WO2023011650A1 - Multispecific antibody, and use thereof - Google Patents

Abstract

A multispecific antibody, comprising a first antigen binding portion and a second antigen binding portion. The light chain and heavy chain of the first antigen binding portion and/or the second antigen binding portion comprise a sequence capable of forming a leucine zipper structure pair. The light chain and heavy chain form a dimer by means of the leucine zipper structure pair, thereby effectively reducing mismatch between the light chain and the heavy chain. Further, the heavy chain of the first antigen binding portion and the heavy chain of the second antigen binding portion can reduce mismatch between the heavy chains by means of a knob-into-hole (KIH) structure, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility. The present invention also relates to a method for treating a plurality of diseases by using the present multispecific antibody.

Description

A kind of multispecific antibody and its use technical field

The present invention relates to a novel multispecific antibody and its use.

Background technique

A bispecific antibody is an antibody that can bind to two different antigens or two different epitopes on an antigen to produce an additive or synergistic effect that is superior to that of a single antibody. At present, bispecific antibodies are gradually becoming A new class of therapeutic antibodies.

Existing antibody formats such as CrossMab, DVD-Ig, TrioMab, FIT-Ig, etc. Among these bispecific antibody formats, the general format of an IgG-like bispecific antibody is: one arm binds to the A antigen, and the other One binds to the B antigen and is structurally composed of one half of the A antibody and one half of the B antibody, with a similar size and shape to native IgG. To facilitate downstream development, it is expected that such bispecific antibodies can be easily produced and correctly assembled from a single host cell at high expression levels like normal IgG. But unfortunately, under normal circumstances, there will be random pairing between the light chain and the heavy chain, and between the heavy chain and the heavy chain, and the final assembled antibody molecule will have 16 possibilities, 10 protein molecules, assuming each pairing In the case of the same probability (in fact, the probability will be somewhat different), the biantibody molecule we need will only account for 1/8 or 12.5% of all products.

The light chain-heavy chain interface comprises variable domains (VH-VL) and constant domains (CH1-CL). Several approaches have been applied to the design of the light chain-heavy chain interface to facilitate homologous pairing. Roche swapped the CH1 and CL domains and created the CrossMab platform; MedImmune introduced disulfide bonds instead; Amgen made further electrostatic modifications in the CH1-CL region; The core of WuxiBody is to replace the constant region of Fab with the constant region of TCR to avoid light chain-heavy chain mismatch. However, selective pairing of individual light chain-heavy chains of individual antibodies remains challenging.

Bispecific antibodies cannot automatically control the correct light chain-heavy chain pairing and the correct assembly of two different antibodies, where various mismatches in a random manner can lead to significant product heterogeneity. Therefore, there is an urgent need in the art to design bispecific antibodies with high expression levels, high affinity for antigens, and correct pairing and assembly.

Contents of the invention

The first aspect of the present invention discloses a polypeptide complex comprising a first antigen-binding portion, the first antigen-binding portion comprising:

A first polypeptide comprising a first light chain variable domain (VL1) from N-terminus to C-terminus and a first paired domain (X1) from N-terminus to C-terminus, said VL1 being operable ground to X1, and

A second polypeptide comprising a first heavy chain variable domain (VH1) from N-terminus to C-terminus and a second paired domain (X2) from N-terminus to C-terminus, said VH1 being operable ground to X2, where,

The first antigen-binding portion specifically binds to the first antigen, and the X1 and X2 can form a leucine zipper structure.

In some embodiments, wherein,

1) the C-terminus of VL1 is operably linked to the N-terminus of X1, and

The C-terminal of VH1 is operably linked to the N-terminal of X2; or

2) the N-terminal of VL1 is operably linked to the C-terminal of X1, and

The N-terminal of VH1 is operably connected to the C-terminal of X2; or

3) the N-terminus of VL1 is operably linked to the C-terminus of X1, and the C-terminus of VL1 is further operably linked to the N-terminus of the first domain, the first domain comprising the first CL, and

The N-terminal of the VH1 is operably connected with the C-terminal of X2, and the C-terminal of the VH1 is further operably connected with the N-terminal of the second domain, and the second domain comprises the first CH1; or

4) The C-terminus of VL1 is operably connected to the N-terminus of the first domain, the C-terminus of the first domain is operatively connected to the N-terminus of X1, and the first domain comprises a first CL ,and

The C-terminus of the VH1 is operably connected to the N-terminus of the second domain, the C-terminus of the second domain is operatively connected to the N-terminus of X2, and the second domain comprises the first CH1.

In some embodiments, wherein,

X1 contains Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP structure domain, X2 comprises the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 structural domain, BASE-p1 structural domain, GCN4 structural domain domain or C/EBP domain forming a leucine zipper domain; or

X2 contains Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP structure domain, X1 comprises the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 structural domain, BASE-p1 structural domain, GCN4 structural domain Domain or C/EBP domain forms a leucine zipper domain.

In some embodiments, wherein,

X1 contains the Jun domain and X2 contains the Fos domain;

X1 contains the Fos domain and X2 contains the Jun domain;

X1 contains the Jun domain and X2 contains the FosW domain;

X1 contains the FosW domain and X2 contains the Jun domain;

X1 contains the Myc domain, and X2 contains the Max domain;

X1 contains the Max domain and X2 contains the Myc domain;

X1 contains the WinzipA2 domain, and X2 contains the WinzipB1 domain;

X1 contains the WinzipB1 domain, and X2 contains the WinzipA2 domain;

X1 contains the ACID-p1 domain, and X2 contains the BASE-p1 domain;

X1 contains the BASE-p1 domain, and X2 contains the ACID-p1 domain;

X1 comprises a GCN4 domain and X2 comprises a GCN4 domain; or

X1 contains the C/EBP domain and X2 contains the C/EBP domain.

In some embodiments, wherein,

The Jun domain is a sequence as shown in SEQ ID NO:9, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:9; and/or

The Fos domain is a sequence as shown in SEQ ID NO: 10, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 10; and/or

The FosW domain is a sequence as shown in SEQ ID NO: 29, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 29; and/or

The Max domain is a sequence as shown in SEQ ID NO: 11, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 11; and/or

The Myc domain is a sequence as shown in SEQ ID NO: 12, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 12; and/or

The WinzipA2 domain is a sequence as shown in SEQ ID NO: 13, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 13; and/or

The WinzipB1 domain is a sequence as shown in SEQ ID NO: 14, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 14; and/or

The ACID-p1 domain is a sequence as shown in SEQ ID NO: 33, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 33; and / or

The BASE-p1 domain is a sequence as shown in SEQ ID NO: 34, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 34; and / or

The GCN4 domain is a sequence as shown in SEQ ID NO:35, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:35; and/or

The C/EBP domain is a sequence as shown in SEQ ID NO:36, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:36.

In some embodiments, wherein said VL1 is operably linked to X1 via a first linker L1 and said VH1 is operably linked to X2 via a second linker L2, L1 and L2 may be present or absent, when When L1 and L2 exist, L1 and L2 are each independently selected from the following polypeptide fragments: (G _x S) _y , (T _x G) _y , and (S _x G) _y , where each occurrence of x is independently is an integer of 1, 2, 3, 4, or 5, each occurrence of y is independently an integer of 1, 2, 3, 4, 5, or 6;

Preferably, L1 and L2 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG.

In some embodiments, wherein the first antigen is selected from the group consisting of exogenous antigens, endogenous antigens, autoantigens, neoantigens, viral antigens and tumor antigens.

The invention discloses a multispecific polypeptide complex, which includes:

A first polypeptide complex, which is a polypeptide complex according to any one of the above, which specifically binds to a first antigen, and

a second polypeptide complex comprising a second antigen-binding moiety that specifically binds to a second antigen,

The first polypeptide complex and the second polypeptide complex bind two different antigens, or bind two different epitopes on the same antigen;

Preferably, said second polypeptide complex comprises a second heavy chain variable domain (VH2) from N-terminal to C-terminal and a second light chain variable domain (VL2) from N-terminal to C-terminal, said Mismatch does not easily occur between the VH1 of the first polypeptide complex and the VL2 of the second polypeptide complex, and/or the VH2 of the second polypeptide complex and the VL1 of the first polypeptide complex; preferably, The multispecific polypeptide complex is a bispecific polypeptide complex; preferably, the VH2 and VL2 of the second antigen-binding portion form a second antigen-binding site that specifically binds to a second antigen.

In some embodiments, the second antigen-binding portion of the second polypeptide complex comprises:

a third polypeptide comprising said VL2, said VL2 being operably linked to a third domain or a third pairing domain (X3), said third domain comprising a second CL, and

A fourth polypeptide, said fourth polypeptide comprising VH2, said VH2 being operably linked to a fourth domain or a fourth pairing domain (X4), said fourth domain comprising a second CH1, wherein

The X3 and X4 can form a leucine zipper structure.

In some embodiments, wherein,

1) the C-terminus of VL2 is operably linked to the N-terminus of X3, and

The C-terminal of VH2 is operably linked to the N-terminal of X4; or

2) the N-terminal of VL2 is operably linked to the C-terminal of X3, and

The N-terminal of VH2 is operably linked to the C-terminal of X4; or

3) the C-terminus of the VL2 is operably linked to the N-terminus of the third domain, and

The C-terminus of the VH2 is operably linked to the N-terminus of the fourth domain; or

4) the N-terminal of the VL2 is operably connected to the C-terminal of X3, and the C-terminal of the VL2 is further operably connected to the N-terminal of the third domain, and

The N-terminal of the VH2 is operably connected to the C-terminal of X4, and the C-terminal of the VH2 is further operably connected to the N-terminal of the fourth domain; or

5) the C-terminus of the VL2 is operably linked to the N-terminus of the third domain, the C-terminus of the third domain is operably linked to the N-terminus of X3, and

The C-terminal of the VH2 is operably connected with the N-terminal of the fourth structural domain, and the C-terminal of the fourth structural domain is operatively connected with the N-terminal of X4.

In some embodiments, the first polypeptide complex further comprises a first dimerization domain, the second polypeptide complex further comprises a second dimerization domain, the first dimerization domain domain and a second dimerization domain.

In some embodiments, the C-terminus of the first dimerization domain is operably linked to the N-terminus of the fifth pairing domain (X5), and the C-terminus of the second dimerization domain is operably linked to the sixth The N-terminus of the pairing domain (X6) is operably linked, wherein

The X5 and X6 can form a dimer, preferably the X5 and X6 can form a leucine zipper structure.

In some embodiments, the C-terminus of X2 of the first polypeptide complex is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of X4 of the second polypeptide complex operably linked to the N-terminus of said second dimerization domain; or

The C-terminus of X2 of the first polypeptide complex is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the fourth domain of the second polypeptide complex is operatively linked to the The N-terminus of the second dimerization domain is operably linked; or

The C-terminus of the second domain of the first polypeptide complex is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the fourth domain of the second polypeptide complex is operably linked to the N-terminus of said second dimerization domain; or

The C-terminal of the VH1 of the first polypeptide complex is operably linked to the N-terminal of the first dimerization domain, and the C-terminal of the VH2 of the second polypeptide complex is operably linked to the second two the N-terminus of the polymerization domain is operably linked;

Preferably, the first dimerization domain and the second dimerization domain are combined by means selected from the following: antibody hinge region or part thereof, linker, disulfide bond, hydrogen bond, electrostatic interaction, Salt bridges, hydrophobic-hydrophilic interactions, or combinations thereof;

More preferably, the first dimerization domain and the second dimerization domain are combined through the following means: antibody hinge region or part thereof, more preferably IgG1, IgG2, IgG3 or IgG4 hinge region or its part.

In some embodiments, the first dimerization domain comprises a first CH2 and/or a first CH3, and/or the second dimerization domain comprises a second CH2 and/or a second CH3;

Preferably, said first CH2, first CH3, second CH2, and second CH3 are derived from IgG1, IgG2, IgG3 or IgG4.

In some embodiments, the first dimerization domain and the second dimerization domain are not identical and associate in a manner that prevents homodimerization and/or favors heterodimerization;

Preferably, the first dimerization domain and the second dimerization domain are combined by means selected from the group consisting of knob-into-hole, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and increased way of flexibility;

Further preferably, the first dimerization domain and the second dimerization domain are combined in a knob-into-hole manner.

In some embodiments, wherein,

X3 and X5 each independently contain Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP structural domain, X4 or X6 each independently comprise the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 structure domain, BASE-p1 domain, GCN4 domain, or C/EBP domain forming a leucine zipper domain; or

X4 and X6 each independently contain Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP structural domain, X3 or X5 each independently comprise the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 structure domain, BASE-p1 domain, GCN4 domain or C/EBP domain forms a leucine zipper domain.

In some embodiments, wherein,

X3 and X5 each independently comprise a Jun domain, and X4 and X6 each independently comprise a Fos domain;

X3 and X5 each independently comprise a Fos domain, and X4 and X6 each independently comprise a Jun domain;

X3 and X5 each independently comprise a Jun domain, and X4 and X6 each independently comprise a FosW domain;

X3 and X5 each independently comprise a FosW domain, and X4 and X6 each independently comprise a Jun domain;

X3 and X5 each independently comprise a Myc domain, and X4 and X6 each independently comprise a Max domain;

X3 and X5 each independently comprise a Max domain, and X4 and X6 each independently comprise a Myc domain;

X3 and X5 each independently comprise a WinzipA2 domain, and X4 and X6 each independently comprise a WinzipB1 domain;

X3 and X5 each independently comprise a WinzipB1 domain, and X4 and X6 each independently comprise a WinzipA2 domain;

X3 and X5 each independently comprise an ACID-p1 domain, and X3 and X5 each independently comprise a BASE-p1 domain;

X3 and X5 each independently comprise a BASE-p1 domain, and X3 and X5 each independently comprise an ACID-p1 domain;

X3 and X5 each independently comprise a GCN4 domain, and X3 and X5 each independently comprise a GCN4 domain; or

X3 and X5 each independently comprise a C/EBP domain, and X3 and X5 each independently comprise a C/EBP domain;

Preferably, the Jun domain, Fos domain, FosW domain, Max domain, Myc domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain and C The /EBP domain is as defined above.

In some embodiments, wherein said VL2 is operably linked to X3 via a third linker L3, said VH2 is operably linked to X4 via a fourth linker L4, each of L3 and L4 is independently present or absent , when L3 and L4 exist, each of L3 and L4 is independently selected from the following polypeptide fragments: (G _x S) _y , (T _x G) _y , and (S _x G) _y , where each occurrence of x is each is independently an integer of 1, 2, 3, 4, or 5, each occurrence of y is independently an integer of 1, 2, 3, 4, 5, or 6;

Preferably, L3 and L4 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG.

In some embodiments, wherein the polypeptide complex is a domain-engineered antibody or an antigen-binding fragment thereof.

Aspects of the invention disclose a multispecific antibody comprising a first antigen binding portion and a second antigen binding portion, wherein,

The first antigen binding moiety comprises:

a first light chain comprising a first light chain variable domain (VL1) operably linked to a first pairing domain (X1), and

A first heavy chain comprising a first heavy chain variable domain (VH1) operably linked to a second paired domain (X2), wherein,

The first antigen-binding portion specifically binds to the first antigen, and the X1 and X2 can form a leucine zipper structure;

said second antigen binding portion comprises a second light chain and a second heavy chain;

The first antigen and the second antigen are not identical, or the first antigen and the second antigen are two different epitopes on the same antigen;

Preferably, mismatches between the first heavy chain and the second light chain, and between the second heavy chain and the first light chain are not prone to occur;

Preferably, said multispecific antibody is a bispecific antibody.

In some embodiments, wherein,

1) the C-terminus of VL1 is operably linked to the N-terminus of X1, and

The C-terminal of VH1 is operably linked to the N-terminal of X2; or

2) the N-terminal of VL1 is operably linked to the C-terminal of X1, and

The N-terminal of VH1 is operably connected to the C-terminal of X2; or

3) the N-terminus of VL1 is operably linked to the C-terminus of X1, and the C-terminus of VL1 is further operably linked to the N-terminus of the first domain, the first domain comprising the first CL, and

The N-terminal of VH1 is operably connected to the C-terminal of X2, and the C-terminal of VH1 is operably connected to the N-terminal of CH1; or

4) The C-terminus of VL1 is operably connected to the N-terminus of the first domain, the C-terminus of the first domain is operatively connected to the N-terminus of X1, and the first domain comprises the first CL, and

The C-terminus of the VH1 is operably connected to the N-terminus of the second domain, the C-terminus of the second domain is operatively connected to the N-terminus of X2, and the second domain comprises the first CH1. In some embodiments, wherein,

said second light chain comprises a second light chain variable domain (VL2), said VL2 being operably linked to a second CL, said second heavy chain comprising a second heavy chain variable domain (VH2), said VH2 is operably linked to a second CH1; or

The second light chain comprises a second light chain variable domain (VL2) operably linked to a third paired domain (X3), the second heavy chain comprises a second heavy chain variable domain (VH2), the VH2 is operably linked to the fourth pairing domain (X4), and the X3 and X4 can form a leucine zipper structure.

