NEW POLYPEPTIDE COMPLEX

The present application claims priority to the Chinese Patent Application (Application No. CN202010020878.7) filed on Jan. 9, 2020.

TECHNICAL FIELD

The present disclosure relates to the field of antibody pharmaceuticals, and specifically comprises a novel antibody in which CH1 and CL structures are substituted and use thereof.

BACKGROUND

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute the prior art.

With the continuous improvement of antibody humanization technology, monoclonal antibodies have been developed rapidly in recent years. Various monoclonal antibodies have been used to treat major diseases such as malignant tumors and autoimmune diseases. However, the immune escape of tumors is often accompanied by a variety of different mechanisms, and a single monoclonal antibody can only bind to one specific target, which can greatly reduce the therapeutic effect of the monoclonal antibody.

The bispecific antibody (BsAb) is an artificial antibody formed by combining antibodies targeting two different antigens or two different epitopes together by using a genetic engineering means. Different from the monoclonal antibody, the bispecific antibody has the ability to target two different epitopes simultaneously, and can play special biological functions, such as immune cell recruitment, receptor co-stimulation or co-inhibition, multivalent virus neutralization, etc., and are expected to obtain better clinical therapeutic effects than single monoclonal antibodies, even antibody combinations.

A wide variety of recombinant bispecific antibodies have been developed, such as tetravalent bispecific antibodies formed by fusion of, for example, an IgG antibody and 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). There are other bispecific forms, such as DVD-Ig, CrossMab and BiTE (Spiess et al., Molecular Immunology, 67 (2), pp. 95-106 (2015)).

Various structural models for bispecific antibodies have also been disclosed, for example, the introduction of the mutation “knobs-into-holes” into the Fc region (Ridgway et al., Protein Engineering, 9 (7), pp. 617-21 (1996)); the introduction of electrostatic design (Gunasekaran et al., Journal of Biological Chemistry, 285, (25), pp. 19637-19646, (2010)), or the introduction of negative state design (Kreudenstein et al., mAbs, 5 (5), pp. 646-654 (2013); Leaver-Fay et al., Structure, 24 (4), pp. 641-651 (2016)), the 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 TCR constant region (WO2019057122A1).

SUMMARY

However, in the preparation of a bispecific antibody, a random combination of different polypeptide chains results in a variety of different products, only one of which is desired, and the resulting byproducts comprise an antibody lacking a light chain, a half-antibody, a heavy chain polymer, light-heavy-chain mispairing, and the like, causing significant challenges to the development of downstream processes. Therefore, the present disclosure provides a novel domain-engineered polypeptide complex. In some embodiments, the present disclosure provides a polypeptide complex, which comprises a first antigen-binding moiety comprising:

A) a first polypeptide, comprising a first heavy chain variable domain (VH1) from an N-terminus to a C-terminus, the VH1 being operably linked to a first domain comprising a Titin-T chain, and

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

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

a second polypeptide, comprising a VL1 from an N-terminus to a C-terminus, the VL1 being operably linked to a second domain comprising a Titin-T chain; or,

C) a first polypeptide, comprising a VH1 from an N-terminus to a C-terminus, the VH1 being operably linked to a first domain comprising an Obscurin-O chain or an Obscurin-like-O chain, and

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

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

a second polypeptide, comprising a VL1 from an N-terminus to a C-terminus, the VL1 being operably linked to a second domain comprising an Obscurin-O chain or an Obscurin-like-O chain;

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

In some embodiments, for the aforementioned polypeptide complex, the domain capable of interacting with the Obscurin-O chain or the Obscurin-like-O chain is the Titin-T chain, and the domain capable of interacting with the Titin-T chain is the Obscurin-O chain or the Obscurin-like-O chain.

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

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

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

In some embodiments, for the aforementioned polypeptide complex, the first domain and the second domain form a dimer through natural inter-chain bonds and/or non-natural inter-chain bonds.

In some embodiments, the first domain and the second domain form a dimer through natural inter-chain bonds; 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 Titin-T chain and one or more residues at positions selected from the group consisting of 3-6, 9, 41, 73, 75 and 80-90 on the Obscurin-O chain 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 Titin-T chain 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 Obscurin-like-O chain are linked to each other; the residue position in the Titin-T chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 32; the residue position in the Obscurin-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 33; the residue position in the Obscurin-like-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 34.

