binding site that specifically binds to the first antigen, wherein the C-terminus of VH1 is operably linked to the N-terminus of the T-chain of titin, the C-terminus of VL1 is operably linked to the N-terminus of the O-chain of obscurin or the O-chain of an obscurin-like protein, or the C-terminus of VL1 is operably linked to the N-terminus of the T-chain of titin, and the C-terminus of VH1 is operably linked to the N-terminus of the O-chain of obscurin or the O-chain of an obscurin-like protein.

In some embodiments for the above-mentioned bispecific antibody, the second heavy chain comprises a second heavy chain variable region (VH2) from the N-terminus to the C-terminus, operably linked to CH1; the second light chain comprises a second light chain variable region (VL2) from the N-terminus to the C-terminus, operably linked to CL.

In some embodiments of the aforementioned bispecific antibody, VH2 and VL2 form an antigen-binding site that specifically binds a second antigen, and the C-terminus of VH2 is operably linked to the N-terminus of CH1; the C-terminus of VL2 is operably linked to the N-terminus of CL.

In some embodiments, for the above-mentioned bispecific antibody, the structural model of the bispecific antibody is selected from the group consisting of F(ab) ₂ , scFv-Fab, (scFv) ₂ -Fab, KiH (knock-in-hole), CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb (bispecific monoclonal antibody), Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCB (T-cell bispecific antibody), 1Fab-IgG TDB, FynomAb, two in one/DAF, scFv-Fab-IgG, DART-Fc (DART - dual affinity reorienting antibody), LP-DART, CODV-Fab-TL, HLE-BiTE (BiTE with extended half-life), F(ab) ₂ -CrossMAb, IgG-(scFv) ₂ , Bs4Ab, DVD-Ig, tetravalent-DART-Fc, (scFv) ₄ -Fc, CODV-Ig, mAb ₂ , F(ab) ₄ -CrossMAb, IgG and Fab-IgG, etc.

In some embodiments of the aforementioned bispecific antibody, the C-termini of the first heavy chain and the second heavy chain comprise a CH2 domain and/or a CH3 domain.

In some embodiments, said CH1, CH2, and CH3 domains are derived from IgG1, IgG2, or IgG4. In some embodiments, said CH1, CH2, and CH3 domains are derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, said first heavy chain and said second heavy chain are linked to form a dimer via an antibody hinge region or a portion thereof, a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, or a combination thereof.

In some embodiments, for the aforementioned bispecific antibody, the first heavy chain sequentially comprises VH1-L1-T chain-L2-CH2-CH3 from the N-terminus to the C-terminus, and the first light chain sequentially comprises VL1-L3-O chain or VL1-L4-OL chain from the N-terminus to the C-terminus; the second heavy chain sequentially comprises VH2-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the second light chain sequentially comprises VL2-CL from the N-terminus to the C-terminus; or

the first light chain sequentially comprises a VL1-L1-T chain from the N-terminus to the C-terminus, and the first heavy chain sequentially comprises VH1-L2-O chain-L3-CH2-CH3 or VH1-L4-OL chain-L5-CH2-CH3 from the N-terminus to the C-terminus; the second heavy chain sequentially comprises VH2-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the second light chain sequentially comprises VL2-CL from the N-terminus to the C-terminus;

wherein L1-L5 are spacers that may be present or absent. Preferably, the spacer is an N-terminal fragment of the T-chain of titin, an N-terminal fragment of the O-chain of obscurin, an N-terminal fragment of the O-chain of an obscurin-like protein, or a linker, such as a (G _x S) _y linker, where X is selected from the group consisting of integers from 1 to 5 and Y is selected from the group consisting of integers from 1 to 6; preferably, the spacer is selected from the group consisting of: "KAGIR", "DQPQF", (G ₄ S) ₁ and (G ₄ S) ₂ .

In some embodiments of the aforementioned bispecific antibody, CH2 and CH3 of the first heavy chain are not identical to CH2 and CH3 of the second heavy chain and are linked in a manner that prevents homodimerization and/or promotes heterodimerization.

In some embodiments, said first heavy chain and said second heavy chain are associated to form a dimer via knob-into-hole technology, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility.

In some embodiments, said first heavy chain and said second heavy chain are linked by knob-into-hole technology to form a dimer. In some embodiments, the CH2-CH3 domain of said first heavy chain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 2, and the CH2-CH3 domain of the second heavy chain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 3; or the CH2-CH3 domain of the first heavy chain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 3, and the CH2-CH3 domain of the second heavy chain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 2.

In some embodiments, for the aforementioned bispecific antibody, the first heavy chain sequentially comprises VH1-L1-T chain-L2-knob-Fc from N-terminus to C-terminus, the first light chain sequentially comprises VL1-L3-O chain or VL1-L4-OL chain from N-terminus to C-terminus, the second heavy chain sequentially comprises VH2-CH1-hole-Fc from N-terminus to C-terminus, and the second light chain sequentially comprises VL1-CL from N-terminus to C-terminus; or the first heavy chain sequentially comprises VH1-L1-T-chain-L2-hole-Fc from N-terminus to C-terminus, the first light chain sequentially comprises VL1-L3-O-chain or VL1-L4-OL-chain from N-terminus to C-terminus, the second heavy chain sequentially comprises VH2-CH1-knob-Fc from N-terminus to C-terminus, and the second light chain sequentially comprises VL2-CL from N-terminus to C-terminus; or

the first light chain sequentially comprises VL1-L1-T chain from the N-terminus to the C-terminus, the first heavy chain sequentially comprises VH1-L2-O chain-L3-knob-Fc or VH1-L4-OL chain-L5-knob-Fc from the N-terminus to the C-terminus, the second heavy chain sequentially comprises VH2-CH1-hole-Fc from the N-terminus to the C-terminus, and the second light chain sequentially comprises VL2-CL chain from the N-terminus to the C-terminus; the first light chain sequentially comprises VL1-L1-chain T from the N-terminus to the C-terminus, the first heavy chain sequentially comprises VH1-L2-chain O or VH1-L4-OL chain-L5-hole-Fc from the N-terminus to the C-terminus, the second heavy chain sequentially comprises VH2-CH1-knob-Fc from the N-terminus to the C-terminus, and the second light chain sequentially comprises VL2-CL-O from the N-terminus to the C-terminus;

L1-L5 are spacers that may be present or absent. Preferably, the spacer is an N-terminal fragment of the T-chain of titin, an N-terminal fragment of the O-chain of obscurin, an N-terminal fragment of the O-chain of an obscurin-like protein, or a linker, for example, a linker (G _x S) _y , where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6; preferably, the spacer is selected from the group consisting of: "KAGIR", "DQPQF", (G ₄ S) ₁ and (G ₄ S) ₂ .

In some embodiments, for any of the above-mentioned antibody with an engineered domain, Fab fragment of an antibody with an engineered domain, or bispecific antibody, the domain having a protein-protein interaction with the titin T-chain is the O-chain of obscurin or the O-chain of an obscurin-like protein; the domain having a protein-protein interaction with the O-chain of obscurin or the O-chain of an obscurin-like protein is the T-chain of titin. For any of the above-mentioned antibody with an engineered domain, Fab fragment of an antibody with an engineered domain, or bispecific antibody, the functional linkage is a linkage of two polypeptide sequences via a spacer or a direct linkage of two polypeptides.

In some embodiments, for the above-mentioned domain engineered antibody or bispecific antibody, or Fab fragment of the domain engineered antibody, the titin T chain and the obscurin O chain, or the titin T chain and the obscurin-like protein O chain are linked via natural interchain linkages and/or non-natural interchain linkages to form a dimer.

In some embodiments, the first domain and the second domain form a dimer through natural interchain linkages, and one or more residues at positions selected from the group consisting of 7-15, 19-24, 26, 55, 59, and 60 on the T-chain of titin, and one or more residues at positions selected from the group consisting of 3-6, 9, 41, 73, 75, and 80-90 on the O-chain of obscurin, are linked to each other, or

one or more residues selected from the group consisting of positions 1, 7-10, 13-16, 19-26, 59-60 and 96 in the T-chain of titin, and one or more residues selected from the group consisting of positions 4-5, 10, 12-13, 74, 76, 78 and 82-91 in the O-chain of an obscurin-like protein, are linked to each other. The position of the residue in the T-chain of titin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the residue in the O-chain of obscurin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the residue in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, for the aforementioned domain-engineered antibody, bispecific antibody, or Fab fragment of an domain-engineered antibody, the titin T chain and the obscurin O chain, or the titin T chain and the obscurin-like protein O chain form a dimer via at least one non-natural interchain linkage. In some embodiments, the non-natural interchain linkage is a disulfide bond; in some embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-natural interchain linkages.

In some embodiments, for the above-mentioned domain engineered antibody, bispecific antibody, or Fab fragment of a domain engineered antibody, wherein

The titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39, and/or the obscurin O-chain has one or more amino acid residue mutations at positions selected from the group consisting of 3, 9, 25, 76 and 88; or

The titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39, and/or the obscurin-like protein O-chain has one or more amino acid residue mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86.

In some embodiments, the titin T-chain/obscurin O-chain or titin T-chain/obscurin-like protein O-chain is as follows:

The titin T-chain is a polypeptide having one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 based on SEQ ID NO: 32, and/or the obscurin O-chain is a polypeptide having one or more amino acid residue mutations at positions selected from the group consisting of 3, 9, 25, 76 and 88 based on SEQ ID NO: 33; or

The titin T-chain is a polypeptide having one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 based on SEQ ID NO: 32, and/or the obscurin-like O chain is a polypeptide having one or more amino acid residue mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34.

In some embodiments, for the above-mentioned domain engineered antibody, bispecific antibody, or Fab fragment of a domain engineered antibody, wherein

The titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of 8C, 20C, 22C, 25S, 26C and 39T, and/or the obscurin O-chain has one or more amino acid residue mutations at positions selected from the group consisting of 3C, 9C, 25S, 76S and 88C; or

The titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of 8C, 20C, 22C, 25S, 26C and 39T, and/or the obscurin-like protein O-chain has one or more amino acid residue mutations at positions selected from the group consisting of 6E, 26S, 74C, 77S, 84C and 86C;

more preferably, the non-natural interstrand linkage is located at a mutation position selected from the group consisting of the following:

The T-chain of titin has mutations 25S, 39T and 8C, and the O-chain of obscurin has mutation 88C;

The T-chain of titin has mutations 25S, 39T and 20C, and the O-chain of obscurin has mutation 3C;

The T-chain of titin has mutations 25S, 39T and 26C, and the O-chain of obscurin has mutation 9C;

The T-chain of titin has mutations 25S, 39T and 8C, and the O-chain of obscurin has mutations 25S, 76S and 88C;

The T-chain of titin has mutations 25S, 39T and 20C, and the O-chain of obscurin has mutations 25S, 76S and 3C;

The T-chain of titin has mutations 25S, 39T and 26C, and the O-chain of obscurin has mutations 25S, 76S and 9C;

The T-chain of titin has mutations 25S, 39T and 8C, and the O-chain of obscurin-like protein has mutations 6E and 74C;

The T-chain of titin has mutations 25S, 39T and 20C, and the O-chain of obscurin-like protein has mutations 6E and 84C;

The T-chain of titin has mutations 25S, 39T and 22C, and the O-chain of obscurin-like protein has mutations 6E and 86C;

The T-chain of titin has mutations 25S, 39T and 8C, and the O-chain of obscurin-like protein has mutations 6E, 26S, 77S and 74C;

The T chain of titin has mutations 25S, 39T, and 20C, and the O chain of obscurin-like protein has mutations 6E, 26S, 77S, and 84C; or

The T-chain of titin has mutations 25S, 39T and 22C, and the O-chain of obscurin-like protein has mutations 6E, 26S, 77S and 86C;

the position of the mutation in the T-chain of titin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, for the above-mentioned domain engineered antibody, bispecific antibody, or Fab fragment of a domain engineered antibody, wherein

The titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and/or the obscurin O-chain has one or more amino acid residue mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C; or

The titin T-chain has one or more mutations of an amino acid residue at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and/or the obscurin-like protein O-chain has one or more mutations of an amino acid residue at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C; the position of the mutation in the titin T-chain is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the obscurin-like protein O-chain is a position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, the aforementioned titin T chain has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T based on SEQ ID NO: 32, and/or the obscurin O chain has one or more amino acid residue mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C based on SEQ ID NO: 33; or the titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T based on SEQ ID NO: 32, and/or the obscurin-like protein O-chain has one or more amino acid residue mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C based on SEQ ID NO: 34. The position of the mutation in the titin T-chain is a position according to the numbering of the natural sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is a position according to the numbering of the natural sequence of SEQ ID NO: 33; the position of the mutation in the obscurin-like protein O-chain is a position according to the numbering of the natural sequence of SEQ ID NO: 34.

In some embodiments, for the above-mentioned domain engineered antibody, bispecific antibody, or Fab fragment of a domain engineered antibody, wherein

The T-chain of titin has mutations C25S, C39T and A8C, and the O-chain of obscurin has mutation A88C;

The T-chain of titin has mutations C25S, C39T and V20C, and the O-chain of obscurin has mutation A3C;

The T-chain of titin has mutations C25S, C39T and A26C, and the O-chain of obscurin has mutation R9C;

The T-chain of titin has mutations C25S, C39T and A8C, and the O-chain of obscurin has mutations C25S, C76S and A88C;

The T-chain of titin has mutations C25S, C39T and V20C, and the O-chain of obscurin has mutations C25S, C76S and A3C;

The T-chain of titin has mutations C25S, C39T and A26C, and the O-chain of obscurin has mutations C25S, C76S and R9C;

The T-chain of titin has mutations C25S, C39T and A8C, and the O-chain of obscurin-like protein has mutations C6E and V74C;

The T-chain of titin has mutations C25S, C39T and V20C, and the O-chain of obscurin-like protein has mutations C6E and G84C;

The T-chain of titin has mutations C25S, C39T and T22C, and the O-chain of obscurin-like protein has mutations C6E and A86C;

The T-chain of titin has mutations C25S, C39T and A8C, and the O-chain of obscurin-like protein has mutations C6E, C26S, C77S and V74C;

The T-chain of titin has mutations C25S, C39T, and V20C, and the O-chain of obscurin-like protein has mutations C6E, C26S, C77S, and G84C;

The T-chain of titin has mutations C25S, C39T and T22C, and the O-chain of obscurin-like protein has mutations C6E, C26S, C77S and A86C;

the position of the mutation in the T-chain of titin is the position of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the O-chain of obscurin is the position of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is the position of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, for the above-mentioned domain engineered antibody, bispecific antibody, or Fab fragment of an domain engineered antibody, the titin T chain has the C25S, C39T, and A8C mutations based on SEQ ID NO: 32, and the obscurin O chain has the A88C mutation based on SEQ ID NO: 33; the titin T chain has the C25S, C39T, and V20C mutations based on SEQ ID NO: 32, and the obscurin O chain has the A3C mutation based on SEQ ID NO: 33; the titin T chain has the C25S, C39T, and A26C mutations based on SEQ ID NO: 32, and the obscurin O chain has the R9C mutation based on SEQ ID NO: 33; The titin T chain has the mutations C25S, C39T and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S and A88C based on SEQ ID NO: 33; the titin T chain has the mutations C25S, C39T and V20C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S and A3C based on SEQ ID NO: 33; the titin T chain has the mutations C25S, C39T and A26C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S and R9C based on SEQ ID NO: 33; The titin T chain has the mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin-like protein O chain has the mutations C6E and V74C based on SEQ ID NO: 34; the titin T chain has the mutations C25S, C39T, and V20C based on SEQ ID NO: 32, and the obscurin-like protein O chain has the mutations C6E and G84C based on SEQ ID NO: 34; the titin T chain has the mutations C25S, C39T, and T22C based on SEQ ID NO: 32, and the obscurin-like protein O chain has the mutations C6E and A86C based on SEQ ID NO: 34; The titin T chain has mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin-like protein O chain has mutations C6E, C26S, C77S, and V74C based on SEQ ID NO: 34; The titin T chain has mutations C25S, C39T, and V20C based on SEQ ID NO: 32, and the obscurin-like protein O chain has mutations C6E, C26S, C77S, and G84C based on SEQ ID NO: 34; The titin T-chain has mutations C25S, C39T and T22C based on SEQ ID NO: 32, and the obscurin-like protein O-chain has mutations C6E, C26S, C77S and A86C based on SEQ ID NO: 34. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33; the position of the mutation in the obscurin-like protein O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 34.

In some embodiments, for the aforementioned domain engineered antibody, bispecific antibody, or domain engineered antibody Fab fragment, the obscurin O chain further has one or more amino acid residue mutations at positions selected from the group consisting of 7, 11, and 62. In some embodiments, the obscurin O chain has one or more amino acid residue mutations at positions selected from the group consisting of 7R and 7K, 62K and 62H, and 11L; most preferably, the titin T chain has the mutations 25S, 39T, and 8C, and the obscurin O chain has the mutations 25S, 76S, 88C, 7K, and 62K; The titin T chain has mutations at 25S, 39T and 8C, and the obscurin O chain has mutations at 25S, 76S, 88C, 7K and 62H; the titin T chain has mutations at 25S, 39T and 8C, and the obscurin O chain has mutations at 25S, 76S, 88C, 11L and 62K; or the titin T chain has mutations at 25S, 39T and 8C, and the obscurin O chain has mutations at 25S, 76S, 88C, 11L and 62H. In some embodiments, the obscurin O chain has one or more amino acid residue mutations at positions selected from the group consisting of L7K and L7R, K11L, T62K and T62H; In some embodiments, the titin T chain has the mutations C25S, C39T, and A8C, and the obscurin O chain has the mutations C25S, C76S, A88C, L7K, and T62K; the titin T chain has the mutations C25S, C39T, and A8C, and the obscurin O chain has the mutations C25S, C76S, A88C, L7K, and T62H; the titin T chain has the mutations C25S, C39T, and A8C, and the obscurin O chain has the mutations C25S, C76S, A88C, K11L, and T62K; the titin T chain has the mutations C25S, C39T, and A8C, and the obscurin O chain has the mutations C25S, C76S, A88C, K11L, and T62H. The position of the mutation in the titin T chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33.

In some embodiments, for the above-mentioned domain engineered antibody, bispecific antibody, Fab fragment of the domain engineered antibody, the obscurin O chain is an obscurin O chain having one or more amino acid residue mutations at positions selected from the group consisting of 7, 11, and 62 based on SEQ ID NO: 45; in some embodiments, the obscurin O chain has one or more amino acid residue mutations at positions selected from the group consisting of L7K and L7R, K11L and T62K and T62H based on SEQ ID NO: 45; in some embodiments, the titin T chain has the mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S, A88C, L7K, and T62K based on SEQ ID NO: 45; the titin T chain has the mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S, A88C, L7K, and T62H based on SEQ ID NO: 45; the titin T chain has the mutations C25S, C39T, and A8C based on SEQ ID NO: 32, and the obscurin O chain has the mutations C25S, C76S, A88C, K11L, and T62K based on SEQ ID NO: 45; The titin T-chain has the mutations C25S, C39T and the mutation A8C based on SEQ ID NO: 32, and the obscurin O-chain has the mutations C25S, C76S, A88C, K11L and T62H based on SEQ ID NO: 45. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 32; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 33.

In some embodiments, for the aforementioned domain engineered antibody, bispecific antibody, Fab fragment of the domain engineered antibody, the titin T chain has one or more amino acid mutations at positions selected from the group consisting of 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 at positions selected from the group consisting of 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. In some embodiments, the T chain of titin has one or more amino acid mutations at positions selected from the group consisting of 3W, 11I, 13L, 22M, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 66S and 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D and 84L, and/or the O chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of 2E, 11K, 12S, 13Y, 14T, 17E, 20L, 22M and 22S, 30D, 32P, 34E, 36T, 41K, 42L, 44I, 45T, 53L, 58V, 62E, 67Q and 67T, 69S, 89L, 92E, 94G and 97G. More preferably, the T-chain of titin has amino acid mutations 66S and 77S, and/or the O-chain of obscurin has amino acid mutations 11K, 12S, 13Y, 14T and 22S; The T chain of titin has amino acid mutations 66K, 70R, 79T, and 81R, and/or the O chain of obscurin has amino acid mutations 2E, 17E, 30D, 32P, 34E, 36T, 44I, 45T, 58V, 62E, 67Q, 69S, and 97G; The T chain of titin has amino acid mutations 3W, 11I, 13L, 22M, and 82M, and/or the O chain of obscurin has amino acid mutations 20L, 22M, and 53L; The titin T chain has amino acid mutations 11I, 66K, 79T, and 81R, and/or the obscurin O chain has amino acid mutations 41K, 45T, 67Q, 69S, and 89L; the titin T chain has amino acid mutations 40S, 42K, 45S, 47E, 49G, 56S, 58E, 75V, 83D, and 84L, and/or the obscurin O chain has amino acid mutations 42L, 45T, 67T, 69S, 92E, and 94G; The T chain of titin has amino acid mutations 47E, 49G, 56S, 58E and 75V, and/or the O chain of obscurin has amino acid mutations 42L, 45T, 67T, 69S, 92E and 94G; The T chain of titin has amino acid mutations 56S, 58E and 75V, and/or the O chain of obscurin has amino acid mutations 42L, 45T, 67T, 69S, 92E and 94G amino acid mutations; or the titin T-chain has 56S, 58E, 66S and 77S amino acid mutations, and/or the obscurin O-chain has 12S, 13Y, 22S, 42L, 45T, 67Q, 69S, 92E and 94G amino acid mutations. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 35; the position of the mutation in the O chain of obscurin is a position according to the natural sequence numbering relative to the sequence of SEQ ID NO: 50. In some embodiments, the T chain of titin has one or more amino acid mutations at positions selected from the group consisting of P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or the O chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G. In some embodiments, the titin T chain has the amino acid mutations M66S and T77S, and/or the obscurin O chain has the amino acid mutations L11K, A12S, F13Y, V14T and T22S; The titin T chain has the amino acid mutations M66K, K70R, S79T, and G81R, and/or the obscurin O chain has the amino acid mutations G2E, V17E, N30D, T32P, Q34E, S36T, V44I, A45T, L58V, K62E, A67Q, G69S, and A97G; the titin T chain has the amino acid mutations P3W, S11I, I13L, T22M, and N82M, and/or the obscurin O chain has the amino acid mutations D20L, T22M, and A53L; The titin T chain has the amino acid mutations S11I, M66K, S79T, and G81R, and/or the obscurin O chain has the amino acid mutations Q41K, A45T, A67Q, G69S, and V89L; the titin T chain has the amino acid mutations G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D, and F84L; and/or the obscurin O chain has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E, and D94G; The T chain of titin has the amino acid mutations Q47E, Q49G, N56S, D58E, and L75V, and/or the O chain of obscurin has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E, and D94G; the T chain of titin has the amino acid mutations N56S, D58E, and L75V, and/or the O chain of obscurin has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E, and D94G; or the titin T-chain has the amino acid mutations N56S, D58E, M66S and T77S, and/or the obscurin O-chain has the amino acid mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E and D94G. The position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 35; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence relative to the sequence of SEQ ID NO: 50.

In some embodiments, for the aforementioned domain engineered antibody, bispecific antibody, or domain engineered antibody Fab fragment, the titin T chain is a polypeptide having one or more amino acid mutations at positions selected from the group consisting of 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84 based on SEQ ID NO: 35, and the obscurin O chain is a polypeptide having one or more amino acid mutations at positions selected from the group consisting of 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 based on SEQ ID NO: 50. The position of the mutation in the titin T chain is the position according to the numbering of the natural sequence of SEQ ID NO: 35; the position of the mutation in the O chain of obscurin is a position according to the numbering of the natural sequence of SEQ ID NO: 50. In some embodiments, the T chain of titin has one or more amino acid mutations at positions selected from the group consisting of P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L based on SEQ ID NO: 35, and/or the O chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G based on SEQ ID NO: 35. In some embodiments, the titin T chain has the amino acid mutations M66S and T77S based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations L11K, A12S, F13Y, V14T and T22S based on SEQ ID NO: 50; The titin T chain has the amino acid mutations M66K, K70R, S79T and G81R based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations G2E, V17E, N30D, T32P, Q34E, S36T, V44I, A45T, L58V, K62E, A67Q, G69S and A97G based on SEQ ID NO: 50; the titin T chain has the amino acid mutations P3W, S11I, I13L, T22M and N82M based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations D20L, T22M and A53L based on SEQ ID NO: 50; The titin T chain has the amino acid mutations S11I, M66K, S79T and G81R based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations Q41K, A45T, A67Q, G69S and V89L based on SEQ ID NO: 50; the titin T chain has the amino acid mutations G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D and F84L based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E and D94G based on SEQ ID NO: 50; The titin T chain has the amino acid mutations Q47E, Q49G, N56S, D58E and L75V based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E and D94G based on SEQ ID NO: 50; the titin T chain has the amino acid mutations N56S, D58E and L75V based on SEQ ID NO: 35, and/or the obscurin O chain has the amino acid mutations Q42L, A45T, A67T, G69S, Q92E and D94G based on SEQ ID NO: 50; or the titin T-chain has the amino acid mutations N56S, D58E, M66S and T77S based on SEQ ID NO: 35, and/or the obscurin O-chain has the amino acid mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E and D94G based on SEQ ID NO: 50; the position of the mutation in the titin T-chain is the position according to the numbering of the natural sequence of SEQ ID NO: 35; the position of the mutation in the obscurin O-chain is the position according to the numbering of the natural sequence of SEQ ID NO: 50.

In some embodiments, for the above-mentioned domain-engineered antibody, bispecific antibody, or Fab fragment of the domain-engineered antibody, the N-termini of the titin T-chain, the obscurin O-chain, or the obscurin-like protein O-chain are operably linked to a VH (heavy chain variable domain) or VL (light chain variable domain) via a linker domain. In some embodiments, the linker domain is selected from the group consisting of: an N-terminal fragment of the T-chain of titin, an N-terminal fragment of the O-chain of obscurin, an N-terminal fragment of the O-chain of an obscurin-like protein, and a linker (G _x S) _y , where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6. In some embodiments, the linker domain is selected from the group consisting of: "KAGIR", "DQPQF", (G ₄ S) ₁ , and (G ₄ S) ₂ .

In some embodiments, the titin T chain is linked to the VH or VL via a KAGIR polypeptide, and/or the obscurin O chain is linked to the VL or VH via a DQPQF polypeptide.

