JPWO2021139758A5 - - Google Patents

Description

According to the experimental results, VL-CL easily bound to the heavy chain in which CH1 was replaced by TCRβ, thereby expressing a mismatched molecule with the same two light chains. However, the VL-CL light chain does not combine with the heavy chain in which CH1 is replaced by the Titin-T chain to form a mismatched molecule of the same two light chains. As a result, the bispecific antibody in which the heavy chain CH1 is replaced with the Titin-T chain of the present disclosure has a better ability to reduce light and heavy chain mismatch than the bispecific antibody in which the heavy chain CH1 is replaced with TCRβ. It has been indirectly proven that
The present disclosure relates to, for example, the following.
[1]
a first antigen-binding portion, the first antigen-binding portion comprising:
A) comprising a first heavy chain variable domain (VH1) from the N-terminus to the C-terminus, said VH1 being operably linked to a first domain, said first domain comprising a first polypeptide comprising a Titin-T chain; ,
a first light chain variable domain (VL1) from the N-terminus to the C-terminus, said VL1 operably linked to a second domain, said second domain comprising a domain capable of interacting with the Titin-T chain; comprising a polypeptide;
Or
B) a first polypeptide comprising a VH1 from the N-terminus to the C-terminus, said VH1 operably linked to a first domain, and said first domain comprising a domain capable of interacting with a Titin-T chain;
a second polypeptide comprising a VL1 from the N-terminus to the C-terminus, the VL1 operably linked to a second domain, and the second domain comprising a Titin-T chain;
Or
C) a first polypeptide comprising a VH1 from the N-terminus to the C-terminus, the VH1 being operably linked to a first domain, and the first domain comprising an Obscurin-O chain or an Obscurin-Like-O chain;
a second polypeptide comprising a VL1 from the N-terminus to the C-terminus, said VL1 operably linked to a second domain, said second domain comprising a domain capable of interacting with an Obscurin-O chain or an Obscurin-Like-O chain; peptide;
Or
D) a VH1 comprising a VH1 from the N-terminus to the C-terminus, said VH1 operably linked to a first domain, said first domain comprising a domain capable of interacting with an Obscurin-O chain or an Obscurin-Like-O chain; 1 polypeptide and
a second polypeptide comprising a VL1 from the N-terminus to the C-terminus, the VL1 being operably linked to a second domain, and the second domain comprising an Obscurin-O chain or an Obscurin-Like-O chain; ,
A polypeptide conjugate, wherein the first antigen-binding portion specifically binds to a first antigen, and the first domain is capable of forming a dimer with a second domain.
[2]
The domain that can interact with the Obscurin-O chain or Obscurin-Like-O chain is a Titin-T chain,
The polypeptide complex according to [1] above, wherein the domain that can interact with the Titin-T chain is an Obscurin-O chain or an Obscurin-Like-O chain.
[3]
VH1 and VL1 of the first antigen-binding portion form a first antigen-binding site that specifically binds to a first antigen, and the C-terminus of the VH1 is operably linked to the N-terminus of the first domain, and the VL1 The polypeptide complex according to [1] or [2] above, wherein the C-terminus of the polypeptide complex is operably linked to the N-terminus of the second domain.
[4]
The first domain forms a dimer with the second domain through a natural interchain bond and/or a non-natural interchain bond,
Preferably, the first domain forms a dimer with the second domain through natural interchain binding,
More preferably, one or more residues selected from positions 7 to 15, 19 to 24, 26, 55, 59, and 60 in the Titin-T chain are 3 to 6, 9, 41, bonding to one or more residues selected from positions 73, 75 and 80 to 90, or
One or more residues selected from positions 1, 7 to 10, 13 to 16, 19 to 26, 59 to 60 and 96 in the Titin-T chain are 4 to 5, 10 in the Obscurin-Like-O chain, bonding to one or more residues selected from positions 12 to 13, 74, 76, 78 and 82 to 91,
The residue site in the Titin-T chain is a natural sequence number site for the sequence SEQ ID NO: 32, the residue site in the Obscurin-O chain is a natural sequence number site for the sequence SEQ ID NO: 33, and the Obscurin-Like The polypeptide complex according to any one of [1] to [3] above, wherein the residue site in the -O chain is a natural sequence number site with respect to the sequence of SEQ ID NO: 34.
[5]
the first domain forms a dimer with the second domain through at least one non-natural interchain bond;
Preferably, the non-natural interchain bond is formed between a specific variant residue of the first domain and a specific variant residue of the second domain,
More preferably, at least one pair of the mutant residues is a cysteine residue,
Most preferably, the non-natural interchain bond is a disulfide bond;
More preferably, any one of [1] to [4] above, wherein the dimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-natural interchain bonds. The polypeptide conjugate described in Section.
[6]
The specific mutated residue of the first domain and the specific mutated residue of the second domain are selected from the following mutations, namely:
(A) the Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26, and 39, and/or the Obscurin-O chain has mutations at positions 3, 9, has one or more amino acid residue mutations selected from positions 25, 76, and 88, or
(B) the Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26, and 39, and/or the Obscurin-Like-O chain has 6, Having one or more amino acid residue mutations selected from positions 26, 74, 77, 84 and 86,
Preferably, the Titin-T chain has one or more amino acid residue mutations selected from A8C, V20C, T22C, C25S, A26C and C39T, and/or the Obscurin-O chain has A3C, R9C, C25S. , has one or more amino acid residue mutations selected from C76S and A88C, or
The Titin-T chain has one or more amino acid residue mutations selected from A8C, V20C, T22C, C25S, A26C and C39T, and/or the Obscurin-Like-O chain has C6E, C26S, C77S, Having one or more amino acid residue mutations selected from V74C, G84C and A86C,
More preferably, the mutated residues of the first domain and the mutated residues of the second domain are selected from the following mutations, namely:
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has A88C mutation,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-O chain has A3C mutation,
Titin-T chain has C25S, C39T and A26C mutations, and Obscurin-O chain has R9C mutation,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S and A88C mutations,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-O chain has C25S, C76S and A3C mutations,
Titin-T chain has C25S, C39T and A26C mutations, and Obscurin-O chain has C25S, C76S and R9C mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-Like-O chain has C6E and V74C mutations,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-Like-O chain has C6E and G84C mutations,
Titin-T chain has C25S, C39T and T22C mutations, and 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,
Titin-T chain has C25S, C39T and V20C mutations and Obscurin-Like-O chain has C6E, C26S, C77S and G84C mutations, or
The Titin-T chain has C25S, C39T and T22C mutations, and the Obscurin-Like-O chain has C6E, C26S, C77S and A86C mutations,
The Titin-T chain mutation site is the natural sequence number site for the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is the natural sequence number site for the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is the natural sequence number site for the sequence SEQ ID NO: 32. The polypeptide complex according to the above [5], which is a naturally numbered site for the sequence numbered 34.
[7]
The Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11, and 62,
Preferably, the Obscurin-O chain has one or more amino acid residue mutations selected from L7R or L7K, T62K or T62H, and K11L,
Most preferably, the Titin-T chain has C25S, C39T and A8C mutations, and the Obscurin-O chain has C25S, C76S, A88C, L7K and T62K mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, L7K and T62H mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, K11L and T62K mutations, or
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, K11L and T62H,
The polypeptide complex according to [6] above, wherein the Titin-T chain mutation site is a natural sequence number site with respect to the sequence of SEQ ID NO: 32, and the Obscurin-O chain mutation site is a natural sequence number site with respect to the sequence of SEQ ID NO: 33. thing.
[8]
The first domain and the second domain have one or more amino acid residue mutations selected from the following, namely:
One Titin-T chain selected from positions 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84 or has multiple amino acid mutations and/or Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53 , having one or more amino acid mutations selected from positions 58, 62, 67, 69, 89, 92, 94 and 97,
Preferably, the first domain and the second domain have one or more residue mutations selected from the following, namely:
1 where the Titin-T chain is selected from P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L has one or more amino acid mutations, and/or Obscurin-O chain is G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, Having one or more amino acid mutations selected from A45T, A53L, L58V, K62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G,
More preferably, the 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, Has 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 amino acid mutations of G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D and F84L, and/or the Obscurin-O chain has Q42L, A45T, A67T, G69S, Q92E and Has the D94G amino acid mutation,
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. ,
[1] to [7] above, wherein the Titin-T chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 35, and the Obscurin-O chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 50. The polypeptide complex according to any one of the above.
[9]
the VH1 is operably linked to a first domain by a first linking domain, and the VL1 is operably linked to a second domain by a second linking domain;
Preferably, the first linking domain and/or the second linking domain are selected from the following: N-terminal fragment of Titin-T chain, N-terminal fragment of Obscurin-O chain, N-terminal fragment of Obscurin-Like-O chain. terminal fragment, and (G _x S) _y connexons (where X is selected from an integer from 1 to 5 and Y is selected from an integer from 1 to 6),
More preferably, the first linking domain and/or the second linking domain are the following polypeptide fragments, namely "KAGIR", "DQPQF", (G _Four S) ₁ and (G _Four S) ₂ selected from
Most preferably, said first domain is a Titin-T chain, said first linking domain is a KAGIR polypeptide, said second domain is an Obscurin-O chain, and said second linking domain is a DQPQF polypeptide. Yes or
the second domain is a Titin-T chain, the second linking domain is a KAGIR polypeptide, the first domain is an Obscurin-O chain, and the first linking domain is a DQPQF polypeptide; 1] to [8].
[10]
(A) The sequence of the Titin-T chain is as shown in SEQ ID NO: 32, or 3, 8, 11, 13, 20, 22, 25, 26, 39, 40 based on the sequence of SEQ ID NO: 32. , 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or This is a sequence that adds a KAGIR amino acid residue to the N-terminus,
Preferably A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R , N82M, E83D, and F84L, and/or has an amino acid mutation at one or more sites selected from N82M, E83D, and F84L, and/or has a KAGIR amino acid residue added to the N-terminus, and/or
(B) The sequence of the Obscurin-O chain is as shown in SEQ ID NO: 33, or 2, 3, 7, 9, 11, 12, 13, 14, 17, 20 based on the sequence of SEQ ID NO: 33. , 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 76, 88, 89, 92, 94 and 97. A sequence that further has an amino acid mutation at the site and/or adds a DQPQF amino acid residue to the N-terminus, preferably A3C, R9C, C25S, C76S, A88C, L7K, T62K or T62H, G2E, L11K, A12S , F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G has an amino acid mutation at one or more sites, and/or has a DQPQF amino acid residue added to the N-terminus, or
The sequence of the Obscurin-Like-O chain is as shown in SEQ ID NO: 34, or one or more sites selected from 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34. A sequence further having an amino acid mutation, preferably a sequence having an amino acid mutation at one or more sites selected from C6E, C26S, C77S, V74C, G84C and A86C,
The Titin-T chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33. It is the natural sequence number site of the sequence SEQ ID NO: 34,
Preferably,
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76 and 88 based on the sequence of SEQ ID NO: 33, preferably A3C, R9C, has one or more amino acid residue mutations selected from C25S, C76S and A88C, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33,
More preferably, the Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11, and 62 based on the sequence of SEQ ID NO: 45, preferably L7K, L7R, or T62K. or has one or more amino acid residue mutations selected from T62H and K11L, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 45,
Most preferably, the Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45 based on the sequence of SEQ ID NO: 50. , 53, 58, 62, 67, 69, 89, 92, 94 and 97, preferably G2E, L11K, A12S, F13Y, V14T, V17E, D20L, One or more amino acid mutations selected from T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G. and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 50,
Or
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34, preferably, having one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C, and A86C, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 34, The polypeptide complex according to any one of [1] to [9] above.