In some embodiments, for any one of the aforementioned polypeptide complexes, the first domain and the second domain form a dimer through at least one non-natural inter-chain bonds.

In some embodiments, the non-natural inter-chain bond is formed between a particular mutated residue of the first domain and a particular mutated residue of the second domain. In some embodiments, at least one of the particular mutated residue of the first domain and the particular mutated residue of the second domain is a cysteine residue.

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

In some embodiments, for the aforementioned polypeptide complex, the particular mutated residue of the first domain and the particular mutated residue of the second domain are located at a contact interface 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 on positions selected from the group consisting of: the Titin-T chain has one or more amino acid residue mutations on 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 on 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 on positions selected from the group consisting of 8, 20, 22, 25, 26 and 39, and/or the Obscurin-like-O chain has one or more amino acid residue mutations on positions selected from the group consisting of 6, 26, 74, 77, 84 and 86. The mutation position in the Titin-T chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 32; the mutation position in the Obscurin-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 33; the mutation position in the Obscurin-like-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 34.

In some specific embodiments, the aforementioned first mutated residue and the aforementioned second mutated residue have mutations on positions selected from the group consisting of: the Titin-T chain has one or more amino acid residue mutations on 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 on 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 on positions selected from the group consisting of 8C, 20C, 22C, 25S, 26C and 39T, and/or the Obscurin-like-O chain has one or more amino acid residue mutations on positions selected from the group consisting of 6E, 26S, 74C, 77S, 84C and 86C. In some embodiments, the Titin-T chain has 25S, 39T and 8C mutations, and the Obscurin-O chain has an 88C mutation;

the Titin-T chain has 25S, 39T and 20C mutations, and the Obscurin-O chain has a 3C mutation;

the Titin-T chain has 25S, 39T and 26C mutations, and the Obscurin-O chain has a 9C mutation;

the Titin-T chain has 25S, 39T and 8C mutations, and the Obscurin-O chain has 25S, 76S and 88C mutations;

the Titin-T chain has 25S, 39T and 20C mutations, and the Obscurin-O chain has 25S, 76S and 3C mutations;

the Titin-T chain has 25S, 39T and 26C mutations, and the Obscurin-O chain has 25S, 76S and 9C mutations;

the Titin-T chain has 25S, 39T and 8C mutations, and the Obscurin-like-O chain has 6E and 74C mutations;

the Titin-T chain has 25S, 39T and 20C mutations, and the Obscurin-like-O chain has 6E and 84C mutations;

the Titin-T chain has 25S, 39T and 22C mutations, and the Obscurin-like-O chain has 6E and 86C mutations;

the Titin-T chain has 25S, 39T and 8C mutations, and the Obscurin-like-O chain has 6E, 26S, 77S and 74C mutations;

the Titin-T chain has 25S, 39T and 20C mutations, and the Obscurin-like-O chain has 6E, 26S, 77S and 84C mutations; or

the Titin-T chain has 25S, 39T and 22C mutations, and the Obscurin-like-O chain has 6E, 26S, 77S and 86C mutations;

the mutation position in the Titin-T chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 32; the mutation position in the Obscurin-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 33; the mutation position in the Obscurin-like-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 34.

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

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

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

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

In some embodiments, for the aforementioned polypeptide complex, the Obscurin-O chain has one or more amino acid residue mutations on 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 on 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 25S, 39T and 8C mutations, and the Obscurin-O chain has 25S, 76S, 88C, 11L and 62K mutations; or the Titin-T chain has 25S, 39T and 8C mutations and the Obscurin-O chain has 25S, 76S, 88C, 11L and 62H mutations; the mutation position in the Titin-T chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 32; the mutation position in the Obscurin-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 33.