In some embodiments, wherein,

1) the C-terminus of VL2 is operably linked to the N-terminus of X3, and

The C-terminal of VH2 is operably linked to the N-terminal of X4; or

2) the N-terminal of VL2 is operably linked to the C-terminal of X3, and

The N-terminal of VH2 is operably linked to the C-terminal of X4; or

3) the C-terminal of VL2 is operably linked to the N-terminal of the second CL, and

The C-terminal of VH2 is operably linked to the N-terminal of CH1; or

4) the N-terminal of VL2 is operably connected to the C-terminal of X3, and the C-terminal of VL2 is further operably connected to the N-terminal of the second CL, and

The N-terminal of VH2 is operably connected to the C-terminal of X4, and the C-terminal of VH2 is further operably connected to the N-terminal of CH1; or

5) the C-terminal of VL2 is operably connected to the N-terminal of the second CL, the C-terminal of the second CL is operatively connected to the N-terminal of X3, and

The C-terminal of VH2 is operably connected with the N-terminal of CH1, and the C-terminal of CH1 is operatively connected with the N-terminal of X4.

In some embodiments, wherein,

1) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), and a second paired domain (X2); and

The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2) and constant domain (CH1); or

2) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), and a second paired domain (X2); and

The second light chain comprises a second light chain variable domain (VL2) and a third pairing domain (X3) from the N-terminus to the C-terminus, and the second heavy chain comprises a second light chain from the N-terminus to the C-terminus a heavy chain variable domain (VH2), and a fourth paired domain (X4); or

3) The first light chain comprises the first pairing domain (X1), the first light chain variable domain (VL1) and the second CL from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to The C-terminus comprises the second pairing domain (X2), the first heavy chain variable domain (VH1) and the first CH1; and

The second light chain comprises a third pairing domain (X3), a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus comprising a fourth paired domain (X4), a second heavy chain variable domain (VH2) and a first CH1; or

4) The first light chain comprises a first pairing domain (X1) and a first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus the second pairing domain (X2), and the first heavy chain variable domain (VH1); and

The second light chain comprises a third pairing domain (X3) and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a fourth a pairing domain (X4), and a second heavy chain variable domain (VH2).

In some embodiments, the first antigen binding moiety further comprises a first dimerization domain, the second antigen binding moiety further comprises a second dimerization domain, the first dimerization domain and The second dimerization domain is associated.

In some embodiments, the C-terminus of the first dimerization domain is operably linked to the N-terminus of the fifth pairing domain (X5), and the C-terminus of the second dimerization domain is operably linked to the sixth The N-terminus of the pairing domain (X6) is operably linked, wherein

The X5 and X6 can form a dimer, preferably the X5 and X6 can form a leucine zipper structure.

In some embodiments,

The C-terminus of X2 of the first antibody is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of CH1 of the second antibody is operably linked to the N-terminus of the second dimerization domain. terminal is operatively connected; or

The C-terminus of the second domain of the first antibody is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the fourth domain of the second antibody is operably linked to the second dimerization domain. The N-terminus of the polymerization domain is operably linked; or

The C-terminus of the VH1 of the first antibody is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the VH2 of the second antibody is operably linked to the N-terminus of the second dimerization domain. terminal is operably connected;

Preferably, the first dimerization domain and the second dimerization domain are combined by means selected from the following: antibody hinge region or part thereof, linker, disulfide bond, hydrogen bond, electrostatic interaction, Salt bridges, hydrophobic-hydrophilic interactions, or combinations thereof;

More preferably, the first dimerization domain and the second dimerization domain are combined through the following means: antibody hinge region or part thereof, more preferably IgG1, IgG2, IgG3 or IgG4 hinge region or its part.

In some embodiments, said first dimerization domain comprises an antibody first CH2 and/or first CH3, and/or said second dimerization domain comprises a second CH2 and/or second CH3;

Preferably, said antibody first CH2, first CH3, second CH2 and second CH3 are derived from IgG1, IgG2, IgG3 or IgG4.

In some embodiments, the first dimerization domain and the second dimerization domain are not identical and associate in a manner that prevents homodimerization and/or favors heterodimerization;

Preferably, the first dimerization domain and the second dimerization domain are combined by means selected from: knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased way of flexibility.

In some embodiments, where

1) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), a second pairing domain (X2), and a first dimerization domain; and

The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2), a second CH1, and a second dimerization domain; or

2) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), a second pairing domain (X2), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises a second light chain variable structure (VL2) domain and a constant domain (CL) from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain can from N-terminus to C-terminus a variable domain (VH2), a constant domain (CH1), a second dimerization domain, and a sixth paired domain (X6); or

3) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), a second pairing domain (X2), and a first dimerization domain; and

The second light chain comprises a second light chain variable domain (VL2) and a third pairing domain (X3) from the N-terminus to the C-terminus, and the second heavy chain comprises a second light chain from the N-terminus to the C-terminus a heavy chain variable domain (VH2), a fourth pairing domain (X4), and a second dimerization domain; or

4) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), a second pairing domain (X2), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises a second light chain variable domain (VL2) and a third pairing domain (X3) from the N-terminus to the C-terminus, and the second heavy chain comprises a second light chain from the N-terminus to the C-terminus a heavy chain variable domain (VH2), a fourth pairing domain (X4), a second dimerization domain, and a sixth pairing domain (X6); or

5) The first light chain comprises a first pairing domain (X1), a first light chain variable domain (VL1) and a first CL from N-terminus to C-terminus, and the first heavy chain is from N-terminus to The C-terminus comprises a second pairing domain (X2), a first heavy chain variable domain (VH1), a first CH1, and a first dimerization domain; and

The second light chain comprises a third pairing domain (X3), a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus comprising a fourth pairing domain (X4), a second heavy chain variable domain (VH2), a second CH1, and a second dimerization domain; or

6) The first light chain comprises a first pairing domain (X1), a first light chain variable domain (VL1) and a first CL from N-terminus to C-terminus, and the first heavy chain is from N-terminus to The C-terminus comprises a second pairing domain (X2), a first heavy chain variable domain (VH1), a first CH1, a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises a third pairing domain (X3), a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus comprising a fourth pairing domain (X4), a second heavy chain variable domain (VH2), a second CH1, a second dimerization domain, and a sixth pairing domain (X6); or

7) The first light chain comprises a first pairing domain (X1) and a first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A second pairing domain (X2), a first heavy chain variable domain (VH1), and a first dimerization domain; and

The second light chain comprises a third pairing domain (X3) and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a fourth A pairing domain (X4), a second heavy chain variable domain (VH2), and a second dimerization domain.

8) The first light chain comprises a first pairing domain (X1) and a first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A second pairing domain (X2), a first heavy chain variable domain (VH1), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises a third pairing domain (X3) and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a fourth A pairing domain (X4), a second heavy chain variable domain (VH2), a second dimerization domain, and a sixth pairing domain (X6).

In some embodiments, wherein,

Each of X1, X3 or X5 independently comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 structural domain or C/EBP structural domain, X2, X4 or X6 each independently comprise the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain forms a leucine zipper domain; or

Each of X2, X4 or X6 independently comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 structural domain or C/EBP structural domain, X1, X3 or X5 each independently comprise the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain forms a domain of a leucine zipper structure.

In some embodiments, wherein,

Each of X1, X3 or X5 independently comprises a Jun domain, and each of X2, X4 or X6 independently comprises a Fos domain;

Each of X1, X3 or X5 independently comprises a Fos domain, and each of X2, X4 or X6 independently comprises a Jun domain;

each of X1, X3 or X5 independently comprises a Jun domain, and each of X2, X4 or X6 comprises a FosW domain;

Each of X1, X3 or X5 independently comprises a FosW domain, and each of X2, X4 or X6 independently comprises a Jun domain;

Each of X1, X3 or X5 independently comprises a Myc domain, and each of X2, X4 or X6 independently comprises a Max domain;

Each of X1, X3 or X5 independently comprises a Max domain, and each of X2, X4 or X6 independently comprises a Myc domain;

Each of X1, X3 or X5 independently comprises a WinzipA2 domain, and each of X2, X4 or X6 independently comprises a WinzipB1 domain;

Each of X1, X3 or X5 independently comprises a WinzipB1 domain, and each of X2, X4 or X6 independently comprises a WinzipA2 domain;

Each of X1, X3 or X5 independently comprises an ACID-p1 domain, and each of X1, X3 or X5 independently comprises a BASE-p1 domain;

Each of X1, X3 or X5 independently comprises a BASE-p1 domain, and each of X1, X3 or X5 independently comprises an ACID-p1 domain;

Each of X1, X3 or X5 is independently a GCN4 domain, and each of X1, X3 or X5 is independently a GCN4 domain; or

Each of X1, X3 or X5 independently comprises a C/EBP domain, and each of X1, X3 or X5 independently comprises a C/EBP domain;

Preferably, the Jun domain, Fos domain, FosW domain, Max domain, Myc domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain and C The /EBP domain is as defined in the first aspect.

In some embodiments, wherein the VL1 is operably linked to X1 via a first linker L1, the VH1 is operably linked to X2 via a second linker L2, and the VL2 is operably linked to a third linker L3 is linked to X3, the VH2 is operably linked to X4 via a fourth linker L4, the C-terminus of the first dimerization domain is linked to X5 via a fifth linker L5, and the second dimerization domain is linked to X5 via a fifth linker L5. The C-terminus of the structural domain is connected to X6 through the sixth linker L6, L1-L6 each independently exists or does not exist, when L1-L6 optionally exists, each L1-L6 is independently selected from the following polypeptide fragments: ( G _x S) _y , (T _x G) _y , and (S _x G) _y , wherein x is an integer selected from 1, 2, 3, 4, or 5, and y is selected from 1, 2, 3, 4, 5 or an integer of 6;

Preferably, L1-L6 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG;

Further preferably, each of L1-L4 is independently connected to a restriction site peptide through its C-terminus, and each of L5-L6 is independently connected to a restriction site peptide through its N-terminal, and the restriction site peptide is selected from factory Xa, thrombin, enterokinase or SUMO, the enzyme cleavage site includes but not limited to the above four polypeptides.

In some embodiments, wherein,

1) The first light chain comprises the first light chain variable domain (VL1), L1 and the first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus comprising a first heavy chain variable domain (VH1), L2, a second pairing domain (X2), and a first dimerization domain; and

The second light chain comprises a second light chain variable structure (VL2) domain and a constant domain (CL) from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain can from N-terminus to C-terminus a variable domain (VH2), a second CH1, and a second dimerization domain; or

2) The first light chain comprises the first light chain variable domain (VL1), L1 and the first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus comprising a first heavy chain variable domain (VH1), L2, a second pairing domain (X2), a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2), the second CH1, the second dimerization domain, L6, and the sixth pairing domain (X6); or

3) The first light chain comprises the first light chain variable domain (VL1), L1 and the first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus comprising a first heavy chain variable domain (VH1), L2, a second pairing domain (X2), and a first dimerization domain; and

The second light chain comprises a second light chain variable domain (VL2), L3 and a third pairing domain (X3) from N-terminus to C-terminus, and the second heavy chain comprises a second light chain variable domain (VL2) from N-terminus to C-terminus A two-heavy chain variable domain (VH2), L4, a fourth pairing domain (X4), and a second dimerization domain; or

4) The first light chain comprises the first light chain variable domain (VL1), L1 and the first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus comprising a first heavy chain variable domain (VH1), L2, a second pairing domain (X2), a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises a second light chain variable domain (VL2), L3 and a third pairing domain (X3) from N-terminus to C-terminus, and the second heavy chain comprises a second light chain variable domain (VL2) from N-terminus to C-terminus Two heavy chain variable domain (VH2), L4, fourth pairing domain (X4), second dimerization domain, L6, and sixth pairing domain (X6); or

5) The first light chain comprises the first pairing domain (X1), L1, the first light chain variable domain (VL1) and the first CL from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminal terminal to C-terminal comprising a second pairing domain (X2), L2, a first heavy chain variable domain (VH1), a first CH1, and a first dimerization domain; and

The second light chain comprises a third paired domain (X3), L3, a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to The C-terminus comprises a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH2), a second CH1, and a second dimerization domain; or

6) The first light chain comprises the first pairing domain (X1), L1, the first light chain variable domain (VL1) and the first CL from the N-terminus to the C-terminus, and the first heavy chain is from N Terminal to C-terminal contains the second pairing domain (X2), L2, the first heavy chain variable domain (VH1), the first CH1, the first dimerization domain, L5, and the fifth pairing domain (X5 );and

The second light chain comprises a third paired domain (X3), L3, a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to The C-terminus comprises a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH2), a second CH1, a second dimerization domain, L6, and a sixth pairing domain (X6); or

7) The first light chain comprises the first pairing domain (X1), L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus The end comprises a second pairing domain (X2), L2, a first heavy chain variable domain (VH1), and a first dimerization domain; and

The second light chain comprises a third pairing domain (X3), L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises from N-terminus to C-terminus A fourth pairing domain (X4), L4, a second heavy chain variable domain (VH2), and a second dimerization domain.

8) The first light chain comprises the first pairing domain (X1), L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus The end comprises a second pairing domain (X2), L2, a first heavy chain variable domain (VH1), a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises a third pairing domain (X3), L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises from N-terminus to C-terminus Fourth pairing domain (X4), L4, second heavy chain variable domain (VH2), second dimerization domain, L6, and sixth pairing domain (X6).

In some embodiments, wherein,

1) The first light chain comprises the first light chain variable domain (VL1), L1 and Fos domain from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Jun domain, and first dimerization domain; and

The second light chain comprises a second light chain variable structure (VL2) domain and a second CL) from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable structure from N-terminus to C-terminus domain (VH2), a second CH1, and a second dimerization domain; or

2) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, first dimerization domain, L5, and Max domain; and

The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2), second CH1, second dimerization domain, L6, and Myc domain; or

3) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, and first dimerization domain; and

The second light chain comprises a second light chain variable domain (VL2), L3 and Fos domain from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus domain (VH2), L4, Jun domain, and a second dimerization domain; or

4) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, first dimerization domain, L5, and Max domain; and

The second light chain comprises a second light chain variable domain (VL2), L3 and Fos domain from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus domain (VH2), L4, Jun domain, second dimerization domain, L6, and Myc domain; or

5) The first light chain comprises Jun domain, L1, the first light chain variable domain (VL1) and the first CL from N-terminus to C-terminus, and the first heavy chain comprises from N-terminus to C-terminus Fos domain, L2, first heavy chain variable domain (VH1), first CH1, and first dimerization domain; and

The second light chain comprises a Fos domain, L3, a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a Jun structure from N-terminus to C-terminus domain, L4, a second heavy chain variable domain (VH2), a second CH1, and a second dimerization domain; or

6) The first light chain comprises Jun domain, L1, the first light chain variable domain (VL1) and the first CL from N-terminus to C-terminus, and the first heavy chain comprises from N-terminus to C-terminus Fos domain, L2, first heavy chain variable domain (VH1), first CH1, first dimerization domain, L5, and WinzipA2 domain; and

The second light chain comprises a Fos domain, L3, a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a Jun structure from N-terminus to C-terminus domain, L4, second heavy chain variable domain (VH2), second CH1, second dimerization domain, L6, and WinzipB1 domain; or

7) The first light chain comprises a Jun domain, L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises a Fos domain from the N-terminus to the C-terminus , L2, the first heavy chain variable domain (VH1), and the first dimerization domain; and

The second light chain comprises a Fos domain, L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a Jun domain, L4 from N-terminus to C-terminus , a second heavy chain variable domain (VH2), and a second dimerization domain; or

8) The first light chain comprises a Jun domain, L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises a Fos domain from the N-terminus to the C-terminus , L2, the first heavy chain variable domain (VH1), the first dimerization domain, L5, and the WinzipA2 domain; and

The second light chain comprises a Fos domain, L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a Jun domain, L4 from N-terminus to C-terminus , the second heavy chain variable domain (VH2), the second dimerization domain, L6, and the WinzipB1 domain; or

9) The first light chain comprises a Jun domain, L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises a FosW domain from the N-terminus to the C-terminus , L2, the first heavy chain variable domain (VH1), and the first dimerization domain; and

The second light chain comprises a Myc domain, L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a Max domain, L4, from N-terminus to C-terminus , a second heavy chain variable domain (VH2), and a second dimerization domain.

