CN117915950A

CN117915950A - Multispecific antibody and application thereof

Info

Publication number: CN117915950A
Application number: CN202280052925.3A
Authority: CN
Inventors: 乔晶; 焦娇; 王娇; 王瑞雪; 孙宇
Original assignee: Gan and Lee Pharmaceuticals Co Ltd
Current assignee: Gan and Lee Pharmaceuticals Co Ltd
Priority date: 2021-08-06
Filing date: 2022-08-05
Publication date: 2024-04-19
Also published as: WO2023011650A1

Abstract

To a multispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, the light and heavy chains of the first antigen-binding portion and/or the second antigen-binding portion comprising sequences capable of forming a leucine zipper structure pair by which the light and heavy chains form a dimer, effective to reduce mismatches between the light and heavy chains; further, the heavy chain of the first antigen binding portion and the heavy chain of the second antigen binding portion reduce mismatches between the heavy chains by way of handle entry structure KIH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility. Also relates to methods of using the multispecific antibodies for treating a variety of diseases.

Description

Multispecific antibody and application thereof

Technical Field

The present invention relates to a novel multispecific antibody and uses thereof.

Background

Bispecific antibodies are an antibody that can bind to two different antigens or two different epitopes on one antigen, thereby producing an additive or synergistic effect over the effect of a single antibody, and are now becoming a new class of therapeutic antibodies.

Existing antibody formats such as cross mab, DVD-Ig, trioMab, FIT-Ig, etc., among which a common format for an IgG-like bispecific antibody is: one arm binds to the a antigen and the other to the B antigen, consisting of half of the a antibody and half of the B antibody, with a size and shape similar to that of natural IgG. To facilitate downstream development, it is desirable that such bispecific antibodies can be readily produced and properly assembled at high expression levels from a single host cell, as normal IgG. Unfortunately, in general, there will be random pairing between the light and heavy chains, 16 possibilities for the final assembled antibody molecule, 10 protein molecules, and, assuming the probability of each pairing is the same (in practice, there will be some difference in probability), the final diabody molecule we need will be only 1/8 or 12.5% of all products.

The interface between the light chain and the heavy chain comprises a variable domain (VH-VL) and a constant domain (CH 1-CL). Several schemes have been applied to the design of the light chain-heavy chain interface to facilitate homologous pairing. Roche exchanged the domains of CH1 and CL and created a CrossMab platform; medImmune alternatively incorporates disulfide linkages; amgen further modifies the electrostatic effect in the CH1-CL region; and Lilly and Genntech introduced mutations in both the variable and constant domains; the core of the medicine's organism WuxiBody is to replace the constant region of Fab by that of TCR, avoiding the mismatch of light chain-heavy chain. However, selective pairing of individual light chain-heavy chain of individual antibodies remains challenging.

The correct pairing of light chain-heavy chain and the correct assembly of two different antibodies cannot be automatically controlled based on bispecific antibodies, wherein various mismatches in a random manner may lead to significant product heterogeneity. Thus, there is a need in the art to design bispecific antibodies with high expression levels, high affinity for antigen, and the ability to pair and assemble correctly.

Disclosure of Invention

In a first aspect, the invention discloses a polypeptide complex comprising a first antigen binding portion comprising:

A first polypeptide comprising a first light chain variable domain from N-terminus to C-terminus (VL 1) and a first pairing domain from N-terminus to C-terminus (X1), said VL1 being operably linked to X1, and

A second polypeptide comprising a first heavy chain variable domain from N-terminus to C-terminus (VH 1) and a second pairing domain from N-terminus to C-terminus (X2), said VH1 being operably linked to X2, wherein,

The first antigen binding portion specifically binds to a first antigen, and the X1 and X2 are capable of forming a leucine zipper structure.

In some embodiments, the method comprises, among other things,

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

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

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

The N-terminus of VH1 is operably linked to the C-terminus of X2; or (b)

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

The N-terminus of VH1 is operably linked to the C-terminus of X2, and the C-terminus of VH1 is further operably linked to the N-terminus of the second domain comprising the first CH1; or (b)

4) The C-terminus of VL1 is operably linked to the N-terminus of a first domain comprising a first CL, the C-terminus of the first domain being operably linked to the N-terminus of X1, and

The C-terminus of VH1 is operably linked to the N-terminus of a second domain comprising the first CH1, which is operably linked to the N-terminus of X2.

In some embodiments, the method comprises, among other things,

X1 comprises a 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 domain, X2 comprises a domain capable of forming a leucine zipper with said 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 domain; or (b)

X2 comprises a 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 domain, and X1 comprises a domain capable of forming a leucine zipper with said 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 domain.