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

In some embodiments, for the aforementioned polypeptide complex, the Obscurin-O chain is an Obscurin-O chain having one or more amino acid residue mutations on positions selected from the group consisting of 7, 11 and 62 on the basis of SEQ ID NO: 45; in some embodiments, the Obscurin-O chain has one or more amino acid residue mutations on positions selected from the group consisting of L7K and L7R, K11L and T62K and T62H on the basis of SEQ ID NO: 45; in some embodiments, the Titin-T chain has C25S, C39T and A8C mutations on the basis of SEQ ID NO: 32, and the Obscurin-O chain has C25S, C76S, A88C, L7K and T62K mutations on the basis of SEQ ID NO: 45; the Titin-T chain has C25S, C39T and A8C mutations on the basis of SEQ ID NO: 32, and the Obscurin-O chain has C25S, C76S, A88C, L7K and T62H mutations on the basis of SEQ ID NO: 45; the Titin-T chain has C25S, C39T and A8C mutations on the basis of SEQ ID NO: 32, and the Obscurin-O chain has C25S, C76S, A88C, K11L and T62K mutations on the basis of SEQ ID NO: 45; the Titin-T chain has C25S, C39T and A8C mutations on the basis of SEQ ID NO: 32, and the Obscurin-O chain has C25S, C76S, A88C, K11L and T62H mutations on the basis of SEQ ID NO: 45. The mutation position in the Titin-T chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 32; the mutation position in the Obscurin-O chain is a natural sequence numbering position relative to a sequence 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 on positions selected from the group consisting of: the Titin-T chain has one or more amino acid mutations on 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 on 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 mutation position in the Titin-T chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 35; the mutation position in the Obscurin-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 50.

In some embodiments, for the aforementioned 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 on 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 on the basis of SEQ ID NO: 35, and the Obscurin-O chain is a polypeptide having one or more amino acid mutations on 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 on the basis of SEQ ID NO: 50. The mutation position in the Titin-T chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 35; the mutation position in the Obscurin-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 50.

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

In some embodiments, the aforementioned first domain and the aforementioned 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 on 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 on the basis of SEQ ID NO: 35, and/or the Obscurin-O chain has one or more amino acid mutations on 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 on the basis of SEQ ID NO: 35. The mutation position in the Titin-T chain is a natural sequence numbering position of a sequence SEQ ID NO: 35; the mutation position in the Obscurin-O chain is a natural sequence numbering position of a sequence SEQ ID NO: 50.

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

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

In some embodiments, for the aforementioned polypeptide complex, the VH1 is operably linked to the first domain through a first linking domain, and the VL1 is operably linked to the second domain through a second linking domain.

In some embodiments, the aforementioned first and/or second linking domain is selected from the group consisting of: an N-terminal fragment of the Titin-T chain, an N-terminal fragment of the Obscurin-O chain and an N-terminal fragment of the Obscurin-like-O chain, and a (G_xS)_ylinker, wherein X is selected from the group consisting of integers from 1 to 5 (for example, 1, 2, 3, 4 or 5), and Y is selected from the group consisting of integers from 1 to 6 (for example, 1, 2, 3, 4, 5 or 6).

In some embodiments, the aforementioned first and/or second linking domain is selected from the group consisting of: “KAGIR”, “DQPQF”, (G₄S)₁and (G₄S)₂.

In some embodiments, for the aforementioned polypeptide complex, when the first domain is a Titin-T chain, the first linking domain is a KAGIR polypeptide; when the second domain is an Obscurin-O chain, the second linking domain is a DQPQF polypeptide. In other embodiments, when the second domain is a Titin-T chain, the second linking domain is a KAGIR polypeptide, and when the first domain is an Obscurin-O chain, the first linking domain is a DQPQF polypeptide.

In some embodiments of the polypeptide complex, wherein, (A) the Titin-T chain has a sequence set forth in SEQ ID NO: 32 or further has a sequence having one or more amino acid mutations on 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 amino acid residues of KAGIR added at the N-terminus on the basis of SEQ ID NO: 32; preferably, the Titin-T chain has a sequence having one or more amino acid mutations on positions selected from the group consisting of ABC, 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 a KAGIR amino acid residue added at the N-terminus; and/or