[11]
The Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or at least 80% of the sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76. and/or the Obscurin-O chain has the sequence shown in any one of SEQ ID NO: 33, 42-58, 77-86 or the sequence of SEQ ID NO: 33, 42-58, 77-86. contains a sequence with at least 80% identity to any one sequence, or
The Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or at least 80% of the sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76. A sequence containing a sequence having identity and/or a sequence in which the Obscurin-Like-O chain is shown in any one of SEQ ID NOs: 34, 59 to 64, or a sequence of any one of SEQ ID NOs: 34, 59 to 64 comprising a sequence having at least 80% identity with
More preferably, 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 Titin-T chain comprises the sequence shown in SEQ ID NO: 73, and the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 83,
the Titin-T chain comprises the sequence shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 84,
the Titin-T chain comprises the sequence shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 86, or
The polypeptide according to any one of [1] to [10] above, wherein the Titin-T chain contains the sequence shown in SEQ ID NO: 75, and the Obscurin-O chain contains the sequence shown in SEQ ID NO: 86. Peptide complex.
[12]
The polypeptide complex according to any one of [1] to [11] above, wherein the first antigen is selected from an exogenous antigen, an endogenous antigen, an autoantigen, a neoantigen, a viral antigen, and a tumor antigen.
[13]
The polypeptide complex according to any one of [1] to [12] above, which specifically binds to the first antigen;
a second polypeptide complex that includes a second antigen-binding portion and specifically binds to the second antigen;
the first polypeptide complex and the second polypeptide complex bind to two different antigens, or bind to two different epitopes on the same antigen,
Preferably, the second polypeptide conjugate comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2), and the VH1 of the first polypeptide conjugate and the second polypeptide conjugate and/or between VH2 of the second polypeptide complex and VL1 of the first polypeptide complex, and more preferably a bispecific polypeptide complex, Most preferably, a multispecific polypeptide complex, wherein VH2 and VL2 of said second antigen-binding portion form a second antigen-binding site that specifically binds to a second antigen.
[14]
The second antigen-binding portion of the second polypeptide conjugate is
a third polypeptide comprising VH2 from the N-terminus to the C-terminus, operably linked to a third domain, said third domain comprising CH1;
a fourth polypeptide comprising VL2 from the N-terminus to the C-terminus, operably linked to a fourth domain, the fourth domain comprising a CL;
Preferably, the multispecific polypeptide according to [13] above, wherein the C-terminus of VH2 is operably linked to the N-terminus of CH1, and the C-terminus of VL2 is operably linked to the N-terminus of CL. Composite.
[15]
the first polypeptide complex further comprises a first dimerization domain, the second polypeptide complex further comprises a second dimerization domain, and the first dimerization domain binds to a second dimerization domain;
Preferably, the C-terminus of the first domain of said first polypeptide conjugate is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the third domain of said second polypeptide conjugate is operably linked to the N-terminus of the first domain of said second polypeptide conjugate. operably linked to the N-terminus of the dimerization domain;
More preferably, the first dimerization domain is formed in a manner selected from the following: an antibody hinge region or a portion thereof, a connexon, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, or by a method selected from a combination thereof, binding to the second dimerization domain,
Most preferably, the first dimerization domain binds the hinge region of an IgG1, IgG2, IgG3 or IgG4 or a portion thereof, preferably by a method selected from the following: binding of an antibody hinge region or a portion thereof. The multispecific polypeptide complex according to [13] or [14] above, which binds to the second dimerization domain by a method selected from among them.
[16]
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,
Preferably, the multispecific polypeptide complex according to [15] above, wherein the antibody CH2 domain and/or the antibody CH3 domain are derived from IgG1, IgG2, IgG3, or IgG4.
[17]
The first dimerization domain is different from the second dimerization domain and binds in a manner that prevents homodimerization and/or promotes heterodimerization,
Preferably, the first dimerization domain is formed by a method selected from the following, namely, by a method selected from knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or a method that improves flexibility. , binds to the second dimerization domain,
More preferably, the first dimerization domain has amino acid residues 104 to 330 in the sequence shown in SEQ ID NO: 2, and the second dimerization domain has amino acid residues 104 to 330 in the sequence shown in SEQ ID NO: 3. th amino acid residue, or
The first dimerization domain has the 104th to 330th amino acid residues in the sequence shown in SEQ ID NO: 3, and the second dimerization domain has the 104th to 330th amino acid residues in the sequence shown in SEQ ID NO: 2. The multispecific polypeptide complex according to [15] or [16] above, which has a group.
[18]
at least one heavy chain constant region domain CH1 and at least one light chain constant region domain CL of said antibody are replaced;
A) said domain CH1 is replaced with a Titin-T chain, and said domain CL is replaced with a domain capable of having protein-protein interactions with the Titin-T chain;
B) said domain CL is replaced with a Titin-T chain, and said domain CH1 is replaced with a domain capable of having protein-protein interactions with the Titin-T chain;
C) the domain CH1 is replaced with an Obscurin-O chain, and the domain CL is replaced with a domain capable of having protein-protein interactions with the Obscurin-O chain;
D) the domain CL is replaced with an Obscurin-O chain, and the domain CH1 is replaced with a domain capable of having protein-protein interactions with the Obscurin-O chain;
E) said domain CH1 is replaced with an Obscurin-Like-O chain, and said domain CL is replaced with a domain capable of having protein-protein interactions with the Obscurin-Like-O chain, or
F) the domain CL is replaced with an Obscurin-Like-O chain, and the domain CH1 is replaced with a domain capable of having protein-protein interactions with the Obscurin-Like-O chain;
Preferably, the domain capable of having protein-protein interaction with the Obscurin-O chain or Obscurin-Like-O chain is a Titin-T chain,
The domain capable of having protein-protein interaction with the Titin-T chain is Obscurin-O chain or Obscurin-Like-O chain,
More preferably, said antibody comprises a heavy chain variable region VH1 and a light chain variable region VL1, the C-terminus of said VH1 is operably linked to the N-terminus of a Titin-T chain, and the C-terminus of said VL1 comprises an Obscurin-T chain. operably linked to the N-terminus of the O chain or Obscurin-Like-O chain, or
A domain modification, wherein the C-terminus of said VL1 is operably linked to the N-terminus of a Titin-T chain, and the C-terminus of said VH1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain. antibody.
[19]
a Fab fragment of a domain-modified antibody, wherein the constant region domain CH1 and constant region domain CL of the Fab fragment are replaced,
the domain CH1 is replaced with a Titin-T chain, the domain CL is replaced with a domain capable of having protein-protein interaction with the Titin-T chain,
the domain CL is replaced with a Titin-T chain, the domain CH1 is replaced with a domain capable of having a protein-protein interaction with the Titin-T chain,
the domain CH1 is replaced with an Obscurin-O chain, the domain CL is replaced with a domain capable of having a protein-protein interaction with the Obscurin-O chain,
the domain CL is replaced with an Obscurin-O chain, the domain CH1 is replaced with a domain capable of having a protein-protein interaction with the Obscurin-O chain,
the domain CH1 is replaced with an Obscurin-Like-O chain, the domain CL is replaced with a domain capable of having protein-protein interaction with the Obscurin-Like-O chain, or
the domain CL is replaced with an Obscurin-Like-O chain, the domain CH1 is replaced with a domain capable of having a protein-protein interaction with the Obscurin-Like-O chain,
Preferably, the domain having protein-protein interaction with the Titin-T chain is an Obscurin-O chain or an Obscurin-Like-O chain,
The domain having protein-protein interaction with the Obscurin-O chain or Obscurin-Like-O chain is a Titin-T chain,
More preferably, the Fab comprises a heavy chain variable region VH1 and a light chain variable region VL1, the VH1 and VL1 form an antigen-binding site, and the C-terminus of the VH1 is operable to the N-terminus of the Titin-T chain. and the C-terminus of said VL1 is operably linked to the N-terminus of the Obscurin-O chain or Obscurin-Like-O chain, or
A domain modification, wherein the C-terminus of said VL1 is operably linked to the N-terminus of a Titin-T chain, and the C-terminus of said VH1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain. Fab fragment of antibody.
[20]
A domain-remodeled antibody comprising the Fab fragment described in [19] above.
[21]
comprising 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;
the CH1 domain of the first heavy chain is replaced with a Titin-T chain, the CL domain of the first light chain is replaced with a domain capable of having a protein-protein interaction with the Titin-T chain,
the CL domain of the first light chain is replaced with a Titin-T chain, the CH1 domain of the first heavy chain is replaced with a domain capable of having a protein-protein interaction with the Titin-T chain,
The CH1 domain of the first heavy chain is replaced with an Obscurin-O chain, and the CL domain of the first light chain is replaced with a domain capable of having a protein-protein interaction with the Obscurin-O chain;
the CL domain of the first light chain is replaced with an Obscurin-O chain, the CH1 domain of the first heavy chain is replaced with a domain capable of having a protein-protein interaction with the Obscurin-O chain,
the CH1 domain of the first heavy chain is replaced with an Obscurin-Like-O chain, the CL domain of the first light chain is replaced with a domain capable of having protein-protein interaction with the Obscurin-Like-O chain, or
the CL domain of the first light chain is replaced with an Obscurin-Like-O chain, and the CH1 domain of the first heavy chain is replaced with a domain capable of having a protein-protein interaction with the Obscurin-Like-O chain;
the first antigen is different from the second antigen, or the first antigen and the second antigen are two different epitopes of the same antigen,
Preferably, mismatches are less likely to occur between the first heavy chain and the second light chain, and between the first light chain and the second heavy chain, and more preferably, the interaction between the Titin-T chain and the protein is prevented. the domain is Obscurin-O chain or Obscurin-Like-O chain,
A bispecific antibody, wherein the domain having protein-protein interaction with the Obscurin-O chain or Obscurin-Like-O chain is a Titin-T chain.