In some embodiments, the method comprises, among other things,

X1 comprises a Jun domain and X2 comprises a Fos domain;

X1 comprises a Fos domain and X2 comprises a Jun domain;

x1 comprises a Jun domain and X2 comprises a FosW domain;

x1 comprises FosW domains and X2 comprises Jun domains;

x1 comprises a Myc domain and X2 comprises a Max domain;

X1 comprises a Max domain and X2 comprises a Myc domain;

X1 comprises WinzipA domains and X2 comprises WinzipB1 domains;

x1 comprises WinzipB1 domains and X2 comprises WinzipA2 domains;

x1 comprises an ACID-p1 domain and X2 comprises a BASE-p1 domain;

X1 comprises a BASE-p1 domain and X2 comprises an ACID-p1 domain;

X1 comprises a GCN4 domain and X2 comprises a GCN4 domain; or (b)

X1 comprises a C/EBP domain and X2 comprises a C/EBP domain.

In some embodiments, the method comprises, among other things,

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 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 to SEQ ID NO. 10; and/or

The FosW domain is a sequence as set forth in SEQ ID NO. 29, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to 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 to SEQ ID NO. 11; and/or

The Myc domain is 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; and/or

The WinzipA domain is a sequence as set forth in SEQ ID NO. 13, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO. 13; and/or

The WinzipB domain is a sequence as set forth in SEQ ID NO. 14, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO. 14; and/or

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

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

The GCN4 domain is the sequence shown as SEQ ID NO. 35, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to 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 to SEQ ID NO. 36.

In some embodiments, 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, L1 and L2 may or may not be present, and when L1 and L2 are present, L1 and L2 are each independently selected from the following polypeptide fragments: (G _xS) _y、(T _xG) _y, and (S _xG) _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.

In some embodiments, wherein the first antigen is selected from the following antigens: foreign antigens, endogenous antigens, autoantigens, neoantigens, viral antigens and tumor antigens.

The invention discloses a multi-specific polypeptide complex, which comprises the following components:

A first polypeptide complex which is a polypeptide complex as defined in any one of the preceding claims which specifically binds to a first antigen, and

A second polypeptide complex comprising a second antigen binding portion that specifically binds to a second antigen,

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

Preferably, the second polypeptide complex comprises a second heavy chain variable domain (VH 2) from N-terminus to C-terminus and a second light chain variable domain (VL 2) from N-terminus to C-terminus, the VH1 of the first polypeptide complex and VL2 of the second polypeptide complex, and/or a mismatch is not likely to occur between VH2 of the second polypeptide complex and VL1 of the first polypeptide complex; preferably, the multispecific polypeptide complex is a bispecific polypeptide complex; preferably, 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 the VL2, the VL2 operably linked to a third domain comprising a second CL or a third mating domain (X3), and

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

The X3 and X4 can form a leucine zipper structure.

In some embodiments, the method comprises, among other things,

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

The C-terminal of VH2 is operably connected to the N-terminal of X4; or (b)

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

The N-terminus of VH2 is operably linked to the C-terminus of X4; or (b)

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

The C-terminal of VH2 is operably linked to the N-terminal of the fourth domain; or (b)

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

The N-terminus of VH2 is operably linked to the C-terminus of X4, and the C-terminus of VH2 is further operably linked to the N-terminus of the fourth domain; or (b)

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

The C-terminus of VH2 is operably linked to the N-terminus of the fourth domain, which is operably linked to the N-terminus of X4.

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

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

The X5 and X6 form dimers, preferably the X5 and X6 form leucine zipper structures.

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 is operably linked to the N-terminus of the second dimerization domain; or (b)

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 operably linked to the N-terminus of the second dimerization domain; or (b)

The C-terminus of the second domain of the first polypeptide complex is operably linked to the N-terminus of the first multimerization domain, and the C-terminus of the fourth domain of the second polypeptide complex is operably linked to the N-terminus of the second multimerization domain; or (b)

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

preferably, the first and second dimerization domains are joined by a means selected from the group consisting of: an antibody hinge region or portion thereof, a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, or a combination thereof;

More preferably, the first and second dimerization domains are bound by: the antibody hinge region or portion thereof binds, more preferably the hinge region or portion thereof of IgG1, igG2, igG3 or IgG 4.

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, the first CH2, first CH3, second CH2, and second CH3 are derived from IgG1, igG2, igG3, or IgG4.