(B) the Obscurin-O chain has a sequence set forth in SEQ ID NO: 33 or further has a sequence having one or more amino acid mutations on 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 on the basis of SEQ ID NO: 33; preferably, the Obscurin-O chain has a sequence having one or more amino acid mutations on 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 a DQPQF amino acid residue added at the N-terminus; or the Obscurin-like-O chain has a sequence set forth in SEQ ID NO: 34 or further has a sequence having one or more amino acid mutations on positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 on the basis of SEQ ID NO: 34; preferably, the Obscurin-like-O chain has a sequence having one or more amino acid mutations on positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C;

the mutation position in the Titin-T chain is a natural sequence numbering position of the sequence SEQ ID NO: 32; the mutation position in the Obscurin-O chain is a natural sequence numbering position of the sequence SEQ ID NO: 33; the mutation position in the Obscurin-like-O chain is a natural sequence numbering position of the sequence 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 on positions selected from the group consisting of 8, 20, 22, 25, 26 and 39 on the basis of SEQ ID NO: 32, and preferably has one or more amino acid residue mutations on positions selected from the group consisting of ABC, V20C, T22C, C25S, A26C and C39T, and the mutation position in the Titin-T chain is a natural sequence numbering position of SEQ ID NO: 32; more preferably, the Titin-T chain has one or more amino acid mutations on 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 on the basis of SEQ ID NO: 35, and preferably has one or more amino acid mutations on 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 mutation position in the Titin-T chain is a natural sequence numbering position of SEQ ID NO: 35, and/or

(B) the Obscurin-O chain has one or more amino acid residue mutations on positions selected from the group consisting of 3, 9, 25, 76 and 88 on the basis of SEQ ID NO: 33, and preferably has one or more amino acid residue mutations on positions selected from the group consisting of A3C, R9C, C25S, C76S and A88C, and the mutation position in the Obscurin-O chain is a natural sequence numbering position of SEQ ID NO: 33; preferably, the Obscurin-O chain has one or more amino acid residue mutations on positions selected from the group consisting of 7, 11 and 62 on the basis of SEQ ID NO: 45, and preferably has one or more amino acid residue mutations on positions selected from the group consisting of L7K and L7R, T62K and T62H, and K11L, and the mutation position in the Obscurin-O chain is a natural sequence numbering position of SEQ ID NO: 45; more preferably, the Obscurin-O chain has one or more amino acid mutations on 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 on the basis of SEQ ID NO: 50, and preferably has one or more amino acid mutations on 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 mutation position in the Obscurin-O chain is a natural sequence numbering position of SEQ ID NO: 50; or the Obscurin-like-O chain has one or more amino acid residue mutations on positions selected from the group consisting of 6, 26, 74, 77, 84 and 86 on the basis of SEQ ID NO: 34, and preferably has one or more amino acid residue mutations on positions selected from the group consisting of C6E, C26S, C77S, V74C, G84C and A86C, and the mutation position in the Obscurin-like-O chain is a natural sequence numbering position of SEQ ID NO: 34.

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

In some embodiments, the aforementioned Titin-T chain comprises a polypeptide having a sequence set forth in SEQ ID NO: 68, and the Obscurin-O chain comprises a polypeptide having a sequence set forth in SEQ ID NO: 80.

In other embodiments, the Titin-T chain comprises a polypeptide having a sequence set forth in SEQ ID NO: 73, and the Obscurin-O chain comprises a polypeptide having a sequence set forth in SEQ ID NO: 83.

In other embodiments, the Titin-T chain comprises a polypeptide having a sequence set forth in SEQ ID NO: 76, and the Obscurin-O chain comprises a polypeptide having a sequence set forth in SEQ ID NO: 84.

In other embodiments, the Titin-T chain comprises a polypeptide having a sequence set forth in SEQ ID NO: 75, and the Obscurin-O chain comprises a polypeptide having a sequence set forth in SEQ ID NO: 86.