[22]
The first heavy chain includes a first heavy chain variable region (VH1), the first light chain includes a first light chain variable region (VL1), and the VH1 and VL1 specifically bind to a first antigen. form an antigen binding site, and
the C-terminus of said VH1 is operably linked to the N-terminus of a Titin-T chain, the C-terminus of said VL1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain, or
[ 21].
[23]
the second heavy chain comprising a second heavy chain variable region (VH2) from the N-terminus to the C-terminus, operably linked to CH1, and the second light chain comprising a second heavy chain variable region (VH2) from the N-terminus to the C-terminus; contains a variable region (VL2) and is operably linked to CL;
Preferably, the VH2 and VL2 form an antigen binding site that specifically binds to a second antigen, the C-terminus of the VH2 is operably linked to the N-terminus of CH1, and the C-terminus of VL2 is operably linked to the N-terminus of CL. The bispecific antibody according to [21] or [22] above, which is operably linked to the terminal.
[24]
The C-terminus of the first heavy chain and the second heavy chain contains a CH2 and/or CH3 domain,
Preferably, said CH1, CH2 and CH3 domains are derived from IgG1, IgG2, IgG3 or IgG4,
More preferably, the first heavy chain is formed in a manner selected from the following: an antibody hinge region or a portion thereof, a connexon, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, or The bispecific antibody according to any one of [21] to [23] above, which binds to the second heavy chain by a method selected from a combination of these.
[25]
The first heavy chain is, in order from the N-terminus to the C-terminus, VH1-L1-Titin-T chain-L2-CH2-CH3,
The first light chain is a VL1-L3-Obscurin-O chain or a VL1-L4-Obscurin-Like-O chain in order from the N-terminus to the C-terminus,
The second heavy chain is VH2-CH1-CH2-CH3 in order from the N-terminus to the C-terminus,
The second light chain is VL2-CL in order from the N-terminus to the C-terminus,
Or,
The first light chain is a VL1-L1-Titin-T chain in order from the N-terminus to the C-terminus,
The first heavy chain is, in order from the N-terminus to the C-terminus, VH1-L2-Obscurin-O chain-L3-CH2-CH3, or VH1-L4-Obscurin-Like-O chain-L5-CH2-CH3. can be,
The second heavy chain is VH2-CH1-CH2-CH3 in order from the N-terminus to the C-terminus,
The second light chain is VL2-CL in order from the N-terminus to the C-terminus,
L1 to L5 are spacer regions, and the spacer region may or may not exist,
Preferably, the spacer region is selected from the following: N-terminal fragment of Titin-T chain, N-terminal fragment of Obscurin-O chain, N-terminal fragment of Obscurin-Like-O chain, or (G _x S) _y connexons (where X is selected from an integer from 1 to 5 and Y is selected from an integer from 1 to 6),
Most preferably, said spacer region is selected from: "KAGIR", "DQPQF", (G _Four S) ₁ and (G _Four S) ₂ The bispecific antibody according to [24] above, selected from:
[26]
CH2 and CH3 of the first heavy chain and CH2 and CH3 of the second heavy chain are different and bind in a manner that prevents homodimerization and/or promotes heterodimerization,
Preferably, the first heavy chain is formed by a method selected from the following, namely, by a method selected from knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or a method that improves flexibility. binds to the second heavy chain,
More preferably, the first heavy chain binds to the second heavy chain in a knob-into-hole manner,
Most preferably, the first heavy chain CH2-CH3 domain has amino acid residues 104 to 330 in the sequence shown in SEQ ID NO: 2, and the second heavy chain CH2-CH3 domain has the sequence shown in SEQ ID NO: 3. It has amino acid residues 104 to 330 in the sequence, or
The first heavy chain CH2-CH3 domain has amino acid residues 104 to 330 in the sequence shown in SEQ ID NO: 3, and the second heavy chain CH2-CH3 domain has amino acid residues 104 to 330 in the sequence shown in SEQ ID NO: 2. The bispecific antibody according to [25] above, which has the 330th amino acid residue.
[27]
Said operably linked means that two polypeptide sequences are linked by a linking domain, or that two polypeptides are directly linked, [18] or [20] above. or the Fab fragment of the domain-modified antibody described in [19] above, or the bispecific antibody described in any one of [21] to [26] above.
[28]
The Titin-T chain is bound to the Obscurin-O chain, or the Titin-T chain is bound to the Obscurin-Like-O chain by a natural interchain bond, and/or forms a dimer by a non-natural interchain bond,
Preferably, the Titin-T chain forms a dimer with the Obscurin-O chain or Obscurin-Like-O chain through a natural interchain bond, and 7-15, 19-24, 26, 55 in the Titin-T chain , one or more residues selected from positions 59 and 60 are bonded to one or more residues selected from positions 3 to 6, 9, 41, 73, 75 and 80 to 90 in the Obscurin-O chain. or
One or more residues selected from positions 1, 7 to 10, 13 to 16, 19 to 26, 59 to 60 and 96 in the Titin-T chain are 4 to 5, 10 in the Obscurin-Like-O chain, bonding to one or more residues selected from positions 12 to 13, 74, 76, 78 and 82 to 91,
The Titin-T chain residue site is the natural sequence number site for the sequence SEQ ID NO: 32, the Obscurin-O chain residue site is the natural sequence number site for the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain residue site is the natural sequence number site for the sequence SEQ ID NO: 32. The domain-modified antibody, bispecific antibody, or Fab fragment of the domain-modified antibody according to [27] above, wherein the base site is a natural sequence number site relative to the sequence of SEQ ID NO: 34.
[29]
The Titin-T chain forms a dimer with the Obscurin-O chain, or the Titin-T chain forms a dimer with the Obscurin-Like-O chain due to at least one non-natural interchain bond,
Preferably, the non-natural interchain bond is a disulfide bond,
More preferably, the domain according to [27] or [28] above, wherein the dimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-natural interchain bonds. Fab fragments of modified antibodies, bispecific antibodies or domain modified antibodies.
[30]
The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39, and/or the Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76. and has one or more amino acid residue mutations selected from position 88, or
The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39, and/or the Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74. , having one or more amino acid residue mutations selected from positions 77, 84 and 86,
Preferably, the Titin-T chain has one or more amino acid residue mutations selected from A8C, V20C, T22C, C25S, A26C and C39T, and/or
The Obscurin-O chain has one or more amino acid residue mutations selected from A3C, R9C, C25S, C76S and A88C, or
the Titin-T chain has one or more amino acid residue mutations selected from A8C, V20C, T22C, C25S, A26C and C39T, and/or
The Obscurin-Like-O chain has one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C and A86C,
More preferably, the Titin-T chain has C25S, C39T and A8C mutations, and the Obscurin-O chain has an A88C mutation,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-O chain has A3C mutation,
Titin-T chain has C25S, C39T and A26C mutations, and Obscurin-O chain has R9C mutation,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S and A88C mutations,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-O chain has C25S, C76S and A3C mutations,
Titin-T chain has C25S, C39T and A26C mutations, and Obscurin-O chain has C25S, C76S and R9C mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-Like-O chain has C6E and V74C mutations,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-Like-O chain has C6E and G84C mutations,
Titin-T chain has C25S, C39T and T22C mutations, and 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,
Titin-T chain has C25S, C39T and V20C mutations and Obscurin-Like-O chain has C6E, C26S, C77S and G84C mutations, or
The Titin-T chain has C25S, C39T and T22C mutations, and the Obscurin-Like-O chain has C6E, C26S, C77S and A86C mutations,
The Titin-T chain mutation site is a naturally ordered site for the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is a naturally ordered site for the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is a naturally ordered site for the sequence SEQ ID NO: 33. A Fab fragment of the domain-reengineered antibody, bispecific antibody, or domain-reengineered antibody according to any one of [27] to [29] above, which is a natural sequence relative to the sequence of No. 34.
[31]
The Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11 and 62,
Preferably, the Obscurin-O chain has one or more amino acid residue mutations selected from L7K or L7R, K11L, and T62K or T62H,
Most preferably, the Titin-T chain has C25S, C39T and A8C mutations, and the Obscurin-O chain has C25S, C76S, A88C, L7K and T62K mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, L7K and T62H mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, K11L and T62K mutations, or
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, K11L and T62H,
[29] or [30] above, wherein the Titin-T chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 32, and the Obscurin-O chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 33. A Fab fragment of the domain-modified antibody, bispecific antibody or domain-modified antibody described above.
[32]
1 in which the Titin-T chain is selected from positions 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84; has one or more amino acid mutations, and/or
Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 89, 92 , having one or more amino acid mutations selected from positions 94 and 97,
Preferably, the Titin-T chain is P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L. has one or more amino acid mutations selected from, and/or
Obscurin-O chain is G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q or A67T, G69S , has one or more amino acid mutations selected from V89L, Q92E, D94G and A97G,
More preferably, the 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, Has 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 amino acid mutations of G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D and F84L, and/or the Obscurin-O chain has Q42L, A45T, A67T, G69S, Q92E and Has the D94G amino acid mutation,
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. ,
[27] to [31] above, wherein the Titin-T chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 35, and the Obscurin-O chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 50. A Fab fragment of the domain-modified antibody, bispecific antibody, or domain-modified antibody according to any one of the above.
[33]
The N-terminus of the Titin-T chain, Obscurin-O chain or Obscurin-Like-O chain is operably linked to VH1 or VL1 by a linking domain,
Preferably, the linking domain is selected from the following: N-terminal fragment of Titin-T chain, N-terminal fragment of Obscurin-O chain, N-terminal fragment of Obscurin-Like-O chain, and (G _x S) _y connexons (where X is selected from an integer from 1 to 5 and Y is selected from an integer from 1 to 6),
More preferably, the linking domain is selected from the following: "KAGIR", "DQPQF", (G _Four S) ₁ and (G _Four S) ₂ selected from
Most preferably, the Titin-T chain is linked to VH1 or VL1 by a KAGIR polypeptide, and/or the Obscurin-O chain is linked to VL1 or VH1 by a DQPQF polypeptide, according to [27] to [32] above. A Fab fragment of the domain-modified antibody, bispecific antibody, or domain-modified antibody according to any one of the above.