In some embodiments, the first and second dimerization domains are not identical and bind in a manner that prevents homodimerization and/or favors heterodimerization.

Preferably, the first and second dimerization domains are joined by a means selected from the group consisting of: knob-into-hole, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and ways of increasing flexibility;

Further preferably, the first dimerization domain and the second dimerization domain are bound by a knob-intoo-hole manner.

In some embodiments, the method comprises, among other things,

X3 and X5 each independently comprise a Jun domain, fos domain, fosW domain, myc domain, max domain, winzipA2 domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain, X4 or X6 each independently comprise a domain capable of forming a leucine zipper with said 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 domain; or (b)

X4 and X6 each independently comprise a Jun domain, fos domain, fosW domain, myc domain, max domain, winzipA2 domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain, and X3 or X5 each independently comprise a domain capable of forming a leucine zipper with said 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 domain.

In some embodiments, the method comprises, among other things,

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 FosW domains, and X4 and X6 each independently comprise Jun domains;

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 WinzipA domain, and X4 and X6 each independently comprise WinzipB domain;

x3 and X5 each independently comprise WinzipB domain, and X4 and X6 each independently comprise WinzipA 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 (b)

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, winzipA domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain and C/EBP domain are as defined above.

In some embodiments, wherein the VL2 is operably linked to X3 via a third linker L3, the VH2 is operably linked to X4 via a fourth linker L4, L3 and L4 are each independently present or absent, and when L3 and L4 are present, L3 and L4 are each independently selected from the following polypeptide fragments: (G _xS) _y、(T _xG) _y, and (S _xG) _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, 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 antigen binding fragment thereof.

Disclosed herein in its aspects is a multispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein,

The first antigen binding portion comprises:

a first light chain comprising a first light chain variable domain (VL 1), said VL1 being operably linked to a first pairing domain (X1), and

A first heavy chain comprising a first heavy chain variable domain (VH 1), said VH1 being operably linked to a second pairing domain (X2), wherein,

The first antigen binding portion specifically binds to a first antigen, and the X1 and X2 are capable of forming a leucine zipper structure;

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

The first antigen and the second antigen are different, 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, between the second heavy chain and the first light chain are not likely to occur;

preferably, the multispecific antibody is a bispecific antibody.

In some embodiments, the method comprises, among other things,

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

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

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

The N-terminus of VH1 is operably linked to the C-terminus of X2; or (b)

The N-terminus of VH1 is operably linked to the C-terminus of X2, and the C-terminus of VH1 is operably linked to the N-terminus of CH 1; or (b)

The C-terminus of VH1 is operably linked to the N-terminus of a second domain comprising the first CH1, which is operably linked to the N-terminus of X2. In some embodiments, the method comprises, among other things,

The second light chain comprises a second light chain variable domain (VL 2), the VL2 being operably linked to a second CL, the second heavy chain comprising a second heavy chain variable domain (VH 2), the VH2 being operably linked to a second CH1; or (b)

The second light chain comprises a second light chain variable domain (VL 2), the VL2 is operably linked to a third mating domain (X3), the second heavy chain comprises a second heavy chain variable domain (VH 2), the VH2 is operably linked to a fourth mating domain (X4), the X3 and X4 are capable of forming a leucine zipper structure.

In some embodiments, the method comprises, among other things,

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

The C-terminal of VH2 is operably connected to the N-terminal of X4; or (b)

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

The N-terminus of VH2 is operably linked to the C-terminus of X4; or (b)

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

The C-terminal of VH2 is operably connected to the N-terminal of CH 1; or (b)

4) The N-terminus of VL2 is operably linked to the C-terminus of X3, the C-terminus of VL2 is further operably linked to the N-terminus of a second CL, and

The N-terminus of VH2 is operably linked to the C-terminus of X4, and the C-terminus of VH2 is further operably linked to the N-terminus of CH 1; or (b)

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

The C-terminal of VH2 is operably linked to the N-terminal of CH1, and the C-terminal of CH1 is operably linked to the N-terminal of X4.