In some embodiments of the above-mentioned polypeptide complex, wherein

(A) The titin T chain has the sequence shown in SEQ ID NO: 32, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 8, 20, 22, 25, 26, 39, 3, 11, 13, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or a KAGIR amino acid residue added at the N-terminus based on SEQ ID NO: 32; preferably, the titin T chain has a sequence having one or more amino acid mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S and M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or the amino acid residue KAGIR added at the N-terminus; and/or

(B) The O-chain of obscurin has the sequence shown in SEQ ID NO: 33, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 3, 9, 25, 76, 88, 7, 11, 62, 2, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 67, 69, 89, 92, 94 and 97, and/or the amino acid residue DQPQF added at the N-terminus based on SEQ ID NO: 33; preferably, the obscurin O-chain has a sequence having one or more amino acid mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S, A88C, L7K and L7R, T62K and T62H, G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G, and/or the amino acid residue DQPQF added at the N-terminus; or

The O-chain of the obscurin-like protein has a sequence as presented in SEQ ID NO: 34, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34; preferably, the O-chain of the obscurin-like protein has a sequence having one or more amino acid mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C;

the position of the mutation in the T-chain of titin is the position according to the numbering of the natural sequence of SEQ ID NO: 32; the position of the mutation in the obscurin-O chain is the position according to the numbering of the natural sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is the position according to the numbering of the natural sequence of SEQ ID NO: 34;

preferably where

(A) The titin T chain has one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 based on SEQ ID NO: 32, and preferably has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and the position of the mutation in the titin T chain is a position according to the numbering of the natural sequence of SEQ ID NO: 32; more preferably, the titin T chain has one or more amino acid mutations at positions selected from the group consisting of 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84 based on SEQ ID NO: 35, and preferably has one or more amino acid mutations at positions selected from the group consisting of P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L; the position of the mutation in the titin T-chain is a position according to the numbering of the natural sequence SEQ ID NO: 35, and/or

(B) The obscurin O-chain has one or more amino acid residue mutations at positions selected from the group consisting of 3, 9, 25, 76 and 88 based on SEQ ID NO: 33, and preferably has one or more amino acid residue mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C, and the position of the mutation in the obscurin O-chain is a position according to the numbering of the natural sequence of SEQ ID NO: 33; preferably, the O-chain of obscurin has one or more mutations of an amino acid residue at positions selected from the group consisting of 7, 11 and 62 based on SEQ ID NO: 45, and preferably has one or more mutations of an amino acid residue at positions selected from the group consisting of L7K and L7R, T62K and T62H and K11L, and the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence of SEQ ID NO: 45; more preferably, the O-chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of 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 based on SEQ ID NO: 50, and preferably has one or more amino acid mutations at positions selected from the group consisting of G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G, and the position of the mutation in the O-chain of obscurin is the position according to the numbering of the natural sequence of SEQ ID NO: 50; or

The O-chain of the obscurin-like protein has one or more mutations of the amino acid residue at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34, and preferably has one or more mutations of the amino acid residue at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C, and the position of the mutation in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence of SEQ ID NO: 34.

In some embodiments, for the aforementioned domain engineered antibody, bispecific antibody, or Fab fragment of the domain engineered antibody, the titin T chain comprises a polypeptide having a sequence as set forth in any of SEQ ID NOs: 32, 35, 41, 65-76, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76, and/or the obscurin O chain comprises a polypeptide having a sequence as set forth in any of SEQ ID NOs: 33, 42-58, 77-86, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 33, 42-58, 77-86; or the T-chain of titin comprises a polypeptide having a sequence as presented in any of SEQ ID NOs: 32, 35-41, 65-76, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76, and/or the O-chain of obscurin-like protein comprises a polypeptide having a sequence as presented in any of SEQ ID NOs: 34, 59 64, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 34, 59 64. Wherein a sequence at least 80% identical to a sequence refers to, for example, a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence.

In some embodiments, the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 68, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 80; the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 73, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 83; the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 76, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 84; the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 76, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 86; The titin T-chain comprises a polypeptide having a sequence presented in SEQ ID NO: 76, and the obscurin O-chain comprises a polypeptide having a sequence presented in SEQ ID NO: 86; or the titin T-chain comprises a polypeptide having a sequence presented in SEQ ID NO: 75, and the obscurin O-chain comprises a polypeptide having a sequence presented in SEQ ID NO: 86.

In some embodiments, the aforementioned bispecific antibody is a bispecific antibody selected from the group consisting of any of the following A-G:

A. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 89, or a sequence at least 85% identical to SEQ ID NO: 89, the first light chain has the sequence presented in SEQ ID NO: 90, or a sequence at least 85% identical to SEQ ID NO: 90, the second heavy chain has the sequence presented in SEQ ID NO: 87, or a sequence at least 85% identical to SEQ ID NO: 87, and the second light chain has the sequence presented in SEQ ID NO: 88, or a sequence at least 85% identical to SEQ ID NO: 88;

B. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 93, or a sequence at least 85% identical to SEQ ID NO: 93, the first light chain has the sequence presented in SEQ ID NO: 94, or a sequence at least 85% identical to SEQ ID NO: 94, the second heavy chain has the sequence presented in SEQ ID NO: 91, or a sequence at least 85% identical to SEQ ID NO: 91, and the second light chain has the sequence presented in SEQ ID NO: 92, or a sequence at least 85% identical to SEQ ID NO: 92;

C. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 97, or a sequence at least 85% identical to SEQ ID NO: 97, the first light chain has the sequence presented in SEQ ID NO: 98, or a sequence at least 85% identical to SEQ ID NO: 98, the second heavy chain has the sequence presented in SEQ ID NO: 95, or a sequence at least 85% identical to SEQ ID NO: 95, and the second light chain has the sequence presented in SEQ ID NO: 96, or a sequence at least 85% identical to SEQ ID NO: 96;

D. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 99, or a sequence at least 85% identical to SEQ ID NO: 99, the first light chain has the sequence presented in SEQ ID NO: 100, or a sequence at least 85% identical to SEQ ID NO: 100, the second heavy chain has the sequence presented in SEQ ID NO: 101, or a sequence at least 85% identical to SEQ ID NO: 101, and the second light chain has the sequence presented in SEQ ID NO: 96, or a sequence at least 85% identical to SEQ ID NO: 96;

E. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 102, or a sequence at least 85% identical to SEQ ID NO: 102, the first light chain has the sequence presented in SEQ ID NO: 100, or a sequence at least 85% identical to SEQ ID NO: 100, the second heavy chain has the sequence presented in SEQ ID NO: 95, or a sequence at least 85% identical to SEQ ID NO: 95, and the second light chain has the sequence presented in SEQ ID NO: 96, or a sequence at least 85% identical to SEQ ID NO: 96;

F. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 103, or a sequence at least 85% identical to SEQ ID NO: 103, the first light chain has the sequence presented in SEQ ID NO: 104, or a sequence at least 85% identical to SEQ ID NO: 104, the second heavy chain has the sequence presented in SEQ ID NO: 95, or a sequence at least 85% identical to SEQ ID NO: 95, and the second light chain has the sequence presented in SEQ ID NO: 96, or a sequence at least 85% identical to SEQ ID NO: 96;

G. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 107, or a sequence at least 85% identical to SEQ ID NO: 107, the first light chain has the sequence presented in SEQ ID NO: 108, or a sequence at least 85% identical to SEQ ID NO: 108, the second heavy chain has the sequence presented in SEQ ID NO: 109, or a sequence at least 85% identical to SEQ ID NO: 109, and the second light chain has the sequence presented in SEQ ID NO: 110, or a sequence at least 85% identical to SEQ ID NO: 110;

H. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 122, or a sequence at least 85% identical to SEQ ID NO: 122, the first light chain has the sequence presented in SEQ ID NO: 123, or a sequence at least 85% identical to SEQ ID NO: 123, the second heavy chain has the sequence presented in SEQ ID NO: 116, or a sequence at least 85% identical to the sequence with SEQ ID NO: 116, and the second light chain has the sequence presented in SEQ ID NO: 117, or a sequence at least 85% identical to SEQ ID NO: 117;

I. The first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 118, or a sequence at least 85% identical to SEQ ID NO: 118, the first light chain has the sequence presented in SEQ ID NO: 119, or a sequence at least 85% identical to SEQ ID NO: 119, the second heavy chain has the sequence presented in SEQ ID NO: 124, or a sequence at least 85% identical to SEQ ID NO: 124, and the second light chain has the sequence presented in SEQ ID NO: 125, or a sequence at least 85% identical to SEQ ID NO: 125;

wherein a sequence that is at least 85% identical to a sequence refers to, for example, a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence.

In some embodiments, the present invention relates to a method for producing a multispecific antibody that comprises replacing the CH1/CL of the antibody with the titin T chain and the obscurin O chain, or the titin T chain and the obscurin-like protein O chain. In some embodiments of the method for producing a multispecific antibody, the multispecific antibody of the present invention reduces mispairing between light chains and heavy chains compared to a wild-type antibody in which the CH1/CL is not replaced with the titin T chain/obscurin O chain or the titin T chain/obscurin-like protein O chain.

In some embodiments, the present invention further relates to a multispecific antibody obtained by the aforementioned method for producing a multispecific antibody. In some embodiments, the multispecific antibody comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 and VL1 form an antigen-binding site that specifically binds to a first antigen; wherein the C-terminus of VH1 is operably linked to the N-terminus of the T-chain of titin, the C-terminus of VL1 is operably linked to the N-terminus of the O-chain of obscurin or the O-chain of obscurin-like protein, or the C-terminus of VL1 is operably linked to the N-terminus of the T-chain of titin, and the C-terminus of VH1 is operably linked to the N-terminus of the O-chain of obscurin or the O-chain of obscurin-like protein.

In some embodiments, for the method of producing a multispecific antibody, the titin T chain comprises a polypeptide having a sequence as set forth in any of SEQ ID NOs: 32, 35, 41, 65-76, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76, and/or the obscurin O chain comprises a polypeptide having a sequence as set forth in any of SEQ ID NOs: 33, 42-58, 77-86, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 33, 42-58, 77-86; or the titin T-chain comprises a polypeptide having a sequence as presented in any of SEQ ID NOs: 32, 35-41, 65-76, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76, and/or the obscurin-like protein O-chain comprises a polypeptide having a sequence as presented in any of SEQ ID NOs: 34, 59 64, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 34, 59 64.

In some embodiments, the titin T chain comprises a polypeptide having the sequence set forth in SEQ ID NO: 68, and the obscurin O chain comprises a polypeptide having the sequence set forth in SEQ ID NO: 80; in some embodiments, the titin T chain comprises a polypeptide having the sequence set forth in SEQ ID NO: 73, and the obscurin O chain comprises a polypeptide having the sequence set forth in SEQ ID NO: 83; in some embodiments, the titin T chain comprises a polypeptide having the sequence set forth in SEQ ID NO: 76, and the obscurin O chain comprises a polypeptide having the sequence set forth in SEQ ID NO: 84; in some embodiments, the titin T chain comprises a polypeptide having the sequence set forth in SEQ ID NO: 76, and the obscurin O chain comprises a polypeptide having the sequence set forth in SEQ ID NO: 86; In some embodiments, the titin T chain comprises a polypeptide having the sequence presented in SEQ ID NO: 75, and the obscurin O chain comprises a polypeptide having the sequence presented in SEQ ID NO: 86.

In some embodiments, where

(A) The titin T chain has the sequence shown in SEQ ID NO: 32, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 8, 20, 22, 25, 26, 39, 3, 11, 13, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or 5 KAGIR amino acid residues added at the N-terminus based on SEQ ID NO: 32; preferably, the titin T chain has a sequence having one or more amino acid mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S and M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or 5 amino acid residues KAGIR added at the N-terminus; and/or

(B) The O-chain of obscurin has the sequence shown in SEQ ID NO: 33, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 3, 9, 25, 76, 88, 7, 11, 62, 2, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 67, 69, 89, 92, 94 and 97, and/or the amino acid residue DQPQF added at the N-terminus based on SEQ ID NO: 33; preferably, the O-chain of obscurin has a sequence having one or more amino acid mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S, A88C, L7K and L7R, T62K and T62H, G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G, and/or the amino acid residue DQPQF added at the N-terminus; or

The O-chain of the obscurin-like protein has a sequence as presented in SEQ ID NO: 34, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34; preferably, the O-chain of the obscurin-like protein has a sequence having one or more amino acid mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C;

the position of the mutation in the T-chain of titin is a position according to the numbering of the natural sequence of SEQ ID NO: 32; the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence of SEQ ID NO: 33; the position of the mutation in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence of SEQ ID NO: 34;

preferably where

(A) The titin T chain has one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 based on SEQ ID NO: 32, and preferably has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and the position of the mutation in the titin T chain is a position according to the numbering of the natural sequence of SEQ ID NO: 32; more preferably, the titin T chain has one or more amino acid mutations at positions selected from the group consisting of 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84 based on SEQ ID NO: 35, and preferably has one or more amino acid mutations at positions selected from the group consisting of P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L; the position of the mutation in the titin T-chain is a position according to the numbering of the natural sequence SEQ ID NO: 35, and/or

(B) The obscurin O-chain has one or more amino acid residue mutations at positions selected from the group consisting of 3, 9, 25, 76 and 88 based on SEQ ID NO: 33, and preferably has one or more amino acid residue mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C, and the position of the mutation in the obscurin O-chain is a position according to the numbering of the natural sequence of SEQ ID NO: 33; preferably, the O-chain of obscurin has one or more amino acid residue mutations at positions selected from the group consisting of 7, 11 and 62 based on SEQ ID NO: 45, and preferably has one or more amino acid residue mutations at positions selected from the group consisting of L7K and L7R, T62K and T62H, and K11L, and the position of the mutation in the O-chain of obscurin is a position according to the numbering of the natural sequence of SEQ ID NO: 45; more preferably, the O-chain of obscurin has one or more amino acid mutations at positions selected from the group consisting of 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 based on SEQ ID NO: 50, and preferably has one or more amino acid mutations at positions selected from the group consisting of G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G, and the position of the mutation in the O-chain of obscurin is the position according to the numbering of the natural sequence of SEQ ID NO: 50; or

The O-chain of the obscurin-like protein has one or more mutations of the amino acid residue at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34, and preferably has one or more mutations of the amino acid residue at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C, and the position of the mutation in the O-chain of the obscurin-like protein is a position according to the numbering of the natural sequence of SEQ ID NO: 34.

In some embodiments, the present invention relates to a conjugate comprising any of the above-mentioned polypeptide complex, multispecific polypeptide complex, domain-engineered antibody, domain-engineered antibody Fab fragment, or bispecific antibody, or multispecific antibody.

In some embodiments, there is provided a polypeptide having the sequence shown in any of SEQ ID NOs: 32, 35-41, 65-76, or a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76, or having the sequence shown in any of SEQ ID NOs: 33, 42-58, 77-86, or a sequence at least 80% identical to any of SEQ ID NOs: 33, 42-58, 77-86, or having the sequence shown in any of SEQ ID NOs: 34, 59-64, or a sequence at least 80% identical to any of SEQ ID NOs: 34, 59-64; preferably, the polypeptide can be used to replace CH1 and/or CL of an antibody. More preferably, the polypeptide is a titin T chain and an obscurin O chain, or a titin T chain and an obscurin-like protein O chain, and the one-sided CH1 and CL of the bispecific antibody are substituted with a titin T chain and an obscurin O chain, or a titin T chain and an obscurin-like protein O chain. After the substitution, mispairing between the first heavy chain and the second light chain, and between the first light chain and the second heavy chain, is less likely to occur.

In some embodiments, the titin T chain has the sequence shown in SEQ ID NO: 32, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 8, 20, 22, 25, 26, 39, 3, 11, 13, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84, based on SEQ ID NO: 32; preferably, the titin T chain has a sequence having one or more amino acid mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S and M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L and/or 5 amino acid residues KAGIR added at the N-terminus; The O-chain of obscurin has the sequence presented in SEQ ID NO: 33, or additionally has a sequence having one or more amino acid mutations at positions selected from the group consisting of 3, 9, 25, 76, 88, 7, 11, 62, 2, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 67, 69, 89, 92, 94 and 97 and/or the amino acid residue DQPQF added at the N-terminus based on SEQ ID NO: 33; preferably, the O-chain of obscurin has a sequence having one or more amino acid mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S, A88C, L7K and L7R, T62K and T62H, G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G and/or the amino acid residue DQPQF added at the N-terminus; The O-chain of the obscurin-like protein has a sequence presented in SEQ ID NO: 34, or additionally has a sequence having one or more amino acid mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34; preferably, the O-chain of the obscurin-like protein has a sequence having one or more amino acid mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C.

A polypeptide is disclosed for use in replacing CH1 and/or CL of an antibody, CH1 and/or CL of an antigen-binding fragment, or CH1 and/or CL of a polypeptide complex, wherein the polypeptide has a sequence as presented in any of SEQ ID NOs: 32, 35-41, 65-76, or a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76; or the polypeptide has a sequence as presented in any of SEQ ID NOs: 33, 42-58, 77-86, or a sequence at least 80% identical to any of SEQ ID NOs: 33, 42-58, 77-86; or the polypeptide has a sequence as set forth in any of SEQ ID NOs: 34, 59, 64, or a sequence at least 80% identical to any of SEQ ID NOs: 34, 59-64; preferably, the polypeptide is a titin T chain and an obscurin O chain, or a titin T chain and an obscurin-like protein O chain. More preferably, after the one-sided CH1 and CL of the bispecific antibody are replaced by the polypeptide, mispairing between the first heavy chain and the second light chain or between the first light chain and the second heavy chain is less likely to occur.

In some embodiments, the present invention relates to a titin T-chain, an obscurin O-chain, and/or an obscurin-like protein O-chain, wherein the titin T-chain has the sequence shown in any of SEQ ID NOs: 32, 35-41, 65-76, or a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76, and the obscurin O-chain has the sequence shown in any of SEQ ID NOs: 33, 42-58, 77-86, or a sequence at least 80% identical to any of SEQ ID NOs: 33, 42-58, 77-86; The O-chain of the obscurin-like protein has a sequence as set forth in any of SEQ ID NOs: 34, 59, 64, or a sequence that is at least 80% identical to any of SEQ ID NOs: 34, 59, 64. In some embodiments, the titin T-chain/obscurin O-chain or the titin T-chain/obscurin-like protein O-chain can be used to replace CH1 and/or CL of an antibody.

In some embodiments, the titin T chain has the sequence shown in SEQ ID NO: 32, or further has a sequence comprising one or more amino acid mutations at positions selected from the group consisting of 8, 20, 22, 25, 26, 39, 3, 11, 13, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83, and 84, based on SEQ ID NO: 32; preferably, the titin T chain has a sequence containing one or more amino acid mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S and M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L; The O-chain of obscurin has the sequence presented in SEQ ID NO: 33, or additionally has a sequence comprising one or more amino acid mutations at positions selected from the group consisting of 3, 9, 25, 76, 88, 7, 11, 62, 2, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 67, 69, 89, 92, 94 and 97 based on SEQ ID NO: 33; preferably, the O-chain of obscurin has a sequence containing one or more amino acid mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S, A88C, L7K and L7R, T62K and T62H, G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G; The O-chain of the obscurin-like protein has a sequence presented in SEQ ID NO: 34, or additionally has one or more amino acid mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34; preferably, the O-chain of the obscurin-like protein has a sequence containing one or more amino acid mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C.

In some embodiments, the present invention further discloses the use of a titin T chain and an obscurin O chain, or a titin T chain and an obscurin-like protein O chain, to reduce light/heavy chain mispairing of a multispecific antibody; in some embodiments, the titin T chain comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 32, 35-41, 65-76, or a polypeptide having a sequence at least 80% identical to any one of SEQ ID NOs: 32, 35-41, 65-76, and/or the obscurin O chain comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 33, 42-58, 77-86, or a polypeptide having a sequence at least 80% identical to any one of SEQ ID NOs: 33, 42-58, 77-86; or the titin T-chain comprises a polypeptide having a sequence as presented in any of SEQ ID NOs: 32, 35-41, 65-76, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 32, 35^-1, 65-76, and/or the obscurin-like protein O-chain comprises a polypeptide having a sequence as presented in any of SEQ ID NOs: 34, 59-64, or a polypeptide having a sequence at least 80% identical to any of SEQ ID NOs: 34, 59-64.

In some embodiments, for the aforementioned use of a titin T chain and an obscurin O chain or a titin T chain and an obscurin-like protein O chain for reducing light/heavy chain mispairing of a multispecific antibody, the titin T chain has a sequence as set forth in SEQ ID NO: 32, or further has a sequence having one or more amino acid mutations at positions selected from the group consisting of 8, 20, 22, 25, 26, 39, 3, 11, 13, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84 and/or 5 KAGIR amino acid residues added at the N-terminus, based on SEQ ID NO: 32; preferably, the titin T chain has a sequence having one or more amino acid mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S and M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L and/or 5 amino acid residues KAGIR added at the N-terminus; The O-chain of obscurin has the sequence presented in SEQ ID NO: 33, or additionally has a sequence having one or more amino acid mutations at positions selected from the group consisting of 3, 9, 25, 76, 88, 7, 11, 62, 2, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 67, 69, 89, 92, 94 and 97 and/or the amino acid residue DQPQF added at the N-terminus based on SEQ ID NO: 33; preferably, the O-chain of obscurin has a sequence having one or more amino acid mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S, A88C, L7K and L7R, T62K and T62H, G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M and T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q and A67T, G69S, V89L, Q92E, D94G and A97G and/or the amino acid residue DQPQF added at the N-terminus; The O-chain of the obscurin-like protein has a sequence presented in SEQ ID NO: 34, or additionally has a sequence having one or more amino acid mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 based on SEQ ID NO: 34; preferably, the O-chain of the obscurin-like protein has a sequence having one or more amino acid mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C.

In some embodiments, the multispecific antibody comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 and VL1 form an antigen-binding site that specifically binds to a first antigen; wherein the C-terminus of VH1 is operably linked to the N-terminus of a titanium T-chain, the C-terminus of VL1 is operably linked to the N-terminus of an obscurin O-chain or an obscurin-like protein O-chain, or the C-terminus of VL1 is operably linked to the N-terminus of a titanium T-chain, and the C-terminus of VH1 is operably linked to the N-terminus of an obscurin O-chain or an obscurin-like protein O-chain.

In some embodiments, the present invention relates to a non-kleinic acid molecule encoding any of the above-mentioned polypeptide complex, or multispecific polypeptide complex, or domain-engineered antibody, or Fab fragment of domain-engineered antibody, or bispecific antibody, or polypeptide, or multispecific antibody.

In some embodiments, the present invention relates to a vector comprising the aforementioned nucleic acid molecule.

In some embodiments, the present invention relates to a host cell obtained by transformation (transduction or transfection) with the aforementioned vector, wherein the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells and more preferably mammalian cells. In some embodiments, the host cell does not include any animal or plant cells capable of developing into a whole individual, such as human embryonic stem cells, fertilized eggs, or germ cells.

In some embodiments, the host cell is a eukaryotic cell, and more preferably a mammalian cell, including, but not limited to, CHO (Chinese hamster ovary cells), 293 (HEK 293 - human embryonic kidney cells), NSO (non-secreting mouse myeloma cells), and a cell in which gene editing can be performed to alter the glycosylation modification of the antibody or antigen-binding fragment thereof, thereby altering the ADCC function (antibody-dependent cell-mediated cytotoxicity) of the antibody or antigen-binding fragment thereof, for example, by knocking out genes such as FUT8 (alpha-(1,6)-fucosyltransferase) or GnT-III (beta-1,4-mannosylglycoprotein-4-beta-N-acetylglucosaminyltransferase). In some embodiments, the mammalian cell does not include a human cell.

In some embodiments, the present invention discloses a method for producing any of the above-mentioned polypeptide complex, multispecific polypeptide complex, domain-engineered antibody, domain-engineered antibody Fab fragment, bispecific antibody, or polypeptide or multispecific antibody, which comprises the following steps:

culturing the above-mentioned host cell followed by purification and isolation of the polypeptide complex, the multispecific polypeptide complex, the domain-engineered antibody, the Fab fragment of the domain-engineered antibody, the bispecific antibody or polypeptide, or the multispecific antibody.

In some embodiments, the present invention relates to a pharmaceutical composition comprising any of the aforementioned polypeptide complex, multispecific polypeptide complex, domain-engineered antibody, domain-engineered antibody Fab fragment, bispecific antibody, nucleic acid, multispecific antibody, or conjugate, and one or more pharmaceutically acceptable carriers, excipients, or diluents. Preferably, a unit dose of the pharmaceutical composition may contain from 0.01 to 99 wt. % of the above-mentioned polypeptide complex, multispecific polypeptide complex, antibody with an engineered domain, Fab fragment of an antibody with an engineered domain, bispecific antibody, multispecific antibody or conjugate, or a unit dose of the pharmaceutical composition contains the above-mentioned polypeptide complex, multispecific polypeptide complex, antibody with an engineered domain, Fab fragment of an antibody with an engineered domain, bispecific antibody, multispecific antibody or conjugate in an amount preferably of 0.1-2000 mg, and more preferably 1-1000 mg.

In some embodiments, the present invention relates to the use of any of the above-mentioned polypeptide complex, multispecific polypeptide complex, domain-engineered antibody, domain-engineered antibody Fab fragment, bispecific antibody, pharmaceutical composition, nucleic acid molecule, or multispecific antibody in the preparation of a medicament for the treatment or prevention of a disease or condition. In some embodiments, the disease or condition is a tumor, an inflammatory disease, an autoimmune disease, or an infectious disease. In some embodiments, the disease or condition is a bone-related disease, such as a disease or disorder of osteoporosis, osteopenia, or osteoarthritis, rheumatoid arthritis, periodontitis, or multiple myeloma. In some embodiments, the tumor is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignancies. 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), neuroglioma, 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 cell lymphoma (SLL), large B-cell lymphoma rich in T cells/histocytes, multiple myeloma, myeloid leukemia protein-1 (Mcl-1), Myelodysplastic syndrome (MDS), gastrointestinal cancer (GI), 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, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic amloidosis light chain, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine tumor, Merkel cell carcinoma, testicular cancer, and skin cancer. In some embodiments, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I 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 disease may be 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 thrombocytopenic symptoms (such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura), hemolytic anemia, mastitis gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omenn syndrome, chronic renal failure, acute infectious mononucleosis, HIV- and herpes virus-related diseases, severe acute respiratory syndrome, chorioretinitis, and immunological diseases caused by viral infection (e.g., diseases caused or mediated by infection of B cells by Epstein-Barr virus (EBV)).