10) The first light chain comprises a Jun domain, L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises a FosW domain from the N-terminus to the C-terminus , L2, the first heavy chain variable domain (VH1), the first dimerization domain, L5, and the WinzipA2 domain; and

The second light chain comprises a Myc domain, L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a Max domain, L4, from N-terminus to C-terminus , the second heavy chain variable domain (VH2), the second dimerization domain, L6, and the WinzipB1 domain.

In some embodiments, where

1) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, and first dimerization domain; and

The second light chain comprises a second light chain variable structure (VL2) domain and a constant domain (CL) from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain can from N-terminus to C-terminus a variable domain (VH2), a second CH1, and a second dimerization domain; or

2) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, first dimerization domain, L5, and Max domain; and

The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2), second CH1, second dimerization domain, L6, and Myc domain; or

3) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, and first dimerization domain; and

The second light chain comprises a second light chain variable domain (VL2), L3 and Fos domain from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus domain (VH2), L4, Jun domain, and a second dimerization domain; or

4) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, first dimerization domain, L5, and Max domain; and

The second light chain comprises a second light chain variable domain (VL2), L3 and Fos domain from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus domain (VH2), L4, Jun domain, second dimerization domain, L6, and Myc domain; or

5) The first light chain comprises Jun domain, L1, enzyme cleavage site peptide, first light chain variable domain (VL1) and first CL from N-terminal to C-terminal, and the first heavy chain is from N-terminal to C-terminal comprising Fos domain, L2, cleavage site peptide, first heavy chain variable domain (VH1), first CH1, and first dimerization domain; and

The second light chain comprises Fos domain, L3, enzyme cleavage site peptide, the second light chain variable domain (VL2) and the second CL from the N-terminal to the C-terminal, and the second heavy chain is from the N-terminal to the C-terminus comprising a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH2), a second CH1, and a second dimerization domain; or

6) The first light chain comprises Jun domain, L1, enzyme cleavage site peptide, first light chain variable domain (VL1) and first CL from N-terminal to C-terminal, and the first heavy chain is from N-terminal to C-terminal contains Fos domain, L2, cleavage site peptide, first heavy chain variable domain (VH1), first CH1, first dimerization domain, cleavage site peptide, L5, and the WinzipA2 domain; and

The second light chain comprises Fos domain, L3, enzyme cleavage site peptide, the second light chain variable domain (VL2) and the second CL from the N-terminal to the C-terminal, and the second heavy chain is from the N-terminal To the C-terminus contains Jun domain, L4, cleavage site peptide, second heavy chain variable domain (VH2), second CH1, second dimerization domain, cleavage site peptide, L6, and WinzipB1 domain; or

7) The first light chain comprises a Jun domain, L1, restriction site peptide, and the first light chain variable domain (VL1) from the N-terminal to the C-terminal, and the first heavy chain is from the N-terminal to the C-terminal The C-terminus comprises a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH1), and a first dimerization domain; and

The second light chain comprises a Fos domain, L3, restriction site peptide, and a second light chain variable domain (VL2) from the N-terminus to the C-terminus, and the second heavy chain is from the N-terminus to the C-terminus comprising a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH2), and a second dimerization domain; or

8) The first light chain comprises a Jun domain, L1, restriction site peptide, and the first light chain variable domain (VL1) from the N-terminal to the C-terminal, and the first heavy chain is from the N-terminal to the C-terminal The C-terminus comprises the Fos domain, L2, cleavage site peptide, first heavy chain variable domain (VH1), first dimerization domain, cleavage site peptide, L5, and WinzipA2 domain; and

The second light chain comprises a Fos domain, L3, restriction site peptide, and a second light chain variable domain (VL2) from the N-terminus to the C-terminus, and the second heavy chain is from the N-terminus to the C-terminus comprising a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH2), a second dimerization domain, a cleavage site peptide, L6, and a WinzipB1 domain; or

9) The first light chain comprises a Jun domain, L1, an enzyme cleavage site peptide, and the first light chain variable domain (VL1) from the N-terminal to the C-terminal, and the first heavy chain is from the N-terminal to the C-terminal The C-terminus comprises a FosW domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH1), and a first dimerization domain; and

The second light chain comprises Myc domain, L3, enzyme cleavage site peptide, and the second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus comprising a Max domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH2), and a second dimerization domain; or

10) The first light chain comprises a Jun domain, L1, an enzyme cleavage site peptide, and the first light chain variable domain (VL1) from the N-terminal to the C-terminal, and the first heavy chain is from the N-terminal to the C-terminal The C-terminus comprises the FosW domain, L2, cleavage site peptide, first heavy chain variable domain (VH1), first dimerization domain, cleavage site peptide, L5, and WinzipA2 domain; and

The second light chain comprises Myc domain, L3, enzyme cleavage site peptide, and the second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus Contains Max domain, L4, cleavage site peptide, second heavy chain variable domain (VH2), second dimerization domain, cleavage site peptide, L6, and WinzipB1 domain.

In some embodiments, where

The first dimerization domain and the second dimerization domain are combined by means selected from the following: handle entry structure KIH, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, ways to increase flexibility, or more combination of methods;

More preferably, the first dimerization domain and the second dimerization domain are combined through a handle entry structure KIH (Knob into Hole), and the first dimerization domain and the second dimerization domain Protrusions or recesses are generated in the interface of the interface, and the protrusions or recesses are positioned in the recesses or protrusions in the interface to form a handle entrance structure; preferably the sequence of the first dimerization domain is as SEQ ID NO :7 or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 10, the sequence of the second polymer is as shown in SEQ ID NO: 8 or A sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:810.

The third aspect of the present invention discloses an isolated polynucleotide encoding the polypeptide complex according to any one of the first aspect or the multispecific antibody according to any one of the second aspect.

The fourth aspect of the present invention discloses an isolated vector comprising the polynucleotide described in the third aspect.

The fifth aspect of the present invention discloses a host cell comprising the isolated polynucleotide described in the third aspect, or the isolated vector described in the fourth aspect.

In some embodiments, the host cell is a prokaryotic cell, preferably E. coli.

In some embodiments, the host cell is a eukaryotic cell, preferably, one selected from prokaryotic cells, animal cells, plant cells and fungal cells.

In some embodiments, the cells are selected from one of CHO cells, COS cells, and yeast cells.

The sixth aspect of the present invention discloses a method for preparing the polypeptide complex described in any one of the first aspect or the multispecific antibody described in any one of the second aspect, which comprises the following steps:

a) introducing into the host cell a nucleotide encoding the polypeptide complex according to any one of the first aspect or the multispecific antibody according to any one of the second aspect;

b) allowing said host cell to express said antibody.

In some embodiments, the method further comprises purifying the antibody.

The seventh aspect of the present invention discloses a pharmaceutical composition, which comprises the polypeptide complex according to any one of the first aspect or the multispecific antibody according to any one of the second aspect, and at least one drug With excipients.

The eighth aspect of the present invention discloses that the polypeptide complex described in any one of the first aspects or the multispecific antibody described in any one of the second aspects is used for immunosuppressive treatment, treatment of autoimmune diseases, treatment of Use in medicaments for inflammatory diseases, for treating infectious diseases, for treating allergies, or for treating cancer.

The present invention also discloses a method for treating a disease in a subject, which comprises administering to the subject a therapeutic amount of the polypeptide complex described in any one of the first aspect or any one of the second aspect multispecific antibodies.

The present invention also discloses the polypeptide complex according to any one of the first aspect or the multispecific antibody according to any one of the second aspect, the polypeptide complex or multispecific antibody is used for immunosuppressive treatment, Treat autoimmune disease, treat inflammatory disease, treat infectious disease, treat allergies, or treat cancer.

definition

The "antibody" of the present invention includes antibodies or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof that have been modified on the basis of immunoglobulins while retaining the ability to bind antigens; including monospecific antibodies, multispecific antibodies ( such as bispecific antibodies); also include monovalent or multivalent antibodies. A natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region (VH) and first, second and third constant regions (CH1, CH2, CH3, respectively), while each light chain consists of a variable region (VL) and a constant Area (CL) composition. Antibodies are in the "Y" shape, the backbone of the "Y" structure consisting of the second and third constant domains of the two heavy chains, joined by disulfide bonds. Each arm of the "Y"-shaped structure contains the variable region and the first constant region of one of the heavy chains, which join the variable and constant regions of the light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDR) (the CDR of the light (L) chain includes LCDR1, LCDR2, LCDR3, and the CDR of the heavy (H) chain includes HCDR1, HCDR2, HCDR3 ). Of these, the three CDRs are separated by lateral contiguous portions called framework regions (FR), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. VH and VL each comprise 4 FRs, and the CDRs and FRs are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding, but have various effector functions. Antibodies can be classified into several classes based on the amino acid sequence of the heavy chain constant region. Mammalian antibody heavy chains can be classified into α, δ, ε, γ, and μ, and mammalian antibody light chains can be classified into λ or κ. Antibodies can be divided into five major classes or isotypes based on the presence or absence of alpha, delta, epsilon, gamma, and mu heavy chains: IgA, IgD, IgE, IgG, and IgM, respectively. Several major antibody classes can be further divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain) and so on. In addition to the above-mentioned antibodies with complete constant regions, the antibodies of the present invention also include antibodies with incomplete constant regions, such as only including CH1, including CH1 and CH2, including CH1 and CH3, including CH2 and CH3, etc. In summary, the term "antibody" used in the present invention includes but is not limited to any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific antibody that can bind to a specific antigen, as long as it shows the desired The antigen-binding activity of all are included in the present invention.

According to the number of different target antigens or different epitopes that antibodies bind, they are divided into monospecific antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies..., multispecific antibodies (combining two or two above different target antigens or different epitopes). For example, the term "bispecific antibody" refers to an antibody capable of specifically binding to two different antigens or two different epitopes of the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art; according to the integrity of IgG molecules, they can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies; according to the number and configuration of antigen-binding regions, they can be divided into Bispecific antibodies with bivalent, trivalent, tetravalent or more valence; according to the symmetry of the structure, they can be divided into symmetrical bispecific antibodies and asymmetric bispecific antibodies.

The terms "antigen" or "antigenic molecule" are used herein interchangeably and refer to all molecules capable of being specifically bound by an antibody, a bivalent bispecific antibody specifically binds a first antigen and a second antigen, wherein the first The antigen and the second antigen can be two different antigens, or different epitopes of the same antigen. The term "antigen" or "antigenic molecule" as used herein includes, for example, proteins, different epitopes on proteins (as different antigens within the meaning of the present invention), polysaccharides and the like. Preferably, the antigen is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors cytokines, cell surface proteins, enzymes and receptors. Antigens targeted by the antibodies of the present invention are selected from but not limited to T cell receptors (TCR), natural killer group 2D (NKG2D) receptors, tumor-associated antigens, infectious disease-associated antigens and autoimmune disease-associated antigens A sort of. Antigen specificity can be for any suitable antigen or epitope, eg, exogenous antigens, endogenous antigens, autoantigens, neoantigens, viral antigens, or tumor antigens. Exogenous antigens enter the body through inhalation, ingestion, or injection, and can be presented by antigen-presenting cells (APCs) through endocytosis or phagocytosis and form MHC class II complexes. Endogenous antigens can be produced in normal cells as a result of cellular metabolism, intracellular viral or bacterial infection, which can form MHC class I complexes. Self-antigens (such as peptides, DNA or RNA, etc.) are recognized by the immune system of patients with autoimmune diseases, whereas under normal conditions, this antigen should not be a target of the immune system. Neoantigens are not present in the normal body at all, but are produced as a result of a disease such as a tumor or cancer. In certain embodiments, the antigen is associated with a disease (eg, tumor or cancer, autoimmune disease, infectious and parasitic disease, cardiovascular disease, neurological disease, neuropsychiatric disorder, injury, inflammation, coagulation disorder). In certain embodiments, the antigen is associated with the immune system (eg, immune cells, such as B cells, T cells, NK cells, macrophages, etc.).

The terms "specific binding" and "selective binding" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity ( _KD ) of less than about 10 ^"7M , eg, about less than 10 ^"8M , 10 ^"9M , 10 ^"10M , 10" ^11M , 10" ^12M or less. _KD refers to the dissociation equilibrium ^constant for a particular antibody-antigen interaction, and generally, ^the antibodies disclosed herein have ^a dissociation equilibrium constant (K _D ) Binding to antigen.

The term "Fab fragment" refers to an antigen-binding fragment that can be produced by enzymatically treating an antibody, the fragment comprising the VL-CL of the light chain and the CH-CH1 structure of the heavy chain.

The term "Fc region" or "Fc fragment" refers to a CH2 constant domain and/or a CH3 constant domain of a first heavy chain bound to a CH2 constant domain and/or a CH3 constant domain of a second heavy chain via a disulfide bond And make up the antibody part. The Fc of an antibody is responsible for several different effector functions, such as ADCC and CDC, but is not involved in antigen binding.

The term "electrostatic interactions" are non-covalent interactions and play an important role in protein folding, stability, flexibility and function, including ionic interactions, hydrogen bonding and halogen bonding. Electrostatic interactions can be formed in polypeptides, for example between Lys and Asp, between Lys and Glu, between Glu and Arg, or Glu on the first chain, Trp and Arg on the second chain, Between Val or Thr.

The term "hydrophilic interaction" refers to a molecule with a polar group, which has a large affinity for water, and the molecule can form a temporary bond with water through hydrogen bonding.

The term "hydrophobic interaction" refers to the phenomenon that hydrophobic groups gather close to each other to avoid water.

The term "leucine zipper" is a structural motif that occurs in DNA-binding proteins and other proteins when the hydrophobic faces (often containing leucine residues) of two α-helices from the same or different polypeptide chains ) interact to form a loop-to-loop dimer structure to form a leucine zipper. Specifically, the leucine zipper is composed of stretched amino acids, the seventh amino acid in every 7 amino acids is leucine, leucine is a hydrophobic amino acid, arranged on one side of the alpha helix, all charged amino acids The residues line up on the other side. When two protein molecules are arranged in parallel, the leucine interacts to form a dimer, forming a "zipper" structure. So the leucine zipper domain is a peptide that promotes the oligomerization of proteins in which the leucine zipper domain is present. Known leucine zippers include dimeric or trimeric naturally occurring peptides and derivatives thereof. The leucine zipper structure pair can be selected from: Fos protein/Jun protein, FosW protein/Jun protein, Myc protein/Max protein, WinzipA/WinzipB1, ACID-p1 protein/BASE-p1 protein, GCN4 protein/GCN4 structure Domain, C/EBP protein/C/EBP protein and functional variants of the above proteins/domains. The leucine zipper structure pair described herein can be selected from any of the above groups but is not limited thereto, and any sequence capable of forming the leucine zipper structure can be applied to the present invention. The Jun domain is a sequence as shown in SEQ ID NO: 9, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 9, and the domain can Forming a leucine zipper structure with the Fos domain; the Fos domain is a sequence as shown in SEQ ID NO: 10, or has at least 80%, 85%, 90%, 95%, or with SEQ ID NO: 10 A sequence of 99% identity, the domain can form a leucine zipper structure with the Jun domain; the FosW domain is the sequence shown in SEQ ID NO: 29, or has at least 80 with SEQ ID NO: 29 %, 85%, 90%, 95% or 99% identity sequence, the domain can form a leucine zipper structure with the Jun domain; the Max domain is the sequence shown in SEQ ID NO:11 , or a sequence having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 11, the domain can form a leucine zipper structure with the Myc domain; the Myc domain For a sequence as shown in SEQ ID NO: 12, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 12, said domain is capable of forming with a Max domain Leucine zipper structure; the WinzipA2 domain is a sequence as shown in SEQ ID NO: 13, or a sequence with at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 13 , the domain can form a leucine zipper structure with the WinzipB1 domain; the WinzipB1 domain is a sequence as shown in SEQ ID NO: 14, or has at least 80%, 85%, 90% with SEQ ID NO: 14 %, 95% or 99% identity sequence, the domain can form a leucine zipper structure with the WinzipA2 domain; the ACID-p1 domain is the sequence shown in SEQ ID NO: 33, or with SEQ ID NO:33 ID NO:33 has a sequence of at least 80%, 85%, 90%, 95% or 99% identity, and the domain can form a leucine zipper structure with the BASE-p1 domain; the BASE-p1 domain For a sequence as shown in SEQ ID NO:34, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO:34, the BASE-p1 domain is capable of interacting with ACID -p1 domain forms a leucine zipper structure; the GCN4 domain is a sequence as shown in SEQ ID NO:35, or has at least 80%, 85%, 90%, 95% or 99% with SEQ ID NO:35 A sequence of % identity, the domain can form a leucine zipper structure with the GCN4 domain; and the C The /EBP domain is a sequence as shown in SEQ ID NO: 36, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 36, said domain can be combined with The C/EBP domain forms a leucine zipper structure.