In some embodiments, the Titin-T chain is a polypeptide having a sequence set forth in SEQ ID NO: 68, and the Obscurin-O chain is a polypeptide having a sequence set forth in SEQ ID NO: 80; the Titin-T chain is a polypeptide having a sequence set forth in SEQ ID NO: 73, and the Obscurin-O chain is a polypeptide having a sequence set forth in SEQ ID NO: 83; the Titin-T chain is a polypeptide having a sequence set forth in SEQ ID NO: 76, and the Obscurin-O chain is a polypeptide having a sequence set forth in SEQ ID NO: 84; the Titin-T chain is a polypeptide having a sequence set forth in SEQ ID NO: 76, and the Obscurin-O chain is a polypeptide having a sequence set forth in SEQ ID NO: 86; the Titin-T chain is a polypeptide having a sequence set forth in SEQ ID NO: 76, and the Obscurin-O chain is a polypeptide having a sequence set forth in SEQ ID NO: 86; or the Titin-T chain is a polypeptide having a sequence set forth in SEQ ID NO: 75, and the Obscurin-O chain is a polypeptide having a sequence set forth in SEQ ID NO: 86.

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

a first polypeptide complex, being any one of the aforementioned polypeptide complexes and specifically binding to a first antigen, and

a second polypeptide complex, comprising a second antigen-binding moiety 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 the VH1 of the first polypeptide complex and the VL2 of the second polypeptide complex, and/or between the VH2 of the second polypeptide complex and the VL1 of the first polypeptide complex is less susceptible to happen. In some embodiments, the VH2 and the VL2 of the second antigen-binding moiety form a second antigen-binding site specifically binding to the second antigen.

In some embodiments, for the aforementioned multispecific polypeptide complex, the second antigen-binding moiety of the second polypeptide complex comprises:

a third polypeptide, comprising a VH2 from an N-terminus to a C-terminus, the VH2 being operably linked to a third domain comprising a CH1, and a fourth polypeptide, comprising a VL2 from an N-terminus to a C-terminus, the VL2 being operably linked to a fourth domain comprising a CL.

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, the first dimerization domain and the second dimerization domain being bound together.

In some embodiments, a C-terminus of the first domain of the first polypeptide complex is operably linked to an N-terminus of the first dimerization domain, and a C-terminus of the third domain of the second polypeptide complex is operably linked to an N-terminus of the second dimerization domain.

In some embodiments, the aforementioned first dimerization domain and the aforementioned second dimerization domain are bound to form a dimer by a means of 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, the aforementioned first dimerization domain and the aforementioned second dimerization domain are bound to form a dimer by an antibody hinge region or a portion thereof, and preferably a 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 to form a dimer.

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, for the aforementioned multispecific polypeptide complex, the antibody CH2 domain and/or the antibody CH3 domain is derived from IgG1, IgG2 or IgG4. In some embodiments, the antibody CH2 domain and/or the antibody CH3 domain is 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 from an N-terminus to a C-terminus, the second polypeptide sequentially comprises VL1-L3-O chain or VL1-L4-OL chain from an N-terminus to a C-terminus, the third polypeptide sequentially comprises VH2-CH1-CH2-CH3 from an N-terminus to a C-terminus, and the fourth polypeptide sequentially comprises VL1-CL from an N-terminus to a C-terminus; or the first polypeptide sequentially comprises VH1-L2-O chain-L3-CH2-CH3 or VH1-L4-OL chain-L5-CH2-CH3 from an N-terminus to a C-terminus, the second polypeptide sequentially comprises VL1-L1-T chain from an N-terminus to a C-terminus, the third polypeptide sequentially comprises VH2-CH1-CH2-CH3 from an N-terminus to a C-terminus, and the fourth polypeptide sequentially comprises VL2-CL from an N-terminus to a C-terminus; the L1 to L5 are spacers (also known as linking domains) which may or may not be present. Preferably, the spacer is selected from the group consisting of: an N-terminal fragment of a Titin-T chain, an N-terminal fragment of an Obscurin-O chain, an N-terminal fragment of an Obscurin-like-O chain, and a linker (e.g., (G_xS)_ylinker), wherein 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”, (G₄S)₁and (G₄S)₂.

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

In some embodiments, the aforementioned first dimerization domain and the aforementioned second dimerization domain are bound to form a dimer by a means of knob-into-hole, a hydrophobic interaction, an electrostatic interaction, a hydrophilic interaction or a flexibility increase.