[34]
A) The sequence of the Titin-T chain is as shown in SEQ ID NO: 32, or 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, based on the sequence of SEQ ID NO: 32, further has an amino acid mutation at one or more sites selected from 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or N A sequence that adds five amino acid residues KAGIR to the end, preferably A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, Has an amino acid mutation at one or more sites selected from D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or has a KAGIR amino acid residue added to the N-terminus is an array and/or
B) The sequence of the Obscurin-O chain is as shown in SEQ ID NO: 33, or based on the sequence of SEQ ID NO: 33, 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, One or more parts selected from 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 76, 88, 89, 92, 94 and 97 It is a sequence that further has an amino acid mutation of A12S, F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G From The sequence has amino acid mutations at one or more selected sites and/or has a DQPQF amino acid residue added to the N-terminus, or
The sequence of the Obscurin-Like-O chain is as shown in SEQ ID NO: 34, or one or more sites selected from 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34. A sequence further having an amino acid mutation, preferably a sequence having an amino acid mutation at one or more sites selected from C6E, C26S, C77S, V74C, G84C and A86C,
The Titin-T chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33. It is the natural sequence number site of the sequence SEQ ID NO: 34,
Preferably,
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76 and 88 based on the sequence of SEQ ID NO: 33, preferably A3C, R9C, It has one or more amino acid residue mutations selected from C25S, C76S and A88C, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33,
Preferably, the Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11, and 62 based on the sequence of SEQ ID NO: 45, preferably L7K or L7R, T62K or has one or more amino acid residue mutations selected from T62H and K11L, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 45,
More preferably, the Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45 based on the sequence of SEQ ID NO: 50. , 53, 58, 62, 67, 69, 89, 92, 94 and 97, preferably G2E, L11K, A12S, F13Y, V14T, V17E, D20L, One or more amino acid mutations selected from T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G. and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 50,
Or
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34, preferably, having one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C, and A86C, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 34, Fab fragment of the domain-modified antibody, bispecific antibody, or domain-modified antibody according to any one of [27] to [33] above.
[35]
The Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or at least 80% of the sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76. contain sequences with identity and/or
The Obscurin-O chain has a sequence shown in any one of SEQ ID NOs: 33, 42-58, 77-86, or at least 80% of the sequence shown in any one of SEQ ID NOs: 33, 42-58, 77-86. contain sequences with identity, or
The Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or at least 80% of the sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76. contain sequences with identity and/or
The Obscurin-Like-O chain has a sequence shown in any one of SEQ ID NOs: 34, 59 to 64, or a sequence having at least 80% identity with any one of SEQ ID NOs: 34, 59 to 64. including,
More preferably,
the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 68, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 80,
the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 73, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 83,
the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 84,
the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 86,
The Titin-T chain includes the polypeptide shown in SEQ ID NO: 75, and the Obscurin-O chain includes the polypeptide shown in SEQ ID NO: 86, according to any one of [27] to [34] above. A domain-modified antibody, a bispecific antibody, or a Fab fragment of a domain-modified antibody.
[36]
selected from the bispecific antibodies described in any one of the following A to G, i.e.
A) The sequence of the first heavy chain is as shown in SEQ ID NO: 89, or has at least 85% sequence identity with SEQ ID NO: 89, and the sequence of the first light chain is as shown in SEQ ID NO: 90. or has at least 85% sequence identity with SEQ ID NO: 90, and the sequence of the second heavy chain is as shown in SEQ ID NO: 87, or has at least 85% sequence identity with SEQ ID NO: 87. , the sequence of the second light chain is as shown in SEQ ID NO: 88, or has at least 85% sequence identity with SEQ ID NO: 88;
B) The sequence of the first heavy chain is as shown in SEQ ID NO: 93, or has at least 85% sequence identity with SEQ ID NO: 93, and the sequence of the first light chain is as shown in SEQ ID NO: 94. or has at least 85% sequence identity with SEQ ID NO: 94, and the sequence of the second heavy chain is as shown in SEQ ID NO: 91, or has at least 85% sequence identity with SEQ ID NO: 91. , the sequence of the second light chain is as shown in SEQ ID NO: 92, or has at least 85% sequence identity with SEQ ID NO: 92;
C) the sequence of the first heavy chain is as shown in SEQ ID NO: 97, or has at least 85% sequence identity with SEQ ID NO: 97, and the sequence of the first light chain is as shown in SEQ ID NO: 98; or has at least 85% sequence identity with SEQ ID NO: 98, and the sequence of the second heavy chain is as shown in SEQ ID NO: 95, or has at least 85% sequence identity with SEQ ID NO: 95. , the sequence of the second light chain is as shown in SEQ ID NO: 96, or has at least 85% sequence identity with SEQ ID NO: 96;
D) The sequence of the first heavy chain is as shown in SEQ ID NO: 99, or has at least 85% sequence identity with SEQ ID NO: 99, and the sequence of the first light chain is as shown in SEQ ID NO: 100. or has at least 85% sequence identity with SEQ ID NO: 100, and the sequence of the second heavy chain is as shown in SEQ ID NO: 101, or has at least 85% sequence identity with SEQ ID NO: 101. , the sequence of the second light chain is as shown in SEQ ID NO: 96, or has at least 85% sequence identity with SEQ ID NO: 96;
E) The sequence of the first heavy chain is as shown in SEQ ID NO: 102, or has at least 85% sequence identity with SEQ ID NO: 102, and the sequence of the first light chain is as shown in SEQ ID NO: 100. or has at least 85% sequence identity with SEQ ID NO: 100, and the sequence of the second heavy chain is as shown in SEQ ID NO: 95, or has at least 85% sequence identity with SEQ ID NO: 95. , the sequence of the second light chain is as shown in SEQ ID NO: 96, or has at least 85% sequence identity with SEQ ID NO: 96;
F) The sequence of the first heavy chain is as shown in SEQ ID NO: 103, or has at least 85% sequence identity with SEQ ID NO: 103, and the sequence of the first light chain is as shown in SEQ ID NO: 104. or has at least 85% sequence identity with SEQ ID NO: 104, and the sequence of the second heavy chain is as shown in SEQ ID NO: 95, or has at least 85% sequence identity with SEQ ID NO: 95. , the sequence of the second light chain is as shown in SEQ ID NO: 96, or has at least 85% sequence identity with SEQ ID NO: 96;
G) The sequence of the first heavy chain is as shown in SEQ ID NO: 107, or has at least 85% sequence identity with SEQ ID NO: 107, and the sequence of the first light chain is as shown in SEQ ID NO: 108. or has at least 85% sequence identity with SEQ ID NO: 108, and the sequence of the second heavy chain is as shown in SEQ ID NO: 109, or has at least 85% sequence identity with SEQ ID NO: 109. , the sequence of the second light chain is as shown in SEQ ID NO: 110, or has at least 85% sequence identity with SEQ ID NO: 110;
H) The sequence of the first heavy chain is as shown in SEQ ID NO: 122, or has at least 85% sequence identity with SEQ ID NO: 122, and the sequence of the first light chain is as shown in SEQ ID NO: 123. or has at least 85% sequence identity with SEQ ID NO: 123, and the sequence of the second heavy chain is as shown in SEQ ID NO: 116, or has at least 85% sequence identity with SEQ ID NO: 116. , the sequence of the second light chain is as shown in SEQ ID NO: 117, or has at least 85% sequence identity with SEQ ID NO: 117;
I) The sequence of the first heavy chain is as shown in SEQ ID NO: 118, or has at least 85% sequence identity with SEQ ID NO: 118, and the sequence of the first light chain is as shown in SEQ ID NO: 119. or has at least 85% sequence identity with SEQ ID NO: 119, and the sequence of the second heavy chain is as shown in SEQ ID NO: 124, or has at least 85% sequence identity with SEQ ID NO: 124. , the sequence of the second light chain is as shown in SEQ ID NO: 125, or has at least 85% sequence identity with SEQ ID NO: 125;
The bispecific antibody according to any one of [21] to [35] above, selected from:
[37]
Including the step of replacing CH1/CL of the antibody with a Titin-T chain and Obscurin-O chain, or a Titin-T chain and Obscurin-Like-O chain,
More preferably, the Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or a sequence of any one of SEQ ID NOs: 32, 35-41, 65-76. contains sequences with at least 80% identity, and/or
The Obscurin-O chain has a sequence shown in any one of SEQ ID NOs: 33, 42-58, 77-86, or at least 80% of the sequence shown in any one of SEQ ID NOs: 33, 42-58, 77-86. contain sequences with identity, or
The Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or at least 80% of the sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76. contain sequences with identity and/or
The Obscurin-Like-O chain has a sequence shown in any one of SEQ ID NOs: 34, 59 to 64, or a sequence having at least 80% identity with any one of SEQ ID NOs: 34, 59 to 64. including,
Most preferably,
The Titin-T chain contains the sequence shown in SEQ ID NO: 68, and the Obscurin-O chain contains the sequence shown in SEQ ID NO: 80,
The Titin-T chain contains the sequence shown in SEQ ID NO: 73, and the Obscurin-O chain contains the sequence shown in SEQ ID NO: 83,
The Titin-T chain contains the sequence shown in SEQ ID NO: 76, and the Obscurin-O chain contains the sequence shown in SEQ ID NO: 84,
the Titin-T chain comprises the sequence shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 86, or
A method for preparing a multispecific antibody, wherein the Titin-T chain contains the sequence shown in SEQ ID NO: 75 and the Obscurin-O chain contains the sequence shown in SEQ ID NO: 86.
[38]
Prepared by the method described in [37] above,
comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1), the VH1 and VL1 forming an antigen-binding site that specifically binds to the first antigen, and
the C-terminus of said VH1 is operably linked to the N-terminus of a Titin-T chain, the C-terminus of said VL1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain, or
the C-terminus of said VL1 is operably linked to the N-terminus of a Titin-T chain, and the C-terminus of said VH1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain; sexual antibodies.
[39]
A) The sequence of the Titin-T chain is as shown in SEQ ID NO: 32, or based on the sequence of SEQ ID NO: 32, 8, 20, 22, 25, 26, 39, 3, 11, 13, 40, further has an amino acid mutation at one or more sites selected from 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or N A sequence that adds five amino acid residues KAGIR to the end, preferably A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, Has an amino acid mutation at one or more sites selected from D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or has a KAGIR amino acid residue added to the N-terminus is an array and/or
B) The sequence of the Obscurin-O chain is as shown in SEQ ID NO: 33, or based on the sequence of SEQ ID NO: 33, 3, 9, 25, 76, 88, 7, 11, 62, 2, 12, One or more parts selected from 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 67, 69, 89, 92, 94 and 97 It is a sequence that further has an amino acid mutation of A12S, F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G From The sequence has amino acid mutations at one or more selected sites and/or has a DQPQF amino acid residue added to the N-terminus, or
The sequence of the Obscurin-Like-O chain is as shown in SEQ ID NO: 34, or one or more sites selected from 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34. A sequence further having an amino acid mutation, preferably a sequence having an amino acid mutation at one or more sites selected from C6E, C26S, C77S, V74C, G84C and A86C,
The Titin-T chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33. It is the natural sequence number site of the sequence SEQ ID NO: 34,
Preferably,
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76 and 88 based on the sequence of SEQ ID NO: 33, preferably A3C, R9C, It has one or more amino acid residue mutations selected from C25S, C76S and A88C, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33,
Preferably, the Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11, and 62 based on the sequence of SEQ ID NO: 45, preferably L7K or L7R, T62K or has one or more amino acid residue mutations selected from T62H and K11L, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 45,
More preferably, the Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45 based on the sequence of SEQ ID NO: 50. , 53, 58, 62, 67, 69, 89, 92, 94 and 97, preferably G2E, L11K, A12S, F13Y, V14T, V17E, D20L, One or more amino acid mutations selected from T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G. and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 50,
Or
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34, preferably, having one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C, and A86C, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 34, The multispecific antibody according to [38] above.