In some embodiments, the method comprises, among other things,

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

The second light chain comprises a second light chain variable domain (VL 2) and a second CL from the N-terminus to the C-terminus, and the second heavy chain comprises a second heavy chain variable domain (VH 2) and a constant domain (CH 1) from the N-terminus to the C-terminus; or (b)

2) The first light chain comprises a first light chain variable domain (VL 1) from the N-terminus to the C-terminus, and a first pairing domain (X1), and the first heavy chain comprises a first heavy chain variable domain (VH 1) from the N-terminus to the C-terminus, and a second pairing domain (X2); and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), and a third pairing domain (X3), and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), and a fourth pairing domain (X4); or (b)

3) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), a first light chain variable domain (VL 1) and a second CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), a first heavy chain variable domain (VH 1) and a first CH1; and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), a second light chain variable domain (VL 2) and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), a second heavy chain variable domain (VH 2) and a first CH1; or (b)

4) The first light chain comprises a first pairing domain (X1) from the N-terminus to the C-terminus, and a first light chain variable domain (VL 1), and the first heavy chain comprises a second pairing domain (X2) from the N-terminus to the C-terminus, and a first heavy chain variable domain (VH 1); and

The second light chain comprises a third pairing domain (X3) from the N-terminus to the C-terminus, and a second light chain variable domain (VL 2), and the second heavy chain comprises a fourth pairing domain (X4) from the N-terminus to the C-terminus, and a second heavy chain variable domain (VH 2).

In some embodiments, the first antigen binding portion further comprises a first dimerization domain and the second antigen binding portion further comprises a second dimerization domain, the first dimerization domain and the second dimerization domain being bound.

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

In some embodiments of the present invention, 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; or (b)

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 N-terminus of the second dimerization domain; or (b)

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

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

Preferably, the antibodies first CH2, first CH3, second CH2 and second CH3 are derived from IgG1, igG2, igG3 or IgG4.

Preferably, the first and second dimerization domains are joined by a means selected from the group consisting of: knob-into-hole, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or ways of increasing flexibility.

In some embodiments, wherein

1) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), and a first mating domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), a second mating domain (X2), and a first multimerization domain; and

The second light chain comprises a second light chain variable domain (VL 2) and a second CL from the N-terminus to the C-terminus, the second heavy chain comprises a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain from the N-terminus to the C-terminus; or (b)

2) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), and a first pairing domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), a second pairing domain (X2), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises from N-terminus to C-terminus a second light chain variable domain (VL 2) and a constant domain (CL), the second heavy chain comprises from N-terminus to C-terminus a second heavy chain variable domain (VH 2), a constant domain (CH 1), a second dimerization domain, and a sixth pairing domain (X6); or (b)

3) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), and a first mating domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), a second mating domain (X2), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), and a third mating domain (X3), and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), a fourth mating domain (X4), and a second dimerization domain; or (b)

4) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), and a first pairing domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), a second pairing domain (X2), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), and a third pairing domain (X3), the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), a fourth pairing domain (X4), a second dimerization domain, and a sixth pairing domain (X6); or (b)

5) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), a first light chain variable domain (VL 1) and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), a first heavy chain variable domain (VH 1), a first CH1, and a first dimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), a second light chain variable domain (VL 2) and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain; or (b)

6) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), a first light chain variable domain (VL 1) and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), a first heavy chain variable domain (VH 1), a first CH1, a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), a second light chain variable domain (VL 2) and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, and a sixth pairing domain (X6); or (b)

7) The first light chain comprises a first pairing domain (X1) from the N-terminus to the C-terminus, and a first light chain variable domain (VL 1), the first heavy chain comprises a second pairing domain (X2), a first heavy chain variable domain (VH 1), and a first multimerization domain from the N-terminus to the C-terminus; and

The second light chain comprises a third pairing domain (X3) from the N-terminus to the C-terminus, and a second light chain variable domain (VL 2), and the second heavy chain comprises a fourth pairing domain (X4), a second heavy chain variable domain (VH 2), and a second dimerization domain from the N-terminus to the C-terminus.

8) The first light chain comprises from N-terminus to C-terminus a first pairing domain (X1), and a first light chain variable domain (VL 1), and the first heavy chain comprises from N-terminus to C-terminus a second pairing domain (X2), a first heavy chain variable domain (VH 1), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises a third pairing domain (X3) from the N-terminus to the C-terminus, and a second light chain variable domain (VL 2), and the second heavy chain comprises a fourth pairing domain (X4), a second heavy chain variable domain (VH 2), a second dimerization domain, and a sixth pairing domain (X6) from the N-terminus to the C-terminus.

In some embodiments, the method comprises, among other things,

Each X1, X3 or X5 independently comprises a Jun domain, a Fos domain, fosW domain, myc domain, max domain, winzipA2 domain, winzipB1 domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain, each X2, X4 or X6 independently comprises a domain capable of forming a leucine zipper structure with said 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 domain; or (b)

X2, X4 or X6 each independently comprises a Jun domain, fos domain, fosW domain, myc domain, max domain, winzipA2 domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain, X1, X3 or X5 each independently comprises a domain capable of forming a leucine zipper with said 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 domain.