In some embodiments, the present invention relates to a method for treating or preventing a disease or condition that comprises administering to a subject a prophylactically or therapeutically effective amount of any of the aforementioned polypeptide complex, multispecific polypeptide complex, domain-engineered antibody, domain-engineered antibody Fab fragment, bispecific antibody, pharmaceutical composition, nucleic acid molecule, or multispecific antibody. In some embodiments, the disease or condition is a tumor, an inflammatory disease, an autoimmune disease, or an infectious disease. In some embodiments, the disease or condition is a bone-related disease, such as a disease or disorder of osteoporosis, osteopenia, or osteoarthritis, rheumatoid arthritis, periodontitis, or multiple myeloma. In some embodiments, the tumor is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignancies. 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), neuroglioma, 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 cell lymphoma (SLL), large B-cell lymphoma rich in T cells/histocytes, multiple myeloma, myeloid leukemia protein-1 (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal cancer (GI), 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, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic amloidosis light chain, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine tumor, Merkel cell carcinoma, testicular cancer, and skin cancer. In some embodiments, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I 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 disease may be 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 thrombocytopenic symptoms (such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura), hemolytic anemia, mastitis gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omenn syndrome, chronic renal failure, acute infectious mononucleosis, HIV- and herpes virus-related diseases, severe acute respiratory syndrome, chorioretinitis, and immunological diseases caused by viral infection (e.g., diseases caused or mediated by infection of B cells by Epstein-Barr virus (EBV)).

In some embodiments, the present invention relates to any of the aforementioned polypeptide complex, multispecific polypeptide complex, domain-engineered antibody, domain-engineered antibody Fab fragment, bispecific antibody, pharmaceutical composition, nucleic acid molecule, or multispecific antibody for use as a medicament. In some embodiments, the medicament is used to treat or prevent a disease or condition. In some embodiments, the disease or condition is a tumor, an inflammatory disease, an autoimmune disease, or an infectious disease. In some embodiments, the disease or condition is a bone-related disease, such as a disease or disorder of osteoporosis, osteopenia, or osteoarthritis, rheumatoid arthritis, periodontitis, or multiple myeloma. In some embodiments, the tumor is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignancies. 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), neuroglioma, 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 cell lymphoma (SLL), large B-cell lymphoma rich in T cells/histocytes, multiple myeloma, myeloid leukemia protein-1 (Mcl-1), Myelodysplastic syndrome (MDS), gastrointestinal cancer (GI), 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, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic amloidosis light chain, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine tumor, Merkel cell carcinoma, testicular cancer, and skin cancer. In some embodiments, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I 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 disease may be 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 thrombocytopenic symptoms (such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura), hemolytic anemia, mastitis gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omenn syndrome, chronic renal failure, acute infectious mononucleosis, HIV- and herpes virus-related diseases, severe acute respiratory syndrome, chorioretinitis, and immunological diseases caused by viral infection (e.g., diseases caused or mediated by infection of B cells by Epstein-Barr virus (EBV)).

In some embodiments, the present invention relates to a detection method that comprises the step of contacting a sample to be tested with any of the above-mentioned polypeptide complex, multispecific polypeptide complex, domain-engineered antibody, domain-engineered antibody Fab fragment, bispecific antibody, or multispecific antibody.

In some embodiments, the present invention relates to a reagent for use in detection or analysis, wherein the reagent comprises any of the above-mentioned polypeptide complex, multispecific polypeptide complex, domain-engineered antibody, domain-engineered antibody Fab fragment, bispecific antibody, or multispecific antibody.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1A and Fig. 1B are structural diagrams of the interaction region between the T-chain domain of titin and the O-chain domain of obscurin or between the T-chain domain of titin and the O-chain domain of an obscurin-like protein;

Fig. 2 is a block diagram of Fab with substituted CH1 and CL;

Fig. 3 is a structural block diagram of a monospecific IgG antibody with substituted bilateral CHI and CL;

Fig. 4 is a structural block diagram of a bispecific IgG antibody with substituted one-sided CH1 and CL;

Fig. 5 is a structural block diagram of the bispecific antibody DI-1;

Fig. 6A-6C are DI-1 mass spectrometry diagrams. Fig. 6A is an LC1/LC2 mass spectrogram, and Fig. 6B is an HC1/HC2 mass spectrogram; Fig. 6C is an LC1+LC2+HC1+HC2 mass spectrogram;

Fig. 7 is a structural block diagram of light/heavy chain mispaired antibody molecules;

Fig. 8 is a structural block diagram of the bispecific antibody BU5;

Fig. 9A - 9C are mass spectrometry diagrams of BU5;

Fig. 10 is an experimental graph of the osteoclast differentiation results in the bispecific antibody DI-1;

Fig. 11 is an experimental graph of the TF1 cell proliferation results with the DI-1 bispecific antibody;

Fig. 12 is a structural block diagram of the bispecific antibodies HJ-1, HJ-2, HJ-3 and HJ-4;

Fig. 13A-13D are mass spectrometry diagrams of four-chain coexpression of bispecific antibodies HJ-1, HJ-2, HJ-3 and HJ-4;

Fig. 14A-14B are mass spectrometry diagrams of three-chain coexpression of HJ-1-H1, HJ-1-H2, and HJ-1-L2 for the HJ-1 bispecific antibody;

Fig. 15A-15B are mass spectrometry diagrams of three-chain coexpression of HJ-3-H1, HJ-3-H2 and HJ-3-L2 for the HJ-3 bispecific antibody; and

Fig. 16A-16B represent interactions between residues.

DETAILED DESCRIPTION OF THE INVENTION

To provide a clearer understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The three-letter codes and single-letter codes of amino acids used in this specification are as described in J. biol. chem., 243, p. 3558 (1968).

An "antibody (Ab)" is typically an immunoglobulin (Ig), and a naturally occurring intact antibody is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of an immunoglobulin heavy chain differ in their amino acid composition and arrangement, and thus in their antigenicity. Accordingly, immunoglobulins can be classified into five classes: IgM, IgD, IgG, IgA, and IgE, with their respective heavy chains being μ chains, δ chains, γ chains, α chains, and ε chains, respectively. The same Ig class can be classified into different subclasses based on differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chains. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. Since light chains are classified into κκ chains or λ chains based on differences in the constant regions, antibodies can be classified into two types. Each of the five Ig classes can have either κ chains or λ chains. Based on differences in individual amino acids in the constant region of the λ chain, the λ chain can be divided into four subtypes: λ1, λ2, λ3, and λ4.

The antibodies disclosed herein include antibodies or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof that have been engineered from immunoglobulins with retention of antigen-binding ability; antibodies or antigen-binding fragments thereof include monospecific antibodies and multispecific antibodies (e.g., bispecific antibodies), and further include monovalent antibodies or multivalent antibodies (e.g., divalent antibodies). An antigen-binding fragment of an antibody, for example, can be an antigen-binding fragment comprising at least one VH-CH1 structure and at least one VL-CL structure, wherein the VH and VL structures are capable of being close to each other based on interchain interactions and retaining antigen-binding ability. In some embodiments, the antigen-binding fragment of the antibody is a monovalent Fab fragment (Fab1 fragment), a divalent Fab fragment (F(ab)2), a trivalent Fab fragment (F(ab)3), a multivalent (two or more) Fab fragment, or other monospecific, bispecific, or multispecific antigen-binding fragments comprising at least one Fab fragment.

An "engineered domain antibody" as described herein refers to an antibody formed after the CH1 and/or CL domains of an antibody have been replaced with other domains or peptide fragments that can be used to replace the CH1/CL domains of the antibody and typically have intermolecular interactions; Alternative domain combinations include, but are not limited to, IL13RA1 Fibronectin type III domain 3/IL4R Fibronectin type III domain 2, IL6ST Fibronectin type III domain 2/IL6R Fibronectin type III domain 2, HFE type C1 Ig-like domain/B2M type C1 Ig-like domain, CD1B Ig-like domain/B2M type C1 Ig-like domain, obscurin Ig-like domain 1/titin Ig-like domain 152, or obscurin-like Ig-like domain 1/titin Ig-like domain 152. In some embodiments, the domain engineered antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody; in some embodiments, the domain engineered antibody is a monovalent antibody, a bivalent antibody, or a multivalent antibody; in some embodiments, the domain engineered antibody is a whole antibody or an antigen-binding fragment thereof; in some embodiments, the antigen-binding fragment of the antibody is a Fab fragment, which may be a monovalent Fab fragment (Fab1 fragment), a divalent Fab fragment (F(ab)2), a trivalent Fab fragment (F(ab)3), or a multivalent (two or more) Fab fragment. As an example, a structural block diagram of a Fab fragment of an antibody with CH1 and CL substituted with titin T chain/obscurin O chain or titin T chain/obscurin-like protein O chain is shown in Fig. 2; The structural block diagram of the antibody with CH1 and CL substituted by the titin T-chain/obscurin O-chain or the titin T-chain/obscurin-like protein O-chain is shown in Fig. 3; the structural block diagram of the bispecific antibody with one-sided CH1 and CL substituted by the titin T-chain/obscurin O-chain or the titin T-chain/obscurin-like protein O-chain is shown in Fig. 4. Other antibodies with at least one CH1 and at least one CL substituted by the titin T chain/obscurin O chain or by the titin T chain/obscurin-like protein O chain are, for example, a bivalent antibody with one-sided CH1 and CL substituted by the titin T chain/obscurin O chain or by the titin T chain/obscurin-like protein O chain, a bispecific antibody with two-sided CH1 and CL substituted by the titin T chain/obscurin O chain or by the titin T chain/obscurin-like protein O chain, an E(ab)2 fragment with two CH1/CL substituted by the titin T chain/obscurin O chain or by the titin T chain/obscurin-like protein O chain, or an F(ab)3 fragment with 1 or 2 or 3 or CH1/CL substituted with titin T-chain/obscurin O-chain or titin T-chain/obscurin-like protein O-chain.

Titin is a large sarcomeric protein with a complex molecular folded structure. It is known to link thick myofilaments to the Z-disc, maintaining the integrity and stability of myofibrils, etc. 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, accounting for approximately half of the sarcomere.

"Titin Ig-like domain 152" is an Ig-like domain on the titin protein, called titin Ig-like domain 152, which is capable of binding to obscurin-like Ig-like domain 1 or obscurin Ig-like domain 1 through intermolecular interactions to form a dimeric complex (available from the RCSB PDB database).

"Titin T-chain" or "T-chain" refers to a peptide fragment of 78-118 amino acids in length containing the Ig-like domain 152 of titin in the titin protein or its functional variant, wherein the titin T-chain is capable of binding to the Ig-like domain 1 of obscurin or the Ig-like domain 1 of obscurin through intermolecular interactions to form a complex. The functional T-chain variant is obtained by mutation of partial amino acids of the wild-type T-chain, but still contains a peptide fragment of the complex formed by binding to the Ig-like domain 1 of obscurin or the Ig-like domain 1 of obscurin through intermolecular interactions. In the present invention, the titin T chain can be used to replace the CH1 or CL domain of an antibody without affecting the VH and VL binding of the antibody and the binding of the antibody to the epitope. Partial amino acids of the titin T chain can be mutated as long as the titin T chain is able to bind to the obscurin Ig-like domain 1 to form a complex. For example, amino acids of an appropriate length are added or truncated at the C-terminus and/or N-terminus of the Ig-like domain 152 of titin; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues can be added or truncated; For example, 5 amino acids of "KAGIR" of titin protein, immediately adjacent to the N-terminus of the Ig-like domain 152 of titin, are added at the N-terminus of the Ig-like domain 152 of titin; a linker sequence of suitable length (for example, a linker (G _x S) _y , where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6, such as "G ₄ S" or "(G ₄ S) ₂ ") can also be added at the N-terminus and/or C-terminus of the Ig-like domain 152 of titanium to ensure the binding of the N-terminus of the Ig-like domain 152 of titanium to VH and/or VL via the linker sequence. Other mutations can also be made on the amino acids of the Ig-like domain 152 of titinav, for example, some amino acids can be mutated to increase interchain disulfide bonds, improving the stability of the complex. In some embodiments of the present invention, the titanium T-chain is a polypeptide having a sequence presented in any of SEQ ID NOs: 32, 35-41, 65-76, or a sequence at least 80% identical to any of SEQ ID NOs: 32, 35-41, 65-76, wherein the polypeptide is capable of binding to the O-chain of obscurin or the O-chain of an obscurin-like protein to form a complex.

"Obscurin" is a protein encoded by the OBSCN gene and belongs to the sarcosine family of giant signaling proteins. Obscurin is expressed in cardiac and skeletal muscle and plays an important role in the organization of myofibrils during sarcomere assembly. Obscurin is the major cytoplasmic ligand of the sarcoplasmic reticulin sANK1 (small ankyrin 1), which can prevent sANK1 degradation (Lange S et al., Molecular Biology of the Cell., 23 (13): 2490-504); Obscurin acts as a signaling link between the sarcoplasmic reticulum domain and the sarcoplasmic reticulum domain (Bagnato P et al., The Journal of Cell Biology., 160 (2): 245-53); Obscurin is involved in the formation of new sarcomeres during the assembly of myofibrils (Borisov AV, et al., Biochemical and Biophysical Research Communications, 310 (3):910 918).

"Obscurin Ig-like domain 1" is a region of the Ig-like domain in the obscurin protein, called obscurin Ig-like domain 1, which is capable of binding to the Ig-like domain of titanium 152 through intermolecular interactions to form a complex (available from the RCSB PDB database).

"Obscurin O-chain" or "O-chain" refers to a peptide fragment of 87-117 amino acids in length containing the Ig-like domain 1 of obscurin in the obscurin protein or a functional variant thereof, wherein the obscurin O-chain is capable of binding to the Ig-like domain 152 of titin through intermolecular interactions to form a complex. The functional variant of the obscurin chain is obtained by mutation on partial amino acids of the wild-type O-chain, but still has a peptide fragment of the complex formed by binding to the Ig-like domain 152 of titin through intermolecular interactions. In the present invention, the obscurin O-chain can replace the CH1 or CL domain of an antibody without affecting the binding of the VH and VL of the antibody and the binding of the antibody to the epitope. Partial amino acids of the obscurin O-chain can be mutated until the obscurin O-chain is capable of binding to the Ig-like domain 152 of titin to form a complex. For example, amino acids of appropriate length are added or truncated at the C-terminus and/or N-terminus of the obscurin O-chain domain; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids were added or truncated; for example, 5 amino acids "DQPQF" of the wild-type obscurin protein, immediately adjacent to the N-terminus of the obscurin Ig-like domain 1, were added at the N-terminus of the obscurin O-chain domain. A linker sequence of suitable length (e.g., a (G _x S) _y linker, where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6, such as "G ₄ S" or "(G ₄ S) ₂ "), can also be added at the N-terminus and/or C-terminus of the obscurin O-chain domain, to ensure the binding of the N-terminus of the obscurin O-chain domain to the VH and/or VL of the antibody via the linker sequence. Other mutations can also be made on partial amino acids of the Ig-like domain 1 of obscurin, for example, some amino acids can be mutated to increase interchain disulfide bonds, improving the stability of the antibody, etc. In some embodiments of the present invention, the O-chain of obscurin is a polypeptide having a sequence as set forth in any of SEQ ID NOs: 33, 42-58, 77-86, or a sequence at least 80% identical to any of SEQ ID NOs: 33, 42-58, 77-86, wherein the polypeptide is capable of binding to the T-chain of titin to form a complex.

Obscurin-like protein 1 (OLP1) is a protein encoded by the OBSL1 gene, located in SPEG on human chromosome 2q35, and is closely related to obscurin. Alternative splicing of OLP1 results in several isoforms with predicted molecular masses ranging from 130 kDa to 230 kDa (Geisler SB et al., (2007). Genomics., 89 (4): 521–31).

"Obscurin-like protein Ig-like domain 1" is a region of the Ig-like domain 1 in obscurin-like protein, called the obscurin-like protein Ig-like domain 1, which is capable of binding to the Ig-like domain 152 of titin through intermolecular interactions to form a complex (available from the RCSB PDB database).

The "O chain of obscurin-like protein" or "OL chain" refers to a peptide fragment of 78-118 amino acids in length containing the Ig-like domain 1 of obscurin-like protein in obscurin-like protein 1 or its functional variant. The O chain of obscurin-like protein is capable of binding to the Ig-like domain 152 of titin through intermolecular interactions to form a complex. The functional variant of obscurin-like protein O chain is obtained by mutation of partial amino acids of the wild-type OL chain, but still contains a peptide fragment of the complex formed by binding to the Ig-like domain 152 of titin through intermolecular interactions. In the present invention, the O-chain of the obscurin-like protein can replace the CH1 or CL domain of the antibody without affecting the binding of the VH and VL of the antibody or the binding of the antibody to the epitope. Partial amino acids of the O-chain of the obscurin-like protein can be mutated until the O-chain of the obscurin-like protein is capable of binding to the Ig-like domain of titin 152 to form a complex. In some embodiments, for example, amino acids of an appropriate length are added or truncated at the C-terminus and/or N-terminus of the obscurin O-chain domain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are added or truncated. A linker sequence of an appropriate length (e.g., a (G _x S) _y linker, where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6, such as "G ₄ S" or "(G ₄ S) ₂ ")), can also be added at the N-terminus and/or C-terminus of the O-chain domain of the obscurin-like protein, to ensure the linkage of the N-terminus of the O-chain domain of the obscurin-like protein to the VH and/or VL of the antibody via the linker sequence. Other mutations can also be made on partial amino acids of the Ig-like domain 1 of the obscurin-like protein, for example, some amino acids can be mutated to increase interchain disulfide bonds, improving the stability of the antibody, etc. In some embodiments of the present invention, the O-chain of the obscurin-like protein is a polypeptide having a sequence presented in any of SEQ ID NOs: 34, 59-64, or a sequence at least 80% identical to any of SEQ ID NOs: 34, 59-64, wherein the polypeptide is capable of binding to the T-chain of titin to form a complex.

In addition, partial amino acids in the Ig-like domain 152 of titin, the Ig-like domain 1 of obscurin, or the Ig-like domain 1 of obscurin-like protein can be mutated until the Ig-like domain 152 of titin and the Ig-like domain 1 of obscurin, or the Ig-like domain 152 of titin and the Ig-like domain 1 of obscurin-like protein are associated with each other to form a complex. In some embodiments, amino acids of appropriate length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the wild-type protein immediately adjacent to the domain) may be added or truncated at the C-terminus and/or N-terminus of the titin Ig-like domain 152, the obscurin Ig-like domain 1, or the obscurin-like protein Ig-like domain 1, until the titin Ig-like domain 152 and the obscurin Ig-like domain 1, or the titin Ig-like domain 152 and the obscurin-like protein Ig-like domain 1, are able to associate with each other to form a complex. For example, the 5 amino acids "KAGIR" of the wild-type titin protein immediately adjacent to the N-terminus of the Ig-like domain 152 of titin were added to the N-terminus of the Ig-like domain 152 of titin; or the 5 amino acids "DQPQF" of the wild-type obscurin protein immediately adjacent to the N-terminus of the Ig-like domain 1 of obscurin were added to the N-terminus of the Ig-like domain 1 of obscurin. In some embodiments, a linker sequence of suitable length (e.g., a (G _x S) _y linker, where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6, such as "G ₄ S" or "(G ₄ S) ₂ "), can be added at the N-terminus and/or C-terminus of the Ig-like domain 152 of titin, the Ig-like domain 1 of obscurin, or the Ig-like domain 1 of obscurin-like protein, to ensure that the N-terminus of the Ig-like domain 152 of titin, the Ig-like domain 1 of obscurin, or the Ig-like domain 1 of obscurin-like protein is linked to the VH or VL of the antibody via the linker sequence. In some embodiments, other mutations may also be made on partial amino acids of the Ig-like domain 152 of titin, the Ig-like domain 1 of obscurin, or the Ig-like domain 1 of obscurin-like protein, as long as the Ig-like domain 152 of titin and the Ig-like domain 1 of obscurin, or the Ig-like domain 152 of titin and the Ig-like domain 1 of obscurin-like protein are able to associate with each other to form a complex, for example, partial amino acids are mutated to increase interchain disulfide bonds, improving the stability of the antibody, and the like.

"Fab fragment" refers to a fragment capable of binding to an antigen that can be obtained by enzymatic treatment of a monoclonal antibody and contains the structures of a light chain (VL-CL) and a heavy chain (VH-CH1).

"Domain-engineered Fab fragment of an antibody" refers to a polypeptide fragment of a Fab fragment of an antibody in which CL and/or CH1 are replaced with other domains or peptide fragments, and still retains the interaction between VH and VL and retains the ability of the antibody to bind to an antigen. In some embodiments, the domain-engineered Fab fragment may be part of a multivalent antibody or a multivalent polypeptide complex. In other embodiments, the domain-engineered Fab fragment is a single protein molecule. The Fab fragment may be a monovalent Fab fragment (Fab fragment 1), a divalent Fab fragment (F(ab)2), a trivalent Fab fragment (F(ab)3), or a multivalent (two or more) Fab fragment. As an example, a block diagram of a Fab fragment with substituted CH1 and CL is shown in Fig. 2.

The term "specific antibody" or "specific polypeptide complex" refers to an antibody or polypeptide complex capable of specifically binding to a target antigen or its epitope. Antibodies are classified as monospecific antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and multispecific antibodies (binding to two or more different target antigens or their different epitopes) according to the number of different target antigens or their different epitopes to which the antibodies bind. Polypeptide complexes are classified into monospecific polypeptide complexes, bispecific polypeptide complexes, trispecific polypeptide complexes, tetraspecific polypeptide complexes, multispecific (binding to two or more different target antigens or their different epitopes) polypeptide complexes according to the number of different target antigens or their different epitopes to which the polypeptide complexes bind. For example, a "bispecific antibody" refers to an antibody (including an antibody or an antigen-binding fragment thereof, such as a single-chain antibody) capable of specifically binding to two different antigens or two different epitopes of the same antigen. The prior art discloses bispecific antibodies of various structures; bispecific antibodies can be classified into IgG-like bispecific antibodies and antibody-fragment bispecific antibodies according to the integrity of the IgG molecules; Bispecific antibodies can be classified into divalent, trivalent, tetravalent or high-valent bispecific antibodies according to the configuration according to the number of antigen-binding regions; bispecific antibodies can be classified into symmetric bispecific antibodies and asymmetric bispecific antibodies depending on whether their structures are symmetric or asymmetric. Among them, bispecific antibodies based on antibody fragments such as Fab fragments lacking Fc fragments, formed by linking 2 or more Fab fragments in one molecule, have low immunogenicity, small molecular weight and high permeability to tumor tissue, and typical antibody structures of this type include bispecific antibodies such as F(ab) ₂ , scFv-Fab and (scFv) ₂ -Fab; IgG-like bispecific antibodies (e.g., having Fc fragments) have a relatively large molecular weight, and Fc fragments facilitate the subsequent purification of the antibody and increase their solubility and stability, and Fc fragments can additionally bind to the FcRn receptor and increase the half-life of the antibody in serum, and typical structural models of bispecific antibodies include bispecific antibodies such as KiH, CrossMAb, quadroma triomab, FcΔAdp, ART-Ig (bispecific antibody production technology), BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCB, 1Fab-IgG 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, vol. 18, p. 585-608 (2019); Chen S1 et al., J Immunol Res., Feb. 11, 2019: 4516041).

The term "monovalent," "bivalent," "trivalent," or "polyvalent" refers to the presence of a specified number of antigen-binding sites in an antibody or polypeptide complex. For example, "monovalent antibody" refers to the presence of one antigen-binding site in an antibody, "monovalent polypeptide complex" refers to the presence of one antigen-binding site in a polypeptide complex; "bivalent antibody" refers to the presence of two antigen-binding sites in an antibody, and "bivalent polypeptide complex" refers to the presence of two antigen-binding sites in a polypeptide complex; "trivalent antibody" refers to the presence of three antigen-binding sites in an antibody, and "trivalent polypeptide complex" refers to the presence of three antigen-binding sites in a polypeptide complex; "Polyvalent antibody" refers to the presence of multiple (e.g., three or more) antigen-binding sites in an antibody, and "polyvalent polypeptide complex" refers to the presence of multiple (e.g., two or more) antigen-binding sites in a polypeptide complex.

The terms "polypeptide," "peptide," or "protein" are used interchangeably herein and refer to a polymer of amino acid residues or a collection of polymers of multiple amino acid residues. These terms are used to describe amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as polymers of natural amino acids and polymers of unnatural amino acids. A polypeptide sequence is typically described as having an amino terminus (N-terminus) at the left end and a carboxyl terminus (C-terminus) at the right end. A "polypeptide complex" as disclosed herein refers to a complex comprising one or more polypeptides that are associated with the performance of specific functions. In some embodiments, the polypeptide complex is an antibody or an antigen-binding fragment thereof capable of specifically binding to an antigen.

The term "binding region" or "binding site" or "binding domain" for an antigen refers to a region or portion of a specific protein molecule (e.g., an antibody molecule) that is capable of specifically binding to an antigen, and the antigen-binding region may be a portion of a ligand-binding domain that is capable of directly binding to an antigen, or may be a domain that contains the variable region of an antibody that is capable of directly binding to an antigen.

The term "operably linked" or "functional linkage" means that two or more biological sequences are linked such that they function as intended, regardless of the presence or absence of a spacer (also called a linker or linker sequence). When used to describe polypeptides, this term means that two polypeptide sequences are linked such that the linked product has the desired biological function, with or without a spacer between the two sequences. For example, the variable region of an antibody can be operably linked to the constant region to form a stable product with antigen-binding activity. This term can also be used to describe polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter sequence, an enhancer sequence, or a repressor sequence), this term means that the polynucleotide sequence is linked in a manner that allows for regulated expression of the polypeptide by the polynucleotide.

The term "dimerization domain" refers to a polypeptide domain capable of promoting binding to form a dimer. In some embodiments, a first dimerization domain may be linked to a second dimerization domain. The linking may be accomplished or mediated by any suitable means, such as a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, or a combination thereof. Exemplary dimerization domains include, but are not limited to, antibody hinge regions, antibody CH2 domains, antibody CH3 domains, and other suitable protein monomers or polypeptides capable of dimerizing and interacting with each other. The CH2 and CH3 hinge regions may be derived from any antibody isotype, such as IgG1, IgG2, IgG3, and IgG4.

The term "interaction domain" refers to a domain of a polypeptide that is capable of facilitating the interaction or binding of two or more homologous or heterologous polypeptides. For example, interaction domains are dimerization domains that facilitate binding to each other to form a dimer. A protein-protein interaction domain is a domain of a polypeptide that interacts or binds to two or more proteins, such as the Ig-like domain 1 of obscurin-like protein in obscurin-like protein, which is capable of forming a complex with the Ig-like domain 152 of titin in titin protein through intermolecular interactions. In some embodiments of the present invention, the domain that interacts with the T-chain of titin is the O-chain of obscurin or the O-chain of obscurin-like protein.