[40]
Contains any substance selected from the following (A) to (F), that is,
A) the polypeptide complex according to any one of [1] to [12] above;
B) the multispecific polypeptide complex according to any one of [13] to [17] above;
C) the domain-modified antibody according to any one of [18], [20], [27] to [35],
D) Fab fragment of the domain-modified antibody according to any one of [19], [27] to [35] above;
E) the bispecific antibody according to any one of [21] to [36] above;
F) A complex comprising the multispecific antibody according to [38] or [39] above, and any substance selected from.
[41]
The sequence is as shown in any one of SEQ ID NO: 32, 35-41, 65-76, or is at least 80% identical to any one of SEQ ID NO: 32, 35-41, 65-76 have sex or
The sequence is as shown in any one of SEQ ID NO: 33, 42-58, 77-86, or is at least 80% identical to any one of SEQ ID NO: 33, 42-58, 77-86 have sex or
the sequence is as shown in any one of SEQ ID NOs: 34, 59 to 64, or has at least 80% identity with any one of SEQ ID NOs: 34, 59 to 64;
Preferably, a polypeptide that can be used to replace antibodies CH1 and/or CL.
[42]
Use of a combination of Titin-T chain and Obscurin-O chain or a combination of Titin-T chain and Obscurin-Like-O chain in reducing multispecific antibody light chain/heavy chain mismatch, comprising:
Preferably, the multispecific antibody includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the VH1 and VL1 form an antigen-binding site that specifically binds to the first antigen. and
the C-terminus of said VH1 is operably linked to the N-terminus of a Titin-T chain, the C-terminus of said VL1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain, or
The C-terminus of the VL1 is operably linked to the N-terminus of a Titin-T chain, the C-terminus of the VH1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain,
More preferably, the Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or a sequence of any one of SEQ ID NOs: 32, 35-41, 65-76. comprising a sequence with at least 80% identity and/or the Obscurin-O chain is represented by any one of SEQ ID NO: 33, 42-58, 77-86, or SEQ ID NO: 33, 42-58 , 77 to 86, or
The Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or at least 80% of the sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76. A sequence containing a sequence having identity and/or a sequence in which the Obscurin-Like-O chain is shown in any one of SEQ ID NOs: 34, 59 to 64, or a sequence of any one of SEQ ID NOs: 34, 59 to 64 comprising a sequence having at least 80% identity with
Most preferably,
The Titin-T chain contains the sequence shown in SEQ ID NO: 68, and the Obscurin-O chain contains the sequence shown in SEQ ID NO: 80,
The Titin-T chain contains the sequence shown in SEQ ID NO: 73, and the Obscurin-O chain contains the sequence shown in SEQ ID NO: 83,
the Titin-T chain contains the sequence shown in SEQ ID NO: 76, the Obscurin-O chain contains the sequence shown in SEQ ID NO: 84, the Titin-T chain contains the sequence shown in SEQ ID NO: 76, and the Obscurin-O chain contains the sequence shown in SEQ ID NO: 76; the strand comprises the sequence shown in SEQ ID NO: 86, or
Use in which the Titin-T chain comprises the sequence shown in SEQ ID NO: 75 and the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 86.
[43]
A) The sequence of the Titin-T chain is as shown in SEQ ID NO: 32, or based on the sequence of SEQ ID NO: 32, 8, 20, 22, 25, 26, 39, 3, 11, 13, 40, further has an amino acid mutation at one or more sites selected from 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or N A sequence that adds five amino acid residues KAGIR to the end, preferably A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, Has an amino acid mutation at one or more sites selected from D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or has a KAGIR amino acid residue added to the N-terminus is an array and/or
B) The sequence of the Obscurin-O chain is as shown in SEQ ID NO: 33, or based on the sequence of SEQ ID NO: 33, 3, 9, 25, 76, 88, 7, 11, 62, 2, 12, One or more parts selected from 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 67, 69, 89, 92, 94 and 97 It is a sequence that further has an amino acid mutation of A12S, F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G From The sequence has amino acid mutations at one or more selected sites and/or has a DQPQF amino acid residue added to the N-terminus, or
The sequence of the Obscurin-Like-O chain is as shown in SEQ ID NO: 34, or one or more sites selected from 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34. A sequence further having an amino acid mutation, preferably a sequence having an amino acid mutation at one or more sites selected from C6E, C26S, C77S, V74C, G84C and A86C,
The Titin-T chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33. It is the natural sequence number site of the sequence SEQ ID NO: 34,
Preferably,
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76 and 88 based on the sequence of SEQ ID NO: 33, preferably A3C, R9C, It has one or more amino acid residue mutations selected from C25S, C76S and A88C, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33,
Preferably, the Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11, and 62 based on the sequence of SEQ ID NO: 45, preferably L7K or L7R, T62K or has one or more amino acid residue mutations selected from T62H and K11L, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 45,
More preferably, the Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45 based on the sequence of SEQ ID NO: 50. , 53, 58, 62, 67, 69, 89, 92, 94 and 97, preferably G2E, L11K, A12S, F13Y, V14T, V17E, D20L, One or more amino acid mutations selected from T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G. and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 50,
Or
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34, preferably, having one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C, and A86C, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 34, The use described in [42] above.
[44]
Selected from (A) to (G) below, i.e.
A) the polypeptide complex according to any one of [1] to [12] above;
B) the multispecific polypeptide complex according to any one of [13] to [17] above;
C) the domain-modified antibody according to any one of [18], [20], [27] to [35],
D) Fab fragment of the domain-modified antibody according to any one of [19], [27] to [35],
E) the bispecific antibody according to any one of [21] to [36] above;
F) the polypeptide described in [41] above;
G) A nucleic acid molecule encoding any substance selected from the multispecific antibody described in [38] or [39] above.
[45]
A vector comprising the nucleic acid molecule according to [44] above.
[46]
A host cell obtained by transformation of the vector described in [45] above, selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, and more preferably mammalian cells.
[47]
Selected from:
The polypeptide complex according to any one of [1] to [12] above, or the multispecific polypeptide complex according to any one of [13] to [17] above, or the first half [18] ], [20], [27] to [35], or the domain modified antibody according to any one of [19], [27] to [35]. Fab fragment, or the bispecific antibody according to any one of [21] to [36] above, or the polypeptide according to [41] above, or the multiplex antibody according to [38] or [39] above. preparing any substance selected from specific antibodies,
It includes the following steps, namely:
After culturing the host cell described in [46] above, a polypeptide complex, a multispecific polypeptide complex, a domain-modified antibody, a Fab fragment of a domain-modified antibody, a bispecific antibody, a polypeptide, or a multispecific A preparation method comprising purifying and recovering a sex antibody.
[48]
one or more pharmaceutically acceptable carriers, excipients or diluents, and selected from:
The polypeptide complex according to any one of [1] to [12] above, or the multispecific polypeptide complex according to any one of [13] to [17] above, or the first half [18] ], [20], [27] to [35], or the domain modified antibody according to any one of [19], [27] to [35]. Fab fragment, or the bispecific antibody according to any one of [21] to [36] above, or the multispecific antibody according to [38] or [39] above, or the multispecific antibody according to [40] above. A pharmaceutical composition comprising any substance selected from the following.
[49]
A polypeptide complex selected from the following, that is, the polypeptide complex according to any one of [1] to [12] above, or the multispecific polypeptide according to any one of [13] to [17] above. A complex, or the domain-modified antibody according to any one of [18], [20], [27] to [35] above, or any one of [19], [27] to [35] above. Fab fragment of the domain-modified antibody described in [21] to [36] above, or the pharmaceutical composition described in [48] above, or [38] above. or the multispecific antibody according to [39], or the conjugate according to [40] above, in the preparation of a medicament for treating or preventing a disease or disorder.
[50]
Selected from:
The polypeptide complex according to any one of [1] to [12] above, or the multispecific polypeptide complex according to any one of [13] to [17] above, or the multispecific polypeptide complex according to any one of [13] to [17] above, or [18] ], [20], [27] to [35], or the domain modified antibody according to any one of [19], [27] to [35]. Any substance selected from the Fab fragment, the bispecific antibody according to any one of [21] to [36] above, or the multispecific antibody according to [38] or [39] above. A detection method comprising contacting a sample awaiting detection.