The term "interface" or "contact interface" as used in this application refers to specific regions on the polypeptides that interact/associate with each other. An interface comprises one or more amino acid residues that interact with corresponding amino acid residues that are in contact or associate when the interaction occurs. The amino acid residues in the interface may or may not be in contiguous sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface may be located separately at different positions on the linear sequence.

"Knobs-into-holes" or "Knob into hole" or "KIH", as used in this application, refers to the interaction between two polypeptides, one of which is due to the presence of a amino acid residues (such as tyrosine or tryptophan) to have a protrusion (i.e. "knob"), while another polypeptide has small side chain amino acid residues (such as alanine or threonine) to form a cavity/cavity hole (ie "hole"), and the protrusion can be placed within the cavity/hole to facilitate the interaction of the two polypeptides to form a heterodimer or complex. Methods of producing polypeptides with knobs-into-holes are known in the art, eg, as described in US Pat. No. 5,731,168. Exemplarily, the Knob sequence is T366W, and the Hole sequence is T366S-L368A-Y407V (the numbering sequence of IgG is compiled according to the Kabat numbering rule).

The term "operably linked" or "operably linked" refers to the joining of two or more biological sequences in such a way that they function in the intended manner, whether or not a linker (also called a linker, linker, linker, sequence). When referring to polypeptides, the term refers to the linkage of polypeptide sequences in such a way that the linked product has the intended biological function, with or without a linker between the two sequences. For example, an antibody variable region can be operably linked to a constant region to form a stable product with antigen binding activity. The term also applies to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to regulatory sequences (e.g., promoters, enhancers, silencer sequences, etc.), the term refers to the sequence of the polynucleotide to allow the expression of the polypeptide by the polynucleotide acid-regulated expression.

The term "linker" or "spacer" or "linker" refers to a junction or border region where two polypeptide sequences are fused or combined. For example, the linker domain can comprise at least a portion of the C-terminal segment of the first fusion polypeptide fused to at least a portion of the N-terminal segment of the second fusion polypeptide of Lydia, with or without other linkers in between. For example, the linker can be rich in glycine and proline residues, such as a linker with a single or repeated sequence of threonine/serine composition, GGS, GGGGS, TGGGG, SGGGG or tandem repeats thereof (such as 2,3, or more repetitions).

The term "dimerization domain" refers to domains capable of facilitating association with each other to form dimers. In some embodiments, a first dimerization domain can associate with a second dimerization domain. The association may be associated or linked or bonded by any suitable means; for example, by linkers, disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, or hydrophobic-hydrophilic interactions, or combinations thereof. Exemplary dimerization domains include, but are not limited to, antibody hinge regions, antibody CH2 domains, antibody CH3 domains, and/or other suitable protein monomers or polypeptides capable of dimerizingly interacting with each other. The hinge region, CH2 constant domain, CH3 constant domain may be derived from any antibody isotype, eg IgGl, IgG2, IgG3 and IgG4.

The term "mismatch" refers to the interaction or association of two or more homologous or heterologous polypeptides to form an undesired dimer or polyspecific pairing. "Mismatch is not easy to occur" means, for example, when polypeptides A1, B2 and B2 are co-expressed, if the desired A1-B1 dimer expression is greater than the A1-B2 dimer, it is considered that the A1-B1 Priority pairing occurs between A1-B2, that is, there is no mismatch between A1-B2. In some embodiments, in the bi/multispecific antibody, mismatches between the VL1 of the first light chain and the VL2 of the second heavy chain are less likely to occur, and/or the VL1 of the first heavy chain and the VL2 of the second light chain Mismatching is not easy to occur between VL2, but preferential pairing between VH1-VL1 and preferential pairing between VH2-VL2.

The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single or double-stranded form. Unless expressly limited, the term encompasses polynucleotides that contain known analogs of natural nucleotides that have binding properties similar to a reference nucleic acid and that are similar to naturally occurring nucleotides. way is metabolized. Unless otherwise indicated, a particular polynucleotide sequence also implies conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, homologous genes, SNPs, and complementary sequences, as well as the explicitly indicated sequence.

The terms "homology" and "identity" refer to sequence similarity between polynucleotides or between two polypeptides. When it refers to a nucleic acid or fragment thereof, it means that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide substitutions, insertions or deletions, any sequence identity calculation program such as At least about 80%, more preferably at least about 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, calculated by FASTA, BLAST or Gap Or 99% nucleotide sequence identity in nucleotide bases. When applied to polypeptides, it means that two peptide sequences have at least 80% sequence identity, more preferably at least 81%, 85%, when optimally aligned using programs such as Gap or BESTFIT with default gap weights , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, the difference in residue positions that are not identical can be amino acid substitution, deletion or insertions, more preferably, residue positions that are not identical differ by conservative amino acid substitutions.

The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise stated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed by an equilibrium dissociation constant (K _D ), a dissociation constant (K _d ), or an association constant (K _a ). Affinity can be measured by general methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

The term "surface plasmon resonance" refers to an optical phenomenon that allows real-time interactive analysis by detecting changes in protein concentration in a biosensor matrix, for example using the Biacore ^TM system.

The term "vector" used in this application refers to a delivery vehicle into which a polynucleotide encoding a protein can be operably inserted and the protein can be expressed. Usually, the construct will also contain appropriate regulatory sequences. Vectors can be used to transform, transduce or transfect host cells, so that the genetic elements carried by them can be expressed in the host cells. The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. A vector may also contain components to facilitate its entry into cells, including but not limited to viral particles, liposomes or protein coats. Vectors include, for example: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage , and animal viruses. Animal virus species used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). In some embodiments, the vector system comprises mammalian, bacterial, yeast systems, etc., and comprises plasmids such as but not limited to pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., and other laboratory use and commercially available vectors. Suitable vectors may comprise plasmid or viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

The vector containing the polynucleotide sequence described in this application can be introduced into a host cell for cloning or gene expression. The term "host cell" used in this application refers to a cell into which an exogenous polynucleotide and/or vector is introduced. Suitable host cells for cloning or expressing the DNA in the vectors described herein are prokaryotic cells, animal cells, plant cells and fungal cells as described above.

The term "pharmaceutical composition" means a mixture containing one or more compounds according to the invention or their physiologically/pharmaceutically acceptable salts or prodrugs and other components, such as physiologically/pharmaceutically acceptable Carriers and excipients used. The purpose of the pharmaceutical composition is to promote the administration of the organism, facilitate the absorption of the active ingredient and thus exert its biological activity.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any inactive substance suitable for use in a formulation/pharmaceutical composition for delivery of an antibody or antigen-binding fragment. Carriers can be antiadherents, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial or antifungal agents), sweeteners, absorption delaying agents, wetting agents Agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and the like Osmotic agents such as sugars, polyols, sorbitol and sodium chloride.

In the multispecific antibody of the present invention, the leucine zipper structure pair sequence contained in the light chain and heavy chain of the first antigen-binding part and/or the second antigen-binding part can effectively reduce the gap between the light chain and the heavy chain. Mismatches help light chains and heavy chains to form heterodimers; further, the handle entrance structure KIH, hydrophobic interaction, Electrostatic interaction, hydrophilic interaction, ways to increase flexibility or a combination of the above methods effectively reduce the mismatch between heavy chains, especially the simultaneous use of KIH and leucine zipper structures to prevent mismatching between heavy chains The effect is better. The antibody structure described above ensures that the antigen-binding sequence is expressed and assembled with minimal or almost no mismatches.

Description of drawings

Figure 1-1: Schematic diagram of the structure of antibody AmF1

Figure 1-2: Schematic diagram of the structure of antibody AmF2

Figure 1-3: Schematic diagram of the structure of antibody AmF3

Figure 1-4: Schematic diagram of the structure of antibody AmF4

Figure 1-5: Schematic diagram of the structure of antibody AmF5

Figure 2-1: SEC-HPLC detection results of antibody AmF1

Figure 2-2: SEC-HPLC detection results of antibody AmF2

Figure 2-3: SEC-HPLC detection results of antibody AmF3

Figure 2-4: SEC-HPLC detection results of antibody AmF5

Fig. 3: The cell killing activity experiment result of antibody AmF2, horizontal axis: Log (antibody concentration, ng/Ml), vertical axis: killing efficiency (%)

Figure 4: Schematic diagram of the structure of antibody BmF

Figure 5: SEC-HPLC detection results of antibody mAb-T

Figure 6: SEC-HPLC detection results of antibody BmF

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Example

The present invention exemplarily constructs a bispecific antibody capable of simultaneously targeting DLL3 and CD3 and a bispecific antibody capable of simultaneously targeting TROP2 and PRLR, to demonstrate the multispecific antibody platform technology according to the concept of the present invention. Examples 1-7 are related experiments on bispecific antibodies that can simultaneously target DLL3 and CD3, and Examples 8-12 are related experiments on bispecific antibodies that can simultaneously target TROP2 and PRLR and monoclonal antibodies that target TROP2.

Target introduction

DLL3: Notch signaling pathway widely exists in various animals and is highly conserved in evolution. It regulates the differentiation of neuroendocrine cells and epithelial cells during embryonic development through the interaction between adjacent cells. Mammals have four Notch receptors (Notch1, Notch2, Notch3, Notch4), and five Notch ligands (DLL1, DLL3, DLL4, Jagged2 and Jagged2). DLL3 (Delta-like protein 3) is one of the five ligands of Notch. DLL3 is usually expressed in the developing central nervous system and plays a key role in somitogenesis. By inhibiting the Notch signaling pathway, DLL3 can not only regulate the process of cell differentiation, growth and proliferation, but also indirectly promote or inhibit the occurrence of tumors. As a ligand of the Notch pathway, DLL3 is highly specifically expressed on the surface of SCLC (small cell lung cancer, small cell lung cancer) cells. DLL3 plays an important role in tumor initiation and epithelial-mesenchymal transition, invasion and metastasis of SCLC.

The CD3:T cell co-receptor is a protein complex composed of four distinct chains (CD3γ, CD3δ and two CD3ε chains). These four chains associate with a molecule known as the T cell receptor (TCR) and the zeta chain to generate an activation signal in T lymphocytes. TCR, zeta chain, and CD3 molecules constitute a TCR complex, where TCR serves as a subunit for recognizing and binding antigens, and CD3 serves as a subunit for transferring and carrying antigen stimuli to signal transduction pathways and ultimately regulating T cell activity. The CD3 protein is present in almost all T cells. The CD3-TCR complex regulates T cell function in innate and adaptive immune responses, as well as cellular and humoral immune functions. These include elimination of pathogenic organisms and control of tumor growth through broad cytotoxic effects. Mouse monoclonal antibodies specific for human CD3, such as OKT3 (Kung et al., Science, 206:347-9 (1979)), were the first generation of CD3 antibodies developed for therapy.

TROP2: Human trophoblast cell surface antigen 2 (TROP2) is a cell surface glycoprotein encoded by the TACSTD2 gene. A large number of clinical studies and literature reports have shown that TROP2 is overexpressed in various epithelial cancers such as gastric cancer, lung cancer, large intestine, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, and esophageal cancer (Tsujikawa et al. (1999) Nat

Genet.21:420–423; Fong et al.(2008) Modpath.21:186–191; Muhlmann et al.(2009) J Clin Path.62:152–158; and Wu et al.(2013) Head&Neck.35 :1373–1378). In contrast, TROP2 is rarely expressed or not expressed in adult normal tissues (Cubas et al. (2009) Biochimica et Biophysica Acta.1796:309–314; Zhang et al. (1997)

Science.276:1268–1272), only a small amount of expression is limited to cells in the epithelial area, and the expression level is lower than that in cancerous tumors, indicating that TROP2 is related to tumor formation. Overexpression of TROP2 in tumor tissue is closely related to poor prognosis of patients (Wang et al. (2008) Mol Cancer Ther7:280-285) and metastasis of cancer cells, and affects the overall survival rate of patients. Therefore, TROP2 has become an attractive target in tumor molecular targeted therapy.

PRLR: The prolactin receptor (PRLR) is able to interact with prolactin binding. PRLR is a single transmembrane receptor belonging to the class I cytokine receptor superfamily. Binding of prolactin to PRLR leads to dimerization of the receptor and intracellular signaling. Signaling through PRLRs has been linked to various processes such as mammary gland development, lactation, reproduction and immune regulation. Furthermore, high levels of PRLR expression have been detected in breast, prostate and other tumor types. Blockade of PRLR signaling has been suggested as a means of treating breast and prostate cancer. (Damiano et al. (2013) Clin. Cancer Res. 19(7):1644-1650).

The present invention is directed at the variable region sequence of DLL3 as follows:

>VL amino acid sequence of anti-DLL3 antibody (VLDLL3)

>VH amino acid sequence of anti-DLL3 antibody (VHDLL3)

The variable region sequence of the present invention for CD3 is as follows:

>VL amino acid sequence of anti-CD3 antibody (VLCD3)

>VH amino acid sequence of anti-CD3 antibody (VHCD3)

The present invention is directed at the variable region sequence of PRLR as follows:

> VL amino acid sequence of anti-PRLR antibody (VLPRLR)

>VH amino acid sequence of anti-PRLR antibody (VHPRLR)

The present invention is directed at the variable region sequence of TROP2 as follows:

> VL amino acid sequence of anti-TROP2 antibody (VLTROP2)

>VH amino acid sequence of anti-TROP2 antibody (VHTROP2)

Other sequences of exemplary bispecific antibodies of the present invention are as follows:

>CL constant domain

>CH1 constant domain

>First dimerization domain

Remarks: In the sequence, the single underlined part is the Hinge hinge region, the ununderlined part is the Fc region, and the bold part is Knob, specifically, T366W (Knob).

>Second dimerization domain

Remarks: In the sequence, the single underlined part is the Hinge hinge region, the ununderlined part is the Fc region, and the bold part is the Hole, specifically, T366S-L368A-Y407V (Hole).

>First light chain sequence of AmF1, AmF2

>First heavy chain sequence of AmF1, AmF2

>Second light chain sequence of AmF1

>Second heavy chain sequence of AmF1

>Second light chain sequence of AmF2

>Second heavy chain sequence of AmF2

>First light chain sequence of AmF3, AmF5

>First heavy chain sequence of AmF3

>Second light chain sequence of AmF3

>Second heavy chain sequence of AmF3

>First light chain sequence of AmF4

>First heavy chain sequence of AmF4

>Second light chain sequence of AmF4

>Second heavy chain sequence of AmF4

>First heavy chain sequence of AmF5

>Second light chain sequence of AmF5

>Second heavy chain sequence of AmF5

>First light chain sequence of BmF

>First heavy chain sequence of BmF

>Second light chain sequence of BmF

>Second heavy chain sequence of BmF

>Light chain sequence of mAb-T

>Heavy chain sequence of mAb-T

>Jun domain

>Fos domain

>Max domain

>Myc domain

>WinzipA2 domain

>WinzipB1 domain

>FosW domain

>ACID-p1 domain

>BASE-p1 domain

>GCN4 domain

>C/EBP domain

Example 1: Design of bispecific antibody molecule DLL3×CD3

The exemplary bispecific antibody of the present invention comprises a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion targets the first antigen DLL3, and the second antigen-binding portion targets the second antigen CD3.

According to the bispecific antibody platform technology of the present invention, the bispecific antibody molecule DLL3×CD3 of the present invention is designed as follows:

Antibody AmF1: combine the C-terminus of the light chain variable domain VLDLL3 (sequence shown in SEQ ID NO: 1) of the monoclonal antibody targeting DLL3 with the N of the Jun domain (sequence shown in SEQ ID NO: 9) The ends are connected through the first linker L1 (GGGGS), and the C-terminal of the heavy chain variable domain VHDLL3 (sequence shown in SEQ ID NO: 2) of the monoclonal antibody targeting DLL3 is connected to the Fos domain (sequence shown in SEQ ID NO: 2). The N-terminus of ID NO: 10) is connected through the second linker L2 (GGGGS); the C of the light chain variable domain VLCD3 (sequence shown in SEQ ID NO: 3) of the monoclonal antibody targeting CD3 The end is connected with the N-terminus of the Fos domain (sequence shown in SEQ ID NO: 10) through the third linker L3 (GGGGS), and the heavy chain variable domain VHCD3 of the monoclonal antibody targeting CD3 (sequence shown in SEQ ID NO: 10) is connected. The C-terminus of ID NO: 4) is connected with the Jun domain (sequence shown in SEQ ID NO: 9) through the fourth linker L4 (GGGGS). The first and second antigen-binding moieties further comprise a first dimerization domain (sequence such as SEQ ID NO: 7), a second dimerization domain (sequence such as SEQ ID NO: 8), the first, second two The polymerization domain comprises a hinge region, a CH2 constant domain, and a CH3 constant domain; and the C-terminus of the CH3 constant domain of the first dimerization domain is connected to the max domain through a fifth linker L5 (GGS) (sequence such as shown in SEQ ID NO: 11), the C-terminal of the CH3 constant domain of the second dimerization domain is connected to the Myc domain (sequence shown in SEQ ID NO: 12) through the sixth linker L6 (GGS), forming As the structure shown in accompanying drawing 1-1, the above-mentioned structure is named AmF1, the first light chain sequence of said AmF1 is shown in SEQ ID NO: 15, and the first heavy chain sequence is shown in SEQ ID NO: 16, so The second light chain sequence of AmF1 is shown in SEQ ID NO:17, and the second heavy chain sequence is shown in SEQ ID NO:18.