The term "dimer" refers to a structure formed by covalent or non-covalent interactions between two molecules (e.g., polypeptides or proteins). A homodimer or homodimerization is formed from two identical molecules, while a heterodimer or heterodimerization is formed from two different molecules. Two molecules can form a dimer through natural interchain linkages, or they can form a dimer through non-natural interchain linkages. In some embodiments of the present invention, the T-chain of titin and the O-chain of obscurin, or the T-chain of titin and the O-chain of an obscurin-like protein, can form a dimer through natural interchain linkages. It is well known to those skilled in the art that for two domains associated with each other to form a dimer, the interface residues within 6 angstroms between the first domain and the second domain (especially the residues within 4.5 angstroms) play a critical 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). By analyzing the complex formed by binding of titin T-chain to obscurin O-chain or binding of titin T-chain to obscurin-like protein O-chain using the MOE (Molecular Working Medium) system (see Fig. 1A and Fig. 1B), it was found that one or more residues at positions 7-15, 19-24, 26, 55, 59, and 60 on titin T-chain and one or more residues at positions 3-6, 9, 41, 73, 75, and 80-90 on obscurin O-chain interact with each other, and one or more residues at positions 1, 7-10, 13-16, 19-26, 59-60, and 96 on titin T-chain and one or more residues at positions 4-5, 10, 12-13, 74, 76, 78, and 82-91 on the O-chain of the obscurin-like protein interact with each other (see Fig. 16A and Fig. 16B), where the contact residues at these sites are separated by a distance of within 4.5 angstroms, playing a critical role in maintaining the binding of these two domains. Furthermore, in some other embodiments, the T-chain of titin and the O-chain of obscurin, or the T-chain of titin and the O-chain of the obscurin-like protein, can form a dimer via non-natural interchain linkages, wherein the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-natural interchain linkages. A disulfide bond may be formed between the titin T-chain and the obscurin O-chain or between the titin T-chain and the obscurin-like protein O-chain by mutating partial amino acids on the titin T-chain, the obscurin O-chain, or the obscurin-like protein O-chain, thereby stabilizing the dimer. In some embodiments, the titin T-chain has one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26, and 39, and/or the obscurin O-chain has one or more amino acid residue mutations at positions selected from the group consisting of 3, 9, 25, 76, and 88; or the T-chain of titin has one or more amino acid residue mutations at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39, and/or the O-chain of the obscurin-like protein has one or more amino acid residue mutations at positions selected from the group consisting of 6, 26, 74, 77, 84 and 86. In some embodiments, the T-chain of titin has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and/or the O-chain of obscurin has one or more amino acid residue mutations at positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C; or the titin T chain has one or more amino acid residue mutations at positions selected from the group consisting of A8C, V20C, T22C, C25S, A26C and C39T, and/or the obscurin-like protein O chain has one or more amino acid residue mutations at positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C. In some embodiments, the titin T chain has the mutations C25S, C39T and A8C, and the obscurin O chain has the mutation A88C; the titin T chain has the mutations C25S, C39T and V20C, and the obscurin O chain has the mutation A3C; The T chain of titin has mutations C25S, C39T, and A26C, and the O chain of obscurin has mutation R9C; The T chain of titin has mutations C25S, C39T, and A8C, and the O chain of obscurin has mutations C25S, C76S, and A88C; The T chain of titin has mutations C25S, C39T, and V20C, and the O chain of obscurin has mutations C25S, C76S, and A3C; The T chain of titin has mutations C25S, C39T, and A26C, and the O chain of obscurin has mutations C25S, C76S, and R9C; The T chain of titin has mutations C25S, C39T, and A8C, and the O chain of obscurin-like protein has mutations C6E and V74C; The T chain of titin has mutations C25S, C39T, and V20C, and the O chain of obscurin-like protein has mutations C6E and G84C; The T chain of titin has mutations C25S, C39T, and T22C, and the O chain of obscurin-like protein has mutations C6E and A86C; The T chain of titin has mutations C25S, C39T, and A8C, and the O chain of obscurin-like protein has mutations C6E, C26S, C77S, and V74C; The T chain of titin has mutations C25S, C39T, and V20C, and the O chain of obscurin-like protein has mutations C6E, C26S, C77S, and G84C; The titin T-chain has the mutations C25S, C39T and T22C, and the obscurin-like protein O-chain has the mutations C6E, C26S, C77S and A86C. In some embodiments, the obscurin O-chain further has one or more amino acid residue mutations at positions selected from the group consisting of 7, 11 and 62, preferably, the obscurin O-chain has one or more amino acid residue mutations at positions selected from the group consisting of L7K and L7R, K11L, T62K and T62H; more preferably, the obscurin O-chain has the mutations C25S, C39T and A8C, and the obscurin O-chain has the mutations C25S, C76S, A88C, L7K and T62K; The titin T chain has mutations C25S, C39T, and A8C, and the obscurin O chain has mutations C25S, C76S, A88C, L7K, and T62H; The titin T chain has mutations C25S, C39T, and A8C, and the obscurin O chain has mutations C25S, C76S, A88C, K11L, and T62K; The titin T chain has mutations C25S, C39T, and A8C, and the obscurin O chain has mutations C25S, C76S, A88C, K11L, and T62H. The position of the mutation in the titin T chain is the position according to the natural sequence numbering relative to the T.O chain (SEQ ID NO: 32); the position of the mutation in the O-chain of obscurin is the position according to the numbering of the natural sequence relative to the O.O chain (SEQ ID NO: 33); the position of the mutation in the O-chain of the obscurin-like protein is the position according to the numbering of the natural sequence relative to the OL.0 chain (SEQ ID NO: 34).

As used herein, "mispairing" means that two or more homologous or heterologous polypeptides interact or associate together to form an undesired mispairing of a dimer or multimer. "Mispairing is less likely to occur" means, for example, that when polypeptides A1, B1, and B2 are coexpressed, if the desired amount of expression of the A1-B1 dimer is greater than the amount of the A1-B2 dimer, it is considered that a preferential mispairing between A1-B1 occurs, i.e., a mispairing between A1-B2 is less likely to occur. In some embodiments of the present invention, in a multispecific polypeptide complex, mispairing between VH1 of the first polypeptide complex and VL2 of the second polypeptide complex is less likely to occur, and/or mispairing between VL1 of the first polypeptide complex and VH2 of the second polypeptide complex is less likely to occur. However, preferential mispairing between VH1-VL1 and preferential mispairing between VH2-VL2 are considered.

A "disulfide bond" refers to the covalent bond formed between sulfur atoms in the structure R-S-S-R'. The amino acid cysteine contains a thiol group that can form a disulfide bond with a second thiol group, such as the thiol group of another cysteine residue. Disulfide bonds can be formed between the thiol groups of two cysteine residues located on two polypeptide chains, respectively, thereby forming an interchain bridge or interchain linkage.

Electrostatic interactions are non-covalent interactions that play a critical role in protein folding, stability, flexibility, and function. These interactions include ionic interactions, hydrogen bonds, and halogen bonds. Electrostatic interactions can form in polypeptides, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu and Trp on the first chain and Arg, Val, or Thr on the second chain.

Salt bridges are close-range electrostatic interactions primarily between the anionic carboxylate Asp or Glu and the cationic ammonium Lys or guanidino group 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 act as "hot spots" for binding. Among these, residues with ionizable side chains, such as His, Tyr, and Ser, can also participate in salt bridge formation.

A hydrophilic interaction means that molecules with polar groups have a strong affinity for water and can form transient bonds with water via hydrogen bonds. A hydrophobic interaction is a non-covalent interaction between non-polar molecules. These non-polar molecules (e.g., certain neutral amino acid residues, also known as hydrophobic residues) tend to aggregate with each other in an aqueous environment and stay away from water. For example, hydrophobic interactions can be formed between one or more of Val, Tyr, and Ala in the first chain and one or more of Val, Leu, and Trp in the second chain, or His and Ala in the first chain and Thr and Phe in the second chain. (See Brinkmann et al., 2017).

The term "hydrogen bond" refers to the formation of an 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 backbone oxygen atoms (e.g., chalcogen groups) and amide hydrogen atoms (nitrogen groups) of two polypeptide residues. For example, a hydrogen bond can be formed between the nitrogen group in Asn and the oxygen group in His, or the oxygen group in Asn and the nitrogen group in Lys. Interactions between hydrogen bonds are stronger than van der Waals interactions but weaker than interactions between covalent or ionic bonds and are crucial for maintaining secondary and tertiary structures. For example, an α-helix is formed when the distance between amino acid residues is regular between positions i and i+4, and a sheet of twisted folds formed by peptide fragments of 3-10 amino acids in length when two peptides are linked via at least two or three backbone hydrogen bonds is a β-sheet.

As used herein, "knob-in-hole" refers to the interaction between two polypeptides, one of which has a bulge (i.e., a "knob") due to the presence of amino acid residues with bulky side chains (e.g., tyrosine or tryptophan). The other polypeptide has a cavity (i.e., a "hole") in which small amino acid residues of the side chain (e.g., alanine or threonine) are present, and the bulge may be located in the cavity to facilitate the interaction of the two polypeptides to form a heterodimer or complex. Methods for producing polypeptides with knobs in holes are known in the art and can be found in US Pat. No. 5,731,169, WO 1996027011, etc.

"Unnatural interchain linkages" refer to interchain linkages not found in wild-type polypeptide polymers. For example, an unnatural interchain linkage may be formed between a mutated amino acid residue of one polypeptide and a wild-type or mutated amino acid residue of another polypeptide. In some embodiments, at least one unnatural interchain linkage is a "disulfide bond" formed following amino acid mutation. In some embodiments of the present invention, the non-natural interchain linkages between the titin T chain and the obscurin O chain, or the titin T chain and the obscurin-like protein O chain are formed by mutation at one or more amino acid residues at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 in the titin T chain and/or positions selected from the group consisting of 3, 9, 25, 76 and 88 in the obscurin O chain, or formed by mutation at one or more amino acid residues at positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 in the titin T chain and/or positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 in the O-chain of obscurin-like protein.

The term "interface" refers to a specific region on polypeptides where the polypeptides are in contact or interact with each other. A contact surface contains one or more amino acid residues, and upon interaction, the amino acid residues of the contact surface on the polypeptide are capable of interacting with the corresponding amino acid residues with which they are in contact. The amino acid residues of the contact surface may be in a continuous or discontinuous sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface may be separated at different positions along a linear sequence.

A "linker domain" or "spacer" refers to a boundary or border region where two polypeptide sequences are fused or joined. For example, a linker domain may comprise at least a portion of a C-terminal fragment from a first fusion polypeptide that is fused to at least a portion of an N-terminal fragment from a second fusion polypeptide, with or without other spacers between them. For example, the spacer may be rich in glycine and proline residues, such as a spacer having a single or repeating sequence consisting of threonine/serine and glycine, GGGGS, TGGGG, SGGGG, or a tandem repeat thereof (e.g., 2, 3, 4, 5, or more repeats). In some embodiments, the spacer comprises GGGGS or 2 or more tandem repeats thereof.

A naturally occurring intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR", VH) and the first, second, and third constant regions (CHI, CH2, and CH3); the second (CH2) and third (CH3) constant regions of the antibody constitute "Fc", and the N-terminal region of CH2 may or may not contain a portion of the hinge region of the antibody, which is rich in proline, including interchain disulfide bonds; each light chain consists of a variable region ("LCVR", VL) and a constant region (CL). The portion near the N-terminus of the heavy and light chains has a variable sequence of approximately 110 amino acids and is the variable region (Fv region); the portion near the C-terminus has a relatively stable sequence of the remaining amino acids and is the constant region. The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions, which determine the specificity of the antibody, are also known as complementarity-determining regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, and the sequence from the amino terminus to the carboxyl terminus is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The three CDRs of the light chain are LCDR1, LCDR2, and LCDR3, and the three CDRs of the heavy chain are HCDR1, HCDR2, and HCDR3.

The antibodies described herein may, for example, be antibodies derived from animals (e.g., mouse, bird, rabbit, camel, and monkey antibodies), chimeric antibodies, humanized antibodies, and fully human antibodies. The antibodies disclosed herein include, in addition to full-length antibodies, antigen-binding fragments that bind to an antigen.

The term "murine antibody" as used herein refers to a monoclonal antibody to an antigen obtained in accordance with the knowledge and experience in the art. During production, the test subject is administered the antigen, and then the antibody hybridoma expressing the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine antibody or antigen-binding fragment thereof may further comprise the constant region of the mouse κ and λ chains or a variant thereof, or further comprise the constant region of the heavy chain of murine IgG1, IgG2, or IgG3, or a variant thereof.

The term "chimeric antibody" is an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species. For example, the creation of a human-mouse chimeric antibody involves creating a hybridoma secreting a specific monoclonal antibody of mouse origin, cloning the variable region gene from a mouse hybridoma cell, then cloning the constant region gene of a human antibody, optionally linking the mouse variable region gene and the human constant region gene into the chimeric gene, then inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present invention, the light chain of the chimeric antibody further comprises the constant region of the light chain of the human κ and λ chains or their variants. The heavy chain of the antigenic chimeric antibody further comprises a constant region of a human IgG1, IgG2, IgG3 or IgG4 heavy chain or a variant thereof, and preferably comprises a constant region of a human IgG1, IgG2 or IgG4 heavy chain or a variant of IgG1, IgG2 or IgG4 using an amino acid mutation (e.g., L234A and/or L235A mutations and/or S228P mutations).

The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences onto the variable region framework of a human antibody, i.e., a different type of human germline antibody framework sequence. Such an antibody can overcome the heterogeneous response induced by a chimeric antibody due to the transfer of a large number of mouse protein components. Such framework sequences can be obtained from publicly available DNA (deoxyribonucleic acid) databases or published references that include germline antibody gene sequences. Germline DNA sequences for the human heavy and light chain variable region genes can be obtained from the VBase human germline sequence database and can be found in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid decreased immunogenicity and simultaneously reduced activity, the framework sequences of the human antibody variable region can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies described herein also include humanized antibodies generated after further affinity-maturation mutations at the yeast-derived CDRs.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof may further comprise a constant region of a human or mouse κ and λ light chain, or a variant thereof, or further comprise a constant region of a human or mouse IgG1, IgG2, IgG3 or IgG4 heavy chain, or a variant thereof, and preferably comprise a constant region of a human IgG1, IgG2 or IgG4 heavy chain, or an IgG1, IgG2 or IgG4 variant using an amino acid mutation (e.g., L234A and/or L235A mutations, and/or S228P mutations).

"Normal variants" of a human antibody heavy chain constant region and a human antibody light chain constant region, including the same, in the present description refer to variants of a heavy chain constant region or a light chain constant region that have been disclosed in the prior art and are obtained from a human without changing the structure and function of the antibody variable region, and exemplary variants include variants of an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region with a heavy chain constant region having a site-directed modification and an amino acid substitution, and in particular the substitutions comprise such YTE mutations known in the prior art, L234A and/or L235A mutations, S228P mutations and/or mutations that are obtained using knob-in-hole technology (such that the antibody heavy chain has a combination of knob-Fc and hole-Fc), all of which have been confirmed to obtain new properties of the antibody without changing the function of the variable region antibodies.

A "human antibody" (HuMAb) and a "fully human antibody" may be used interchangeably and may be either an antibody of human origin or an antibody obtained from a transgenic organism that is "engineered" to produce specific human antibodies in response to antigen stimulation and may be produced by any method known in the art. A transgenic organism can synthesize human antibodies, and this organism can be used to produce hybridomas that secrete human antibodies. A human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies may also be constructed by gene or chromosomal transfection methods, phage display methods, or in vitro activated B cells, all of which are known in the art.

The terms "full-length antibody," "intact antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to the substantially intact form of an antibody, as distinct from the antigen-binding fragments defined below. This term specifically refers to antibodies in which the light and heavy chains contain constant regions. An "antibody" as described herein includes a "full-length antibody" and its antigen-binding fragment.

In some embodiments, the full-length antibody described herein comprises a full-length antibody formed by a light chain having a light chain variable region linked to a light chain constant region, and a heavy chain having a heavy chain variable region linked to a heavy chain constant region. Those skilled in the art can select light chain constant regions and heavy chain constant regions derived from different antibodies according to actual needs, such as light chain constant regions and heavy chain constant regions of human antibodies.

The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to bind an antigen (e.g., an antigen). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) the Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) the P(ab') ₂ fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the Fd fragment, consisting of the VH and CHI domains; (iv) the Fv fragment, consisting of the VH and VL domains of one arm of an antibody; (v) dsFv (disulfide-stabilized Fv fragment), a stable antigen-binding fragment formed by interchain disulfide bonds from VH and VL; and (vi) diabodies, bispecific antibodies, and multispecific antibodies containing scFv, dsFv, Fab, and other fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using a recombinant technique so that it is capable of generating a single protein chain in which the VL and VH regions are paired to form monovalent molecules (called single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science, 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci USA 85: 5879 5883). Such single-chain antibodies are also encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are produced using conventional techniques known to those skilled in the art, and the fragments are screened for suitability in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be of various types, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 isotypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

F(ab') ₂ is an antibody fragment with a molecular weight of approximately 100,000 and antigen-binding activity, containing two Fab regions linked at a hinge position, which is obtained by enzymatic cleavage of the lower portion of two disulfide bonds in the hinge region of IgG.

Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, which is obtained by cleavage of a disulfide bond in the hinge region of F(ab') ₂ . Fab' disclosed herein can be obtained by treating F(ab') ₂ disclosed herein, which specifically recognizes an antigen, with a reducing agent such as dithiothreitol.

In addition, Fab' can be produced by inserting DNA encoding a Fab' fragment into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express Fab'.

The term "single chain antibody", "single chain Fv" or "scFv" refers to a molecule comprising the variable domain (or VH) of an antibody heavy chain and the variable domain (or VL) of an antibody light chain linked by a linker. Such scFv molecules may have the general structure: IN ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. A suitable linker in the prior art consists of repeating amino acid sequences GGGGS or variants thereof, for example using variants 1-4 repeats (Holliger et al., (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers used herein are described in Alfthan et al., (1995), Protein Eng., 8: 725-731, Choi et al., (2001), Eur. J. Immunol., 31: 94-106, Ni et al., (1996), Cancer Res., 56: 3055 3061, Kipriyanov et al., (1999), J. Mol. PJiol., 293: 41 56 and Roovers et al., (2001), Cancer Immunol.

Diabodies are antibody fragments in which scFv or Fab are dimerized and are antibody fragments that exhibit bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens may be the same or different.

The diabodies disclosed herein can be produced by the following steps: obtaining cDNA (complementary DNA) encoding VH and VL of the antibodies disclosed herein, constructing DNA encoding scFv, inserting the DNA into an expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the diabody.

DsFv is obtained by linking polypeptides in which one amino acid residue in each of the VH and VL is substituted with a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue substituted with the cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).

A full-length antibody, antigen-binding fragment, bispecific antibody, multispecific antibody, polypeptide, polypeptide complex, or multispecific polypeptide complex disclosed herein can be produced by the following steps: obtaining cDNA encoding an antibody of the present invention, constructing DNA encoding a dsFv, inserting the DNA into an expression vector, and then introducing the expression vector into prokaryotic or eukaryotic cells to express the dsFv.

The term "amino acid change" or "amino acid mutation" refers to the presence of a change or mutation of an amino acid in a variant protein or polypeptide compared to a parent protein or polypeptide, including the implementation of 1, 2, 3 or more amino acid insertions, deletions or substitutions based on the parent protein or polypeptide.

The term "antibody framework" or "FR region" refers to the portion of the VL or VH variable domain that serves as a scaffold for the variable domain's antigen-binding loops (CDRs). This is essentially the variable domain without the CDRs. The term "complementarity-determining regions" (CDRs) refers to one of six hypervariable regions within the antibody variable domain that primarily contribute to antigen binding. In general, there are three CDRs (HCDR1, HCDR2, and HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region. Any of a number of well-known protocols can be used to determine the amino acid sequence boundaries of CDRs, including the Kabat numbering scheme (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 scheme (see Al-Lazikani et al., (1997) JMB 273: 927–948), and the ImMunoGenTics (IMGT) numbering scheme (Lefranc M.P., Immunologist, 7, 132–136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55–77 (2003)), etc.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on an antigen molecule). Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

The term "specific binding,""selectivebinding,""selectivelybinds," or "specifically binds" refers to the binding of an antibody to an epitope on a particular antigen. Typically, the antibody binds with an affinity (KD (dissociation constant)) of less than about ^10-7 M, such as less than about ^10-8 M, ^10-9 M, ^10-10 M, ^10-11 M, ^10-12 M, or less.

The term "KD" refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. Typically, the antibody disclosed herein binds to an antigen with an equilibrium dissociation constant (KD) of less than about ^10-7 M, such as less than about ^10-8 M or ^10-9 M. For example, the affinity of an antibody to an antigen in the present invention is determined by FACS (fluorescence-activated cell sorting) analysis for KD values.

The term "competition," when used in the context of antigen-binding proteins that compete for the same epitope (e.g., neutralizing antigen-binding proteins or neutralizing antibodies), refers to competition between antigen-binding proteins as determined by the following assay: in such an assay, the antigen-binding protein (e.g., an antibody or an immunologically functional fragment thereof) to be detected prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen. Numerous types of competitive binding assays are available to determine whether an antigen-binding protein competes with another, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme-linked immunosorbent assay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol., 137: 3614–3619), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual (antibody, Lab Manual), Cold Spring Harbor Press); solid-phase direct labeling 1-125 RIA (see, e.g., Morel et al., 1988, Molec. Immunol., 25: 7–15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virogy, 17: 546–552); and direct-labeling RIAs (Molderhau et al., 1990, Scand I. Immunol., 322-8). Typically, the assay involves the use of purified antigen bound to a solid surface or cell bearing either unlabeled test antigen-binding protein or labeled reference antigen-binding protein. Competitive inhibition was measured by measuring the amount of label that binds to the solid surface or cell in the presence of the antigen-binding protein being measured. Typically, the antigen-binding protein being measured is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include an antigen-binding protein binding to the same epitope as the reference antigen-binding protein and an antigen-binding protein binding to a proximal epitope sufficiently close to the binding epitope of the reference antigen-binding protein that the two epitopes sterically hinder binding. Further details regarding methods for determining competitive binding are provided in the examples provided herein. Typically, when a competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen-binding protein to the common antigen by at least 40-5%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, the term "nucleic acid molecule" as used herein refers to DNA molecules and RNA (ribonucleic acid) molecules in which binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. A nucleic acid molecule can be single-stranded or double-stranded, and preferably double-stranded DNA, single-stranded mRNA, or modified mRNA (messenger RNA). Nucleic acid is "effectively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is effectively linked to a coding sequence if it influences the transcription of the coding sequence.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments can be linked. In another embodiment, the vector is a viral vector, wherein additional DNA fragments can be linked to the viral genome. The vectors disclosed herein are capable of autonomous replication in the host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors) or are integrated into the host cell genome upon introduction into the host cell and thus replicate along with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold Spring Harbor Laboratory Antibody Technical Manual, Chapters 5-8 and 15. For example, a mouse can be immunized with an antigen, and the resulting antibody can be annealed, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be produced using conventional methods. The antibody or antigen-binding fragment disclosed herein is genetically engineered to include one or more human FR regions in a non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website by aligning the IMGT human antibody variable region germline gene database with MOE software or from Immunoglobulin Journal, 2001 ISBN 012441351.

The term "host cell" refers to the cell into which the expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Bacteria susceptible to transformation include Enterobacteriaceae species such as Escherichia coli or Salmonella strains; Bacillus species such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, 293 cells, and NS0 cells.

The engineered antibodies or antigen-binding fragments disclosed herein can be produced and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant expression vector can stably transfect CHO cells. As more recommended in the prior art, mammalian expression systems can result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to the antigen. Positive clones are expanded in serum-free medium of an antibody bioreactor. The antibody-secreting culture solution can be purified using conventional techniques. For example, purification was performed on an A or G Sepharose FF column. Non-specifically bound fractions were eluted. The bound antibody was eluted using a pH gradient, and antibody fragments were detected by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and collected. The antibody could be filtered and concentrated using standard methods. Soluble mixtures and polymers could also be removed using standard methods, such as molecular sieves and ion exchange. The resulting product was immediately frozen, for example, at -70°C, or lyophilized.

"Administration," "treatment," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "treatment," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cell treatment includes contacting a reagent with cells and contacting a reagent with a fluid, where the fluid contacts the cells. "Administration," "treatment," and "treating" also refer to treatment, for example, of cells in vitro and ex vivo, using an agent, diagnostic agent, binding composition, or another cell. "Treatment" when applied to human, veterinary, or research subjects refers to therapeutic treatment, prophylactic or preventative measures, investigative and diagnostic uses.

"Treatment" refers to the administration of a therapeutic agent, such as a composition comprising any of the binding compositions described herein, either internally or externally to a subject experiencing one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the subject or population being treated, to induce regression of such symptoms, or to inhibit the progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") may vary depending on factors such as the disease state, the age and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Alleviation of a disease symptom may be assessed using any clinical trial method commonly used by physicians or other healthcare professionals to assess the severity or progression of a symptom. Although embodiments of the present invention (e.g., methods of treatment or producing an article) may not be effective in alleviating the symptoms of every disease of interest, they should alleviate the symptoms of the disease of interest in a statistically significant number of subjects, as determined by any statistical test method known in the art, such as the Student t-test, the chi-square test, the Mann-Whitney U-test, the Kruskal-Wallis test (H-test), the Jonker-Terpstra test, and the Wilcoxon test.

"Conservative modifications" or "conservative substitutions or replacements" refer to the replacement of amino acids in a protein with other amino acids that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, conformation, and backbone rigidity), such that the changes can often be made without altering the biological activity of the protein. Those skilled in the art recognize that, in general, the replacement of a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al., (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., p. 224, (4th edition)). Furthermore, substitution of structurally or functionally similar amino acids is unlikely to neutralize biological activity. Illustrative conservative substitutions are provided in the following table, "Illustrative Amino Acid Conservative Substitutions." Illustrative Amino Acid Conservative Substitutions:

"An effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include the elimination or reduction of the risk, reduction in the severity, or delay in the onset of a condition, including the biochemical, histological, and/or behavioral symptoms of the conditions, their complications, and intermediate pathological phenotypes manifested during the development of the condition. For therapeutic use, beneficial or desired results include clinical results, such as a reduction in the incidence or amelioration of one or more symptoms of various conditions associated with the target antigen of the present invention, a reduction in the dosage of other agents required to treat the conditions, an enhancement of the therapeutic effect of another agent, and/or a slowing of the progression of the condition associated with the target antigen of the present invention in a subject.