Claims (32)

a first antigen-binding portion, the first antigen-binding portion comprising:
A) comprising a first heavy chain variable domain (VH1) from the N-terminus to the C-terminus, said VH1 being operably linked to a first domain, said first domain comprising a first polypeptide comprising a Titin-T chain; ,
a first light chain variable domain (VL1) from the N-terminus to the C-terminus, said VL1 operably linked to a second domain, said second domain comprising a domain capable of interacting with the Titin-T chain; comprising a polypeptide;
Or
B) a first polypeptide comprising a VH1 from the N-terminus to the C-terminus, said VH1 operably linked to a first domain, and said first domain comprising a domain capable of interacting with a Titin-T chain;
a second polypeptide comprising a VL1 from the N-terminus to the C-terminus, the VL1 operably linked to a second domain, and the second domain comprising a Titin-T chain;
Or
C) a first polypeptide comprising a VH1 from the N-terminus to the C-terminus, the VH1 being operably linked to a first domain, and the first domain comprising an Obscurin-O chain or an Obscurin-Like-O chain;
a second polypeptide comprising a VL1 from the N-terminus to the C-terminus, said VL1 operably linked to a second domain, said second domain comprising a domain capable of interacting with an Obscurin-O chain or an Obscurin-Like-O chain; peptide;
Or
D) a VH1 comprising a VH1 from the N-terminus to the C-terminus, said VH1 operably linked to a first domain, said first domain comprising a domain capable of interacting with an Obscurin-O chain or an Obscurin-Like-O chain; 1 polypeptide and
a second polypeptide comprising a VL1 from the N-terminus to the C-terminus, the VL1 being operably linked to a second domain, and the second domain comprising an Obscurin-O chain or an Obscurin-Like-O chain; ,
the first antigen-binding portion specifically binds to a first antigen, and the first domain is capable of forming a dimer with a second domain ;
The domain that can interact with the Obscurin-O chain or Obscurin-Like-O chain is a Titin-T chain,
The domain that can interact with the Titin-T chain is an Obscurin-O chain or an Obscurin-Like-O chain,
VH1 and VL1 of the first antigen-binding portion form a first antigen-binding site that specifically binds to a first antigen, and the C-terminus of the VH1 is operably linked to the N-terminus of the first domain, and the VL1 a polypeptide conjugate, the C-terminus of which is operably linked to the N-terminus of a second domain . The first domain forms a dimer with the second domain through a natural interchain bond and/or a non-natural interchain bond,
Preferably, the first domain forms a dimer with the second domain through natural interchain binding,
More preferably, one or more residues selected from positions 7 to 15, 19 to 24, 26, 55, 59, and 60 in the Titin-T chain are 3 to 6, 9, 41, Bonds with one or more residues selected from positions 73, 75 and 80 to 90, or positions 1, 7 to 10, 13 to 16, 19 to 26, 59 to 60 and 96 in the Titin-T chain and one or more residues selected from positions 4 to 5, 10, 12 to 13, 74, 76, 78, and 82 to 91 in the Obscurin-Like-O chain. combine,
The residue site in the Titin-T chain is a natural sequence number site for the sequence SEQ ID NO: 32, the residue site in the Obscurin-O chain is a natural sequence number site for the sequence SEQ ID NO: 33, and the Obscurin-Like 2. The polypeptide conjugate according to claim 1 , wherein the residue positions in the -O chain are naturally numbered positions relative to the sequence of SEQ ID NO: 34. the first domain forms a dimer with the second domain through at least one non-natural interchain bond;
Preferably, the non-natural interchain bond is formed between a specific variant residue of the first domain and a specific variant residue of the second domain,
More preferably, at least one pair of the mutant residues is a cysteine residue,
Most preferably, the non-natural interchain bond is a disulfide bond;
More preferably, the dimer comprises 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10 non-natural interchain bonds. polypeptide complex. The specific mutated residue of the first domain and the specific mutated residue of the second domain are selected from the following mutations, namely:
(A) the Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26, and 39, and/or the Obscurin-O chain has mutations at positions 3, 9, has one or more amino acid residue mutations selected from positions 25, 76, and 88, or
(B) the Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26, and 39, and/or the Obscurin-Like-O chain has 6, Having one or more amino acid residue mutations selected from positions 26, 74, 77, 84 and 86,
Preferably, the Titin-T chain has one or more amino acid residue mutations selected from A8C, V20C, T22C, C25S, A26C and C39T, and/or the Obscurin-O chain has A3C, R9C, C25S. , C76S and A88C, or the Titin-T chain has one or more amino acid residue mutations selected from A8C, V20C, T22C, C25S, A26C and C39T. and/or the Obscurin-Like-O chain has one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C and A86C,
More preferably, the mutated residues of the first domain and the mutated residues of the second domain are selected from the following mutations, namely:
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has A88C mutation,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-O chain has A3C mutation,
Titin-T chain has C25S, C39T and A26C mutations, and Obscurin-O chain has R9C mutation,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S and A88C mutations,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-O chain has C25S, C76S and A3C mutations,
Titin-T chain has C25S, C39T and A26C mutations, and Obscurin-O chain has C25S, C76S and R9C mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-Like-O chain has C6E and V74C mutations,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-Like-O chain has C6E and G84C mutations,
Titin-T chain has C25S, C39T and T22C mutations, and 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,
Titin-T chain has C25S, C39T and V20C mutations and Obscurin-Like-O chain has C6E, C26S, C77S and G84C mutations, or
The Titin-T chain has C25S, C39T and T22C mutations, and the Obscurin-Like-O chain has C6E, C26S, C77S and A86C mutations,
The Titin-T chain mutation site is the natural sequence number site for the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is the natural sequence number site for the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is the natural sequence number site for the sequence SEQ ID NO: 32. 4. The polypeptide conjugate of claim 3 , wherein the polypeptide conjugate is a naturally numbered site relative to the sequence numbered 34. The Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11, and 62,
Preferably, the Obscurin-O chain has one or more amino acid residue mutations selected from L7R or L7K, T62K or T62H, and K11L,
Most preferably, the Titin-T chain has C25S, C39T and A8C mutations, and the Obscurin-O chain has C25S, C76S, A88C, L7K and T62K mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, L7K and T62H mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, K11L and T62K mutations, or
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, K11L and T62H,
The polypeptide complex according to claim 4 , wherein the Titin-T chain mutation site is a natural sequence number site relative to the sequence SEQ ID NO: 32, and the Obscurin O chain mutation site is a natural sequence number site relative to the sequence SEQ ID NO: 33. . The first domain and the second domain have one or more amino acid residue mutations selected from the following, namely:
One Titin-T chain selected from positions 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84 or has multiple amino acid mutations and/or Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53 , having one or more amino acid mutations selected from positions 58, 62, 67, 69, 89, 92, 94 and 97,
Preferably, the first domain and the second domain have one or more residue mutations selected from the following, namely:
1 where the Titin-T chain is selected from P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L has one or more amino acid mutations, and/or Obscurin-O chain is G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, Having one or more amino acid mutations selected from A45T, A53L, L58V, K62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G,
More preferably, the 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, Has 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 amino acid mutations of G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D and F84L, and/or the Obscurin-O chain has Q42L, A45T, A67T, G69S, Q92E and Has the D94G amino acid mutation,
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,
Any one of claims 1 to 5 , wherein the Titin-T chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 35, and the Obscurin O chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 50. The polypeptide conjugate described in Section. the VH1 is operably linked to a first domain by a first linking domain, and the VL1 is operably linked to a second domain by a second linking domain;
Preferably, the first linking domain and/or the second linking domain are selected from the following: N-terminal fragment of Titin-T chain, N-terminal fragment of Obscurin-O chain, N-terminal fragment of Obscurin-Like-O chain. a terminal fragment, and a (G _x S) _y connexon (wherein X is selected from an integer from 1 to 5 and Y is selected from an integer from 1 to 6),
More preferably, the first linking domain and/or the second linking domain are selected from the following polypeptide fragments, namely "KAGIR", "DQPQF", (G ₄ S) ₁ and (G ₄ S) ₂ ,
Most preferably, said first domain is a Titin-T chain, said first linking domain is a KAGIR polypeptide, said second domain is an Obscurin-O chain, and said second linking domain is a DQPQF polypeptide. or, the second domain is a Titin-T chain, the second linking domain is a KAGIR polypeptide, the first domain is an Obscurin-O chain, and the first linking domain is a DQPQF polypeptide. , the polypeptide complex according to any one of claims 1 to 6 . (A) The sequence of the Titin-T chain is as shown in SEQ ID NO: 32, or 3, 8, 11, 13, 20, 22, 25, 26, 39, 40 based on the sequence of SEQ ID NO: 32. , 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or This is a sequence that adds a KAGIR amino acid residue to the N-terminus,
Preferably A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R , N82M, E83D, and F84L, and/or has an amino acid mutation at one or more sites selected from N82M, E83D, and F84L, and/or has a KAGIR amino acid residue added to the N-terminus, and/or (B) the Obscurin-O chain. The sequence is as shown in SEQ ID NO: 33, or 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, 22, 25, 30, 32 based on the sequence of SEQ ID NO: 33. , 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 76, 88, 89, 92, 94 and 97. , and/or a sequence that adds a DQPQF amino acid residue to the N-terminus, preferably A3C, R9C, C25S, C76S, A88C, L7K, T62K or T62H, G2E, L11K, A12S, F13Y, V14T, V17E, D20L , T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G. The sequence has an amino acid mutation and/or has a DQPQF amino acid residue added to the N-terminus, or the sequence of the Obscurin-Like-O chain is as shown in SEQ ID NO: 34, or the sequence is as shown in SEQ ID NO: 34. A sequence that further has an amino acid mutation at one or more sites selected from 6, 26, 74, 77, 84 and 86 based on the sequence, preferably selected from C6E, C26S, C77S, V74C, G84C and A86C. A sequence having amino acid mutations at one or more sites,
The Titin-T chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33. It is the natural sequence number site of the sequence SEQ ID NO: 34,
Preferably,
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76 and 88 based on the sequence of SEQ ID NO: 33, preferably A3C, R9C, It has one or more amino acid residue mutations selected from C25S, C76S and A88C, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33,
More preferably, the Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11, and 62 based on the sequence of SEQ ID NO: 45, preferably L7K, L7R, or T62K. or has one or more amino acid residue mutations selected from T62H and K11L, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 45,
Most preferably, the Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45 based on the sequence of SEQ ID NO: 50. , 53, 58, 62, 67, 69, 89, 92, 94 and 97, preferably G2E, L11K, A12S, F13Y, V14T, V17E, D20L, One or more amino acid mutations selected from T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G. and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 50,
Or