Antibody AmF2: combine the C-terminus of the light chain variable domain VLDLL3 (sequence shown in SEQ ID NO: 1) of the monoclonal antibody targeting DLL3 with the N of the Jun domain (sequence shown in SEQ ID NO: 9) The ends are connected through the first linker L1 (GGGGS), and the C-terminal of the heavy chain variable domain VHDLL3 (sequence shown in SEQ ID NO: 2) of the monoclonal antibody targeting DLL3 is connected to the Fos domain (sequence shown in SEQ ID NO: 2). The N-terminus of ID NO:10) is connected through the second linker L2 (GGGGS); the C-terminus of the light chain variable domain VLCD3 (sequence such as SEQ ID NO:3) of the monoclonal antibody targeting CD3 is connected with The N-terminus of the CL constant domain (sequence such as SEQ ID NO:5) is connected, and the C-terminal of the heavy chain variable domain VHCD3 (sequence such as SEQ ID NO:4) of the monoclonal antibody targeting CD3 is connected to the CH1 constant structure Domains (sequence such as SEQ ID NO:6) are connected at the N-terminus; the first and second antigen-binding portions further comprise a first dimerization domain (sequence such as SEQ ID NO:7), a second dimerization domain ( Sequence such as SEQ ID NO: 8), the first and second dimerization domains comprise hinge region, CH2 domain and CH3 domain; and the C-terminus of the CH3 domain of the first dimerization domain is connected by the fifth Body L5 (GGS) connects the max domain (sequence shown in SEQ ID NO: 11), and the C-terminus of the CH3 domain of the second dimerization domain connects the Myc domain (sequence) through the sixth linker L6 (GGS) As shown in SEQ ID NO: 12), form the structure shown in accompanying drawing 1-2, above-mentioned structure is named as AmF2, the first light chain sequence of described AmF2 is shown in SEQ ID NO: 15, the first heavy chain The sequence is shown in SEQ ID NO:16, the second light chain sequence of AmF2 is shown in SEQ ID NO:19, and the second heavy chain sequence is shown in SEQ ID NO:20.

Antibody AmF3: The first light chain contains the Jun domain (sequence such as SEQ ID NO:9)+L1(GGS)+Factor Xa+VLDLL3 (sequence such as SEQ ID NO:1)+CL (sequence Such as SEQ ID NO:5); the first heavy chain comprises Fos structural domain (sequence such as SEQ ID NO:10)+L2(GGS)+Factor Xa+VHDLL3 (sequence such as SEQ ID NO:2) successively from N terminal to C terminal )+CH1 (sequence such as SEQ ID NO:6)+the first dimerization domain (sequence such as SEQ ID NO:7)+FactorXa+L5(GGS)+WinzipA2 domain (sequence such as SEQ ID NO:13); The second light chain comprises Fos domain (sequence such as SEQ ID NO: 10)+L3 (GGS)+FactorXa+VLCD3 (sequence such as SEQ ID NO:3)+CL (sequence such as SEQ ID NO: 3)+CL (sequence such as SEQ ID NO: 3)+CL (sequence such as SEQ ID NO:10)+CL (sequence such as SEQ ID NO:10)+CL from N terminal to C terminal :5); the second heavy chain comprises sequentially from N-terminus to C-terminus: Jun domain (sequence such as SEQ ID NO:9)+L4(GGS)+FactorXa+VHCD3 (sequence such as SEQ ID NO:4)+CH1( Sequence such as SEQ ID NO:6)+second dimerization domain (sequence such as SEQ ID NO:8)+Factor Xa+L6(GGS)+WinzipB1 (sequence such as SEQ ID NO:14). The above antibody forms the structure shown in Figures 1-3, the above structure is named AmF3, the first light chain sequence of AmF3 is shown in SEQ ID NO: 21, and the first heavy chain sequence is shown in SEQ ID NO: 22 As shown, the second light chain sequence of the AmF2 is shown in SEQ ID NO:23, and the second heavy chain sequence is shown in SEQ ID NO:24.

Antibody AmF4: Its structure is shown in Figures 1-4, and the structure is named AmF4. The difference between it and AmF3 is that the light chain of its first and second antigen-binding parts does not contain a CL constant domain, and the first and second antigen-binding parts The heavy chain does not contain a CH1 constant domain. The first light chain sequence of the AmF4 is shown in SEQ ID NO:25, the first heavy chain sequence is shown in SEQ ID NO:26, and the second light chain sequence of the AmF4 is shown in SEQ ID NO:27, The second heavy chain sequence is shown in SEQ ID NO: 28.

Antibody AmF5: Its structure is shown in Figures 1-5, and the structure is named AmF5. The difference between it and AmF3 is that the second pairing domain is the FosW domain (sequence shown in SEQ ID NO: 29) instead of Fos domain, another aspect is that the third paired domain X3 is a myc domain (as shown in SEQ ID NO: 12), and the fourth paired domain and X4 are a max domain (as shown in SEQ ID NO: 11) . The first light chain sequence of the AmF5 is shown in SEQ ID NO:21, the first heavy chain sequence is shown in SEQ ID NO:30, and the second light chain sequence of the AmF5 is shown in SEQ ID NO:31, The second heavy chain sequence is shown in SEQ ID NO:32.

Example 2: Construction of bispecific antibody vectors p-AmF1, p-AmF2, p-AmF3, p-AmF4 and p-AmF5

gene synthesis

The genes expressing the anti-DLL3 and CD3 antibody sequences in Example 1 were constructed, and after codon optimization, they were handed over to Gene Synthesis Company for whole gene synthesis.

Construction of pCGS3 expression vector

Using a seamless cloning approach, the heavy and light chains of the first antigen-binding portion were cloned into the multiple cloning site of the pCGS3 vector, and the heavy and light chains of the second antigen-binding portion were also cloned into the multiple cloning site of the pCGS3 vector point, where the heavy chain was cloned into the BstBI and PacI restriction sites, and the light chain was cloned into the HindIII and XhoI restriction sites. The specific method is as follows:

Mix the enzyme-cut components in Table 1 evenly, and incubate the resulting reaction solution in a metal bath at 37°C for 3 hours; use the ordinary agarose gel DNA recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to carry out the digestion products respectively. Recycle.

Table 1 Heavy chain double enzyme digestion system

T4 DNA ligase (NEB, USA) was used to connect the DNA recovery product and the pCGS3 carrier enzyme digestion recovery product. The specific ligation reaction system is shown in Table 2. The ligation reaction components were mixed, and the evenly mixed reaction solution was placed in a metal bath at 16°C overnight.

Table 2 Ligation reaction system

Transformation and inoculation were performed according to the following steps: Add 5 μL of the above ligation product to 50 μL Trans 5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd.), mix well and place on ice for 30 minutes, then place in a 42°C constant temperature water bath Heat shock for 90s, then put on ice for 1min, finally add 250μL LB medium, shake culture at 37℃, 220rpm for 1h, take 100μL and apply to ampicillin ⁺ (Amp+, Tiangen Biochemical Technology (Beijing) Co., Ltd.) LB culture plate, cultured overnight at 37°C.

Pick a single clone from each LB culture plate, put them into 1.5mL EP tubes, add 300μL Amp+ LB medium to each tube, shake and culture at 37°C and 220rpm for 8h; culture cells for electrophoresis after PCR , Sequence the bacterial solution with the correct band.

Plasmid DNA was extracted according to the following steps: after sequencing the correct bacterial solution, after shaking the bacteria for amplification, the plasmid DNA was extracted using a plasmid mini-extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.).

Continue to carry out the double enzyme digestion and connection of the light chain, and the double enzyme digestion system of the light chain is shown in Table 3:

Table 3 Light chain double enzyme digestion system

The remaining steps are the same as above.

The endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.) extracted the constructed plasmids, named p-AmF1, p-AmF2, p-AmF3, p-AmF4, p-AmF5, and used NanoDrop UV spectrophotometer (U.S. Thermo Fisher Scientific Company) was used to determine the concentration and purity of the plasmid DNA and set aside.

Example 3: Expression of bispecific antibodies p-AmF1, p-AmF2, p-AmF3, p-AmF4 and p-AmF5

Express bispecific antibodies p-AmF1, p-AmF2, p-AmF3, p-AmF4 and p-AmF5 as follows:

HEK-293 cells (Zhuhai Kairui Biotechnology Co., Ltd.) were placed in an 8% _CO2 constant temperature shaker (Shanghai Yiheng Scientific Instrument Co., Ltd.), and cultured at 37°C with constant temperature and vibration at a speed of 125rpm, counted, and determined For the cell density and survival rate, use cells that are in the exponential phase (density of about 4-6×106 cells/ml) and have a survival rate greater than 98% to prepare for transfection;

Prepare two 15ml sterile centrifuge tubes, add 5mL KPM (supplement manufacturer's information) and 100μg sterile plasmid DNA to one of them, gently blow and mix; take the other and add 5mL KPM and 500μL TA-293 (supplement manufacturer's information) information) transfection reagent, pipetting and mixing;

Transfer all the liquid in the above-mentioned centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, mix well by pipetting; stand at room temperature for 10 minutes to prepare the plasmid-carrier complex;

Take out the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and put them back into the _CO2 constant temperature shaker for culture; 24 hours after transfection, add 600 μL 293 cell protein expression enhancer (KE-293, manufacturer) and transient transfection nutrient supplement (KT-Feed 50×, Zhuhai Kairui Biotechnology Co., Ltd.) to increase the expression of the product; on the 5th day after transfection, the cell supernatant was collected.

Example 4: Purification and concentration of bispecific antibodies AmF1, AmF2, AmF3, AmF4, AmF5

Antibody purification

The antibody was purified using an AKTA purifier chromatography, specifically, the above antibody was purified using a Protein A affinity chromatography column.

Prepare Protein A affinity chromatography column buffer (equilibrium buffer: 9.5mM sodium dihydrogen phosphate, 40.5mM disodium hydrogen phosphate, 200mM sodium chloride, pH 7.0; elution buffer: 100mM glycine, 100mM chloride Sodium, adjust the pH value to 3.0; neutralization buffer: 1M Tris, adjust the pH value to 8.0), the buffer solution is filtered through a 0.45 μm filter membrane; after the sample is centrifuged, take the supernatant and filter through a 0.45 μm filter membrane; wash and prepare Pipeline; install the Protein A column, after the column is equilibrated, prepare to load the sample, and adjust the UV to zero; during the sample loading, ensure that no air bubbles enter the pipeline; then wash the breakthrough peak with the equilibration buffer until the UV value drops to the baseline level; Adjust the flow rate to 5.0 mL/min, use the elution buffer to elute the protein from the column and collect it; finally use the neutralization buffer to adjust the pH of the collected solution to about 7.0.

ultrafiltration concentration

Collect the purified product in a 50mL centrifuge tube, add it to the ultrafiltration tube in portions, and centrifuge to 1mL at a speed of 3,800rpm and a temperature of 4°C; Fill the tube with 1xPBS for replacement, centrifuge to 1mL at 3,800rpm and 4°C, and repeat 3 times; collect the liquid on the ultrafiltration tube, aliquot the protein, and store it in a -80°C refrigerator for later use.

Example 5: Purity detection of bispecific antibodies AmF1, AmF2, AmF3, AmF5

BCA (biquinolinecarboxylic acid) quantitative protein concentration

The protein concentration of the ultrafiltration concentrated product obtained in Example 4 was measured with a BCA quantitative kit (Shanghai Beyontian Biotechnology Co., Ltd.).

Antibody Purity Detection by High Performance Liquid Chromatography (SEC-HPLC)

The purity of the antibody was tested as follows: prepare mobile phase (50mM PBS (phosphate buffer) + 150mM NaCl, pH6.8), filter with 0.45um filter membrane, and degas with ultrasonic; filter the protein after ultrafiltration with a syringe filter ;Insert the inner cannula into the glass vial, add 200 μL of the sample to be tested; put the sample into the HPLC sample tank, open the HPLC working software, flush the system with the equilibrium buffer, and observe the baseline change. When the system has reached the equilibrium state, set program to start testing samples. Detection conditions: flow rate 0.5mL/min; time 30min, injection volume 20μL, column temperature 25°C, detection wavelength 214nm; 280nm; isocratic elution; after sample detection, analyze the results.

Example 6: Purity test results of bispecific antibodies AmF1, AmF2, AmF3, AmF5

BCA quantitative protein concentration results ( see Table 4)

Table 4 BCA quantitative protein concentration results

It can be seen from the above BCA quantitative protein concentration results that the supernatant expression level of the antibody obtained in the present invention is relatively high.

High performance liquid chromatography (SEC-HPLC) detection results

After the samples were detected by SEC-HPLC, the SEC-HPLC results of antibodies AmF1, AmF2, AmF3, and AmF5 are shown in Figure 2-1, 2-2, 2-3, 2-4, and details are shown in Table 5:

Table 5 SEC-HPLC detection results

ND means not determined.

It can be seen from the above SEC-HPLC detection results that the purity of the antibody obtained in the present invention is very high.

Example 7: Detection of cell killing activity of bispecific antibody AmF2

Use the DLL3-positive SHP-77 cell line (human small cell lung cancer cells, ATCC) to detect the cell killing activity of CD3/DLL3, the specific method is as follows:

Activated T cells were co-cultured with SHP-77GFP&luc cell line (SHP-77 cell line overexpressing green fluorescent protein (GFP) gene and luciferase (luciferase) gene), and the effect-target ratio of T cells and cancer cells (E: T=5:1). The number of SHP-77GFP&luc cells was 2x10 ⁴ cells/well, the number of T cells was 1x10 ⁵ cells/well, the co-culture system was 200 μl, and the co-culture was 24 hours. The antibody concentration is set to 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml, and the co-culture group (without antibody), the co-culture group (with antibody), and the cancer cell group alone (with or without antibody can be set respectively) ), a separate T cell group, each group set up three replicate wells, and the culture container was selected as a 96-U plate. The specific operation is as follows:

Use R10F (RPMI-1640 medium containing 10% FBS (Gibco), where RPMI-1640 medium was purchased from ATCC) to prepare 8ml 200ng/ml antibody solution, filter it through a 0.22μM filter, and then serially dilute to obtain 500μl 20ng/ml ml antibody solution (50ul+450ul R10F), 500μl 2ng/ml antibody solution;

Resuspend the corresponding number of SHP-77luc&GFP with different concentrations of antibody solution, the concentration is 2x10 ^{^5} cells/ml, prepare T cells with a concentration of 1x10 ^{^6} cells/ml in R10F medium, and add 100 μl of T cells and cancer cells to each well of the co-culture group 100μl of cancer cells and 100μl of R10F were added to the single culture group, and finally mixed evenly with a row gun.

After 24 hours of co-cultivation, the detection was carried out. The substrate detection method was used to detect the killing rate, centrifuged at 500g for 5 minutes, and the supernatant of 100ul was discarded with a pipette (less than 80ul of the supernatant would remain in the well), and One- ^LumiTM was added to restore room temperature. Substrate of Firefly Luciferase Reporter Gene Detection Kit (Beyontian product number: RG055M), 100ul/well, mixed with a pipette and lysed at room temperature for 5min, then transferred all the lysate to a black 96-well plate, enzyme-labeled Calculate the kill rate after the instrument detects.

Graphpad Prism 5.0 analyzes and organizes data, killing rate%=1-luminescence value of co-culture group/luminescence value of individual cancer cells*100%

The experimental results are shown in Figure 3 and Table 6, and the results show that the bispecific antibody AmF2 of the present invention has a strong killing effect on DLL3-positive SHP-77 cells. It further demonstrates that the antibody assembly platform of the present invention can effectively prevent mismatching of bispecific antibodies, and the produced multispecific antibodies have strong specificity.

Table 6 The killing results of bispecific antibody AmF2 on DLL3 positive SHP-77 cells

Example 8: Design of bispecific antibody molecule TROP2×PRLR and monoclonal antibody molecule TROP2

The exemplary bispecific antibody of the present invention comprises a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion targets the first antigen TROP2, and the second antigen-binding portion targets the second antigen PRLR.