The term "exogenous" refers to a substance produced outside the organism, cell, or person, respectively.

The term "endogenous" refers to a substance produced within an organism, cell, or person, respectively.

"Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both compared sequences are occupied by the same subunit of a base monomer or amino acid, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of paired or homologous positions common to the two sequences divided by the number of positions compared × 100%. For example, if there are 6 matches or homologies at 10 positions in two sequences when the sequences are optimally aligned, then the two sequences are 60% homologous; two sequences are 95% homologous if there are 95 matches or homologies at 100 positions in the two sequences. Typically, two sequences in an alignment were compared to obtain the maximum percentage of homology. For example, the comparison can be performed using the BLAST algorithm, where the algorithm parameters are chosen to ensure maximum correspondence between the reference sequences over the entire length of each sequence. The following references refer to the BLAST algorithm, which is frequently used for sequence analysis: BLAST algorithms: Altschul, S. F., et al., (1990) J. Mol. Biol., 215: 403–410; Gish, W, et al., (1993) Nature Genet., 3: 266–272; Madden, T. L., et al., (1996) Meth. Enzymol., 266: 131–141; Altschul, S. F., et al., (1997) Nucleic Acids Res., 25: 3389–3402; Zhang, J. et al., (1997) Genome Res., 7: 649-656. Other conventional BLAST algorithms, such as the one proposed by NCBI BLAST, are also well known to those skilled in the art. In the present invention, for example, "a numbering position of a natural sequence relative to an XX sequence" includes both a position corresponding to a natural sequence number in an XX sequence and a position in a mutated sequence of said XX sequence at a position equivalent to the XX sequence, where the equivalent position can be obtained by aligning the mutated sequence in said XX sequence with the XX sequence to obtain the highest percent identity, and the position on the mutated sequence in said XX sequence corresponding to the XX sequence is the equivalent position.

As used herein, the terms "cells," "cell lines," and "cell cultures" may be used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the subject's primary cells and cultures derived therefrom, regardless of the number of metastases. Furthermore, it should be understood that all progeny may not be exactly identical in DNA content due to intentional or unintentional mutations. Mutated progeny possessing the same function or biological activity as those screened in the originally transformed cells are included. Where different names are intended, they are used regardless of context.

"Polymerase chain reaction" or "PCR" as used herein refers to a procedure or technique in which minute amounts of a specific portion of nucleic acid, RNA, and/or DNA are amplified, as described, for example, in U.S. Pat. No. 4,683,195. Typically, it is desirable to obtain sequence information from or beyond the end of the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5'-terminal nucleotide of the 2 primers may coincide with the end of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See, generally, Mullis et al., (1987) Cold Spring Harbor Symp. Ouant. Biol., 51: 263; ed. Erlich, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR, as used herein, is considered one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample that involves using a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specified portion of the nucleic acid.

The terms "conjugate" and "drug conjugate" refer to a new drug formed by linking a ligand to a biologically active drug via a stable linking unit. For example, an "antibody drug conjugate" (ADC) is a drug formed by linking an antibody or antibody fragment to a biologically active drug via a linker unit. The antibody can be linked directly to the drug or via a linker. The average number of drug moieties per antibody can vary, for example, from about 0 to about 20 drug moieties per antibody, in some embodiments from 1 to about 10 drug moieties per antibody, and in some embodiments from 1 to about 8 drug moieties per antibody. For the conjugate disclosed herein, the average drug load per antibody is from about 2 to about 5 or from about 3 to about 4.

"Isolated" refers to a state of purification, and in this case means that the molecule in question is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cellular debris and growth medium. Generally, the term "isolated" is not intended to denote the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

The term "optional" or "optional" means that the event or circumstance subsequently described may, but need not, occur, and that the description includes instances in which the event or circumstance does or does not occur.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration, facilitating absorption of the active ingredient, thereby mediating biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in antibody or antigen-binding fragment delivery formulations. The carrier may be an anti-adhesive, binder, coating, disintegrating agent, filler or diluent, preservative (e.g., antioxidant, antibacterial, or antifungal), sweetener, absorption delaying agent, wetting agent, emulsifying agent, buffer, or the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyhydric alcohols (e.g., glycerol, propylene glycol, and polyethylene glycol), dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyhydric alcohols, sorbitol, and sodium chloride.

Furthermore, the present invention includes a pharmaceutical preparation for use in the treatment of a disease or condition, for example, an agent for the treatment of a disease associated with an antigen-positive cell of interest, wherein the preparation comprises a polypeptide complex, a multispecific polypeptide complex, an antibody with an engineered domain, a Fab fragment of an antibody with an engineered domain, a bispecific antibody, a pharmaceutical composition, a nucleic acid molecule, or a multispecific antibody disclosed herein as an active ingredient.

The disease disclosed herein is not limited, for example, a therapeutic response induced using the molecules described herein can be achieved by binding to a human antigen, then blocking the binding of the antigen to its receptor/ligand, or killing tumor cells that overexpress the antigen.

Furthermore, the present invention relates to a method for immunodetection or analysis of a target antigen, a reagent for use in immunodetection or analysis of a target antigen, a method for immunodetection or analysis of a cell expressing a target antigen, and a diagnostic agent for diagnosing a disease associated with an antigen-positive target cell, which comprises a polypeptide complex, a multispecific polypeptide complex, an antibody with an engineered domain, a Fab fragment of an antibody with an engineered domain, a bispecific antibody, a pharmaceutical composition, a nucleic acid molecule, or a multispecific antibody disclosed herein as an active ingredient.

In the present description, the method for detecting or analyzing the amount of a target antigen may be any known method. For example, it includes immunodetection or assay methods.

An immunodetection or assay method is a method for detecting or analyzing the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of immunodetection or assay methods include radiolabeled immunoantibody (RIA) methods, enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays (FIAs), luminescent immunoassays, Western blotting, and physicochemical methods.

The above-mentioned diseases associated with antigen-positive cells can be diagnosed by detecting or analyzing cells expressing the antigen with the antibody described herein.

Known immunodetection methods, preferably immunoprecipitation, fluorescent cell staining, immunohistological staining, and other methods, can be used to detect cells expressing the polypeptide. Additionally, fluorescent antibody staining using the FMAT8100HTS system (Applied Biosystems) and the like can be used.

In the present description, the living sample for use in detecting or analyzing the target antigen is not limited, in particular, as long as it has the ability to contain cells expressing the target antigen, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, interstitial fluid or culture fluid.

A diagnostic agent comprising an antibody or fragment thereof described herein may further comprise a reagent for use in conducting an antigen-antibody reaction or a reagent for use in detecting the reaction, depending on the desired diagnostic method. The reagent for use in conducting the antigen-antibody reaction includes buffers, salts, and the like. The reagent for use in detection includes reagents commonly used in immunodetection or assay methods, such as a labeled second antibody recognizing a monoclonal antibody, an antibody fragment thereof, or a binding agent thereof and a substrate corresponding to the label, and the like.

The details of one or more embodiments of the present invention are set forth in the foregoing description. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or study of the present invention, the preferred methods and materials are described below. Other features, objects, and advantages of the invention will be apparent from the description and claims. As used in the present description and the appended claims, the singular includes the plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same commonly understood meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. All patents and publications cited in the description are incorporated by reference. The following examples are provided to more fully illustrate the preferred embodiments of the present invention. These examples should not be construed as limiting the scope of the present invention in any way.

The present invention is further described below with reference to examples, but these examples are not intended to limit the scope of the present invention. Experimental methods in the examples of the present invention, in which specific conditions are not indicated, were generally performed under standard conditions, such as the Cold Spring Harbor Laboratory Antibody and Molecular Cloning Manual, or under conditions recommended by the manufacturer of the raw materials or products. Reagents not specifically indicated are common commercially available reagents.

Although the antibodies used in the examples target specific antigens, it will be understood by those skilled in the art, in light of the disclosure of the present invention, that the technical effect is achieved regardless of the specific CDR sequences and regardless of the specific antigen sequences, but with the advantage of replacing CH1/CL with the titin T-chain/obscurin O-chain or the titin T-chain/obscurin-like protein O-chain to reduce mispairing between heavy/light chains.

Example 1: Method for producing an antibody or polypeptide protein

The method comprises: designing a PCR primer to construct a gene fragment (such as a VH/VK antibody gene fragment), performing homologous recombination on the gene fragment and an expression vector (such as pHr (with a signal peptide and a constant region gene fragment (such as CH1-Fc/CL))) to construct an expression vector (such as VH-CH1-Fc-pHr/VK-CL-pHr), introducing the constructed expression vector into a prokaryotic or eukaryotic cell for expression, and finally purifying the product to obtain the desired antibody or polypeptide protein. Without limitation, the constant region of the antibody can be selected from the group consisting of the constant region of the human κ and λ chain light chain and the constant region of the heavy chain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Non-limiting examples further include optimization of human antibody constant regions, such as mutations at positions L234A/L235A or L234F/L235E of the heavy chain constant region. As an example, the sequences of the light/heavy chain constant region of an antibody are as follows:

IgG1 heavy chain constant region (abbreviated hlgG1):

Note: In the sequence, the section underlined with a single line represents CH1, the section underlined with a dotted line represents CH2, and the section in italics represents CH3.

IgG1 heavy chain constant region with knob (abbreviated as knob-IgG1):

Note: In the sequence, the single underlined region represents CH1, the dotted underlined region represents CH2, and the italicized region represents CH3.

IgG1 heavy chain constant region with a hole (abbreviated as hole-IgG1):

Note: In the sequence, the section underlined with a single line represents CH1, the section underlined with a dotted line represents CH2, and the section in italics represents CH3.

The constant region of the kappa light chain (abbreviated kappa or hκ):

The constant region of the light chain lambda (abbreviated lambda or hλ):

The amino acid sequences of the variable regions of the light and heavy chains of antibodies to various antigens, such as B7H3 and CD3, mentioned in the examples or test examples of the present invention are as follows:

Amino acid sequence of VH antibody F0:

Amino acid sequence of VL antibody F0:

Amino acid sequence of VH antibody N0:

Amino acid sequence of VL antibody N0:

Amino acid sequence of VH antibody S0:

Amino acid sequence of VL antibody S0:

Amino acid sequence of VH antibody V0:

Amino acid sequence of VL antibody V0:

Amino acid sequence of VH antibody J0:

Amino acid sequence of VL antibody J0:

Amino acid sequence of VH antibody H0:

Amino acid sequence of VL antibody H0:

Amino acid sequence of VH antibody R0:

Amino acid sequence of VL antibody R0:

The amino acid sequence of the VH antibody Bmab (abbreviated BO):

Amino acid sequence of the VL antibody Bmab (abbreviated BO):

The amino acid sequence of the VH antibody Umab (abbreviated U0):

The amino acid sequence of the VL antibody Umab (abbreviated U0):

The amino acid sequence of the VH antibody Dmab (abbreviated DO):

The amino acid sequence of the VL antibody Dmab (abbreviated DO):

The amino acid sequence of the VH antibody Tmab (abbreviated 10):

The amino acid sequence of the VL antibody Tmab (abbreviated 10):

Amino acid sequence of VH antibody CO:

Amino acid sequence of VL antibody CO:

Amino acid sequence of VH antibody AO:

Amino acid sequence of the VL antibody AO:

The heavy chain constant region sequences of antibodies F0, N0, S0, V0, J0, H0, R0, B0, U0, D0, I0, C0 and A0 are IgG1 heavy chain constant regions (SEQ ID NO: 1), and the light chain constant region sequences of antibodies N0, S0, H0, R0, U0, I0 and C0 are kappa light chain constant regions (SEQ ID NO: 4); the light chain constant region sequences of antibodies F0, V0, J0, B0, D0 and A0 are lambda light chain constant regions (SEQ ID NO: 5).

In addition, the sequence of the hB7H3 antigen protein is as follows:

The hCD3 antigen protein is a heterodimer consisting of the δ subunit and the ε subunit of the hCD3 antigen, where the sequence of the δ subunit of the hCD3 antigen is as follows:

The sequence of the ε-subunit of the hCD3 antigen is as follows:

Example 2: Substitution of CH1/CL domains of an antibody

Antibodies with engineered CH1/CL domains were generated by replacing the CH1/CL domains of the antibody with Ig-like domains of the natural titin/obscurin complex, titin/obscurin-like protein complex, and IL6Ra/IL6Rb complex, which have natural intermolecular interactions, respectively.

In the IL6Ra/IL6Rb complex, the Ig-like domain in the IL6Ra protein for replacing CH1 or CL of the antibody is the IL6Ra.0 chain, and the Ig-like domain in the IL6Rb protein for replacing CL or CH1 of the antibody is the IL6Rb.0 chain; in the titin/obscurin complex, the Ig-like domain (titanium Ig-like domain 152) in the titin protein used to replace CH1 or CL of the antibody is the T.0 chain, and the Ig-like domain (obscurin Ig-like domain 1) in the obscurin protein used to replace CL or CH1 of the antibody is the O.0 chain; in the titin/obscurin-like protein complex, the Ig-like domain (Ig-like domain 1 of obscurin-like protein) in the obscurin-like protein used to replace the CH1 or CL antibody is an OL.0 chain, and the Ig-like domain (Ig-like domain 152 of titin) in the titin protein used to replace the CL or CH1 antibody is a T.0 chain, where:

The IL6Ra.0 chain sequence is:

The IL6Rb.0 chain sequence is:

the sequence of the T.0 chain is:

the sequence of chain O.0 is:

the sequence of chain OL.0 is:

A variety of antibodies with substituted CH1/CL were constructed, and the purity and binding activity to the corresponding antigen of the test antibodies were detected according to the methods described in Test Examples 2 and 4. The experimental results are shown in Table 1 below, and the experimental results showed that after antibodies such as F0, V0, S0, N0, J0, H0, R0, and C0 were bound to various antigens, CH1/CL was simultaneously substituted with the T.0/O.0 chain or the T.0/OL.0 chain, the antibodies still retained good binding activity and had high antibody purity.

Example 3: Optimization of the design of engineered CH1/CL antibody domains

Individual amino acids of titin Ig-like domain 152 (T.0 chain), obscurin Ig-like domain 1 (O.0 chain), and obscurin-like protein Ig-like domain 1 (OL.0 chain) were mutated, including amino acid insertion, deletion, or substitution, to test the activity of the engineered antibody domain.

As an example, first, interchain disulfide bonds were increased by mutation at a small number of amino acids on the T.0 chain, the O.0 chain, and the OL.0 chain; second, other individual amino acids in the domain were also mutated, such as amino acid mutations were added at positions 7, 62, and 11 of the obscurin Ig-like domain 1; amino acids were extended or truncated at the N-terminus of the titin Ig-like domain 152 (e.g., the 5 "KAGIR" amino acids of the wild-type titin protein immediately adjacent to the N-terminus of the titin Ig-like domain 152); amino acids were extended or truncated at the N-terminus of the obscurin Ig-like domain 1 (e.g., the 5 "DQPQF" amino acids of the wild-type obscurin protein immediately adjacent to the N-terminus of the obscurin Ig-like domain 1); In addition, a linker sequence was added at the N-terminus or C-terminus of the Ig-like domain 152 of titin, Ig-like domain 1 of obscurin, or Ig-like domain 1 of obscurin-like protein (e.g., a linker sequence of “G ₄ S” or “(G ₄ S) ₂ ” was added at the N-terminus), and the C-terminus of the VH or VL antibody was linked to the Ig-like domain 152 of titin, Ig-like domain 1 of obscurin, or Ig-like domain 1 of obscurin-like protein through a linker sequence. The optimization design of a specific domain is shown in Table 2.

Sequences of the T-chain of titin, the O-chain of obscurin, and the O-chain

obscurin-like protein after mutation of partial amino acids are as follows:

T.1 (T.0 has mutations C25S, C39T and A8C)

T.2 (T.0 has mutations C25S, C39T and V20C)

T.3 (T.0 has mutations C25S, C39T and A26C)

T.4 (T.0 has mutations C25S, C39T and T22C)

T.5 (T.0 has mutations C25S, C39T and A8C; KAGIR is added at the N-terminus)

T.6 (T.0 has mutations C25S, C39T and A8C; linker (G ₄ S) ₁ added at the N-terminus)

T.7 (T.0 has mutations C25S, C39T and A8C; linker (G ₄ S) ₂ added at the N-terminus)

O.1 (O.0 has the A88C mutation)

O.2 (O.0 has the A3C mutation)

O.3 (O.0 has the R9C mutation)

O.4 (O.0 has mutations C25S, C76S and A88C)

O.5 (O.0 has mutations C25S, C76S and A3C)

O.6 (O.0 has mutations C25S, C76S and R9C)

O.7 (O.0 has mutations C25S, C76S, A88C, L7K and T62K)

O.8 (O.0 has mutations C25S, C76S, A88C, L7K and T62H)

O.9 (O.0 has mutations C25S, C76S, A88C, K11L and T62K)

O.10(0.0 has mutations C25S, C76S, A88C, K11L and T62H)

O.11 (O.0 has mutations C25S, C76S and A88C; DQPQF added at the N-terminus)

O.12 (O.0 has mutations C25S, C76S, A88C, L7K, and T62K; DQPQF is added at the N-terminus)

O.13 (O.0 has mutations C25S, C76S, A88C, L7K, and T62H; DQPQF is added at the N-terminus)

O.14 (O.0 has mutations C25S, C76S, A88C, L7R, and T62K; DQPQF added at the N-terminus)

O.15 (O.0 has mutations C25S, C76S, A88C, L7R, and T62H; DQPQF is added at the N-terminus)

O.16 (O.0 has mutations C25S, C76S and A88C; linker (G ₄ S) ₁ added at the N-terminus)

O.17 (O.0 has mutations C25S, C76S and A88C; linker (G ₄ S) ₂ added at the N-terminus)

OL. 1 (OL.0 has mutations C6E and V74C)

OL.2 (OL.0 has mutations C6E and G84C)

OL.3 (OL.0 has mutations C6E and A86C)

OL.4 (OL.0 has mutations C6E, C26S, C77S and V74C)

OL.5 (OL.0 has mutations C6E, C26S, C77S and G84C)

OL.6 (OL.0 has mutations C6E, C26S, C77S and A86C)

A plurality of antibodies with substituted CH1/CL were constructed by mutating or engineering the domain of the titanium T chain, the obscurin O chain, and the obscurin-like protein O chain, the binding activity of each optimized mutated antibody and the corresponding antigen was detected using the method disclosed in Test Example 4 herein, and the experimental results are shown in Table 3. The results showed that the antibody modified with a disulfide bond retains good antigen-binding activity.

In addition, antibodies with substituted CH1/CL were constructed by mutating or constructing the domain of the titanium T chain, the obscurin O chain, and the obscurin-like protein O chain, and the functional activity of antibodies with other substituted CH1/CLs was detected using the methods disclosed in Test Example 2 and Test Example 4 herein. The experimental results are shown in Table 4, and the experimental results showed that the antibodies with substituted CH1/CLs still retain good antigen-binding activity, and the purity of the antibody by SEC (%) was relatively high.

In addition, mutations were made at other amino acids in the Ig-like domain 152 of titin and the Ig-like domain 1 of obscurin, and specific amino acid mutations were designed as shown in Table 5.

The mutant sequences of the T.1 chain and the O.9 chain are as follows: T.8 (T.1 has mutations M66S and T77S)

T.9 (T.1 has mutations M66K, K70R, S79T and G81R)

T.10 (T.1 has mutations P3W, S11I, II3L, T22M and N82M)

T.11 (T.1 has mutations S11I, M66K, S79T and G81R)

T.12 (T.1 has mutations G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D and F84L)

T.13 (T.1 has mutations Q47E, Q49G, N56S, D58E and L75V)

T.14 (T.1 has mutations N56S, D58E and L75V)

T.15 (T.1 has mutations N56S, D58E, M66S and T77S)

T.16 (T.1 has mutations S11L M66K, S79T and G81R + linker sequence (G ₄ S) ₁ )

T.17 (T.1 has mutations N56S, D58E, M66S and T77S, + KAGIR)

T.18 (T.1 has mutations N56S, D58E, M66S and T77S, + KAGIR, + linker sequence (G ₄ S) ₁ )

T.19 (T.1 has mutations N56S, D58E, M66S and T77S, + linker sequence (G ₄ S) ₁ )

O.18 (O.9 has mutations L11K, A12S, F13Y, V14T and T22S)

O.19 (O.9 has mutations G2E, V17E, N30D, T32P, Q34E, S36T, V44I, A45T, L58V, K62E, A67Q, G69S and A97G)

O.20 (O.9 has mutations D20L, T22M and A53L)

O.21 (O.9 has mutations Q41K, A45T, A67Q, G69S and V89L)

O.22 (O.9 has mutations Q42L, A45T, A67T, G69S, Q92E and D94G)

O.23 (O.9 has mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E and

D94G)

O.24 (O.9 has mutations Q41K, A45T, A67Q, G69S and V89L; + linker sequence (G ₄ S) ₁ )

O.25 (O.9 has mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E and D94G, + linker sequence (G ₄ S) ₁ )

O.26 (O.9 has mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E and D94G; + DQPQF)

O.27 (O.9 has mutations A12S, F13Y, T22S, Q42L, A45T, A67Q, G69S, Q92E, and D94G; + DQPQF; and + linker sequence (C ₄ S) ₁ )

The antibodies with substituted CH1/CL were constructed by mutating or engineering the domain of titin T chain and obscurin O chain, the antibodies with substituted CH1/CL were detected using the method disclosed in Test Example 4 herein, and the experimental results are shown in Table 6. The results showed that the antibodies still retained good antigen-binding activity after CH1/CL was substituted with the optimized titin T chain and obscurin O chain.

In addition, the protein expression level of the CH1/CL-substituted antibody was measured using the method described in Test Example 1 herein, and the experimental results are shown in Table 7. The results showed that the expression level of the antibody was significantly improved after partial amino acid mutations of the Ig-like domain 152 of titin and the Ig-like domain 1 of obscurin.

Example 4: Half-life of an antibody with engineered CH1/CL domains

To assess the effect of replacing the CH1/CL antibody with the titin T chain/obscurin O chain or the titin T chain/obscurin-like protein O chain on antibody stability in rats, the authors studied the antibody half-life in rats before and after the replacement. As an example, the authors studied the half-lives of the F20 antibody, in which the CH1/CL of the F0 antibody was replaced with T.1/O.9, and the C3 antibody, in which the CH1/CL of the C0 antibody was replaced with T.1/O.9, respectively. The specific method includes the following steps:

The antibody of 3 mg/kg was intravenously administered to rats, and 0.3 ml of whole blood was collected at 5 min, 8 h, 1 d, 2 d, 4 d, 7 d, 10 d, 14 d, 21 d, and 28 d after the administration, respectively, without anticoagulation, and after collecting the blood, the blood was left at 4°C for 30 min and centrifuged at 1000 g for 15 min, and the supernatant (serum) was placed in an EP tube and stored at -80°C. As a method for detecting the antibody concentrations in the drug at each time point, a sandwich ELISA was used, namely, the corresponding antigens were applied to the ELISA plate, and the plate was incubated overnight at 4°C, washed with washing liquid, then blocking buffer was added and incubated at room temperature for 13 h, then washed with washing liquid, 100 μl of a standard substance or serum sample for detection was added and incubated at 37°C for 3 h, then washed with washing liquid, pre-adsorbed anti-human FC (HRP) mouse secondary antibody (Abeam, product no. ab98624, 1:10000 dilution) was added and incubated at 37°C for 1-1.5 h, then washed with washing buffer, TMB was added (3,3',5,5'-tetramethylbenzidine) and incubated in the dark at room temperature for 10 min, then stop buffer was added; OD450 values were read.

The experimental results are shown in Table 8, and the results showed that after the CH1/CL antibodies were substituted with titin T chain/obscurin O chain, the half-life of the original antibody in the rat body was still maintained, indicating that the CH1/CL-substituted antibody had good stability in the body as the original antibody.

Example 5: Construction and detection of bispecific antibodies A bispecific antibody was constructed by bridge PCR of an antibody binding to a first antigen and an antibody binding to a second antigen. As an example, the authors constructed multiple bispecific antibodies.

I. Construction of bispecific antibodies

Firstly, the inventors constructed a bispecific antibody DI-1, which is an IgG-like bispecific antibody consisting of a heavy chain (with a modification of the knob in the Fc region) and a light chain of the DO antibody, and a heavy chain (with a modification of the hole in the Fc region) and a light chain of a modified antibody obtained by replacing CH1/CL of the IO antibody with titin T chain/obscurin O chain; its structural diagram is shown in Fig. 5, and its sequence is shown as follows:

Amino acid sequence of chain 1 of DI-1 (DI-1-H1):

Note: In the sequence, the region in bold represents VH DO, the region underlined with a single line represents CH1, the region underlined with a dotted line represents CH2, and the region in italics represents CH3.

Amino acid sequence of chain 2 of DI-1 (DI-1-L1):

Note: In the sequence, the region highlighted in bold represents VL D0, and the region underlined with a single line represents the light chain constant region CL D0.

Amino acid sequence of chain 3 of DI-1 (DI-1-H2):

Note: In the sequence, the bold section represents VH I0, the single-line underline represents T.16, the dotted section represents CH2, and the italicized section represents CH3.

Amino acid sequence of chain 4DI-1 (DI-1-L2):

Note: In the sequence, the section in bold represents VL I0, and the section underlined with one line represents O.24.

In addition, an IgG-like bispecific antibody was constructed from B0 or B0 and U0 antibodies with engineered CH1/CL domains or U0 antibody with engineered CH1/CL domains (as an example, the schematic block diagram of BU5 is shown in Fig. 8), and the four chain sequences of the resulting bispecific antibody are shown in Table 9 below:

In addition, a bispecific antibody FA-1 was constructed, which is an IgG-like bispecific antibody consisting of the heavy chain (modification of the Fc region knob) and light chain of the A0 antibody, and the heavy chain (modification of the Fc region knob, substitution of CH1 with T.16) and light chain (substitution of CL with O.24) of the F0 antibody. The four-chain sequences of FA-1 are shown in Table 10 below:

II. Detection of bispecific antibodies

DI-1 was detected using the method described in Test Example 3 herein, and four DI-1 chains were co-transfected into expression cells and then subjected to mass spectrometry. The experimental results are shown in Table 11 and Figs. 6A–6C. Homodimers and mismatched molecules were not detected in the mass spectrometry results, and DI-1 was correctly assembled.