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

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

the domain CH1 is substituted with a Titin-T chain, and the domain CL is substituted with a domain capable of having an inter-protein interaction with the Titin-T chain;

the domain CL is substituted with the Titin-T chain, and the domain CH1 is substituted with a domain capable of having an inter-protein interaction with the Titin-T chain;

the domain CH1 is substituted with an Obscurin-O chain, and the domain CL is substituted with a domain capable of having an inter-protein interaction with the Obscurin-O chain;

the domain CL is substituted with the Obscurin-O chain, and the domain CH1 is substituted with a domain capable of having an inter-protein interaction with the Obscurin-O chain;

the domain CH1 is substituted with an Obscurin-like-O chain, and the domain CL is substituted with a domain capable of having an inter-protein interaction with the Obscurin-like-O chain; or

the domain CL is substituted with the Obscurin-like-O chain, and the domain CH1 is substituted with a domain capable of having an inter-protein interaction with the Obscurin-like-O chain.

In some embodiments, for the aforementioned domain engineered antibody, the domain capable of interacting with the Obscurin-O chain or the Obscurin-like-O chain is the Titin-T chain, and the domain capable of interacting with the Titin-T chain is the Obscurin-O chain or the Obscurin-like-O chain.

In some embodiments, the antibody comprises a heavy chain variable region VH1 and a light chain variable region VL1, the VH1 and the VL1 forming a primary binding site specifically binding to an antigen thereof; a C-terminus of the VH1 is operably linked to an N-terminus of the Titin-T chain, a C-terminus of the VL1 is operably linked to an N-terminus of the Obscurin-O chain or the Obscurin-like-O chain, or the C-terminus of the VL1 is operably linked to the N-terminus of the Titin-T chain, and the C-terminus of the VH1 is operably linked to the N-terminus of the Obscurin-O chain or the Obscurin-like-O chain.

In some embodiments, the present disclosure provides a Fab fragment of a domain engineered antibody in which a constant region domain CH1 and a constant region domain CL are substituted, wherein,

the domain CH1 is substituted with a Titin-T chain, and the domain CL is substituted with a domain capable of having an inter-protein interaction with the Titin-T chain; or

the domain CL is substituted with the Titin-T chain, and the domain CH1 is substituted with a domain capable of having an inter-protein interaction with the Titin-T chain; or

the domain CH1 is substituted with an Obscurin-O chain, and the domain CL is substituted with a domain capable of having an inter-protein interaction with the Obscurin-O chain; or

the domain CL is substituted with the Obscurin-O chain, and the domain CH1 is substituted with a domain capable of having an inter-protein interaction with the Obscurin-O chain; or

the domain CH1 is substituted with the Obscurin-like-O chain, and the domain CL is substituted with a domain capable of having an inter-protein interaction with the Obscurin-like-O chain; or

the domain CL is substituted with the Obscurin-like-O chain, and the domain CH1 is substituted with a domain capable of having an inter-protein interaction with the Obscurin-like-O chain.

In some embodiments, for the aforementioned Fab fragment, the domain having an inter-protein interaction with the Titin-T chain is the Obscurin-O chain or the Obscurin-like-O chain; the domain having an inter-protein interaction with the Obscurin-O chain or the Obscurin-like-O chain is the Titin-T chain.

In some embodiments, the Fab comprises a heavy chain variable region VH1 and a light chain variable region VL1, the VH1 and the VL1 forming a primary binding site specifically binding to an antigen thereof; a C-terminus of the VH1 is operably linked to an N-terminus of the Titin-T chain, a C-terminus of the VL1 is operably linked to an N-terminus of the Obscurin-O chain or the Obscurin-like-O chain, or the C-terminus of the VL1 is operably linked to the N-terminus of the Titin-T chain, and the C-terminus of the VH1 is operably linked to the N-terminus of the Obscurin-O chain or the Obscurin-like-O chain.

In some embodiments, the present disclosure provides a domain engineered antibody comprising the aforementioned Fab fragment.