According to the bispecific antibody platform technology of the present invention, the bispecific antibody molecule TROP2×PRLR of the present invention is designed as follows:

Antibody BmF: the C-terminus and the Fos domain (sequence shown in SEQ ID NO: 10) of the second light chain variable domain VLPRLR (sequence shown in SEQ ID NO: 37) of the monoclonal antibody targeting PRLR The N-terminal of the VHPRLR (sequence shown in SEQ ID NO: 38) and the C-terminal of the second heavy chain variable domain VHPRLR (sequence shown in SEQ ID NO: 38) of the monoclonal antibody targeting PRLR are connected to the Jun domain by the first linker L1 (GGGGS). (Sequence shown in SEQ ID NO: 9) N-terminus is connected through the second linker L2 (GGGGS); The first light chain variable domain VLTROP2 (sequence such as SEQ ID NO: 39) of the monoclonal antibody targeting TROP2 Shown) and the first heavy chain variable domain VHTROP2 (sequence shown in SEQ ID NO: 40), form the structure shown in Figure 4, the above-mentioned structure is named BmF. The first light chain sequence is shown in SEQ ID NO:41, the first heavy chain sequence is shown in SEQ ID NO:42, the second light chain sequence is shown in SEQ ID NO:43, and the second heavy chain sequence is shown in SEQ ID As shown in NO:44, amino acids T366S-L368A-Y407V (Hole) on the first heavy chain sequence and amino acid T366W (Knob) on the second heavy chain sequence form a KIH (knob-into-hole) structure.

Antibody mAb-T: The monoclonal antibody targeting TROP2 is named mAb-T, the amino acid sequence of the light chain is SEQ ID NO:45, and the amino acid sequence of the heavy chain is SEQ ID NO:46.

Example 9: Construction of bispecific antibody p-BmF and monoclonal antibody p-mAb-T

gene synthesis

The sequences expressing the bispecific antibody BmF and the monoclonal antibody mAb-T in Example 7 were constructed, and after codon optimization, the whole gene synthesis was carried out by Nanjing GenScript, a gene synthesis company.

Construction of pCGS3 expression vector

For bispecific antibody BmF:

Using a seamless cloning approach, the heavy and light chains of the first antigen-binding portion were cloned into the multiple cloning site of the pCGS3 vector, and the heavy and light chains of the second antigen-binding portion were also cloned into the multiple cloning site of the pCGS3 vector point, where the heavy chain was cloned into the BstBI and PacI restriction sites, and the light chain was cloned into the HindIII and XhoI restriction sites.

The specific method is as follows:

Mix the enzyme-cut components in Table 8 evenly, and incubate the resulting reaction solution in a metal bath at 37°C for 3 hours; use the ordinary agarose gel DNA recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to carry out the digestion products respectively. Recycle.

Table 7 Heavy chain double enzyme digestion system

T4 DNA ligase (NEB, USA) was used to connect the DNA recovery product and the pCGS3 carrier digestion recovery product. The specific ligation reaction system is shown in Table 8. Mix the ligation reaction components, and put the evenly mixed reaction solution in a metal bath at 16°C overnight.

Table 8 Ligation reaction system

Transformation and inoculation were carried out as follows: Add 5 μL of the above ligation product to 50 μL Trans 5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd.), mix well and place on ice for 30 minutes, then place in a constant temperature water bath at 42°C Heat shock for 90s, then put on ice for 1min, finally add 250μL LB medium, shake culture at 37℃, 220rpm for 1h, take 100μL and apply to ampicillin+ (Amp+, Tiangen Biochemical Technology (Beijing) Co., Ltd.) LB culture plate (Luria-Bertani medium, broth medium), cultured overnight at 37°C.

Pick single clones from each LB culture plate, put them into 1.5mL EP tubes, add 300μL Amp+ LB medium to each tube, shake and culture at 37°C and 220rpm for 8 hours; perform electrophoresis after culturing cells for PCR. The bacterial solution with the correct band was sequenced. Plasmid DNA was extracted according to the following steps: after sequencing the correct bacterial solution, after shaking the bacteria for amplification, the plasmid DNA was extracted using a plasmid mini-extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.).

Continue to carry out double enzyme digestion and connection of the light chain, and the double enzyme digestion system of the light chain is shown in Table 9:

Table 9 Light chain double enzyme digestion system

The remaining steps are the same as above.

The endotoxin-free plasmid extraction kit (DP117, Tiangen Biochemical Technology Co., Ltd.) extracted the constructed plasmid and named it p-BmF. The concentration and purity of the plasmid DNA were determined with a NanoDrop UV spectrophotometer (Thermo Fisher Scientific, USA). ,spare.

The monoclonal antibody mAb-T sequence was constructed into the pCGS3 expression vector according to the same method as above, and the constructed plasmid was named p-mAb-T.

Example 10: Expression of bispecific antibody p-BmF and monoclonal antibody p-mAb-T

Express bispecific antibody p-BmF and monoclonal antibody p-mAb-T according to the following steps:

293f cells (Zhuhai Kairui Biotechnology Co., Ltd.) were used KOP293 cell culture medium (Zhuhai Kairui, K03252), placed in a 5% _CO2 shaker at 37°C, with a rotation speed of 100-130rpm and a humidity control of more than 75%. nourish. The day before transfection, subculture 293f cells in logarithmic growth phase to 2×10 ⁶ /mL, culture overnight on a shaker (110rpm, 37°C, 5% CO2), and transfect the next day . Before transfection, preheat TA-293 (293 cell suspension chemical transfection reagent) and KPM (serum-free cell transfection buffer solution) at room temperature, measure the cell density and viability, the density is 4×10 ⁶ /mL, the viability More than 97% can be used. Transfection was performed according to the KOP293 Transient Transfection Protein Expression System User Guide. The expression supernatant was harvested by centrifugation 5 days after transfection.

Example 11: Purification of bispecific antibody BmF and monoclonal antibody mAb-T

The expression supernatants of the bispecific antibody p-BmF and the monoclonal antibody p-mAb-T were purified by the following steps: filter with a 0.45uM filter membrane, and use a ProteinA affinity chromatography column to obtain the Fc structure from the expression supernatant Domain antibodies. Prepare Protein A affinity chromatography column buffer: equilibration buffer is 9.5mM sodium dihydrogen phosphate, 40.5mM disodium hydrogen phosphate, pH 7.4; elution buffer is 0.1M glycine, pH 3.0. The eluted antibody was replaced with PBS buffer. Purified bispecific antibody BmF and monoclonal antibody mAb-T were obtained.

Example 12: Purity detection of bispecific antibody BmF and monoclonal antibody mAb-T

Purified bispecific antibody BmF and monoclonal antibody mAb-T were detected by high performance liquid chromatography (SEC-HPLC): prepare mobile phase (50mM PBS (phosphate buffer) + 150mM NaCl, pH6.8), and use 0.45 um filter membrane filtration, ultrasonic degassing; filter the protein after ultrafiltration with a needle filter; insert the inner cannula into the glass vial, add 200 μL of the sample to be tested; put the sample into the HPLC sample tank, and turn on the HPLC work The software flushes the system with the equilibrium buffer, observes the baseline change, and when the system has reached the equilibrium state, sets the program and starts to detect the sample. Detection conditions: flow rate 0.5mL/min; time 30min, injection volume 20μL, column temperature 25°C, detection wavelength 214nm; 280nm; isocratic elution; after sample detection, analyze the results.

Calculated from the results in Fig. 5 and Fig. 6: the purity of the monoclonal antibody mAb-T is above 96% (see Fig. 5), and the purity of the bispecific antibody BmF is above 94% (see Fig. 6). It can be seen from the above results that the purity of the antibody obtained in the present invention is relatively high, and the bispecific antibody BmF of the present invention can effectively prevent the mismatch and assembly between light chain-heavy chain and heavy chain-heavy chain.

Example 13: Affinity of bispecific antibody BmF and monoclonal antibody mAb-T to antigen

Surface plasmon resonance biosensors were used to measure the binding affinities of bispecific antibody BmF and monoclonal antibody mAb-T to TROP2 and PRLR, respectively. Affinity analysis was analyzed by SPR, and the instrument used was Biacore, T200 (purchased from GE Healthcare); mobile phase (GE, BR-1006-69). All reagents and materials were purchased from GE, unless otherwise stated.

Table 10 Specific information of instruments and reagents

Experimental procedure

According to the method described in the instructions of the human Fab capture kit (Cat.#28-9583-25, GE), the human Fab capture molecules were covalently coupled to the CM5 biosensor chip (Cat.#BR-1000-12, GE) to affinity capture the antibody to be tested. The antigen used was human TROP2-his (CAT#10428-H08H-100, Sino biological) antigen or human PRLR antigen (purchased from: Bapses, catalog number: PRR-H52Ha); bispecific antibody BmF and monoclonal antibody mAb- T is an analyte, and the affinity between it and TROP2 or PRLR is detected. After analyte serial dilution (100nM, 50nM, 25nM, 10nM, 5nM), proceed as follows:

Capture antibody: Capture antibody for 60s at a flow rate of 10 μl/min.

Analysis: After the analyte was serially diluted (100nM, 50nM, 25nM, 10nM, 5nM), flowed in at a flow rate of 30μl/min, bound to the antigen for 120s, and then dissociated at a flow rate of 30μl/min for 900s.

Regeneration: Glycine-HCl, pH3.0 solution (GE, BR-1003-57) was used for regeneration at a flow rate of 30 μl/min for 30 seconds.

Processing results: wait for the end of the experiment, and open the analysis software to process the results.

Experimental results

Table 11 Affinity results of bispecific antibody BmF and monoclonal antibody mAb-T to antigen

The results in Table 11 show that the bispecific antibody BmF and monoclonal antibody mAb-T obtained in the present invention have strong affinity to TROP2, and the bispecific antibody BmF has strong affinity to PRLR. This also shows that the antibody assembly platform of the present invention can correctly assemble multispecific antibodies and effectively prevent mismatches.

The above experimental results show that the antibody produced by the antibody assembly platform of the present invention can effectively prevent the mismatch and assembly between light chain-heavy chain and heavy chain-heavy chain.

The present invention has been described through the above-mentioned embodiments, but it should be understood that the above-mentioned embodiments are only for the purpose of illustration and description, and are not intended to limit the present invention to the scope of the described embodiments. In addition, those skilled in the art can understand that the present invention is not limited to the above-mentioned embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications all fall within the scope of the present invention. within. The protection scope of the present invention is defined by the appended claims and their equivalent scope.

Claims (49)