In addition, BU5 expression was detected using the methods described in Test Example 3 and Test Example 2 herein. First, four BU5 chains were co-transfected into cells to express the bispecific antibody, with B0 and U0 serving as controls. Then, the expressed product was analyzed for purity and mass spectrometry. The experimental results are shown in Table 12 and Figs. 9A–9C, and the results indicate that the BU5 bispecific antibody was successfully expressed, and four BU5 chains were successfully assembled into target molecules without mispairing. In addition, the purity of the BU5 bispecific molecule was high, and the % SEC reached 88%.

In addition, the designed bispecific antibody BU1-BU4 was detected for antibody purity and antigen-binding activity using the methods described in Test Example 2 and Test Example 4 herein. The experimental results are shown in Table 13, and the results showed that the bispecific antibody with substituted CH1/CL still retains good antigen-binding activity and has high antibody purity.

Test examples

Test Example 1: Method for Measuring the Antibody Expression Level HEK293E cells were transfected with monoclonal antibody or bispecific antibody expression plasmids, and the expression supernatants were collected after 6 days 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 (phosphate-buffered saline) until the A280 reading dropped to the initial level, and the column was washed with 100 mM acetate buffer (pH 3.5) and neutralized with 1 M Tris-HCl (Tris - tris(hydroxymethyl)aminomethane), pH 8.0. The antibody expression level was quantified by the antibody/expression volume obtained in the final purification.

Test Example 2: Method for Determining the Purity of Antibody

Antibody purity was monitored using size-exclusion SEC-HPLC (HPLC - high-performance liquid chromatography). Detection was performed according to the instrument's manual using a Waters e2695 chromatograph with a Waters Xbridge BEH 200A SEC column. PBS served as the mobile phase (pH adjusted to 6.8 with dilute hydrochloric acid). 100 μg of protein was injected, and gradient elution was performed at a flow rate of 0.5 ml/min. Antibody purity was expressed as a percentage of the peak area of the primary peak relative to the total peak area (SEC (%)).

Test Case 3: Antibody Detection Method Using Mass Spectrometry

(I) Sample preparation:

1. Determination of total molecular weight after deglycosylation: 30 μg of the expressed antibody was collected, 10 μl of 8 M Gua-HCl (guanidinium chloride) was added after lyophilization, and denaturation was carried out by means of a water bath at 70°C for 10 min; 90 μl of distilled water was added, 30 μl was collected and 0.8 μl of F PNG enzyme (peptide N-glycosidase F) was added, the resulting solution was placed in a water bath at 37°C for incubation for 2 h, and 0.5 μg was collected to determine the total molecular weight after deglycosylation.

2. Determination of molecular weight after deglycosylation and reduction: 30 μg of the expressed antibody was collected, 10 μl of 8 M Gua-HCl after lyophilization was added, and denaturation was carried out by means of a water bath at 70°C for 10 min; 90 μl of distilled water was added, 30 μl was collected, and 0.8 μl of PNG F (peptide N-glycosidase F) enzyme was added and placed in a water bath at 37°C for incubation for 2 h; and then 2 μl of 0.025 M DTT (dithiothreitol) was added, reduced for 10 min in a water bath at 70°C, and 0.5 μg was collected for determination of the molecular weight after deglycosylation and reduction.

(II) Chromatographic mass spectrometry conditions:

The chromatographic conditions are as follows:

Chromatographic column: Poroshell 300SB-C8 5 µm 2.1×75 mm

Mobile phase: A: 0.1% HCOOH/H ₂ O B: 0.1% HCOOH/CAN

Column temperature: 75°C

The conditions for mass spectrometry are as follows:

Mass spectrometer: Agilent 6530 Q-TOF LC-MS

ESI (electrospray ionization) mode

Data acquisition mode: 1 GHz (mass range: 500-5000 m/z)

Drying gas temperature: 325°C

Drying gas consumption: 10 l/min

Spray Nozzle: 40 psi (pounds per square inch)

Shielding gas temperature: 350°C

Shielding gas flow rate: 12 l/min

Spray voltage: 500V

Capillary voltage: 3500 V

Splitting voltage: 200 V

Skimmer voltage: 65 V

Rf voltage (radio frequency): 7500 V

Mass spectrometry was performed on the detected antibody, and the experimental results showed that the antibody remained correctly assembled after replacing CH1/CL with titin T-chain/obscurin O-chain.

Test Example 4: Method for Detecting Antibody Binding Activity The binding activity of an antibody to a target membrane protein can be detected using FACS. For example, C0, N0, F0, V0, and S0 antibodies with engineered CH1/CL domains can be detected using the FACS method. Resuspended cells (2× ¹⁰⁶ cells/mL, 90 μL) in FACS buffer (98% PBS, 2% FBS (fetal bovine serum) were added to a 96-well U-bottom plate (Corning, 3795) and 10 μL of gradient diluted antibody were added; the reaction mixture was incubated at 4°C for 1 hour, washed 2 times with FACS buffer, then goat anti-human IgG with Alexa Fluor 488 (H+L) (Invitrogen, cat. No. 2015982, 1:1000 dilution) was added per well, incubated at 4°C for 1 hour, and then washed 2 times, the cells were resuspended with FACS buffer, and finally, the fluorescence signal values The data were read using a FACS CantoII (BD). Finally, the data were processed and analyzed using FlowJo 7.6 and Graphpad Prism 5.

For antibodies targeting soluble proteins, the binding activity of antibodies to soluble proteins was measured by ELISA, for example, CH1/CL domain-engineered antibodies D0, I0, B0, U0, H0, R0, and J0 can be measured by ELISA. The method comprises the following steps: diluting the protein to 1 μg/ml with PBS buffer (Basalmedia, B320) (pH 7.4), adding the resulting solution to a 96-well microplate (Corning, 9018) at a volume of 100 μl/well and incubating at 4°C overnight; after discarding the solution, adding 300 μl of 5% skim milk (BD, 232100) diluted with PBS to each well for blocking and incubating at 37°C for 2 hours; After blocking was completed, discarding the blocking solution, and after the plate was washed 3 times with PBST buffer (pH 7.4, PBS containing 0.1% Tween-20), adding 100 μl of the gradient diluted antibody solution to each well and incubating at 37°C for 1 h; after incubation was completed, washing the plate 3 times with PBST, adding 100 μl of mouse anti-human IgG (H+L) (Jackson ImmunoResearch, 209-035-088, 1:8000 dilution) to each well, and incubating at 37°C for 1 h; and after the plate was washed 3 times with PBST, adding 100 μl of the chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine) (KPL, 5120-0077) to each well and incubating at room temperature for 10-15 min, adding 50 μl _of 1 M _H2SO4 to stop the reaction, reading the absorbance value at 450 nm using a microplate reader, and fitting the antibody-antigen binding curve using software to calculate EC50 values.

The antigens corresponding to the antibodies in this Test Example are as follows: the antigen binding to the CH1/CL engineered antibody D0 is hRANKL (available from Sino Biological, 11682-HNCH); the antigen binding to the CH1/CL engineered antibody 10 is hNGF (available from Sino Biological, 11050-HNAC); the antigen binding to the CH1/CL engineered antibody BO is hBAFF (available from Sino Biological, 10056-HNCH); the antigen binding to the CH1/CL engineered antibody U0 is hP40 (available from Sino Biological, 10052-H08H); The antigen binding to CH1/CL domain-engineered antibodies H0 and R0 is hIL-5 (available from R&D systems, 205-IL-025/CF); The antigen binding to CH1/CL domain-engineered antibody J0 is hTSLP (available from Sino Biological, 16135-H08H); The antigen binding to CH1/CL domain-engineered antibodies C0 and N0 is CEA (CEA (carcinoembryonic antigen)-overexpressing MKN45 cells (gastric cancer cell line) (available from Nanjing Cobioer, CBP60488)); The antigen binding to the F0, V0, and S0 antibodies with engineered CH1/CL domains is B7H3 (overexpressed B7H3 in the recombinant cell lines CT26-B7H3 (CT26 was obtained from the China Academy of Cell Bank, and the sequence of B7H3 is shown in ID: XP_005254757.1)).

Test Case 5: Experimental Method for Reducing Mismatch in an Antibody with Engineered CH1/CL Domains

I. Method for detecting the expression of various light/heavy chain cross-matched antibody molecules

The experiment confirmed that CH1/CL could effectively reduce heavy/light chain mispairing after substitution with titin T chain/obscurin O chain or titin T chain/obscurin-like protein O chain through the analysis of paired expression of different light/heavy chain antibodies. Specifically, when the transfected cells expressed antibodies, the heavy/light chains of D0 and I0 were respectively substituted (i.e., I/D-1 (heavy chain DO + light chain I0); I/D-2 (heavy chain I0 + light chain D0)) to detect whether miscross-paired molecules would be formed in wild-type CH1 and CL; The heavy and light chains of antibody D3 (the heavy chain of CHI DO was substituted with T. 11, and the light chain CL was substituted with O.21) and antibody 13 (the heavy chain of CH1 10 was substituted with T.11, and the light chain CL was substituted with O.21), respectively, were substituted after CH1/CL D0 and I0 were substituted with titin T chain/obscurin O O chain, and mispaired molecules I/D-3 (heavy chain D0 + light chain I3), I/D-4 (heavy chain D3 + light chain I0), I/D-5 (heavy chain I0 + light chain I3), and I/D-6 (heavy chain I3 + light chain D0) were generated; the structural block diagrams of the designed antibodies are shown in Fig. 7.

The expression level of the antibodies was measured using the method described in Test Example 1, and whether mispaired molecules were formed was determined by mass spectrometry of the expression product using the method described in Test Example 3. The experimental results are shown in Table 14, and the experimental results showed that when D0 heavy chain + I0 light chain or I0 heavy chain + D0 light chain were co-expressed, complete IgG molecules (150 kDa) with high expression levels of 74.4 mg/L and 73.8 mg/L, respectively, could be formed; and the LC-MS (liquid chromatography with mass spectrometry) results showed that the resulting proteins were mispaired IgG molecules, indicating that if CH1-CL is not designed or substituted, a large number of mispaired light/heavy chain molecules may be formed when four different heavy and light chains of the bispecific antibody are co-expressed. After CH1/CL were substituted with titin T chain/obscurin O chain, no target molecules (150 kDa) were obtained in four forms 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) through the expression of cross-matching mispairing, and no mispaired molecules were obtained in the other three forms, except that a small number of mispaired molecules (molecular weight was 100 kDa) of two heavy chains were formed by the combination of D3 heavy chain and I0 light chain, indicating that non-homologous pairing after modification could not form mispaired molecules in the structural form of IgG. Thus, substitution of CH1-CL with titin T chain/obscurin O chain effectively prevents or reduces the formation of non-homologous cross-pairing molecules between the light/heavy chains of IgG-like bispecific antibodies.

II. Method for detecting expression of mismatched light/heavy chains of bispecific antibodies

To test the absence of mispairing or the reduction of mispairing in CH1/CL that were substituted with titin T-chain/obscurin O-chain, three forms of light/heavy chain cross-pairing expression were constructed in the test case, namely, two different heavy chains and one identical VL-CL light chain for bispecific antibody expression, or two different heavy chains and one identical light chain with CL substituted with obscurin O-chain for expression, or two different heavy chains and two different light chains for normal expression as a control (see Table 15 for specific pairing).

Then, the expression product was subjected to mass spectrometry (for the method, see Test Example 3 disclosed in the present description) and the purity of the antibody was determined by SEC (for the method, see Test Example 2 disclosed in the present description). The experimental results are shown in Table 15, and the results showed that the correct molecules were formed when the light chain substituted with the O-chain of obscurin and the light chain not substituted with CL were added together, and the correct target molecules were not detected in the cross-pairing when only one light chain not substituted with CL was added, or only one light chain with CL substituted with the O-chain of obscurin was added. This indicates that a light chain with CL substituted by the O-chain of obscurin can be more easily associated with a heavy chain with CH1 substituted by the T-chain of titin to form a molecule, but will not miscross-pair with a heavy chain with CH1 not substituted by the T-chain of titin; a light chain with CL not substituted by the O-chain of obscurin will more easily associate with a heavy chain with CH1 not substituted by the T-chain of titin, but will not associate with a heavy chain with CH1 substituted by the T-chain of titin.

Test Example 6: Detection of the Affinity of a Bispecific Antibody to an Antigen

The affinity of the detectable antibody to the antigen protein was determined using a Biacore T200 instrument (GE). According to the method described in the instruction manual of the human anti-capture antibody kit (GE, cat. No. BR-1008-39), a Protein A biosensor chip (cat. No. 29127556, GE) was used for affinity capture of the detectable antibody. Soluble antigens were then passed across the chip surface with a series of concentration gradients, and the Biacore T200 instrument was used to detect the reaction signals in real time, thereby obtaining a binding-dissociation curve. After completion of dissociation for each cycle, the biosensor chip was washed with a glycine-hydrochloric acid solution (pH 1.5, cat. No. BR-1003-54, GE) for recovery. The data obtained in the experiment were fit to the data using BIAevaluation version 4.1, GE software using the Langmuir model (1:1) to obtain affinity values, and the experimental results are shown in Table 16.

Experimental results show that the domain-engineered bispecific antibody disclosed herein fully retains the affinity of the two parent monoclonal antibodies to their antigens. The antigens included in this experiment were hRANKL (available from Sino Biological, 11682-HNCH), hNGF (available from Sino Biological, 11050-HNAC), hBAFF (available from Sino Biological, 10056-HNCH), hP40 (available from Sino Biological, 10052-H08H), hB7H3 (produced in-house, for sequence, see SEQ ID NO: 111), and hCD3 (produced in-house, a heterodimer consisting of the δ subunit (for sequence, see SEQ ID NO: 112) and the ε subunit (for sequence, see SEQ ID NO: 113) of hCD3).

Test Case 7: Simultaneous Binding Experiment of CH1-CL Domain-Engineered Diabodies to Two Target Antigens

The CH1-CL domain-engineered bispecific antibodies disclosed herein were tested for binding to two target antigens simultaneously by determining the affinity of the antibody to be detected to the antigen protein using a Biacore T200 instrument (GE). The specific experimental steps were as follows: the BU5 antibody was affinity captured using a Protein A biosensor chip (Cat. No. 29127556, GE), then the first hP40 antigen molecule (available from Sino Biological, 10052-H08H) of the bispecific antibody was flowed across the chip surface to achieve saturation, and then the second hBAFF antigen molecule (available from Sino Biological, 10056-HNCH) was introduced at a certain concentration, and the reaction signals were detected in real time using a Biacore T200 instrument to obtain a binding-dissociation curve. After completion of dissociation for each cycle, the biosensor chip was washed with glycine-hydrochloric acid solution for regeneration (pH 1.5, Cat. No. BR-1003-54, GE), and a data fitting model was obtained using BIAevaluation version 4.1, GE software, using the (1:1) Langmuir model. Then, the BU5 antibody was affinity captured by a protein A biosensor chip (Cat. No. 29127556, GE), the first hP40 antigen molecule (available from Sino Biological, 10052-H08H) of the bispecific antibody was passed across the chip surface to achieve saturation, then the second hBAFF antigen molecule (available from Sino Biological, 10056-HNCH) was introduced at a certain concentration, and the reaction signal was detected in real time by a Biacore T200 instrument to obtain a binding-dissociation curve. After completion of dissociation for each cycle, the biosensor chip was washed with a glycine-hydrochloric acid solution for regeneration (pH 1.5, Cat. No. BR-1003-54, GE), and a data fitting model was obtained using BIAevaluation version 4.1, GE software, using a (1:1) Langmuir model.

The experimental results are shown in Table 17, and the results showed that the bispecific antibody BU5 can continue to bind to the hBAFF antigen after binding to the hP40 antigen to achieve saturation, and has the ability to re-bind to hBAFF comparable to the monoclonal antibody B0. Similarly, the bispecific antibody BU5 can continue to bind to the hP40 antigen after binding to the hBAFF antigen to achieve saturation, and has the ability to re-bind to hP40 comparable to U0. This indicates that the bispecific antibody disclosed in the present description with CH1/CL substituted with the titin T chain/obscurin O chain had the feature of dual targeting and could bind to two target antigens simultaneously.

Test Case 8: Osteoclast differentiation experiment of bispecific antibody DI-1

Raw264.7 cells (China Academy of Cell Bank, SCSP-5036) were split and counted in resuspension, seeded in a 24-well cell culture plate (Corning, 3524), and cultured overnight in a cell incubator at 37°C. The next day, the antibody solution was diluted to different concentrations and homogeneously mixed with RANKL (Sino Biological, 11682-HNCH) in the cell culture plate to a final concentration of 50 ng/mL, and the cells were incubated at 37°C. After 96 h, the solution in the 24-well plate was removed, 100 μl of cell lysate (Beyotime, P0013J) was added to each well, the mixture was pipetted and mixed uniformly, the lysate was transferred to an EP tube and centrifuged at 12,000 g for 5 min, the supernatant was collected and measured using an anti-tartaric acid phosphatase test kit (Beyotime, P0332) according to the method described in the instruction manual, and the absorbance values at 405 nm were read using a microplate reader. The measured values were curve-fitted using the software, and the IC50 values were calculated. The experimental results are shown in Fig. 10, and the experimental results showed that the CH1/CL-substituted bispecific antibody DI-1 retained good activity and effectively inhibited osteoclast differentiation.

Test Case 9: Cell Proliferation Experiment of TF1 Bispecific Antibody DI-1

The antibody activity at the cellular level was assessed in an experiment on the proliferation of TF-1 cells induced by NGF (nerve growth factor) (ATCC, CRL-2003). The experimental procedure was as follows: TF1 cells were digested and collected, resuspended, then counted, seeded in a 96-well plate (Corning, 3903) and incubated overnight in an incubator at 37°C. The next day, the antibody solution was diluted to various concentrations and homogeneously mixed with NGF (Sino Biological, 11050-HNAC) and added to the cell culture plate to a final concentration of 10 ng/ml, and the cells were incubated in an incubator. After 72 h, the cell culture plate was removed, 50 μl of Cell-titer Glo assay solution (Promega, G755B) was added to each well, the reaction solution was incubated on a shaker for 10 min and left to stand at room temperature for 10 min. The luminescent signals were detected using a microplate reader (PerkinElmer, Victor 3). The detected signal values were plotted using software, and the IC50 values were calculated. The experimental results are shown in Fig. 11, and the results showed that the CH1/CL-substituted bispecific antibody DI-1 had good activity.

Test Case 10: Detection of mispairing of light and heavy chain of bispecific antibody with CH1/CL substituted with different domains

The following experiments were performed to analyze mispairing of the light and heavy chains of bispecific antibodies with CH1/CL substituted with titin T chain/obscurin O chain or TCRα/TCRβ, and the specific experiments were as follows:

I. Experiment on mispairing of light and heavy chains during coexpression of four chains of a bispecific antibody

Bispecific antibodies HJ-1 and HJ-2, each consisting of four chains, were constructed by replacing CH1/CL H0 or J1 with TCRα/TCRβ, and bispecific antibodies HJ-3 and HJ-4, each consisting of four chains, were constructed by replacing CH1/CL H0 or J1 with titin T chain/obscurin O chain; schematic block diagrams of the bispecific antibodies are shown in Fig. 12, and the full-length sequences of the bispecific antibodies are shown in Table 18:

Note: In the sequence, the region highlighted in bold represents the substituted portion, the region underlined with a dotted line represents the constant region, and the region underlined with a single line represents the variable region.

Heavy Chain J1:

Light chain J1:

The four chains HJ-1, HJ-2, HJ-3, and HJ-4 were cotransfected for expression, respectively, and then mass spectrometry was performed on the expression products (for the method, see Test Example 3 disclosed herein) to detect the presence of mispaired light and heavy chain molecules. The experimental results are shown in Figs. 13A–13D and Table 19.

Note: In the table, “√” means that the molecule was detected, “×” means that the molecule was not detected, “HJ (1+2+3+4)” means the bispecific antibody formed by four corresponding chains numbered 1, 2, 3 and 4 on its left side, “mispaired molecule 1 (1+2+3+3)” means the mispaired molecule 1 formed by four corresponding chains numbered 1, 2, 3 and 3 on its left side; and “mispaired molecule 2 (1+2+4+4)” means the mispaired molecule 2 formed by four corresponding chains numbered 1, 2, 4 and 4 on its left side.

Experimental results showed that when four chains of the bispecific antibody with CH1/CL substituted with TCRβ/TCRα were co-transfected for expression, the light and heavy chains of the antibody were mispaired. However, the bispecific antibody with CH1/CL substituted with the titin T chain/obscurin O chain did not exhibit mispairing of the light and heavy chains. This indicates that the bispecific antibody disclosed herein with CH1/CL substituted with the titin T chain/obscurin O chain exhibited excellent ability to reduce mispairing of the light and heavy chains.

II. Experiment with three-chain coexpression of a bispecific antibody

The experiment was carried out by co-transfecting and expressing three chains (1 heavy chain with CH1 substituted by TCRβ or titanium T chain, 1 heavy chain with unsubstituted CH1, and 1 wild-type light chain (VL-CL) with unsubstituted CL, and the specific sequences are shown in Table 18), then performing mass spectrometry on the expression product (for the method, see Test Example 3 disclosed in the present specification) and detecting the purity of the antibody by SEC (for the method, see Test Example 2 disclosed in the present specification) to check whether the wild-type light chain (VL-CL) and the heavy chain with CH1 substituted by TCRβ or titanium T chain were associated to form a mismatched molecule. The experimental results are shown in Fig. 14A, Fig. 14B, Fig. 15A, Fig. 15B and Table 20.

Note: In the table, “√” means that the molecule was detected, “x” means that the molecule was not detected, “molecule (1+2+3)” means the molecule formed by three corresponding chains numbered 1, 2 and 3 on their left side, and “mispaired molecule (1+2+3+3)” means the mispaired molecule formed by four corresponding chains numbered 1, 2, 3 and 3 on its left side.

Experimental results showed that VL-CL readily binds to the heavy chain with CH1 substituted by TCRβ to express a mispaired molecule with two identical light chains. However, the VL-CL light chain did not bind to the heavy chain with CH1 substituted by titin T chain to form a mispaired molecule with two identical light chains, further indirectly indicating that the bispecific antibody with CH1 of the heavy chain substituted by titin T chain disclosed herein had a superior ability to reduce mispairing of light and heavy chains compared to the bispecific antibody with CH1 of the heavy chain substituted by TCRβ.

--->

SEQUENCE LIST

<110> JIANGSU HANGRUI MEDICAL CO., LTD.

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> NEW POLYPEPTIDE COMPLEX

<130> 702150CPCT

<150> CN202010020878.7

<151> January, 09, 2020

<160> 126

<170> SIPOSequenceListing 1.0

<210> 1

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Constant region of the heavy chain of IgG1

<400> 1

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 2

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IgG1 heavy chain constant region with knob

<400> 2

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 3

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IgG 1 heavy chain constant region with a pit

<400> 3

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 4

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Kappa light chain constant region

<400> 4

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 5

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> lambda light chain constant region

<400> 5

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 6

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody F0

<400> 6

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Ala Leu Val Thr Val Ser Ser

115

<210> 7

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody F0

<400> 7

Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser

20 25 30

His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met

35 40 45

Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg

85 90 95

Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 8

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody NO

<400> 8

Asp Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Ser Ser Leu Ser

20 25 30

Trp Met Ser Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Asn Gln Asp Gly Ser Glu Lys Asn Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Trp Thr Phe Asp Ser Trp Gly Gln Gly Thr Leu Leu

100 105 110

Thr Val Ser Pro

115

<210> 9

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody N0

<400> 9

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Asn Asn Arg

20 25 30

Phe Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Ala Ala Ser Ser Arg Ala Thr Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Ala Glu Asp Phe Ala Met Tyr His Cys Gln Gln Tyr Gly Ser Phe Pro

85 90 95

Arg Thr Phe Gly Gln Gly Thr Met Val Asp Ile Lys

100 105

<210> 10

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody S0

<400> 10

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Arg Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Gly Gly Ile Thr Tyr Ala Pro Ser Thr Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody S0

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val

35 40 45

Tyr Asn Thr Arg Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody V0

<400> 12

Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Asn Ser Arg Asn Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Glu Thr Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ile Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Ser Gly Ser Thr Pro Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 13

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody V0

<400> 13

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Val Ser

50 55 60

Val Ser Gly Thr Ser His Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 14

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody J0

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly His Ile Lys Ser Lys Thr Asp Ala Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Ile Tyr Tyr Tyr Ala Phe Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody J0

<400> 15

Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Asn Ile Gly Ser Lys Tyr Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Asp Asn Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Ala Asp Trp Val Asp Phe Tyr

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 16

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody H0

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Ser Ile Ser Tyr Glu Gly Asp Ile Thr Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Gln Thr Leu Arg Glu Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 17

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody H0

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ala Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Lys Glu Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 18

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody R0

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Ser Leu Thr Ser Asn

20 25 30

Ser Val Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Trp Ser Asn Gly Asp Thr Asp Tyr Asn Ser Ala Ile Lys

50 55 60

Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 19

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody R0

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Glu Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Ala Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Lys Phe Pro Asn

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys

100 105

<210> 20

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody B0

<400> 20

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro

100 105 110

Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 21

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody B0

<400> 21

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu

100 105

<210> 22

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody U0

<400> 22

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile

35 40 45

Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr

65 70 75 80

Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 23

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody U0

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody D0

<400> 24

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 25

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody D0

<400> 25

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody I0

<400> 26

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr

20 25 30

Asp Leu Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Tyr Trp Tyr Ala Thr Ser Tyr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 27

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody I0

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Phe His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Glu His Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody C0

<400> 28

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

His Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Asp Ile Asn Pro Asp Ile Gly Gly Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Asp Ser Phe Ala Asn Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 29

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody C0

<400> 29

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Ser Ile Ile

20 25 30

Gly Thr Asn Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Asn Leu Glu Thr Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Lys Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 30

<211> 104

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IL6Ra.0 chain sequence

<400> 30

Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val Thr Ala Val

1 5 10 15

Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp Pro His Ser

20 25 30

Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg Tyr Arg Ala

35 40 45

Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp Leu Gln His

50 55 60

His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His Val Val Gln

65 70 75 80

Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser Glu Trp Ser

85 90 95

Pro Glu Ala Met Gly Thr Pro Trp

100

<210> 31

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IL6Rb.0 chain sequence

<400> 31

Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro Pro His Asn Leu Ser

1 5 10 15

Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu Lys Leu Thr Trp Thr

20 25 30

Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys Tyr Asn Ile Gln Tyr

35 40 45

Arg Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile Pro Pro Glu Asp Thr

50 55 60

Ala Ser Thr Arg Ser Ser Phe Thr Val Gln Asp Leu Lys Pro Phe Thr

65 70 75 80

Glu Tyr Val Phe Arg Ile Arg Cys Met Lys Glu Asp Gly Lys Gly Tyr

85 90 95

Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile Thr

100 105

<210> 32

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.0

<400> 32

Gly Ile Pro Pro Lys Ile Glu Ala Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Cys Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Cys Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 33