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34, preferably, having one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C, and A86C, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 34, The polypeptide complex according to any one of claims 1 to 7 . The Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or at least 80% of the sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76. and/or the Obscurin-O chain has the sequence shown in any one of SEQ ID NO: 33, 42-58, 77-86 or the sequence of SEQ ID NO: 33, 42-58, 77-86. a sequence having at least 80% identity with any one sequence, or the Titin-T chain has a sequence shown in any one of SEQ ID NO: 32, 35-41, 65-76, or SEQ ID NO: 32 , 35-41, 65-76, and/or the Obscurin-Like-O chain is any one of SEQ ID NOs: 34, 59-64. or a sequence having at least 80% identity with any one of SEQ ID NOs: 34, 59 to 64,
More preferably, 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 Titin-T chain comprises the sequence shown in SEQ ID NO: 73, and the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 83,
the Titin-T chain comprises the sequence shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 84,
the Titin-T chain comprises the sequence shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 86, or the Titin-T chain comprises the sequence shown in SEQ ID NO: 75, The polypeptide complex according to any one of claims 1 to 8 , wherein the Obscurin-O chain comprises the sequence shown in SEQ ID NO: 86. The polypeptide complex according to any one of claims 1 to 9 , wherein the first antigen is selected from an exogenous antigen, an endogenous antigen, an autoantigen, a neoantigen, a viral antigen, and a tumor antigen. Polypeptide conjugate according to any one of claims 1 to 10, which is a multispecific polypeptide conjugate . 11. The polypeptide conjugate according to any one of claims 1 to 10, which is an F ab fragment. comprising 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;
the CH1 domain of the first heavy chain is replaced with a Titin-T chain, the CL domain of the first light chain is replaced with a domain capable of having a protein-protein interaction with the Titin-T chain,
the CL domain of the first light chain is replaced with a Titin-T chain, the CH1 domain of the first heavy chain is replaced with a domain capable of having a protein-protein interaction with the Titin-T chain,
The CH1 domain of the first heavy chain is replaced with an Obscurin-O chain, and the CL domain of the first light chain is replaced with a domain capable of having a protein-protein interaction with the Obscurin-O chain;
the CL domain of the first light chain is replaced with an Obscurin-O chain, the CH1 domain of the first heavy chain is replaced with a domain capable of having a protein-protein interaction with the Obscurin-O chain,
the CH1 domain of the first heavy chain is replaced with an Obscurin-Like-O chain, the CL domain of the first light chain is replaced with a domain capable of having protein-protein interaction with the Obscurin-Like-O chain, or the CL domain of the first light chain is replaced with an Obscurin-Like-O chain, and the CH1 domain of the first heavy chain is replaced with a domain capable of having a protein-protein interaction with the Obscurin-Like-O chain;
the first antigen is different from the second antigen, or the first antigen and the second antigen are two different epitopes of the same antigen,
Preferably, mismatches are less likely to occur between the first heavy chain and the second light chain, and between the first light chain and the second heavy chain, and more preferably, the interaction between the Titin-T chain and the protein is prevented. the domain is Obscurin-O chain or Obscurin-Like-O chain,
The domain having protein-protein interaction with the Obscurin-O chain or Obscurin-Like-O chain is a Titin-T chain ,
The first heavy chain includes a first heavy chain variable region (VH1), the first light chain includes a first light chain variable region (VL1), and the VH1 and VL1 specifically bind to a first antigen. form an antigen binding site, and
the C-terminus of said VH1 is operably linked to the N-terminus of a Titin-T chain, the C-terminus of said VL1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain, or
the C-terminus of said VL1 is operably linked to the N-terminus of a Titin-T chain, and the C-terminus of said VH1 is operably linked to the N-terminus of an Obscurin-O chain or an Obscurin-Like-O chain; The polypeptide conjugate according to any one of claims 1 to 10, which is a specific 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, and the second light chain comprises a second heavy chain variable region (VH2) from the N-terminus to the C-terminus; contains a variable region (VL2) and is operably linked to CL;
Preferably, the VH2 and VL2 form an antigen binding site that specifically binds to a second antigen, the C-terminus of the VH2 is operably linked to the N-terminus of CH1, and the C-terminus of VL2 is operably linked to the N-terminus of CL. 14. The bispecific antibody of claim 13 , wherein the bispecific antibody is operably linked to a terminus. The C-terminus of the first heavy chain and the second heavy chain contains a CH2 and/or CH3 domain,
Preferably, said CH1, CH2 and CH3 domains are derived from IgG1, IgG2, IgG3 or IgG4,
More preferably, the first heavy chain is formed in a manner selected from the following: an antibody hinge region or a portion thereof, a connexon, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, or The bispecific antibody according to any one of claims 13 to 14 , which binds to the second heavy chain in a manner selected from a combination of these. The first heavy chain is, in order from the N-terminus to the C-terminus, VH1-L1-Titin-T chain-L2-CH2-CH3,
The first light chain is a VL1-L3-Obscurin-O chain or a VL1-L4-Obscurin-Like-O chain in order from the N-terminus to the C-terminus,
The second heavy chain is VH2-CH1-CH2-CH3 in order from the N-terminus to the C-terminus,
The second light chain is VL2-CL in order from the N-terminus to the C-terminus,
Or,
The first light chain is a VL1-L1-Titin-T chain in order from the N-terminus to the C-terminus,
The first heavy chain is, in order from the N-terminus to the C-terminus, VH1-L2-Obscurin-O chain-L3-CH2-CH3, or VH1-L4-Obscurin-Like-O chain-L5-CH2-CH3. can be,
The second heavy chain is VH2-CH1-CH2-CH3 in order from the N-terminus to the C-terminus,
The second light chain is VL2-CL in order from the N-terminus to the C-terminus,
L1 to L5 are spacer regions, and the spacer region may or may not exist,
Preferably, said spacer region is selected from: 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 (G _x S) _y connexons (where X is selected from an integer from 1 to 5 and Y is selected from an integer from 1 to 6),
Most preferably, the bispecific according to claim 15 , wherein the spacer region is selected from: "KAGIR", "DQPQF", ( _G4S ) ₁ and ( _G4S ) ₂ . antibody. CH2 and CH3 of the first heavy chain and CH2 and CH3 of the second heavy chain are different and bind in a manner that prevents homodimerization and/or promotes heterodimerization,
Preferably, the first heavy chain is formed by a method selected from the following, namely, by a method selected from knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or a method that improves flexibility. binds to the second heavy chain,
More preferably, the first heavy chain binds to the second heavy chain in a manner including a knob-into-hole,
Most preferably, the first heavy chain CH2-CH3 domain has amino acid residues 104 to 330 in the sequence shown in SEQ ID NO: 2, and the second heavy chain CH2-CH3 domain has the sequence shown in SEQ ID NO: 3. 104th to 330th amino acid residue in the sequence, or the first heavy chain CH2-CH3 domain has amino acid residues 104th to 330th in the sequence shown in SEQ ID NO: 3, and the second heavy chain 17. The bispecific antibody according to claim 16 , wherein the CH2-CH3 domain has amino acid residues 104 to 330 in the sequence shown in SEQ ID NO:2. Any of claims 13 to 17, wherein said operably linked means that two polypeptide sequences are linked by a linking domain, or that two polypeptides are directly linked. The polypeptide conjugate according to item 1 . The Titin-T chain is bound to the Obscurin-O chain, or the Titin-T chain is bound to the Obscurin-Like-O chain by a natural interchain bond, and/or forms a dimer by a non-natural interchain bond,
Preferably, the Titin-T chain forms a dimer with the Obscurin-O chain or Obscurin-Like-O chain through a natural interchain bond, and 7-15, 19-24, 26, 55 in the Titin-T chain , one or more residues selected from positions 59 and 60 are bonded to one or more residues selected from positions 3 to 6, 9, 41, 73, 75 and 80 to 90 in the Obscurin-O chain. or one or more residues selected from positions 1, 7 to 10, 13 to 16, 19 to 26, 59 to 60 and 96 in the Titin-T chain are 4 to 5 in the Obscurin-Like-O chain. , 10, 12 to 13, 74, 76, 78, and 82 to 91, bonding to one or more residues,
The Titin-T chain residue site is the natural sequence number site for the sequence SEQ ID NO: 32, the Obscurin-O chain residue site is the natural sequence number site for the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain residue site is the natural sequence number site for the sequence SEQ ID NO: 32. 19. The polypeptide conjugate of claim 18 , wherein the base site is a natural sequence number site relative to the sequence of SEQ ID NO:34. The Titin-T chain forms a dimer with the Obscurin-O chain, or the Titin-T chain forms a dimer with the Obscurin-Like-O chain due to at least one non-natural interchain bond,
Preferably, the non-natural interchain bond is a disulfide bond,
More preferably, the polypeptide conjugate according to claim 18 or 19 , wherein the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-natural interchain linkages. . The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39, and/or the Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76. and one or more amino acid residue mutations selected from positions 88, or the Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26, and 39. and/or the Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74, 77, 84 and 86,
Preferably, the Titin-T chain has one or more amino acid residue mutations selected from A8C, V20C, T22C, C25S, A26C and C39T, and/or the Obscurin-O chain has A3C, R9C, C25S. , C76S and A88C, or the Titin-T chain has one or more amino acid residue mutations selected from A8C, V20C, T22C, C25S, A26C and C39T. and/or the Obscurin-Like-O chain has one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C and A86C,
More preferably, the Titin-T chain has C25S, C39T and A8C mutations, and the Obscurin-O chain has an A88C mutation,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-O chain has A3C mutation,
Titin-T chain has C25S, C39T and A26C mutations, and Obscurin-O chain has R9C mutation,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S and A88C mutations,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-O chain has C25S, C76S and A3C mutations,
Titin-T chain has C25S, C39T and A26C mutations, and Obscurin-O chain has C25S, C76S and R9C mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-Like-O chain has C6E and V74C mutations,
Titin-T chain has C25S, C39T and V20C mutations, and Obscurin-Like-O chain has C6E and G84C mutations,
Titin-T chain has C25S, C39T and T22C mutations, and 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,
Titin-T chain has C25S, C39T and V20C mutations and Obscurin-Like-O chain has C6E, C26S, C77S and G84C mutations, or
The Titin-T chain has C25S, C39T and T22C mutations, and the Obscurin-Like-O chain has C6E, C26S, C77S and A86C mutations,
The Titin-T chain mutation site is a naturally ordered site for the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is a naturally ordered site for the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is a naturally ordered site for the sequence SEQ ID NO: 33. 21. A polypeptide conjugate according to any one of claims 18 to 20 , which is a naturally ordered site for a sequence of 34. The Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11 and 62,
Preferably, the Obscurin-O chain has one or more amino acid residue mutations selected from L7K or L7R, K11L, and T62K or T62H,
Most preferably, the Titin-T chain has C25S, C39T and A8C mutations, and the Obscurin-O chain has C25S, C76S, A88C, L7K and T62K mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, L7K and T62H mutations,
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, K11L and T62K mutations, or
Titin-T chain has C25S, C39T and A8C mutations, and Obscurin-O chain has C25S, C76S, A88C, K11L and T62H,
The polypeptide according to claim 20 or 21, wherein the Titin-T chain mutation site is a natural sequence number site with respect to the sequence SEQ ID NO: 32, and the Obscurin-O chain mutation site is a natural sequence number site with respect to the sequence SEQ ID NO: 33. Peptide complex . 1 in which the Titin-T chain is selected from positions 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84; has one or more amino acid mutations, and/or
Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 89, 92 , having one or more amino acid mutations selected from positions 94 and 97,
Preferably, the Titin-T chain is P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L. has one or more amino acid mutations selected from, and/or
Obscurin-O chain is G2E, L11K, A12S, F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q or A67T, G69S , has one or more amino acid mutations selected from V89L, Q92E, D94G and A97G,
More preferably, the 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, Has 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 amino acid mutations of G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, L75V, E83D and F84L, and/or the Obscurin-O chain has Q42L, A45T, A67T, G69S, Q92E and Has the D94G amino acid mutation,
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,
Any one of claims 18 to 22, wherein the Titin-T chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 35, and the Obscurin-O chain mutation site is a natural sequence number site for the sequence SEQ ID NO: 50. The polypeptide conjugate described in Section. The N-terminus of the Titin-T chain, Obscurin-O chain or Obscurin-Like-O chain is operably linked to VH1 or VL1 by a linking domain,
Preferably, said linking domain is selected from: N-terminal fragment of Titin-T chain, N-terminal fragment of Obscurin-O chain, N-terminal fragment of Obscurin-Like-O chain, and (G _x S) _y connexons (where X is selected from an integer from 1 to 5 and Y is selected from an integer from 1 to 6),
More preferably, the linking domain is selected from the following: "KAGIR", "DQPQF", (G ₄ S) ₁ and (G ₄ S) ₂ ,
Most preferably, said Titin-T chain is linked to VH1 or VL1 by a KAGIR polypeptide and/or said Obscurin-O chain is linked to VL1 or VH1 by a DQPQF polypeptide. The polypeptide conjugate described in Section. A) The sequence of the Titin-T chain is as shown in SEQ ID NO: 32, or 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, based on the sequence of SEQ ID NO: 32, further has an amino acid mutation at one or more sites selected from 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79, 81, 82, 83 and 84, and/or N A sequence that adds five amino acid residues KAGIR to the end, preferably A8C, V20C, T22C, C25S, A26C, C39T, P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, Has an amino acid mutation at one or more sites selected from D58E, M66S or M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D and F84L, and/or has a KAGIR amino acid residue added to the N-terminus is an array and/or
B) The sequence of the Obscurin-O chain is as shown in SEQ ID NO: 33, or based on the sequence of SEQ ID NO: 33, 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, One or more parts selected from 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 53, 58, 62, 67, 69, 76, 88, 89, 92, 94 and 97 It is a sequence that further has an amino acid mutation of A12S, F13Y, V14T, V17E, D20L, T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, T62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G From The sequence has amino acid mutations at one or more selected sites and/or has a DQPQF amino acid residue added to the N-terminus, or the sequence of the Obscurin-Like-O chain is shown in SEQ ID NO: 34. or a sequence further having amino acid mutations at one or more sites selected from 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34, preferably C6E, C26S, A sequence having an amino acid mutation at one or more sites selected from C77S, V74C, G84C and A86C,
The Titin-T chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 32, the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33. It is the natural sequence number site of the sequence SEQ ID NO: 34,
Preferably,
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-O chain has one or more amino acid residue mutations selected from positions 3, 9, 25, 76 and 88 based on the sequence of SEQ ID NO: 33, preferably A3C, R9C, It has one or more amino acid residue mutations selected from C25S, C76S and A88C, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 33,
Preferably, the Obscurin-O chain further has one or more amino acid residue mutations selected from positions 7, 11, and 62 based on the sequence of SEQ ID NO: 45, preferably L7K or L7R, T62K or has one or more amino acid residue mutations selected from T62H and K11L, and the Obscurin-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 45,
More preferably, the Obscurin-O chain is 2, 11, 12, 13, 14, 17, 20, 22, 30, 32, 34, 36, 41, 42, 44, 45 based on the sequence of SEQ ID NO: 50. , 53, 58, 62, 67, 69, 89, 92, 94 and 97, preferably G2E, L11K, A12S, F13Y, V14T, V17E, D20L, One or more amino acid mutations selected from T22M or T22S, N30D, T32P, Q34E, S36T, Q41K, Q42L, V44I, A45T, A53L, L58V, K62E, A67Q or A67T, G69S, V89L, Q92E, D94G and A97G. and the Obscurin-O chain mutation site is the natural sequence number site of the sequence SEQ ID NO: 50,
Or
A) The Titin-T chain has one or more amino acid residue mutations selected from positions 8, 20, 22, 25, 26 and 39 based on the sequence of SEQ ID NO: 32, preferably A8C, It has one or more amino acid residue mutations selected from V20C, T22C, C25S, A26C and C39T, and the titin-T chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 32,
More preferably, the Titin-T chain is 3, 11, 13, 22, 40, 42, 45, 47, 49, 56, 58, 66, 70, 75, 77, 79 based on the sequence of SEQ ID NO: 35. , 81, 82, 83 and 84, preferably P3W, S11I, I13L, T22M, G40S, R42K, H45S, Q47E, Q49G, N56S, D58E, M66S, It has one or more amino acid mutations selected from M66K, K70R, L75V, T77S, S79T, G81R, N82M, E83D, and F84L, and the titin-T chain mutation site is at the natural sequence number site of the sequence of SEQ ID NO: 35. Yes, and/or
B) The Obscurin-Like-O chain has one or more amino acid residue mutations selected from positions 6, 26, 74, 77, 84 and 86 based on the sequence of SEQ ID NO: 34, preferably, having one or more amino acid residue mutations selected from C6E, C26S, C77S, V74C, G84C, and A86C, and the Obscurin-Like-O chain mutation site is the natural sequence number site of the sequence of SEQ ID NO: 34, The polypeptide complex according to any one of claims 18 to 24 . The Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or at least 80% of the sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76. and/or the Obscurin-O chain has a sequence shown in any one of SEQ ID NO: 33, 42-58, 77-86, or SEQ ID NO: 33, 42-58, 77-86. or the Titin-T chain has a sequence shown in any one of SEQ ID NOs: 32, 35-41, 65-76, or SEQ ID NO: 32, 35-41, 65-76, and/or the Obscurin-Like-O chain is any one of SEQ ID NOs: 34, 59-64. or a sequence having at least 80% identity with any one of SEQ ID NOs: 34, 59 to 64,
More preferably,
the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 68, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 80,
the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 73, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 83,
the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 84,
the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 76, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 86,
The polypeptide according to any one of claims 18 to 25 , wherein the Titin-T chain comprises the polypeptide shown in SEQ ID NO: 75, and the Obscurin-O chain comprises the polypeptide shown in SEQ ID NO: 86. Composite . selected from the bispecific antibodies described in any one of the following A to G, i.e.
A) The sequence of the first heavy chain is as shown in SEQ ID NO: 89, or has at least 85% sequence identity with SEQ ID NO: 89, and the sequence of the first light chain is as shown in SEQ ID NO: 90. or has at least 85% sequence identity with SEQ ID NO: 90, and the sequence of the second heavy chain is as shown in SEQ ID NO: 87, or has at least 85% sequence identity with SEQ ID NO: 87. , the sequence of the second light chain is as shown in SEQ ID NO: 88, or has at least 85% sequence identity with SEQ ID NO: 88;
B) The sequence of the first heavy chain is as shown in SEQ ID NO: 93, or has at least 85% sequence identity with SEQ ID NO: 93, and the sequence of the first light chain is as shown in SEQ ID NO: 94. or has at least 85% sequence identity with SEQ ID NO: 94, and the sequence of the second heavy chain is as shown in SEQ ID NO: 91, or has at least 85% sequence identity with SEQ ID NO: 91. , the sequence of the second light chain is as shown in SEQ ID NO: 92, or has at least 85% sequence identity with SEQ ID NO: 92;
C) the sequence of the first heavy chain is as shown in SEQ ID NO: 97, or has at least 85% sequence identity with SEQ ID NO: 97, and the sequence of the first light chain is as shown in SEQ ID NO: 98; or has at least 85% sequence identity with SEQ ID NO: 98, and the sequence of the second heavy chain is as shown in SEQ ID NO: 95, or has at least 85% sequence identity with SEQ ID NO: 95. , the sequence of the second light chain is as shown in SEQ ID NO: 96, or has at least 85% sequence identity with SEQ ID NO: 96;
D) The sequence of the first heavy chain is as shown in SEQ ID NO: 99, or has at least 85% sequence identity with SEQ ID NO: 99, and the sequence of the first light chain is as shown in SEQ ID NO: 100. or has at least 85% sequence identity with SEQ ID NO: 100, and the sequence of the second heavy chain is as shown in SEQ ID NO: 101, or has at least 85% sequence identity with SEQ ID NO: 101. , the sequence of the second light chain is as shown in SEQ ID NO: 96, or has at least 85% sequence identity with SEQ ID NO: 96;
E) The sequence of the first heavy chain is as shown in SEQ ID NO: 102, or has at least 85% sequence identity with SEQ ID NO: 102, and the sequence of the first light chain is as shown in SEQ ID NO: 100. or has at least 85% sequence identity with SEQ ID NO: 100, and the sequence of the second heavy chain is as shown in SEQ ID NO: 95, or has at least 85% sequence identity with SEQ ID NO: 95. , the sequence of the second light chain is as shown in SEQ ID NO: 96, or has at least 85% sequence identity with SEQ ID NO: 96;
F) The sequence of the first heavy chain is as shown in SEQ ID NO: 103, or has at least 85% sequence identity with SEQ ID NO: 103, and the sequence of the first light chain is as shown in SEQ ID NO: 104. or has at least 85% sequence identity with SEQ ID NO: 104, and the sequence of the second heavy chain is as shown in SEQ ID NO: 95, or has at least 85% sequence identity with SEQ ID NO: 95. , the sequence of the second light chain is as shown in SEQ ID NO: 96, or has at least 85% sequence identity with SEQ ID NO: 96;
G) The sequence of the first heavy chain is as shown in SEQ ID NO: 107, or has at least 85% sequence identity with SEQ ID NO: 107, and the sequence of the first light chain is as shown in SEQ ID NO: 108. or has at least 85% sequence identity with SEQ ID NO: 108, and the sequence of the second heavy chain is as shown in SEQ ID NO: 109, or has at least 85% sequence identity with SEQ ID NO: 109. , the sequence of the second light chain is as shown in SEQ ID NO: 110, or has at least 85% sequence identity with SEQ ID NO: 110;
H) The sequence of the first heavy chain is as shown in SEQ ID NO: 122, or has at least 85% sequence identity with SEQ ID NO: 122, and the sequence of the first light chain is as shown in SEQ ID NO: 123. or has at least 85% sequence identity with SEQ ID NO: 123, and the sequence of the second heavy chain is as shown in SEQ ID NO: 116, or has at least 85% sequence identity with SEQ ID NO: 116. , the sequence of the second light chain is as shown in SEQ ID NO: 117, or has at least 85% sequence identity with SEQ ID NO: 117;
I) The sequence of the first heavy chain is as shown in SEQ ID NO: 118, or has at least 85% sequence identity with SEQ ID NO: 118, and the sequence of the first light chain is as shown in SEQ ID NO: 119. or has at least 85% sequence identity with SEQ ID NO: 119, and the sequence of the second heavy chain is as shown in SEQ ID NO: 124, or has at least 85% sequence identity with SEQ ID NO: 124. , the sequence of the second light chain is as shown in SEQ ID NO: 125, or has at least 85% sequence identity with SEQ ID NO: 125;
27. The polypeptide conjugate according to any one of claims 13 to 26 , selected from: A conjugate comprising a polypeptide conjugate according to any one of claims 1 to 27 . A nucleic acid molecule encoding a polypeptide complex according to any one of claims 1 to 27 . A vector comprising a nucleic acid molecule according to claim 29 . A host cell obtained by transformation of the vector according to claim 30 , selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells. one or more pharmaceutically acceptable carriers, excipients or diluents, and selected from:
A pharmaceutical composition comprising any substance selected from the polypeptide complex according to any one of claims 1 to 27 or the complex according to claim 28 .