In some embodiments, the present disclosure provides 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,

a CH1 domain of the first heavy chain is substituted with a Titin-T chain, and a CL domain of the first light chain is substituted with a domain capable of having an inter-protein interaction with the Titin-T chain; or

the CL domain of the first light chain is substituted with the Titin-T chain, and the CH1 domain of the first heavy chain is substituted with a domain capable of having an inter-protein interaction with the Titin-T chain; or

the domain CH1 of the first heavy chain is substituted with the Obscurin-O chain, and the domain CL of the first light chain is substituted with a domain capable of having an inter-protein interaction with the Obscurin-O chain; or

the domain CL of the first light chain is substituted with the Obscurin-O chain, and the domain CH1 of the first heavy chain is substituted with a domain capable of having an inter-protein interaction with the Obscurin-O chain; or

the domain CH1 of the first heavy chain is substituted with the Obscurin-like-O chain, and the domain CL of the first light chain is substituted with a domain capable of having an inter-protein interaction with the Obscurin-like-O chain; or

the domain CL of the first light chain is substituted with the Obscurin-like-O chain, and the domain CH1 of the first heavy chain is substituted with a domain capable of having an inter-protein interaction with the Obscurin-like-O chain.

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

In specific 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 susceptible to happen.

In some embodiments, for the aforementioned bispecific antibody, the domain having an inter-protein interaction with the Titin-T chain is the Obscurin-O chain or the Obscurin-like-O chain; the domain having an inter-protein interaction with the Obscurin-O chain or the Obscurin-like-O chain is the Titin-T chain.

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), the VH1 and the VL1 forming an antigen-binding site specifically binding to a first antigen, wherein a C-terminus of the VH1 is operably linked to an N-terminus of the Titin-T chain, a C-terminus of the VL1 is operably linked to an N-terminus of the Obscurin-O chain or the Obscurin-like-O chain, or the C-terminus of the VL1 is operably linked to the N-terminus of the Titin-T chain, and the C-terminus of the VH1 is operably linked to the N-terminus of the Obscurin-O chain or the Obscurin-like-O chain. In some embodiments, for the aforementioned bispecific antibody, the second heavy chain comprises a second heavy chain variable region (VH2) from an N-terminus to a C-terminus operably linked to the CH1; the second light chain comprises a second light chain variable region (VL2) from an N-terminus to a C-terminus operably linked to the CL.

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

In some embodiments, for the aforementioned bispecific antibody, the bispecific antibody structural model is selected from the group consisting of F(ab)₂, scFv-Fab, (scFv)₂-Fab, KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 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, F(ab)₄-CrossMAb, IgG and Fab-IgG, and the like.

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

In some embodiments, the aforementioned CH1, CH2 and CH3 domains are derived from IgG1, IgG2 or IgG4. In some embodiments, the aforementioned CH1, CH2 and CH3 domains are derived from IgG1, IgG2, IgG3 or IgG4. In some embodiments, the aforementioned first heavy chain and the aforementioned second heavy chain are bound to form a dimer by a means of 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 an N-terminus to a C-terminus, and the first light chain sequentially comprises VL1-L3-O chain or VL1-L4-OL chain from an N-terminus to a C-terminus; the second heavy chain sequentially comprises VH2-CH1-CH2-CH3 from an N-terminus to a C-terminus, and the second light chain sequentially comprises VL2-CL from an N-terminus to a C-terminus; or the first light chain sequentially comprises VL1-L1-T chain from an N-terminus to a 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 an N-terminus to a C-terminus; the second heavy chain sequentially comprises VH2-CH1-CH2-CH3 from an N-terminus to a C-terminus, and the second light chain sequentially comprises VL2-CL from an N-terminus to a C-terminus;

wherein L1 to L5 are spacers which may or may not be present. Preferably, the spacer is an N-terminal fragment of a Titin-T chain, an N-terminal fragment of an Obscurin-O chain, an N-terminal fragment of an Obscurin-like-O chain or a linker, for example, a (G_xS)_ylinker, wherein 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”, (G₄S)₁and (G₄S)₂.

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

In some embodiments, the aforementioned first heavy chain and the aforementioned second heavy chain are bound to form a dimer by a means of knob-into-hole, a hydrophobic interaction, an electrostatic interaction, a hydrophilic interaction or a flexibility increase.