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence O.0

<400> 33

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Cys Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Thr Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Cys Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Ala Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 34

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence OL.0

<400> 34

Gln Gly Ser Pro Pro Cys Phe Leu Arg Phe Pro Arg Pro Val Arg Val

1 5 10 15

Val Ser Gly Ala Glu Ala Glu Leu Lys Cys Val Val Leu Gly Glu Pro

20 25 30

Pro Pro Val Val Val Trp Glu Lys Gly Gly Gln Gln Leu Ala Ala Ser

35 40 45

Glu Arg Leu Ser Phe Pro Ala Asp Gly Ala Glu His Gly Leu Leu Leu

50 55 60

Thr Ala Ala Leu Pro Thr Asp Ala Gly Val Tyr Val Cys Arg Ala Arg

65 70 75 80

Asn Ala Ala Gly Glu Ala Tyr Ala Ala Ala Ala Val Thr Val Leu Glu

85 90 95

Pro Pro

<210> 35

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence T.1

<400> 35

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 36

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> T.2 Circuit Sequence

<400> 36

Gly Ile Pro Pro Lys Ile Glu Ala Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Cys Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 37

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> T.3 Circuit Sequence

<400> 37

Gly Ile Pro Pro Lys Ile Glu Ala Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Cys Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 38

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> T.4 chain sequence

<400> 38

Gly Ile Pro Pro Lys Ile Glu Ala Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Cys Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 39

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> T.5 Circuit Sequence

<400> 39

Lys Ala Gly Ile Arg Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser

1 5 10 15

Asp Ile Ser Ile Asp Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe

20 25 30

Thr Gly Glu Pro Thr Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys

35 40 45

Ile His Ser Gln Glu Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp

50 55 60

Leu Thr Thr Leu Ile Ile Met Asp Val Gln Lys Gln Asp Gly Gly Leu

65 70 75 80

Tyr Thr Leu Ser Leu Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val

85 90 95

Asn Ile His Ile Arg Ser Ile

100

<210> 40

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> T.6 Circuit Sequence

<400> 40

Gly Gly Gly Gly Ser Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser

1 5 10 15

Asp Ile Ser Ile Asp Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe

20 25 30

Thr Gly Glu Pro Thr Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys

35 40 45

Ile His Ser Gln Glu Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp

50 55 60

Leu Thr Thr Leu Ile Ile Met Asp Val Gln Lys Gln Asp Gly Gly Leu

65 70 75 80

Tyr Thr Leu Ser Leu Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val

85 90 95

Asn Ile His Ile Arg Ser Ile

100

<210> 41

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence T.7

<400> 41

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro Lys Ile

1 5 10 15

Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp Glu Gly Lys Val Leu Thr

20 25 30

Val Ala Ser Ala Phe Thr Gly Glu Pro Thr Pro Glu Val Thr Trp Ser

35 40 45

Thr Gly Gly Arg Lys Ile His Ser Gln Glu Gln Gly Arg Phe His Ile

50 55 60

Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile Ile Met Asp Val Gln Lys

65 70 75 80

Gln Asp Gly Gly Leu Tyr Thr Leu Ser Leu Gly Asn Glu Phe Gly Ser

85 90 95

Asp Ser Ala Thr Val Asn Ile His Ile Arg Ser Ile

100 105

<210> 42

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.1

<400> 42

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Cys Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Thr Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Cys Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 43

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.2

<400> 43

Ser Gly Cys Pro Arg Phe Leu Thr Arg Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Cys Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Thr Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Cys Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Ala Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 44

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.3

<400> 44

Ser Gly Ala Pro Arg Phe Leu Thr Cys Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Cys Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Thr Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Cys Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Ala Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 45

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.4

<400> 45

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Thr Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 46

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.5

<400> 46

Ser Gly Cys Pro Arg Phe Leu Thr Arg Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Thr Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Ala Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 47

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.6

<400> 47

Ser Gly Ala Pro Arg Phe Leu Thr Cys Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Thr Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Ala Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 48

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.7

<400> 48

Ser Gly Ala Pro Arg Phe Lys Thr Arg Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 49

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.8

<400> 49

Ser Gly Ala Pro Arg Phe Lys Thr Arg Pro Lys Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu His Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 50

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.9

<400> 50

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 51

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.10

<400> 51

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu His Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 52

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.11

<400> 52

Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Lys

1 5 10 15

Ala Phe Val Val Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro

35 40 45

Val Ala Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu Thr Ile Leu Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu

85 90 95

Gln Val Asp Ala Glu Ala

100

<210> 53

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.12

<400> 53

Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe Lys Thr Arg Pro Lys

1 5 10 15

Ala Phe Val Val Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro

35 40 45

Val Ala Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu Lys Ile Leu Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu

85 90 95

Gln Val Asp Ala Glu Ala

100

<210> 54

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.13

<400> 54

Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe Lys Thr Arg Pro Lys

1 5 10 15

Ala Phe Val Val Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro

35 40 45

Val Ala Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu His Ile Leu Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu

85 90 95

Gln Val Asp Ala Glu Ala

100

<210> 55

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.14

<400> 55

Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe Arg Thr Arg Pro Lys

1 5 10 15

Ala Phe Val Val Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro

35 40 45

Val Ala Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu Lys Ile Leu Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu

85 90 95

Gln Val Asp Ala Glu Ala

100

<210> 56

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.15

<400> 56

Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe Arg Thr Arg Pro Lys

1 5 10 15

Ala Phe Val Val Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro

35 40 45

Val Ala Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu His Ile Leu Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu

85 90 95

Gln Val Asp Ala Glu Ala

100

<210> 57

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.16

<400> 57

Gly Gly Gly Gly Ser Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Lys

1 5 10 15

Ala Phe Val Val Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro

35 40 45

Val Ala Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu Thr Ile Leu Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu

85 90 95

Gln Val Asp Ala Glu Ala

100

<210> 58

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.17

<400> 58

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Ala Pro Arg Phe

1 5 10 15

Leu Thr Arg Pro Lys Ala Phe Val Val Ser Val Gly Lys Asp Ala Thr

20 25 30

Leu Ser Ser Gln Ile Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu

35 40 45

Lys Asp Gln Gln Pro Val Ala Ala Gly Ala Arg Phe Arg Leu Ala Gln

50 55 60

Asp Gly Asp Leu Tyr Arg Leu Thr Ile Leu Asp Leu Ala Leu Gly Asp

65 70 75 80

Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe

85 90 95

Ala Cys Val Gly Leu Gln Val Asp Ala Glu Ala

100 105

<210> 59

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence of chain OL.1

<400> 59

Gln Gly Ser Pro Pro Glu Phe Leu Arg Phe Pro Arg Pro Val Arg Val

1 5 10 15

Val Ser Gly Ala Glu Ala Glu Leu Lys Cys Val Val Leu Gly Glu Pro

20 25 30

Pro Pro Val Val Val Trp Glu Lys Gly Gly Gln Gln Leu Ala Ala Ser

35 40 45

Glu Arg Leu Ser Phe Pro Ala Asp Gly Ala Glu His Gly Leu Leu Leu

50 55 60

Thr Ala Ala Leu Pro Thr Asp Ala Gly Cys Tyr Val Cys Arg Ala Arg

65 70 75 80

Asn Ala Ala Gly Glu Ala Tyr Ala Ala Ala Ala Val Thr Val Leu Glu

85 90 95

Pro Pro

<210> 60

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence OL.2

<400> 60

Gln Gly Ser Pro Pro Glu Phe Leu Arg Phe Pro Arg Pro Val Arg Val

1 5 10 15

Val Ser Gly Ala Glu Ala Glu Leu Lys Cys Val Val Leu Gly Glu Pro

20 25 30

Pro Pro Val Val Val Trp Glu Lys Gly Gly Gln Gln Leu Ala Ala Ser

35 40 45

Glu Arg Leu Ser Phe Pro Ala Asp Gly Ala Glu His Gly Leu Leu Leu

50 55 60

Thr Ala Ala Leu Pro Thr Asp Ala Gly Val Tyr Val Cys Arg Ala Arg

65 70 75 80

Asn Ala Ala Cys Glu Ala Tyr Ala Ala Ala Ala Val Thr Val Leu Glu

85 90 95

Pro Pro

<210> 61

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence of chain OL.3

<400> 61

Gln Gly Ser Pro Pro Glu Phe Leu Arg Phe Pro Arg Pro Val Arg Val

1 5 10 15

Val Ser Gly Ala Glu Ala Glu Leu Lys Cys Val Val Leu Gly Glu Pro

20 25 30

Pro Pro Val Val Val Trp Glu Lys Gly Gly Gln Gln Leu Ala Ala Ser

35 40 45

Glu Arg Leu Ser Phe Pro Ala Asp Gly Ala Glu His Gly Leu Leu Leu

50 55 60

Thr Ala Ala Leu Pro Thr Asp Ala Gly Val Tyr Val Cys Arg Ala Arg

65 70 75 80

Asn Ala Ala Gly Glu Cys Tyr Ala Ala Ala Ala Val Thr Val Leu Glu

85 90 95

Pro Pro

<210> 62

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence OL.4

<400> 62

Gln Gly Ser Pro Pro Glu Phe Leu Arg Phe Pro Arg Pro Val Arg Val

1 5 10 15

Val Ser Gly Ala Glu Ala Glu Leu Lys Ser Val Val Leu Gly Glu Pro

20 25 30

Pro Pro Val Val Val Trp Glu Lys Gly Gly Gln Gln Leu Ala Ala Ser

35 40 45

Glu Arg Leu Ser Phe Pro Ala Asp Gly Ala Glu His Gly Leu Leu Leu

50 55 60

Thr Ala Ala Leu Pro Thr Asp Ala Gly Cys Tyr Val Ser Arg Ala Arg

65 70 75 80

Asn Ala Ala Gly Glu Ala Tyr Ala Ala Ala Ala Val Thr Val Leu Glu

85 90 95

Pro Pro

<210> 63

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence OL.5

<400> 63

Gln Gly Ser Pro Pro Glu Phe Leu Arg Phe Pro Arg Pro Val Arg Val

1 5 10 15

Val Ser Gly Ala Glu Ala Glu Leu Lys Ser Val Val Leu Gly Glu Pro

20 25 30

Pro Pro Val Val Val Trp Glu Lys Gly Gly Gln Gln Leu Ala Ala Ser

35 40 45

Glu Arg Leu Ser Phe Pro Ala Asp Gly Ala Glu His Gly Leu Leu Leu

50 55 60

Thr Ala Ala Leu Pro Thr Asp Ala Gly Val Tyr Val Ser Arg Ala Arg

65 70 75 80

Asn Ala Ala Cys Glu Ala Tyr Ala Ala Ala Ala Val Thr Val Leu Glu

85 90 95

Pro Pro

<210> 64

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence OL.6

<400> 64

Gln Gly Ser Pro Pro Glu Phe Leu Arg Phe Pro Arg Pro Val Arg Val

1 5 10 15

Val Ser Gly Ala Glu Ala Glu Leu Lys Ser Val Val Leu Gly Glu Pro

20 25 30

Pro Pro Val Val Val Trp Glu Lys Gly Gly Gln Gln Leu Ala Ala Ser

35 40 45

Glu Arg Leu Ser Phe Pro Ala Asp Gly Ala Glu His Gly Leu Leu Leu

50 55 60

Thr Ala Ala Leu Pro Thr Asp Ala Gly Val Tyr Val Ser Arg Ala Arg

65 70 75 80

Asn Ala Ala Gly Glu Cys Tyr Ala Ala Ala Ala Val Thr Val Leu Glu

85 90 95

Pro Pro

<210> 65

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> T.8 Circuit Sequence

<400> 65

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Ser Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Ser Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 66

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence T.9

<400> 66

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Lys Asp Val Gln Arg Gln Asp Gly Gly Leu Tyr Thr Leu Thr Leu

65 70 75 80

Arg Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 67

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence T.10

<400> 67

Gly Ile Trp Pro Lys Ile Glu Cys Leu Pro Ile Asp Leu Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Met Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Ser Leu

65 70 75 80

Gly Met Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 68

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.11

<400> 68

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ile Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

50 55 60

Ile Lys Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Thr Leu

65 70 75 80

Arg Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 69

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence T.12

<400> 69

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Ser Gly Lys Lys Ile Ser Ser Glu Glu

35 40 45

Gly Gly Arg Phe His Ile Glu Ser Thr Glu Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Val Tyr Thr Leu Ser Leu

65 70 75 80

Gly Asn Asp Leu Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 70

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.13

<400> 70

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Glu Glu

35 40 45

Gly Gly Arg Phe His Ile Glu Ser Thr Glu Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Val Tyr Thr Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 71

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.14

<400> 71

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Ser Thr Glu Asp Leu Thr Thr Leu Ile

50 55 60

Ile Met Asp Val Gln Lys Gln Asp Gly Gly Val Tyr Thr Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 72

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.15

<400> 72

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

1 5 10 15

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

20 25 30

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

35 40 45

Gln Gly Arg Phe His Ile Glu Ser Thr Glu Asp Leu Thr Thr Leu Ile

50 55 60

Ile Ser Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Ser Leu Ser Leu

65 70 75 80

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

85 90 95

Ser Ile

<210> 73

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.16

<400> 73

Gly Gly Gly Gly Ser Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ile

1 5 10 15

Asp Ile Ser Ile Asp Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe

20 25 30

Thr Gly Glu Pro Thr Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys

35 40 45

Ile His Ser Gln Glu Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp

50 55 60

Leu Thr Thr Leu Ile Ile Lys Asp Val Gln Lys Gln Asp Gly Gly Leu

65 70 75 80

Tyr Thr Leu Thr Leu Arg Asn Glu Phe Gly Ser Asp Ser Ala Thr Val

85 90 95

Asn Ile His Ile Arg Ser Ile

100

<210> 74

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.17

<400> 74

Lys Ala Gly Ile Arg Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser

1 5 10 15

Asp Ile Ser Ile Asp Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe

20 25 30

Thr Gly Glu Pro Thr Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys

35 40 45

Ile His Ser Gln Glu Gln Gly Arg Phe His Ile Glu Ser Thr Glu Asp

50 55 60

Leu Thr Thr Leu Ile Ile Ser Asp Val Gln Lys Gln Asp Gly Gly Leu

65 70 75 80

Tyr Ser Leu Ser Leu Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val

85 90 95

Asn Ile His Ile Arg Ser Ile

100

<210> 75

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.18

<400> 75

Gly Gly Gly Gly Ser Lys Ala Gly Ile Arg Gly Ile Pro Pro Lys Ile

1 5 10 15

Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp Glu Gly Lys Val Leu Thr

20 25 30

Val Ala Ser Ala Phe Thr Gly Glu Pro Thr Pro Glu Val Thr Trp Ser

35 40 45

Thr Gly Gly Arg Lys Ile His Ser Gln Glu Gln Gly Arg Phe His Ile

50 55 60

Glu Ser Thr Glu Asp Leu Thr Thr Leu Ile Ile Ser Asp Val Gln Lys

65 70 75 80

Gln Asp Gly Gly Leu Tyr Ser Leu Ser Leu Gly Asn Glu Phe Gly Ser

85 90 95

Asp Ser Ala Thr Val Asn Ile His Ile Arg Ser Ile

100 105

<210> 76

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain Sequence T.19

<400> 76

Gly Gly Gly Gly Ser Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser

1 5 10 15

Asp Ile Ser Ile Asp Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe

20 25 30

Thr Gly Glu Pro Thr Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys

35 40 45

Ile His Ser Gln Glu Gln Gly Arg Phe His Ile Glu Ser Thr Glu Asp

50 55 60

Leu Thr Thr Leu Ile Ile Ser Asp Val Gln Lys Gln Asp Gly Gly Leu

65 70 75 80

Tyr Ser Leu Ser Leu Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val

85 90 95

Asn Ile His Ile Arg Ser Ile

100

<210> 77

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.18

<400> 77

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Lys Ser Tyr Thr Val Ser

1 5 10 15

Val Gly Lys Asp Ala Ser Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 78

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.19

<400> 78

Ser Glu Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

1 5 10 15

Glu Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asp Pro Pro

20 25 30

Pro Glu Val Thr Trp Glu Lys Asp Gln Gln Pro Ile Thr Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Val Tyr Arg Leu Glu Ile Leu

50 55 60

Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Gly

<210> 79

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.20

<400> 79

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Leu Ala Met Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Gln Pro Val Ala Ala Gly Ala

35 40 45

Arg Phe Arg Leu Leu Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

50 55 60

Asp Leu Ala Leu Gly Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 80

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.21

<400> 80

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Lys Gln Pro Val Thr Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

50 55 60

Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Leu Gly Leu Gln Val Asp Ala Glu

85 90 95

Ala

<210> 81

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.22

<400> 81

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Leu Pro Val Thr Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

50 55 60

Asp Leu Thr Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Glu Val Gly Ala Glu

85 90 95

Ala

<210> 82

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.23

<400> 82

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ser Tyr Val Val Ser

1 5 10 15

Val Gly Lys Asp Ala Ser Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

20 25 30

Pro Gln Val Ser Trp Glu Lys Asp Gln Leu Pro Val Thr Ala Gly Ala

35 40 45

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

50 55 60

Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

65 70 75 80

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Glu Val Gly Ala Glu

85 90 95

Ala

<210> 83

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.24

<400> 83

Gly Gly Gly Gly Ser Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu

1 5 10 15

Ala Phe Val Val Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Lys Gln Pro

35 40 45

Val Thr Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu Lys Ile Leu Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Leu Gly Leu

85 90 95

Gln Val Asp Ala Glu Ala

100

<210> 84

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.25

<400> 84

Gly Gly Gly Gly Ser Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu

1 5 10 15

Ser Tyr Val Val Ser Val Gly Lys Asp Ala Ser Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Leu Pro

35 40 45

Val Thr Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu Lys Ile Leu Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu

85 90 95

Glu Val Gly Ala Glu Ala

100

<210> 85

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.26

<400> 85

Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu

1 5 10 15

Ser Tyr Val Val Ser Val Gly Lys Asp Ala Ser Leu Ser Ser Gln Ile

20 25 30

Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Leu Pro

35 40 45

Val Thr Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr

50 55 60

Arg Leu Lys Ile Leu Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val

65 70 75 80

Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu

85 90 95

Glu Val Gly Ala Glu Ala

100

<210> 86

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Chain sequence O.27

<400> 86

Gly Gly Gly Gly Ser Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe

1 5 10 15

Leu Thr Arg Pro Leu Ser Tyr Val Val Ser Val Gly Lys Asp Ala Ser

20 25 30

Leu Ser Ser Gln Ile Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu

35 40 45

Lys Asp Gln Leu Pro Val Thr Ala Gly Ala Arg Phe Arg Leu Ala Gln

50 55 60

Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu Asp Leu Gln Leu Ser Asp

65 70 75 80

Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe

85 90 95

Ala Cys Val Gly Leu Glu Val Gly Ala Glu Ala

100 105

<210> 87

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 1 of DI-1 (DI-1-H1)

<400> 87

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 88

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 2 of DI-1 (DI-1-L1)

<400> 88

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 89

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 3 of DI-1 (DI-1-H2)

<400> 89

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr

20 25 30

Asp Leu Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Tyr Trp Tyr Ala Thr Ser Tyr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Ile

115 120 125

Pro Pro Lys Ile Glu Cys Leu Pro Ile Asp Ile Ser Ile Asp Glu Gly

130 135 140

Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr Pro Glu

145 150 155 160

Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu Gln Gly

165 170 175

Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile Ile Lys

180 185 190

Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Thr Leu Arg Asn

195 200 205

Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg Ser Ile

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 90

<211> 209

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 4 of DI-1 (DI-1-L2)

<400> 90

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Phe His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Glu His Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

115 120 125

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

130 135 140

Pro Gln Val Ser Trp Glu Lys Asp Lys Gln Pro Val Thr Ala Gly Ala

145 150 155 160

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

165 170 175

Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

180 185 190

Ala Ile Gly Glu Ala Phe Ala Cys Leu Gly Leu Gln Val Asp Ala Glu

195 200 205

Ala

<210> 91

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 1 of BU1 (BU1-H1)

<400> 91

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro

100 105 110

Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

225 230 235 240

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 92

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of BU1 chain 2 (BU1-L1)

<400> 92

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 93

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 3 of BU1 (BU1-H2)

<400> 93

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile

35 40 45

Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr

65 70 75 80

Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro

115 120 125

Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp Glu Gly Lys Val

130 135 140

Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr Pro Glu Val Thr

145 150 155 160

Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu Gln Gly Arg Phe

165 170 175

His Ile Glu Ser Thr Glu Asp Leu Thr Thr Leu Ile Ile Ser Asp Val

180 185 190

Gln Lys Gln Asp Gly Gly Leu Tyr Ser Leu Ser Leu Gly Asn Glu Phe

195 200 205

Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg Ser Ile Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 94

<211> 209

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 4 of BU1 (BU1-L2)

<400> 94

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ser Tyr Val Val Ser

115 120 125

Val Gly Lys Asp Ala Ser Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

130 135 140

Pro Gln Val Ser Trp Glu Lys Asp Gln Leu Pro Val Thr Ala Gly Ala

145 150 155 160

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

165 170 175

Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

180 185 190

Ala Ile Gly Glu Ala Phe Ala Cys Val Gly Leu Glu Val Gly Ala Glu

195 200 205

Ala

<210> 95

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 1 BU2

(BU2-H1)/chain 1 BU4 (chain BU4-H1)/chain 3 BU5 (BU5-H2)

<400> 95

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile

35 40 45

Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr

65 70 75 80

Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 96

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 2 BU2

(BU2-L1)/chain 4 BU3 (chain 4 BU3-L2)/chain 2 BU4

(chain 2 BU4-L1)/chain 4 BU5 (BU5-L2)

<400> 96

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 97

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 3 of BU2 (BU2-H2)

<400> 97

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro

100 105 110

Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

130 135 140

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

145 150 155 160

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

165 170 175

Gln Gly Arg Phe His Ile Glu Ser Thr Glu Asp Leu Thr Thr Leu Ile

180 185 190

Ile Ser Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Ser Leu Ser Leu

195 200 205

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

210 215 220

Ser Ile Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

225 230 235 240

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 98

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 4 of BU2 (BU2-L2)

<400> 98

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly Gly Gly Gly

100 105 110

Ser Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro

115 120 125

Leu Ser Tyr Val Val Ser Val Gly Lys Asp Ala Ser Leu Ser Ser Gln

130 135 140

Ile Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Leu

145 150 155 160

Pro Val Thr Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu

165 170 175

Tyr Arg Leu Lys Ile Leu Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr

180 185 190

Val Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly

195 200 205

Leu Glu Val Gly Ala Glu Ala

210 215

<210> 99

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 1 of BU3 (BU3-H1)

<400> 99

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro

100 105 110

Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser Asp Ile Ser Ile Asp

130 135 140

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

145 150 155 160

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

165 170 175

Gln Gly Arg Phe His Ile Glu Ser Thr Glu Asp Leu Thr Thr Leu Ile

180 185 190

Ile Ser Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Ser Leu Ser Leu

195 200 205

Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

210 215 220

Ser Ile Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

225 230 235 240

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 100

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 2 BU3

(BU3-L1)/chain 4 BU4 (BU4-L2)

<400> 100

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly Gly Gly Gly

100 105 110

Ser Asp Gln Pro Gln Phe Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro

115 120 125

Leu Ser Tyr Val Val Ser Val Gly Lys Asp Ala Ser Leu Ser Ser Gln

130 135 140

Ile Val Gly Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Gln Leu

145 150 155 160

Pro Val Thr Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu

165 170 175

Tyr Arg Leu Lys Ile Leu Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr

180 185 190

Val Ser Arg Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Val Gly

195 200 205

Leu Glu Val Gly Ala Glu Ala

210 215

<210> 101

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 3 BU3 (BU3-H2)

<400> 101

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile

35 40 45

Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr

65 70 75 80

Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 102

<211> 458

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 3 of BU4 (BU4-H2)

<400> 102

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro

100 105 110

Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Lys Ala Gly Ile Arg Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ser

130 135 140

Asp Ile Ser Ile Asp Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe

145 150 155 160

Thr Gly Glu Pro Thr Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys

165 170 175

Ile His Ser Gln Glu Gln Gly Arg Phe His Ile Glu Ser Thr Glu Asp

180 185 190

Leu Thr Thr Leu Ile Ile Ser Asp Val Gln Lys Gln Asp Gly Gly Leu

195 200 205

Tyr Ser Leu Ser Leu Gly Asn Glu Phe Gly Ser Asp Ser Ala Thr Val

210 215 220

Asn Ile His Ile Arg Ser Ile Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu

355 360 365

Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

435 440 445

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 103

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 1 of BU5 (BU5-H1)

<400> 103

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro

100 105 110

Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Ile Pro Pro Lys Ile Glu Cys Leu Pro Ile Asp Ile Ser Ile Asp

130 135 140

Glu Gly Lys Val Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr

145 150 155 160

Pro Glu Val Thr Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu

165 170 175

Gln Gly Arg Phe His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile

180 185 190

Ile Lys Asp Val Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Thr Leu

195 200 205

Arg Asn Glu Phe Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg

210 215 220

Ser Ile Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

225 230 235 240

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 104

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of BU5 chain 2 (BU5-L1)

<400> 104

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly Gly Gly Gly

100 105 110

Ser Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val

115 120 125

Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro

130 135 140

Thr Pro Gln Val Ser Trp Glu Lys Asp Lys Gln Pro Val Thr Ala Gly

145 150 155 160

Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile

165 170 175

Leu Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg

180 185 190

Asn Ala Ile Gly Glu Ala Phe Ala Cys Leu Gly Leu Gln Val Asp Ala

195 200 205

Glu Ala

210

<210> 105

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VH antibody A0

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Glu Tyr Ile Ser Tyr Trp

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 106

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of VL antibody A0

<400> 106

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 107

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 1 of FA-1 (FA-1-H1)

<400> 107

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Ala Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro

115 120 125

Lys Ile Glu Cys Leu Pro Ile Asp Ile Ser Ile Asp Glu Gly Lys Val

130 135 140

Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr Pro Glu Val Thr

145 150 155 160

Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu Gln Gly Arg Phe

165 170 175

His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile Ile Lys Asp Val

180 185 190

Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Thr Leu Arg Asn Glu Phe

195 200 205

Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg Ser Ile Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 108

<211> 212

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of FA-1 chain 2 (FA-1-L1)

<400> 108

Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser

20 25 30

His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met

35 40 45

Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg

85 90 95

Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

100 105 110

Gly Gly Ser Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe

115 120 125

Val Val Ser Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly

130 135 140

Asn Pro Thr Pro Gln Val Ser Trp Glu Lys Asp Lys Gln Pro Val Thr

145 150 155 160

Ala Gly Ala Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu

165 170 175

Lys Ile Leu Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg

180 185 190

Ala Arg Asn Ala Ile Gly Glu Ala Phe Ala Cys Leu Gly Leu Gln Val

195 200 205

Asp Ala Glu Ala

210

<210> 109

<211> 455

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 3 of FA-1 (FA-1-H2)