A polypeptide complex comprising a first antigen binding portion comprising: A first polypeptide comprising a first light chain variable domain (VL1) from N-terminus to C-terminus and a first paired domain (X1) from N-terminus to C-terminus, said VL1 being operable ground to X1, and A second polypeptide comprising a first heavy chain variable domain (VH1) from N-terminus to C-terminus and a second paired domain (X2) from N-terminus to C-terminus, said VH1 being operable ground to X2, where, The first antigen-binding portion specifically binds to the first antigen, and the X1 and X2 can form a leucine zipper structure. The polypeptide complex as claimed in claim 1, wherein, 1) the C-terminus of VL1 is operably linked to the N-terminus of X1, and The C-terminal of VH1 is operably linked to the N-terminal of X2; or 2) the N-terminal of VL1 is operably linked to the C-terminal of X1, and The N-terminal of VH1 is operably connected to the C-terminal of X2; or 3) the N-terminus of VL1 is operably linked to the C-terminus of X1, and the C-terminus of VL1 is further operably linked to the N-terminus of the first domain, the first domain comprising the first CL, and The N-terminus of the VH1 is operably connected to the C-terminus of X2, and the C-terminus of the VH1 is further operably connected to the N-terminus of the second domain, the second domain comprising the first CH1; or 4) The C-terminus of VL1 is operably linked to the N-terminus of the first domain, the C-terminus of the first domain is operably linked to the N-terminus of X1, and the first domain comprises a first CL ,and The C-terminus of the VH1 is operably connected to the N-terminus of the second domain, the C-terminus of the second domain is operatively connected to the N-terminus of X2, and the second domain comprises the first CH1. The polypeptide complex as claimed in claim 1 or 2, wherein, X1 contains Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP structure domain, X2 comprises the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 structural domain, BASE-p1 structural domain, GCN4 structural domain domain or C/EBP domain forming a leucine zipper domain; or X2 contains Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP structure domain, X1 comprises the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 structural domain, BASE-p1 structural domain, GCN4 structural domain Domain or C/EBP domain forms a leucine zipper domain. The polypeptide complex according to any one of claims 1-3, wherein, X1 contains the Jun domain and X2 contains the Fos domain; X1 contains the Fos domain and X2 contains the Jun domain; X1 contains the Jun domain and X2 contains the FosW domain; X1 contains the FosW domain and X2 contains the Jun domain; X1 contains the Myc domain, and X2 contains the Max domain; X1 contains the Max domain and X2 contains the Myc domain; X1 contains the WinzipA2 domain, and X2 contains the WinzipB1 domain; X1 contains the WinzipB1 domain, and X2 contains the WinzipA2 domain; X1 contains the ACID-p1 domain, and X2 contains the BASE-p1 domain; X1 contains the BASE-p1 domain, and X2 contains the ACID-p1 domain; X1 comprises a GCN4 domain and X2 comprises a GCN4 domain; or X1 contains the C/EBP domain and X2 contains the C/EBP domain. The polypeptide complex as claimed in claim 3 or 4, wherein, The Jun domain is a sequence as shown in SEQ ID NO:9, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:9; and/or The Fos domain is a sequence as shown in SEQ ID NO: 10, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 10; and/or The FosW domain is a sequence as shown in SEQ ID NO: 29, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 29; and/or The Max domain is a sequence as shown in SEQ ID NO: 11, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 11; and/or The Myc domain is a sequence as shown in SEQ ID NO: 12, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 12; and/or The WinzipA2 domain is a sequence as shown in SEQ ID NO: 13, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 13; and/or The WinzipB1 domain is a sequence as shown in SEQ ID NO: 14, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 14; and/or The ACID-p1 domain is a sequence as shown in SEQ ID NO: 33, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 33; and / or The BASE-p1 domain is a sequence as shown in SEQ ID NO: 34, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 34; and / or The GCN4 domain is a sequence as shown in SEQ ID NO:35, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:35; and/or The C/EBP domain is a sequence as shown in SEQ ID NO:36, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:36. The polypeptide complex according to any one of claims 1-5, wherein the VL1 is operably linked to X1 through a first linker L1, and the VH1 is operably linked to X2 through a second linker L2, L1 and L2 may or may not exist, and when L1 and L2 exist, L1 and L2 are each independently selected from the following polypeptide fragments: (G _x S) _y , (T _x G) _y , and (S _x G) _y , wherein each occurrence of x is independently an integer of 1, 2, 3, 4, or 5, and each occurrence of y is independently an integer of 1, 2, 3, 4, 5, or 6; Preferably, L1 and L2 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG. The polypeptide complex according to any one of claims 1-6, wherein the first antigen is selected from the following antigens: exogenous antigens, endogenous antigens, autoantigens, neoantigens, viral antigens and tumor antigens. A multispecific polypeptide complex comprising: A first polypeptide complex, which is a polypeptide complex according to any one of claims 1-7, which specifically binds to a first antigen, and a second polypeptide complex comprising a second antigen-binding moiety that specifically binds to a second antigen, The first polypeptide complex and the second polypeptide complex bind two different antigens, or bind two different epitopes on the same antigen; Preferably, said second polypeptide complex comprises a second heavy chain variable domain (VH2) from N-terminal to C-terminal and a second light chain variable domain (VL2) from N-terminal to C-terminal, said Mismatch does not easily occur between the VH1 of the first polypeptide complex and the VL2 of the second polypeptide complex, and/or the VH2 of the second polypeptide complex and the VL1 of the first polypeptide complex; preferably, The multispecific polypeptide complex is a bispecific polypeptide complex; preferably, the VH2 and VL2 of the second antigen-binding portion form a second antigen-binding site that specifically binds to a second antigen. The multispecific polypeptide complex of claim 8, the second antigen-binding portion of the second polypeptide complex comprising: a third polypeptide comprising said VL2, said VL2 being operably linked to a third domain or a third pairing domain (X3), said third domain comprising a second CL, and A fourth polypeptide, said fourth polypeptide comprising VH2, said VH2 being operably linked to a fourth domain or a fourth pairing domain (X4), said fourth domain comprising a second CH1, wherein The X3 and X4 can form a leucine zipper structure. The multispecific polypeptide complex of claim 9, wherein, 1) the C-terminus of VL2 is operably linked to the N-terminus of X3, and The C-terminal of VH2 is operably linked to the N-terminal of X4; or 2) the N-terminal of VL2 is operably linked to the C-terminal of X3, and The N-terminal of VH2 is operably linked to the C-terminal of X4; or 3) the C-terminus of the VL2 is operably linked to the N-terminus of the third domain, and The C-terminus of the VH2 is operably linked to the N-terminus of the fourth domain; or 4) the N-terminal of the VL2 is operably connected to the C-terminal of X3, and the C-terminal of the VL2 is further operably connected to the N-terminal of the third domain, and The N-terminal of the VH2 is operably connected to the C-terminal of X4, and the C-terminal of the VH2 is further operably connected to the N-terminal of the fourth domain; or 5) the C-terminus of the VL2 is operably linked to the N-terminus of the third domain, the C-terminus of the third domain is operably linked to the N-terminus of X3, and The C-terminal of the VH2 is operably connected with the N-terminal of the fourth structural domain, and the C-terminal of the fourth structural domain is operatively connected with the N-terminal of X4. The multispecific polypeptide complex according to any one of claims 8-10, wherein the first polypeptide complex further comprises a first dimerization domain, and the second polypeptide complex further comprises a second two a polymerization domain, the first dimerization domain and the second dimerization domain associate. The multispecific polypeptide complex according to claim 11, the C-terminus of the first dimerization domain is operably linked to the N-terminus of the fifth pairing domain (X5), the second dimerization The C-terminus of the domain is operably linked to the N-terminus of the sixth paired domain (X6), wherein The X5 and X6 can form a dimer, preferably the X5 and X6 can form a leucine zipper structure. The multispecific polypeptide complex as claimed in claim 11 or 12, The C-terminus of X2 of the first polypeptide complex is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of X4 of the second polypeptide complex is operably linked to the second dimerization domain. The N-terminus of the polymerization domain is operably linked; or The C-terminus of X2 of the first polypeptide complex is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the fourth domain of the second polypeptide complex is operatively linked to the The N-terminus of the second dimerization domain is operably linked; or The C-terminus of the second domain of the first polypeptide complex is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the fourth domain of the second polypeptide complex is operably linked to the N-terminus of said second dimerization domain; or The C-terminal of the VH1 of the first polypeptide complex is operably linked to the N-terminal of the first dimerization domain, and the C-terminal of the VH2 of the second polypeptide complex is operably linked to the second two the N-terminus of the polymerization domain is operably linked; Preferably, the first dimerization domain and the second dimerization domain are combined by means selected from the following: antibody hinge region or part thereof, linker, disulfide bond, hydrogen bond, electrostatic interaction, Salt bridges, hydrophobic-hydrophilic interactions, or combinations thereof; More preferably, the first dimerization domain and the second dimerization domain are combined through the following means: antibody hinge region or part thereof, more preferably IgG1, IgG2, IgG3 or IgG4 hinge region or its part. The multispecific polypeptide complex according to claim 12 or 13, wherein the first dimerization domain comprises the first CH2 and/or the first CH3, and/or the second dimerization domain comprises the second two CH2 and/or second CH3; Preferably, said first CH2, first CH3, second CH2, and second CH3 are each independently derived from IgG1, IgG2, IgG3 or IgG4. The multispecific polypeptide complex according to any one of claims 12-14, the first dimerization domain and the second dimerization domain are not identical, and to prevent homodimerization and/or Or combine in a way that favors heterodimerization; Preferably, the first dimerization domain and the second dimerization domain are combined by means selected from the group consisting of knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, and increased way of flexibility; Further preferably, the first dimerization domain and the second dimerization domain are combined in a knob-into-hole manner. The multispecific polypeptide complex according to any one of claims 8-15, wherein, X3 and X5 each independently contain Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain Or C/EBP structural domain, X4 and X6 each independently comprise and can be combined with described Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 structure domain, BASE-p1 domain, GCN4 domain, or C/EBP domain forming a leucine zipper domain; or X4 and X6 each independently contain Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain Or C/EBP structural domain, X3 and X5 each independently comprise and can be combined with described Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 structure domain, BASE-p1 domain, GCN4 domain or C/EBP domain forms a leucine zipper domain. The multispecific polypeptide complex according to any one of claims 8-16, wherein, X3 and X5 each independently comprise a Jun domain, and X4 and X6 each independently comprise a Fos domain; X3 and X5 each independently comprise a Fos domain, and X4 and X6 each independently comprise a Jun domain; X3 and X5 each independently comprise a Jun domain, and X4 and X6 each independently comprise a FosW domain; X3 and X5 each independently comprise a FosW domain, and X4 and X6 each independently comprise a Jun domain; X3 and X5 each independently comprise a Myc domain, and X4 and X6 each independently comprise a Max domain; X3 and X5 each independently comprise a Max domain, and X4 and X6 each independently comprise a Myc domain; X3 and X5 each independently comprise a WinzipA2 domain, and X4 and X6 each independently comprise a WinzipB1 domain; X3 and X5 each independently comprise a WinzipB1 domain, and X4 and X6 each independently comprise a WinzipA2 domain; X3 and X5 each independently comprise an ACID-p1 domain, and X3 and X5 each independently comprise a BASE-p1 domain; X3 and X5 each independently comprise a BASE-p1 domain, and X3 and X5 each independently comprise an ACID-p1 domain; X3 and X5 each independently comprise a GCN4 domain, and X3 and X5 each independently comprise a GCN4 domain; or X3 and X5 each independently comprise a C/EBP domain, and X3 and X5 each independently comprise a C/EBP domain; Preferably, the Jun domain, Fos domain, FosW domain, Max domain, Myc domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain and C The /EBP domain is as defined in claim 5. The multispecific polypeptide complex according to any one of claims 8-16, wherein the VL2 is operably linked to X3 through a third linker L3, and the VH2 is operably linked through a fourth linker L4 To X4, L3 and L4 are each independently present or absent, and when L3 and L4 exist, each of L3 and L4 is independently selected from the following polypeptide fragments: (G _x S) _y , (T _x G) _y , and ( S _x G) _y , wherein each occurrence of x is independently an integer of 1, 2, 3, 4, or 5, and each occurrence of y is independently 1, 2, 3, 4, 5, or 6 integer; Preferably, L3 and L4 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG. The polypeptide complex according to any one of claims 1-18, wherein the polypeptide complex is a domain engineered antibody or an antigen-binding fragment thereof. A multispecific antibody comprising a first antigen binding portion and a second antigen binding portion, wherein, The first antigen binding moiety comprises: a first light chain comprising a first light chain variable domain (VL1) operably linked to a first pairing domain (X1), and A first heavy chain comprising a first heavy chain variable domain (VH1) operably linked to a second paired domain (X2), wherein, The first antigen-binding portion specifically binds to the first antigen, and the X1 and X2 can form a leucine zipper structure; said second antigen binding portion comprises a second light chain and a second heavy chain; The first antigen and the second antigen are not identical, or the first antigen and the second antigen are two different epitopes on the same antigen; Preferably, mismatches between the first heavy chain and the second light chain, and between the second heavy chain and the first light chain are not prone to occur; Preferably, said multispecific antibody is a bispecific antibody. The antibody of claim 20, wherein, 1) the C-terminus of VL1 is operably linked to the N-terminus of X1, and The C-terminal of VH1 is operably linked to the N-terminal of X2; or 2) the N-terminal of VL1 is operably linked to the C-terminal of X1, and The N-terminal of VH1 is operably connected to the C-terminal of X2; or 3) the N-terminus of VL1 is operably linked to the C-terminus of X1, and the C-terminus of VL1 is further operably linked to the N-terminus of the first domain, the first domain comprising the first CL, and The N-terminal of VH1 is operably connected to the C-terminal of X2, and the C-terminal of VH1 is operably connected to the N-terminal of CH1; or 4) The C-terminus of VL1 is operably connected to the N-terminus of the first domain, the C-terminus of the first domain is operatively connected to the N-terminus of X1, and the first domain comprises the first CL, and The C-terminus of the VH1 is operably connected to the N-terminus of the second domain, the C-terminus of the second domain is operatively connected to the N-terminus of X2, and the second domain comprises the first CH1. The antibody of claim 20 or 21, wherein, said second light chain comprises a second light chain variable domain (VL2), said VL2 being operably linked to a second CL, said second heavy chain comprising a second heavy chain variable domain (VH2), said VH2 is operably linked to a second CH1; or The second light chain comprises a second light chain variable domain (VL2) operably linked to a third paired domain (X3), the second heavy chain comprises a second heavy chain variable domain (VH2), the VH2 is operably linked to the fourth pairing domain (X4), and the X3 and X4 can form a leucine zipper structure. The antibody of claim 22, wherein, 1) the C-terminus of VL2 is operably linked to the N-terminus of X3, and The C-terminal of VH2 is operably linked to the N-terminal of X4; or 2) the N-terminal of VL2 is operably linked to the C-terminal of X3, and The N-terminal of VH2 is operably linked to the C-terminal of X4; or 3) the C-terminal of VL2 is operably linked to the N-terminal of the second CL, and The C-terminal of VH2 is operably linked to the N-terminal of CH1; or 4) the N-terminal of VL2 is operably connected to the C-terminal of X3, and the C-terminal of VL2 is further operably connected to the N-terminal of the second CL, and The N-terminal of VH2 is operably connected to the C-terminal of X4, and the C-terminal of VH2 is further operably connected to the N-terminal of CH1; or 5) the C-terminal of VL2 is operably connected to the N-terminal of the second CL, the C-terminal of the second CL is operatively connected to the N-terminal of X3, and The C-terminal of VH2 is operably connected with the N-terminal of CH1, and the C-terminal of CH1 is operatively connected with the N-terminal of X4. The antibody of any one of claims 20-23, wherein, 1) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), and a second paired domain (X2); and The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2) and constant domain (CH1); or 2) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), and a second paired domain (X2); and The second light chain comprises a second light chain variable domain (VL2) and a third pairing domain (X3) from the N-terminus to the C-terminus, and the second heavy chain comprises a second light chain from the N-terminus to the C-terminus a heavy chain variable domain (VH2), and a fourth paired domain (X4); or 3) The first light chain comprises the first pairing domain (X1), the first light chain variable domain (VL1) and the second CL from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to The C-terminus comprises the second paired domain (X2), the first heavy chain variable domain (VH1) and the constant domain (CH1); and The second light chain comprises a third pairing domain (X3), a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus comprising a fourth paired domain (X4), a second heavy chain variable domain (VH2) and a constant domain (CH1); or 4) The first light chain comprises a first pairing domain (X1) and a first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus the second pairing domain (X2), and the first heavy chain variable domain (VH1); and The second light chain comprises a third pairing domain (X3) and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a fourth a pairing domain (X4), and a second heavy chain variable domain (VH2). The antibody of any one of claims 20-24, said first antigen-binding portion further comprising a first dimerization domain, said second antigen-binding portion further comprising a second dimerization domain, said The first dimerization domain and the second dimerization domain associate. The antibody according to any one of claim 25, the C-terminus of the first dimerization domain is operably linked to the N-terminus of the fifth pairing domain (X5), the second dimerization domain The C-terminus of is operably linked to the N-terminus of the sixth pairing domain (X6), wherein The X5 and X6 can form a dimer, preferably the X5 and X6 can form a leucine zipper structure. The antibody of claim 25 or 26, The C-terminus of X2 of the first antibody is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of CH1 of the second antibody is operably linked to the N-terminus of the second dimerization domain. terminal is operatively connected; or The C-terminus of the second domain of the first antibody is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the fourth domain of the second antibody is operably linked to the second dimerization domain. The N-terminus of the polymerization domain is operably linked; or The C-terminus of the VH1 of the first antibody is operably linked to the N-terminus of the first dimerization domain, and the C-terminus of the VH2 of the second antibody is operably linked to the N-terminus of the second dimerization domain. terminal is operatively connected; Preferably, the first dimerization domain and the second dimerization domain are combined by means selected from the following: antibody hinge region or part thereof, linker, disulfide bond, hydrogen bond, electrostatic interaction, Salt bridges, hydrophobic-hydrophilic interactions, and combinations thereof; More preferably, the first dimerization domain and the second dimerization domain are combined by means of: antibody hinge region or part thereof, more preferably IgG1, IgG2, IgG3 or IgG4 hinge region or part thereof . The antibody of any one of claims 24-27, wherein the first dimerization domain comprises a first CH2 and/or CH3, and/or the second dimerization domain comprises a second CH2 and/or or CH3; Preferably, said antibody first CH2, first CH3, second CH2 and second CH3 are each independently derived from IgGl, IgG2, IgG3 or IgG4. The antibody of any one of claims 24-28, wherein the first dimerization domain and the second dimerization domain are different, and to prevent homodimerization and/or to facilitate heterodimerization Combined in a way of aggregation; Preferably, the first dimerization domain and the second dimerization domain are combined by means selected from: knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased way of flexibility. The antibody of any one of claims 20-29, wherein 1) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), a second pairing domain (X2), and a first dimerization domain; and The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2), a second CH1, and a second dimerization domain; or 2) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), a second pairing domain (X2), a first dimerization domain, and a fifth pairing domain (X5); and The second light chain comprises a second light chain variable structure (VL2) domain and a constant domain (CL) from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain can from N-terminus to C-terminus a variable domain (VH2), a constant domain (CH1), a second dimerization domain, and a sixth paired domain (X6); or 3) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), a second pairing domain (X2), and a first dimerization domain; and The second light chain comprises a second light chain variable domain (VL2) and a third pairing domain (X3) from the N-terminus to the C-terminus, and the second heavy chain comprises a second light chain from the N-terminus to the C-terminus a heavy chain variable domain (VH2), a fourth pairing domain (X4), and a second dimerization domain; or 4) The first light chain comprises a first light chain variable domain (VL1) and a first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A first heavy chain variable domain (VH1), a second pairing domain (X2), a first dimerization domain, and a fifth pairing domain (X5); and The second light chain comprises a second light chain variable domain (VL2) and a third pairing domain (X3) from the N-terminus to the C-terminus, and the second heavy chain comprises a second light chain from the N-terminus to the C-terminus a heavy chain variable domain (VH2), a fourth pairing domain (X4), a second dimerization domain, and a sixth pairing domain (X6); or 5) The first light chain comprises a first pairing domain (X1), a first light chain variable domain (VL1) and a first CL from N-terminus to C-terminus, and the first heavy chain is from N-terminus to The C-terminus comprises a second pairing domain (X2), a first heavy chain variable domain (VH1), a first CH1, and a first dimerization domain; and The second light chain comprises a third pairing domain (X3), a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus comprising a fourth pairing domain (X4), a second heavy chain variable domain (VH2), a second CH1, and a second dimerization domain; or 6) The first light chain comprises a first pairing domain (X1), a first light chain variable domain (VL1) and a first CL from N-terminus to C-terminus, and the first heavy chain is from N-terminus to The C-terminus comprises a second pairing domain (X2), a first heavy chain variable domain (VH1), a first CH1, a first dimerization domain, and a fifth pairing domain (X5); and The second light chain comprises a third pairing domain (X3), a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus comprising a fourth pairing domain (X4), a second heavy chain variable domain (VH2), a second CH1, a second dimerization domain, and a sixth pairing domain (X6); or 7) The first light chain comprises a first pairing domain (X1) and a first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus A second pairing domain (X2), a first heavy chain variable domain (VH1), and a first dimerization domain; and The second light chain comprises a third pairing domain (X3) and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a fourth A pairing domain (X4), a second heavy chain variable domain (VH2), and a second dimerization domain. 