In some embodiments, the aforementioned first heavy chain and the aforementioned second heavy chain are bound by knob-into-hole to form a dimer. In some embodiments, the CH2-CH3 domain of the aforementioned first heavy chain has amino acid residues at positions 104 to 330 of a sequence set forth in SEQ ID NO: 2, and the CH2-CH3 domain of the second heavy chain has amino acid residues at positions 104 to 330 of a sequence set forth in SEQ ID NO: 3; or the CH2-CH3 domain of the first heavy chain has amino acid residues at positions 104 to 330 of a sequence set forth in SEQ ID NO: 3, and the CH2-CH3 domain of the second heavy chain has amino acid residues at positions 104 to 330 of a sequence set forth 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 an N-terminus to a C-terminus, the first light chain sequentially comprises VL1-L3-O chain or VL1-L4-OL chain from an N-terminus to a C-terminus, the second heavy chain sequentially comprises VH2-CH1-hole-Fc from an N-terminus to a C-terminus, and the second light chain sequentially comprises VL1-CL from an N-terminus to a C-terminus; or the first heavy chain sequentially comprises VH1-L1-T chain-L2-hole-Fc from an N-terminus to a C-terminus, the first light chain sequentially comprises VL1-L3-O chain or VL1-L4-OL chain from an N-terminus to a C-terminus, the second heavy chain sequentially comprises VH2-CH1-knob-Fc from an N-terminus to a C-terminus, and the second light chain sequentially comprises VL2-CL from an N-terminus to a C-terminus; or

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

the L1 to L5 are spacers which may or may not be present. Preferably, the spacer is an N-terminal fragment of a Titin-T chain, an N-terminal fragment of an Obscurin-O chain, an N-terminal fragment of an Obscurin-like-O chain or a linker, for example, a (G_xS)_ylinker, wherein 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”, (G₄S)₁and (G₄S)₂.

In some embodiments, for any one of the aforementioned domain engineered antibody, the Fab fragment of a domain engineered antibody, or the bispecific antibody, the domain having an inter-protein interaction with a Titin-T chain is an Obscurin-O chain or an Obscurin-like-O chain; the domain having an inter-protein interaction with an Obscurin-O chain or an Obscurin-like-O chain is a Titin-T chain. For any one of the aforementioned domain engineered antibody, or the Fab fragment of a domain engineered antibody, or the bispecific antibody, the operable linkage is a linkage of two polypeptide sequences through a spacer or a direct linkage of two polypeptides.

In some embodiments, for the aforementioned domain engineered antibody, or the bispecific antibody, or the Fab fragment of a domain engineered antibody, the Titin-T chain and the Obscurin-O chain, or the Titin-T chain and the Obscurin-like-O chain, are bound through natural inter-chain bonds and/or unnatural inter-chain bonds to form a dimer.

In some embodiments, the first domain and the second domain form a dimer through natural inter-chain bonds, and one or more residues at positions selected from the group consisting of 7-15, 19-24, 26, 55, 59 and 60 on the Titin-T chain and one or more residues at positions selected from the group consisting of 3-6, 9, 41, 73, 75, and 80-90 on the Obscurin-O chain 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 Titin-T chain 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 Obscurin-like-O chain are linked to each other. the residue position in the Titin-T chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 32; the residue position in the Obscurin-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 33; the residue position in the Obscurin-like-O chain is a natural sequence numbering position relative to a sequence SEQ ID NO: 34.

In some embodiments, for the aforementioned domain engineered antibody, the bispecific antibody, or the Fab fragment of a domain engineered antibody, the Titin-T chain and the Obscurin-O chain, or the Titin-T chain and the Obscurin-like-O chain, form a dimer through at least one non-natural inter-chain bonds. In some embodiments, the non-natural inter-chain bond is a disulfide bond; in some embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-natural inter-chain bonds.

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

the Titin-T chain has one or more amino acid residue mutations on 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 on 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 on positions selected from the group consisting of 8, 20, 22, 25, 26 and 39, and/or the Obscurin-like-O chain has one or more amino acid residue mutations on 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 the Titin-T chain/Obscurin-like-O chain, is as follows:

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

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

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

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

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

the Titin-T chain has 25S, 39T and 8C mutations, and the Obscurin-O chain has a 88C mutation;