<400> 109

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Glu Tyr Ile Ser Tyr Trp

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

355 360 365

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 110

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Amino acid sequence of chain 4 of FA-1 (FA-1-L2)

<400> 110

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 111

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Sequence of the hB7H3 antigen protein

<400> 111

Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr

1 5 10 15

Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu

20 25 30

Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val

35 40 45

His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg

50 55 60

Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg

65 70 75 80

Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val

85 90 95

Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala

100 105 110

Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg

115 120 125

Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro

130 135 140

Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly

145 150 155 160

Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val

165 170 175

His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys

180 185 190

Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr

195 200 205

Ile Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro

210 215 220

Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys

225 230 235 240

Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile

245 250 255

Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly

260 265 270

Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp

275 280 285

Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val

290 295 300

Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly

305 310 315 320

Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser

325 330 335

Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr

340 345 350

Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp

355 360 365

Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln

370 375 380

Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val

385 390 395 400

Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val

405 410 415

Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met

420 425 430

Thr Asp Tyr Lys Asp Asp Asp Asp Lys His His His His His

435 440 445

<210> 112

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Sequence of the δ-subunit of the hCD3 antigen

<400> 112

Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys

1 5 10 15

Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser

20 25 30

Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly

35 40 45

Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr

50 55 60

Val Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp Pro

65 70 75 80

Ala Thr Val Ala Ser Ala Glu Asn Ala Gln Cys Glu Lys Glu Leu Gln

85 90 95

Ala Leu Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu

100 105 110

Glu Lys Glu Leu Ala Gln Asp Tyr Lys Asp Asp Asp Asp Lys

115 120 125

<210> 113

<211> 143

<212> PRT

<213> Artificial sequence

<220>

<223> Sequence of the ε-subunit of the hCD3 antigen

<400> 113

Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val

1 5 10 15

Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly

20 25 30

Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu

35 40 45

Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu

50 55 60

Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly

65 70 75 80

Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val

85 90 95

Cys Glu Asn Cys Met Glu Met Asp Ala Lys Asn Ala Gln Cys Lys Lys

100 105 110

Lys Leu Gln Ala Leu Lys Lys Lys Asn Ala Gln Leu Lys Trp Lys Leu

115 120 125

Gln Ala Leu Lys Lys Lys Leu Ala Gln His His His His His

130 135 140

<210> 114

<211> 482

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-1-H1

<400> 114

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Ser Ile Ser Tyr Glu Gly Asp Ile Thr Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Gln Thr Leu Arg Glu Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Leu Glu Asp Leu Lys Asn Val Phe Pro Pro

115 120 125

Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln

130 135 140

Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val

145 150 155 160

Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys

165 170 175

Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg

180 185 190

Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn

195 200 205

Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu

210 215 220

Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val

225 230 235 240

Ser Ala Glu Ala Trp Gly Arg Ala Ser Asp Lys Thr His Thr Cys Pro

245 250 255

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

260 265 270

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

275 280 285

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

290 295 300

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

305 310 315 320

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

325 330 335

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

340 345 350

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

355 360 365

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg

370 375 380

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

385 390 395 400

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

405 410 415

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

420 425 430

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

435 440 445

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

450 455 460

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His His His

465 470 475 480

His His

<210> 115

<211> 203

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-1-L1

<400> 115

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ala Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Lys Glu Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Lys Pro Asp Ile Gln

100 105 110

Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp

115 120 125

Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Gln Val Ser

130 135 140

Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp

145 150 155 160

Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Gln

165 170 175

Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn Ser Ile Ile Pro

180 185 190

Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 116

<211> 457

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-1-H2 and HJ-3-H2

<400> 116

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Val Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys Asp Tyr Lys Asp Asp Asp Asp Lys

450 455

<210> 117

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-1-L2 and HJ-3-L2

<400> 117

Ser Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Glu His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 118

<211> 456

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-2-H1 and HJ-4-H1

<400> 118

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Ser Ile Ser Tyr Glu Gly Asp Ile Thr Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Gln Thr Leu Arg Glu Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

Asp Tyr Lys Asp Asp Asp Asp Lys

450 455

<210> 119

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-2-L1 and HJ-4-L1

<400> 119

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ala Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Lys Glu Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 120

<211> 483

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-1-H2

<400> 120

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Val Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Leu Glu Asp Leu Lys Asn Val Phe Pro

115 120 125

Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr

130 135 140

Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His

145 150 155 160

Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val

165 170 175

Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp Ser

180 185 190

Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln

195 200 205

Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser

210 215 220

Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile

225 230 235 240

Val Ser Ala Glu Ala Trp Gly Arg Ala Ser Asp Lys Thr His Thr Cys

245 250 255

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

260 265 270

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

275 280 285

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

290 295 300

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

305 310 315 320

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

325 330 335

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

340 345 350

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

355 360 365

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys

370 375 380

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

385 390 395 400

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

405 410 415

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

420 425 430

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

435 440 445

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

450 455 460

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His His

465 470 475 480

His His His

<210> 121

<211> 203

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-2-L2

<400> 121

Ser Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Glu His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Lys Pro Asp Ile Gln

100 105 110

Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp

115 120 125

Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Gln Val Ser

130 135 140

Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp

145 150 155 160

Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Gln

165 170 175

Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn Ser Ile Ile Pro

180 185 190

Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 122

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-3-H1

<400> 122

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Ser Ile Ser Tyr Glu Gly Asp Ile Thr Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Gln Thr Leu Arg Glu Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro Lys

115 120 125

Ile Glu Cys Leu Pro Ile Asp Ile Ser Ile Asp Glu Gly Lys Val Leu

130 135 140

Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr Pro Glu Val Thr Trp

145 150 155 160

Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu Gln Gly Arg Phe His

165 170 175

Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile Ile Lys Asp Val Gln

180 185 190

Lys Gln Asp Gly Gly Leu Tyr Thr Leu Thr Leu Arg Asn Glu Phe Gly

195 200 205

Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg Ser Ile Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

His His His His His

450

<210> 123

<211> 209

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-3-L1

<400> 123

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ala Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Asn Leu Glu Val Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Lys Glu Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

115 120 125

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

130 135 140

Pro Gln Val Ser Trp Glu Lys Asp Lys Gln Pro Val Thr Ala Gly Ala

145 150 155 160

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

165 170 175

Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

180 185 190

Ala Ile Gly Glu Ala Phe Ala Cys Leu Gly Leu Gln Val Asp Ala Glu

195 200 205

Ala

<210> 124

<211> 455

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-4-H2

<400> 124

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Val Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro

115 120 125

Lys Ile Glu Cys Leu Pro Ile Asp Ile Ser Ile Asp Glu Gly Lys Val

130 135 140

Leu Thr Val Ala Ser Ala Phe Thr Gly Glu Pro Thr Pro Glu Val Thr

145 150 155 160

Trp Ser Thr Gly Gly Arg Lys Ile His Ser Gln Glu Gln Gly Arg Phe

165 170 175

His Ile Glu Asn Thr Asp Asp Leu Thr Thr Leu Ile Ile Lys Asp Val

180 185 190

Gln Lys Gln Asp Gly Gly Leu Tyr Thr Leu Thr Leu Arg Asn Glu Phe

195 200 205

Gly Ser Asp Ser Ala Thr Val Asn Ile His Ile Arg Ser Ile Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys His His His His His His

450 455

<210> 125

<211> 209

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HJ-4-L2

<400> 125

Ser Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Ser Asp

20 25 30

Val Thr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Val Ser Glu His Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His His Arg Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Ser Gly Ala Pro Arg Phe Leu Thr Arg Pro Leu Ala Phe Val Val Ser

115 120 125

Val Gly Lys Asp Ala Thr Leu Ser Ser Gln Ile Val Gly Asn Pro Thr

130 135 140

Pro Gln Val Ser Trp Glu Lys Asp Lys Gln Pro Val Thr Ala Gly Ala

145 150 155 160

Arg Phe Arg Leu Ala Gln Asp Gly Asp Leu Tyr Arg Leu Lys Ile Leu

165 170 175

Asp Leu Gln Leu Ser Asp Ser Gly Gln Tyr Val Ser Arg Ala Arg Asn

180 185 190

Ala Ile Gly Glu Ala Phe Ala Cys Leu Gly Leu Gln Val Asp Ala Glu

195 200 205

Ala

<210> 126

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> heavy chain J1

<400> 126

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Gly Val Gly Asp Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Asn Thr Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr

245 250 255

Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<---

Claims (98)

1. An antigen-binding polypeptide complex comprising a first antigen-binding fragment comprising: A) a first polypeptide comprising a first heavy chain variable domain (VH1) from the N-terminus to the C-terminus, wherein VH1 is operably linked to a first domain containing the T-chain of titin, and a second polypeptide comprising a first variable light chain domain (VL1) from the N-terminus to the C-terminus, wherein VL1 is operably linked to a second domain comprising an obscurin O-chain or an obscurin-like protein O-chain; or B) a first polypeptide comprising VH1 from the N-terminus to the C-terminus, wherein VH1 is operably linked to a first domain comprising an obscurin O-chain or an obscurin-like protein O-chain, and a second polypeptide comprising VL1 from the N-terminus to the C-terminus, wherein VL1 is operably linked to a second domain containing the T-chain of titin; wherein the first antigen-binding fragment specifically binds to a first antigen, and the first domain and the second domain are capable of forming a dimer; wherein VH1 and VL1 of the first antigen-binding fragment form a first antigen-binding site that specifically binds to the first antigen; the C-terminus of VH1 is operably linked to the N-terminus of the first domain, and the C-terminus of VL1 is operably linked to the N-terminus of the second domain; the first domain and the second domain form a dimer through natural interchain linkages and/or non-natural interchain linkages; the non-natural interchain linkage is formed between a particular mutated residue of the first domain and a particular mutated residue of the second domain; wherein the titin T chain comprises a sequence as shown in any one of SEQ ID NOs: 32, 35-41, 65-76; The O-chain of obscurin comprises a sequence as shown in any one of SEQ ID NOs: 33, 42-58, 77-86; The O-chain of the obscurin-like protein comprises a sequence as represented in any one of SEQ ID NOs: 34, 59-64. 2. The antigen-binding polypeptide complex of claim 1, wherein VH1 is operably linked to the first domain via a first linker domain, and VL1 is operably linked to the second domain via a second linker domain; the first linker domain and/or the second linker domain is selected from the group consisting of: an N-terminal fragment of the T-chain of titin, an N-terminal fragment of the O-chain of obscurin, an N-terminal fragment of the O-chain of an obscurin-like protein and a linker (G _x S) _y , where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6. 3. The antigen-binding polypeptide complex according to claim 2, wherein the first linker domain and/or the second linker domain is selected from the group consisting of the following polypeptide fragments: “KAGIR”, “DQPQF”, (G ₄ S) ₁ and (G ₄ S) ₂ . 4. The antigen-binding polypeptide complex according to any one of claims 1-3, wherein the titin T-chain comprises the sequence presented in SEQ ID NO: 68, and the obscurin O-chain comprises the sequence presented in SEQ ID NO: 80; The T-chain of titin comprises the sequence presented in SEQ ID NO: 73, and the O-chain of obscurin comprises the sequence presented in SEQ ID NO: 83; The T-chain of titin comprises the sequence presented in SEQ ID NO: 76, and the O-chain of obscurin comprises the sequence presented in SEQ ID NO: 84; The T-chain of titin comprises the sequence presented in SEQ ID NO: 76, and the O-chain of obscurin comprises the sequence presented in SEQ ID NO: 86; or The titin T chain comprises the sequence presented in SEQ ID NO: 75, and the obscurin O chain comprises the sequence presented in SEQ ID NO: 86. 5. An antigen-binding polypeptide complex according to any one of claims 1-4, wherein the first antigen is selected from the group consisting of: a foreign antigen, an endogenous antigen, an autoantigen, a neoantigen, a viral antigen, and a tumor antigen. 6. An antigen-binding polypeptide complex according to any one of claims 1-5, which is an engineered domain antibody or a Fab fragment thereof comprising a first antigen-binding fragment as defined in any one of claims 1-5. 7. A bispecific antigen-binding polypeptide complex containing: a first polypeptide complex that is an antigen-binding polypeptide complex according to any one of claims 1-6 and specifically binds to a first antigen, and a second polypeptide complex containing a second antigen-binding fragment and specifically binding to a second antigen, a first polypeptide complex and a second polypeptide complex that bind to two different antigens or that bind to two different epitopes on the same antigen; where the second polypeptide complex comprises a second variable domain of the heavy chain (VH2) and a second variable domain of the light chain (VL2), and mispairing between VH1 of the first polypeptide complex and VL2 of the second polypeptide complex and/or between VH2 of the second polypeptide complex and VL1 of the first polypeptide complex is less likely to occur. 8. The bispecific antigen-binding polypeptide complex according to claim 7, wherein the second antigen-binding fragment of the second polypeptide complex comprises: a third polypeptide comprising VH2 from the N-terminus to the C-terminus, wherein VH2 is operably linked to a third domain containing CH1 (heavy chain constant domain 1), and a fourth polypeptide comprising VL2 from the N-terminus to the C-terminus, wherein VL2 is operably linked to a fourth domain containing CL (light chain constant domain); where The C-terminus of VH2 is functionally linked to the N-terminus of CH1, and the C-terminus of VL2 is functionally linked to the N-terminus of CL. 9. The bispecific antigen-binding polypeptide complex of claim 7 or 8, wherein the first polypeptide complex further comprises a first dimerization domain and the second polypeptide complex further comprises a second dimerization domain, wherein the first dimerization domain and the second dimerization domain are linked together; The C-terminus of the first domain of the first polypeptide complex is functionally linked to the N-terminus of the first dimerization domain, and the C-terminus of the third domain of the second polypeptide complex is functionally linked to the N-terminus of the second dimerization domain. 10. The bispecific antigen-binding polypeptide complex of claim 9, wherein the first dimerization domain and the second dimerization domain are linked by means selected from the group consisting of: a hinge region of an antibody and a portion thereof, a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, and a combination thereof; where the hinge region of the antibody and its portion are the hinge regions and portions thereof of IgG1 (immunoglobulin G1), IgG2, IgG3 and IgG4. 11. The bispecific antigen-binding polypeptide complex according to claim 9 or 10, wherein the first dimerization domain comprises an antibody CH2 domain and/or an antibody CH3 domain, and/or the second dimerization domain comprises an antibody CH2 domain and/or an antibody CH3 domain; wherein the CH2 domain of the antibody and/or the CH3 domain of the antibody are derived from IgG1, IgG2, IgG3 or IgG4. 12. The bispecific antigen-binding polypeptide complex of any one of claims 9-11, wherein the first dimerization domain and the second dimerization domain are not identical and are linked in a manner that prevents homodimerization and/or promotes heterodimerization; wherein the first dimerization domain and the second dimerization domain are linked by means selected from the group consisting of: a knob-in-hole technology, a hydrophobic interaction, an electrostatic interaction, a hydrophilic interaction, and an increase in flexibility. 13. The bispecific antigen-binding polypeptide complex of any one of claims 9-12, wherein the first dimerization domain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 2, and the second dimerization domain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 3; or the first dimerization domain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 3, and the second dimerization domain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 2. 14. A bispecific antibody for binding to an antigen, comprising a first antigen-binding fragment, wherein said first antigen-binding fragment comprises a first heavy chain and a first light chain that specifically bind to a first antigen, and further comprising a second heavy chain and a second light chain that specifically bind to a second antigen, wherein the CH1 domain of the first heavy chain is replaced by the O-chain of obscurin, and the CL domain of the first light chain is replaced by the T-chain of titin; the CL domain of the first light chain is replaced by the O-chain of obscurin, and the CH1 domain of the first heavy chain is replaced by the T-chain of titin; the CH1 domain of the first heavy chain is replaced by the O-chain of obscurin-like protein, and the CL domain of the first light chain is replaced by the T-chain of titin; or the CL domain of the first light chain is replaced by the O-chain of obscurin-like protein, and the CH1 domain of the first heavy chain is replaced by the T-chain of titin; the first antigen and the second antigen are not identical, or the first antigen and the second antigen represent two different epitopes on the same antigen; wherein the first heavy chain comprises a first heavy chain variable region (VH1), and the first light chain comprises a first light chain variable region (VL1), wherein VH1 and VL1 form an antigen-binding site that specifically binds to the first antigen, and The C-terminus of VH1 is functionally linked to the N-terminus of the T-chain of titin, and the C-terminus of VL1 is functionally linked to the N-terminus of the O-chain of obscurin or the O-chain of an obscurin-like protein, or The C-terminus of VL1 is functionally linked to the N-terminus of the T-chain of titin, and the C-terminus of VH1 is functionally linked to the N-terminus of the O-chain of obscurin or the O-chain of obscurin-like protein; wherein the titin T chain comprises a sequence as shown in any one of SEQ ID NOs: 32, 35-41, 65-76; The O-chain of obscurin comprises a sequence as shown in any one of SEQ ID NOs: 33, 42-58, 77-86; The O-chain of the obscurin-like protein comprises a sequence as represented in any one of SEQ ID NOs: 34, 59-64. 15. Bispecific antibody according to claim 14, where The titin T chain comprises the sequence shown in SEQ ID NO: 68 and the obscurin O chain comprises the sequence shown in SEQ ID NO: 80; The T-chain of titin comprises the sequence presented in SEQ ID NO: 73, and the O-chain of obscurin comprises the sequence presented in SEQ ID NO: 83; The T-chain of titin comprises the sequence presented in SEQ ID NO: 76, and the O-chain of obscurin comprises the sequence presented in SEQ ID NO: 84; The T-chain of titin comprises the sequence presented in SEQ ID NO: 76, and the O-chain of obscurin comprises the sequence presented in SEQ ID NO: 86; or The titin T chain comprises the sequence presented in SEQ ID NO: 75, and the obscurin O chain comprises the sequence presented in SEQ ID NO: 86. 16. The bispecific antibody of claim 14 or 15, wherein the second heavy chain comprises a second heavy chain variable region (VH2) from the N-terminus to the C-terminus, wherein VH2 is operably linked to CH1; the second light chain comprises a second light chain variable region (VL2) from the N-terminus to the C-terminus, wherein VL2 is operably linked to CL; where VH2 and VL2 form an antigen-binding site that specifically binds to a second antigen, with the C-terminus of VH2 being operably linked to the N-terminus of CH1; and the C-terminus of VL2 being operably linked to the N-terminus of CL. 17. The bispecific antibody of any one of claims 14-16, wherein the C-termini of the first heavy chain and the second heavy chain comprise a CH2 domain and/or a CH3 domain; the CH1 domain, CH2 domain, and CH3 domain are derived from IgG1, IgG2, IgG3, or IgG4; the first heavy chain and the second heavy chain are linked by means selected from the group consisting of: a hinge region of the antibody and a portion thereof, a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, and a combination thereof. 18. Bispecific antibody according to claim 17, where the first heavy chain sequentially contains VH1-L1-T-chain titin-L2-CH2-CH3 from the N-terminus to the C-terminus, and the first light chain sequentially comprises a VL1-L3-O-chain of obscurin or a VL1-L4-O-chain of obscurin-like protein from the N-terminus to the C-terminus; the second heavy chain contains VH2–CH1–CH2–CH3 sequentially from the N-terminus to the C-terminus, and the second light chain contains VL2–CL sequentially from the N-terminus to the C-terminus; or the first light chain contains the VL1-L1-T chain of titin sequentially from the N-terminus to the C-terminus, and the first heavy chain sequentially contains VH1-L2-O-chain of obscurin-L3-CH2-CH3 or VH1-L4-O-chain of obscurin-like protein-L5-CH2-CH3 from the N-terminus to the C-terminus; the second heavy chain contains VH2–CH1–CH2–CH3 sequentially from the N-terminus to the C-terminus, and the second light chain contains VL2–CL sequentially from the N-terminus to the C-terminus; L1-L5 are spacers that may or may not be present; where the spacer is selected from the group consisting of: an N-terminal fragment of the T-chain of titin, an N-terminal fragment of the O-chain of obscurin, an N-terminal fragment of the O-chain of an obscurin-like protein and a linker (G _x S) _y , where X is selected from the group consisting of integers from 1 to 5, and Y is selected from the group consisting of integers from 1 to 6. 19. The bispecific antibody of claim 18, wherein the spacer is selected from the group consisting of: "KAGIR", "DQPQF", (G ₄ S) ₁ and (G ₄ S) ₂ . 20. The bispecific antibody of claim 18 or 19, wherein the CH2 and CH3 of the first heavy chain are not identical to the CH2 and CH3 of the second heavy chain and are linked in a manner that prevents homodimerization and/or promotes heterodimerization; the first heavy chain and the second heavy chain are linked by means selected from the group consisting of: a tongue-and-groove technology, a hydrophobic interaction, an electrostatic interaction, a hydrophilic interaction, and an increase in flexibility. 21. The bispecific antibody of claim 20, wherein the CH2-CH3 domain of the first heavy chain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 2, and the CH2-CH3 domain of the second heavy chain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 3; or the CH2-CH3 domain of the first heavy chain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 3, and the CH2-CH3 domain of the second heavy chain has amino acid residues at positions 104-330 of the sequence presented in SEQ ID NO: 2. 22. The bispecific antibody of any one of claims 14-21, wherein the antigen-binding bispecific antibody is selected from the group consisting of any of the following A-I: A) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 89, or a sequence at least 85% identical to SEQ ID NO: 89, the first light chain has the sequence presented in SEQ ID NO: 90, or a sequence at least 85% identical to SEQ ID NO: 90, the second heavy chain has the sequence presented in SEQ ID NO: 87, or a sequence at least 85% identical to SEQ ID NO: 87, and the second light chain has the sequence presented in SEQ ID NO: 88, or a sequence at least 85% identical to SEQ ID NO: 88; B) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 93, or a sequence at least 85% identical to SEQ ID NO: 93, the first light chain has the sequence presented in SEQ ID NO: 94, or a sequence at least 85% identical to SEQ ID NO: 94, the second heavy chain has the sequence presented in SEQ ID NO: 91, or a sequence at least 85% identical to SEQ ID NO: 91, and the second light chain has the sequence presented in SEQ ID NO: 92, or a sequence at least 85% identical to SEQ ID NO: 92; C) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 97, or a sequence at least 85% identical to SEQ ID NO: 97, the first light chain has the sequence presented in SEQ ID NO: 98, or a sequence at least 85% identical to SEQ ID NO: 98, the second heavy chain has the sequence presented in SEQ ID NO: 95, or a sequence at least 85% identical to SEQ ID NO: 95, and the second light chain has the sequence presented in SEQ ID NO: 96, or a sequence at least 85% identical to SEQ ID NO: 96; D) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 99, or a sequence at least 85% identical to SEQ ID NO: 99, the first light chain has the sequence presented in SEQ ID NO: 100, or a sequence at least 85% identical to SEQ ID NO: 100, the second heavy chain has the sequence presented in SEQ ID NO: 101, or a sequence at least 85% identical to SEQ ID NO: 101, and the second light chain has the sequence presented in SEQ ID NO: 96, or a sequence at least 85% identical to SEQ ID NO: 96; E) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 102, or a sequence at least 85% identical to SEQ ID NO: 102, the first light chain has the sequence presented in SEQ ID NO: 100, or a sequence at least 85% identical to SEQ ID NO: 100, the second heavy chain has the sequence presented in SEQ ID NO: 95, or a sequence at least 85% identical to SEQ ID NO: 95, and the second light chain has the sequence presented in SEQ ID NO: 96, or a sequence at least 85% identical to SEQ ID NO: 96; F) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 103, or a sequence at least 85% identical to SEQ ID NO: 103, the first light chain has the sequence presented in SEQ ID NO: 104, or a sequence at least 85% identical to SEQ ID NO: 104, the second heavy chain has the sequence presented in SEQ ID NO: 95, or a sequence at least 85% identical to SEQ ID NO: 95, and the second light chain has the sequence presented in SEQ ID NO: 96, or a sequence at least 85% identical to SEQ ID NO: 96; and G) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 107, or a sequence at least 85% identical to SEQ ID NO: 107, the first light chain has the sequence presented in SEQ ID NO: 108, or a sequence at least 85% identical to SEQ ID NO: 108, the second heavy chain has the sequence presented in SEQ ID NO: 109, or a sequence at least 85% identical to SEQ ID NO: 109, and the second light chain has the sequence presented in SEQ ID NO: 110, or a sequence at least 85% identical to SEQ ID NO: 110; H) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 122, or a sequence at least 85% identical to SEQ ID NO: 122, the first light chain has the sequence presented in SEQ ID NO: 123, or a sequence at least 85% identical to SEQ ID NO: 123, the second heavy chain has the sequence presented in SEQ ID NO: 116, or a sequence at least 85% identical to SEQ ID NO: 116, and the second light chain has the sequence presented in SEQ ID NO: 117, or a sequence at least 85% identical to SEQ ID NO: 117; I) the first heavy chain of the bispecific antibody has the sequence presented in SEQ ID NO: 118, or a sequence at least 85% identical to SEQ ID NO: 118, the first light chain has the sequence presented in SEQ ID NO: 119, or a sequence at least 85% identical to SEQ ID NO: 119, the second heavy chain has the sequence presented in SEQ ID NO: 124, or a sequence at least 85% identical to SEQ ID NO: 124, and the second light chain has the sequence presented in SEQ ID NO: 125, or a sequence at least 85% identical to SEQ ID NO: 125. 23. A nucleic acid molecule for expressing an antigen-binding polypeptide complex, a bispecific antigen-binding polypeptide complex or a bispecific antibody, encoding the antigen-binding polypeptide complex according to any one of claims 1-6, the bispecific antigen-binding polypeptide complex according to any one of claims 7-13 or the bispecific antibody according to any one of claims 14-22. 24. A vector for expressing an antigen-binding polypeptide complex, a bispecific antigen-binding polypeptide complex, or a bispecific antibody, comprising a nucleic acid molecule according to claim 23. 25. A host cell for expressing an antigen-binding polypeptide complex, a bispecific antigen-binding polypeptide complex, or a bispecific antibody, transformed with the vector of claim 24, wherein the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells. 26. The host cell of claim 25, wherein the eukaryotic cells are mammalian cells. 27. A method for producing an antigen-binding polypeptide complex according to any one of claims 1-6, a bispecific antigen-binding polypeptide complex according to any one of claims 7-13, or a bispecific antibody according to any one of claims 14-22, where The method includes the following steps: culturing the host cell according to claim 25 or 26, followed by purification and isolation of the antigen-binding polypeptide complex according to any one of claims 1-6, the bispecific antigen-binding polypeptide complex according to any one of claims 7-13, or the bispecific antibody according to any one of claims 14-22.