8) The first light chain comprises a first pairing domain (X1) and a first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises from the N-terminus to the C-terminus a second pairing domain (X2), a first heavy chain variable domain (VH1), a first dimerization domain, and a fifth pairing domain (X5); and The second light chain comprises a third pairing domain (X3) and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a fourth A pairing domain (X4), a second heavy chain variable domain (VH2), a second dimerization domain, and a sixth pairing domain (X6). The antibody of any one of claims 20-30, wherein, X1, X3 and X5 each independently contain Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 structural domain or C/EBP structural domain, X2, X4 or X6 each independently comprise the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain forms a leucine zipper domain; or X2, X4 and X6 each independently contain Jun domain, Fos domain, FosW domain, Myc domain, Max domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 structural domain or C/EBP structural domain, X1, X3 or X5 each independently comprise the Jun structural domain, Fos structural domain, FosW structural domain, Myc structural domain, Max structural domain, WinzipA2 structural domain, WinzipB1 structural domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain forms a domain of a leucine zipper structure. The antibody of any one of claims 20-31, wherein, X1, X3 and X5 each independently comprise a Jun domain, and X2, X4 and X6 each independently comprise a Fos domain; X1, X3 and X5 each independently comprise a Fos domain, and X2, X4 and X6 each independently comprise a Jun domain; X1, X3 and X5 each independently comprise a Jun domain, and X2, X4 and X6 each independently comprise a FosW domain; X1, X3 and X5 each independently comprise a FosW domain, and X2, X4 and X6 each independently comprise a Jun domain; X1, X3 and X5 each independently comprise a Myc domain, and X2, X4 and X6 each independently comprise a Max domain; X1, X3 and X5 each independently comprise a Max domain, and X2, X4 and X6 each independently comprise a Myc domain; X1, X3 and X5 each independently comprise a WinzipA2 domain, and X2, X4 and X6 each independently comprise a WinzipB1 domain; X1, X3 and X5 each independently comprise a WinzipB1 domain, and X2, X4 and X6 each independently comprise a WinzipA2 domain; X1, X3 and X5 each independently comprise an ACID-p1 domain, and X1, X3 and X5 each independently comprise a BASE-p1 domain; X1, X3 and X5 each independently comprise a BASE-p1 domain, and X1, X3 and X5 each independently comprise an ACID-p1 domain; X1, X3, and X5 are each independently a GCN4 domain, and X1, X3, and X5 are each independently a GCN4 domain; or X1, X3 and X5 each independently comprise a C/EBP domain, and X1, X3 and X5 each independently comprise a C/EBP domain; Preferably, the Jun domain, Fos domain, FosW domain, Max domain, Myc domain, WinzipA2 domain, WinzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain and C The /EBP domain is as defined in claim 5. The antibody of any one of claims 20-32, wherein the VL1 is operably linked to X1 through a first linker L1, the VH1 is operably linked to X2 through a second linker L2, and the VL2 is operably linked to X3 via a third linker L3, the VH2 is operably linked to X4 via a fourth linker L4, and the C-terminus of the first dimerization domain is linked to X5 via a fifth linker L5 , the C-terminus of the second dimerization domain is connected to X6 through a sixth linker L6, each of L1-L6 independently exists or does not exist, and when L1-L6 optionally exists, each of L1-L6 independently Polypeptide fragments selected from the following: (G _x S) _y , (T _x G) _y , and (S _x G) _y , where x is an integer of 1, 2, 3, 4, or 5 at each occurrence, and y an integer of 1, 2, 3, 4, 5 or 6 in each occurrence; Preferably, L1-L6 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG; Further preferably, each of L1-L4 is independently connected to a restriction site peptide through its C-terminus, and each of L5-L6 is independently connected to a restriction site peptide through its N-terminal, and the restriction site peptide is selected from factory Xa, thrombin, enterokinase, or SUMO. The antibody of any one of claims 20-33, wherein, 1) The first light chain comprises the first light chain variable domain (VL1), L1 and the first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus comprising a first heavy chain variable domain (VH1), L2, a second pairing domain (X2), and a first dimerization domain; and The second light chain comprises a second light chain variable structure (VL2) domain and a constant domain (CL) from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain can from N-terminus to C-terminus a variable domain (VH2), a constant domain (CH1), and a second dimerization domain; or 2) The first light chain comprises the first light chain variable domain (VL1), L1 and the first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus comprising a first heavy chain variable domain (VH1), L2, a second pairing domain (X2), a first dimerization domain, L5, and a fifth pairing domain (X5); and The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2), the second CH1, the second dimerization domain, L6, and the sixth pairing domain (X6); or 3) The first light chain comprises the first light chain variable domain (VL1), L1 and the first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus comprising a first heavy chain variable domain (VH1), L2, a second pairing domain (X2), and a first dimerization domain; and The second light chain comprises a second light chain variable domain (VL2), L3 and a third pairing domain (X3) from N-terminus to C-terminus, and the second heavy chain comprises a second light chain variable domain (VL2) from N-terminus to C-terminus A two-heavy chain variable domain (VH2), L4, a fourth pairing domain (X4), and a second dimerization domain; or 4) The first light chain comprises the first light chain variable domain (VL1), L1 and the first pairing domain (X1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus comprising a first heavy chain variable domain (VH1), L2, a second pairing domain (X2), a first dimerization domain, L5, and a fifth pairing domain (X5); and The second light chain comprises a second light chain variable domain (VL2), L3 and a third pairing domain (X3) from N-terminus to C-terminus, and the second heavy chain comprises a second light chain variable domain (VL2) from N-terminus to C-terminus Two heavy chain variable domain (VH2), L4, fourth pairing domain (X4), second dimerization domain, L6, and sixth pairing domain (X6); or 5) The first light chain comprises the first pairing domain (X1), L1, the first light chain variable domain (VL1) and the first CL from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminal terminal to C-terminal comprising a second pairing domain (X2), L2, a first heavy chain variable domain (VH1), a first CH1, and a first dimerization domain; and The second light chain comprises a third paired domain (X3), L3, a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to The C-terminus comprises a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH2), a second CH1, and a second dimerization domain; or 6) The first light chain comprises the first pairing domain (X1), L1, the first light chain variable domain (VL1) and the first CL from the N-terminus to the C-terminus, and the first heavy chain is from N Terminal to C-terminal contains the second pairing domain (X2), L2, the first heavy chain variable domain (VH1), the first CH1, the first dimerization domain, L5, and the fifth pairing domain (X5 );and The second light chain comprises a third paired domain (X3), L3, a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain is from N-terminus to The C-terminal comprises a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH2), a second CH1, a second dimerization domain, L6, and a sixth pairing domain (X6); or 7) The first light chain comprises the first pairing domain (X1), L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus The end comprises a second pairing domain (X2), L2, a first heavy chain variable domain (VH1), and a first dimerization domain; and The second light chain comprises a third pairing domain (X3), L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises from N-terminus to C-terminus A fourth pairing domain (X4), L4, a second heavy chain variable domain (VH2), and a second dimerization domain. 8) The first light chain comprises the first pairing domain (X1), L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain is from the N-terminus to the C-terminus The end comprises a second pairing domain (X2), L2, a first heavy chain variable domain (VH1), a first dimerization domain, L5, and a fifth pairing domain (X5); and The second light chain comprises a third pairing domain (X3), L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises from N-terminus to C-terminus Fourth pairing domain (X4), L4, second heavy chain variable domain (VH2), second dimerization domain, L6, and sixth pairing domain (X6). The antibody of any one of claims 20-34, wherein, 1) The first light chain comprises the first light chain variable domain (VL1), L1 and Fos domain from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Jun domain, and first dimerization domain; and The second light chain comprises a second light chain variable structure (VL2) domain and a second CL) from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable structure from N-terminus to C-terminus domain (VH2), a second CH1, and a second dimerization domain; or 2) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, first dimerization domain, L5, and Max domain; and The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2), second CH1, second dimerization domain, L6, and Myc domain; or 3) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, and first dimerization domain; and The second light chain comprises a second light chain variable domain (VL2), L3 and Fos domain from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus domain (VH2), L4, Jun domain, and a second dimerization domain; or 4) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, first dimerization domain, L5, and Max domain; and The second light chain comprises a second light chain variable domain (VL2), L3 and Fos domain from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus domain (VH2), L4, Jun domain, second dimerization domain, L6, and Myc domain; or 5) The first light chain comprises Jun domain, L1, the first light chain variable domain (VL1) and the first CL from N-terminus to C-terminus, and the first heavy chain comprises from N-terminus to C-terminus Fos domain, L2, first heavy chain variable domain (VH1), first CH1, and first dimerization domain; and The second light chain comprises a Fos domain, L3, a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a Jun structure from N-terminus to C-terminus domain, L4, a second heavy chain variable domain (VH2), a second CH1, and a second dimerization domain; or 6) The first light chain comprises Jun domain, L1, the first light chain variable domain (VL1) and the first CL from N-terminus to C-terminus, and the first heavy chain comprises from N-terminus to C-terminus Fos domain, L2, first heavy chain variable domain (VH1), first CH1, first dimerization domain, L5, and WinzipA2 domain; and The second light chain comprises a Fos domain, L3, a second light chain variable domain (VL2) and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a Jun structure from N-terminus to C-terminus domain, L4, second heavy chain variable domain (VH2), second CH1, second dimerization domain, L6, and WinzipB1 domain; or 7) The first light chain comprises a Jun domain, L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises a Fos domain from the N-terminus to the C-terminus , L2, the first heavy chain variable domain (VH1), and the first dimerization domain; and The second light chain comprises a Fos domain, L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a Jun domain, L4 from N-terminus to C-terminus , a second heavy chain variable domain (VH2), and a second dimerization domain; or 8) The first light chain comprises a Jun domain, L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises a Fos domain from the N-terminus to the C-terminus , L2, the first heavy chain variable domain (VH1), the first dimerization domain, L5, and the WinzipA2 domain; and The second light chain comprises a Fos domain, L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a Jun domain, L4 from N-terminus to C-terminus , the second heavy chain variable domain (VH2), the second dimerization domain, L6, and the WinzipB1 domain; or 9) The first light chain comprises a Jun domain, L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises a FosW domain from the N-terminus to the C-terminus , L2, the first heavy chain variable domain (VH1), and the first dimerization domain; and The second light chain comprises a Myc domain, L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a Max domain, L4, from N-terminus to C-terminus , a second heavy chain variable domain (VH2), and a second dimerization domain. 10) The first light chain comprises a Jun domain, L1, and the first light chain variable domain (VL1) from the N-terminus to the C-terminus, and the first heavy chain comprises a FosW domain from the N-terminus to the C-terminus , L2, the first heavy chain variable domain (VH1), the first dimerization domain, L5, and the WinzipA2 domain; and The second light chain comprises a Myc domain, L3, and a second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain comprises a Max domain, L4, from N-terminus to C-terminus , the second heavy chain variable domain (VH2), the second dimerization domain, L6, and the WinzipB1 domain. The antibody of any one of claims 20-35, wherein, 1) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, and first dimerization domain; and The second light chain comprises a second light chain variable structure (VL2) domain and a constant domain (CL) from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain can from N-terminus to C-terminus a variable domain (VH2), a second CH1, and a second dimerization domain; or 2) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, first dimerization domain, L5, and Max domain; and The second light chain comprises a second light chain variable structure (VL2) domain and a second CL from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus (VH2), second CH1, second dimerization domain, L6, and Myc domain; or 3) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, and first dimerization domain; and The second light chain comprises a second light chain variable domain (VL2), L3 and Fos domain from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus domain (VH2), L4, Jun domain, and a second dimerization domain; or 4) The first light chain comprises the first light chain variable domain (VL1), L1 and Jun domains from the N-terminus to the C-terminus, and the first heavy chain comprises the first heavy chain from the N-terminus to the C-terminus Variable domain (VH1), L2, Fos domain, first dimerization domain, L5, and Max domain; and The second light chain comprises a second light chain variable domain (VL2), L3 and Fos domain from N-terminus to C-terminus, and the second heavy chain comprises a second heavy chain variable domain from N-terminus to C-terminus domain (VH2), L4, Jun domain, second dimerization domain, L6, and Myc domain; or 5) The first light chain comprises Jun domain, L1, enzyme cleavage site peptide, first light chain variable domain (VL1) and first CL from N-terminal to C-terminal, and the first heavy chain is from N-terminal to C-terminal comprising Fos domain, L2, cleavage site peptide, first heavy chain variable domain (VH1), first CH1, and first dimerization domain; and The second light chain comprises Fos domain, L3, enzyme cleavage site peptide, the second light chain variable domain (VL2) and the second CL from the N-terminal to the C-terminal, and the second heavy chain is from the N-terminal to the C-terminus comprising a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH2), a second CH1, and a second dimerization domain; or 6) The first light chain comprises Jun domain, L1, enzyme cleavage site peptide, first light chain variable domain (VL1) and first CL from N-terminal to C-terminal, and the first heavy chain is from N-terminal to C-terminal contains Fos domain, L2, cleavage site peptide, first heavy chain variable domain (VH1), first CH1, first dimerization domain, cleavage site peptide, L5, and the WinzipA2 domain; and The second light chain comprises Fos domain, L3, enzyme cleavage site peptide, the second light chain variable domain (VL2) and the second CL from the N-terminal to the C-terminal, and the second heavy chain is from the N-terminal To the C-terminus contains Jun domain, L4, cleavage site peptide, second heavy chain variable domain (VH2), second CH1, second dimerization domain, cleavage site peptide, L6, and WinzipB1 domain; or 7) The first light chain comprises a Jun domain, L1, restriction site peptide, and the first light chain variable domain (VL1) from the N-terminal to the C-terminal, and the first heavy chain is from the N-terminal to the C-terminal The C-terminus comprises a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH1), and a first dimerization domain; and The second light chain comprises a Fos domain, L3, restriction site peptide, and a second light chain variable domain (VL2) from the N-terminus to the C-terminus, and the second heavy chain is from the N-terminus to the C-terminus comprising a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH2), and a second dimerization domain; or 8) The first light chain comprises a Jun domain, L1, restriction site peptide, and the first light chain variable domain (VL1) from the N-terminal to the C-terminal, and the first heavy chain is from the N-terminal to the C-terminal The C-terminus comprises the Fos domain, L2, cleavage site peptide, first heavy chain variable domain (VH1), first dimerization domain, cleavage site peptide, L5, and WinzipA2 domain; and The second light chain comprises a Fos domain, L3, restriction site peptide, and a second light chain variable domain (VL2) from the N-terminus to the C-terminus, and the second heavy chain is from the N-terminus to the C-terminus comprising a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH2), a second dimerization domain, a cleavage site peptide, L6, and a WinzipB1 domain; or 9) The first light chain comprises a Jun domain, L1, an enzyme cleavage site peptide, and the first light chain variable domain (VL1) from the N-terminal to the C-terminal, and the first heavy chain is from the N-terminal to the C-terminal The C-terminus comprises a FosW domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH1), and a first dimerization domain; and The second light chain comprises Myc domain, L3, enzyme cleavage site peptide, and the second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus comprising a Max domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH2), and a second dimerization domain; or 10) The first light chain comprises a Jun domain, L1, an enzyme cleavage site peptide, and the first light chain variable domain (VL1) from the N-terminal to the C-terminal, and the first heavy chain is from the N-terminal to the C-terminal The C-terminus comprises the FosW domain, L2, cleavage site peptide, first heavy chain variable domain (VH1), first dimerization domain, cleavage site peptide, L5, and WinzipA2 domain; and The second light chain comprises Myc domain, L3, enzyme cleavage site peptide, and the second light chain variable domain (VL2) from N-terminus to C-terminus, and the second heavy chain is from N-terminus to C-terminus Contains Max domain, L4, cleavage site peptide, second heavy chain variable domain (VH2), second dimerization domain, cleavage site peptide, L6, and WinzipB1 domain. The antibody of any one of claims 20-36, wherein, The first dimerization domain and the second dimerization domain are combined by means selected from the following: handle entry structure KIH, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, ways to increase flexibility, or more combination of methods; More preferably, the first dimerization domain and the second dimerization domain are combined through a handle entry structure KIH (Knob into Hole), and the first dimerization domain and the second dimerization domain Protrusions or recesses are generated in the interface of the interface, and the protrusions or recesses are positioned in the recesses or protrusions in the interface to form a handle entrance structure; preferably the sequence of the first dimerization domain is as SEQ ID NO :7 or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:7, the sequence of the second polymer is as shown in SEQ ID NO:8 or A sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:8. An isolated polynucleotide encoding the polypeptide complex of any one of claims 1-19 or encoding the multispecific antibody of any one of claims 20-37. An isolated vector comprising the polynucleotide of claim 38. A host cell comprising the isolated polynucleotide of claim 38, or the isolated vector of claim 38. The host cell according to claim 40, which is a prokaryotic cell, preferably Escherichia coli. The host cell according to claim 40, which is a eukaryotic cell, preferably, one selected from prokaryotic cells, animal cells, plant cells and fungal cells. The host cell according to claim 40, said cell is selected from one of CHO cells, COS cells and yeast cells. A method for preparing the polypeptide complex according to any one of claims 1-19 or the multispecific antibody according to any one of 20-37, comprising the following steps: a) introducing the nucleotide encoding as claimed in claim 37 into the host cell; b) allowing said host cell to express said antibody. The method of claim 44, further comprising purifying the antibody. A pharmaceutical composition comprising the polypeptide complex according to any one of claims 1-19 or the multispecific antibody according to any one of 20-37 and at least one pharmaceutically acceptable excipient. The polypeptide complex according to any one of claims 1-20 or the multispecific antibody described in any one of claims 20-37 is used in the preparation of immunosuppressive therapy, treatment of autoimmune diseases, treatment of inflammatory diseases, treatment of infectious diseases Use in medicines for disease, treatment of allergies or for the treatment of cancer. A method of treating a disorder in a subject, comprising administering to the subject a therapeutic amount of the polypeptide complex of any one of claims 1-19 or the multispecific antibody of any one of claims 20-37 . The polypeptide complex according to claims 1-19 or the multispecific antibody according to any one of claims 20-37, the polypeptide complex or multispecific antibody is used for immunosuppressive therapy, treatment of autoimmune diseases, Treat inflammatory diseases, treat infectious diseases, treat allergies, or treat cancer.

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