In some embodiments, the method comprises, among other things,

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

x1, X3 or X5 each independently comprises a Fos domain, and X2, X4 or X6 each independently comprises a Jun domain;

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

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

x1, X3 or X5 each independently comprises a Myc domain, and X2, X4 or X6 each independently comprises a Max domain;

x1, X3 or X5 each independently comprises a Max domain, and X2, X4 or X6 each independently comprises a Myc domain;

X1, X3 or X5 each independently comprises WinzipA2 domain, X2, X4 or X6 each independently comprises WinzipB domain;

X1, X3 or X5 each independently comprises WinzipB1 domain, and X2, X4 or X6 each independently comprises WinzipA domain;

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

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

x1, X3 or X5 are each independently a GCN4 domain, and X1, X3 or X5 are each independently a GCN4 domain; or (b)

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

Preferably, the Jun domain, fos domain, fosW domain, max domain, myc domain, winzipA domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain and C/EBP domain are as defined in the first aspect.

In some embodiments, 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, the VL2 is operably linked to X3 through a third linker L3, the VH2 is operably linked to X4 through a fourth linker L4, the C-terminus of the first dimerization domain is linked to X5 through a fifth linker L5, the C-terminus of the second dimerization domain is linked to X6 through a sixth linker L6, each of L1-L6 is independently present or absent, and each of L1-L6 is independently selected from the following polypeptide fragments when L1-L6 are optionally present: (G _xS) _y、(T _xG) _y, and (S _xG) _y, wherein x is an integer selected from 1, 2, 3, 4, or 5, and y is an integer selected from 1, 2, 3, 4, 5, or 6;

preferably, each of L1-L6 is independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG;

Further preferably, L1-L4 are each independently linked by their C-terminus to a cleavage site peptide selected from the group consisting of factor Xa, thrombin, enterokinase, and SUMO, and L5-L6 are each independently linked by their N-terminus to a cleavage site peptide including but not limited to the four polypeptides described above.

In some embodiments, the method comprises, among other things,

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

The second light chain comprises a second light chain variable domain (VL 2) and a constant domain (CL) from the N-terminus to the C-terminus, the second heavy chain comprises a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain from the N-terminus to the C-terminus; or (b)

2) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), L1, and a first pairing domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), 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 (VL 2) and a second CL from the N-terminus to the C-terminus, the second heavy chain comprising a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and a sixth pairing domain (X6); or (b)

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

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), L3, and a third pairing domain (X3), and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), L4, a fourth pairing domain (X4), and a second dimerization domain; or (b)

4) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), L1, and a first pairing domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), L2, a second pairing domain (X2), a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), L3, and a third pairing domain (X3), and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), L4, a fourth pairing domain (X4), a second dimerization domain, L6, and a sixth pairing domain (X6); or (b)

5) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), L1, a first light chain variable domain (VL 1) and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), L2, a first heavy chain variable domain (VH 1), a first CH1, and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), L3, a second light chain variable domain (VL 2) and a second CL, the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain; or (b)

6) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), L1, a first light chain variable domain (VL 1) and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), L2, a first heavy chain variable domain (VH 1), a first CH1, a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), L3, a second light chain variable domain (VL 2) and a second CL, the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and a sixth pairing domain (X6); or (b)

7) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), L2, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises a third pairing domain (X3), L3, and a second light chain variable domain (VL 2) from the N-terminus to the C-terminus, and the second heavy chain comprises a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH 2), and a second dimerization domain from the N-terminus to the C-terminus.

8) The first light chain comprises from N-terminus to C-terminus a first pairing domain (X1), L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises from N-terminus to C-terminus a second pairing domain (X2), L2, a first heavy chain variable domain (VH 1), a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH 2), a second dimerization domain, L6, and a sixth pairing domain (X6).

In some embodiments, the method comprises, among other things,

1) The first light chain comprises a first light chain variable domain (VL 1), L1, and Fos domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, jun domain, and a first multimerization domain from the N-terminus to the C-terminus; and

2) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, a first dimerization domain, L5, and Max domain from the N-terminus to the C-terminus; and

The second light chain comprises a second light chain variable domain (VL 2) and a second CL from the N-terminus to the C-terminus, the second heavy chain comprising a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and a Myc domain; or (b)

3) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, and a first multimerization domain from the N-terminus to the C-terminus; and

The second light chain comprises a second light chain variable domain (VL 2), L3, and Fos domain from the N-terminus to the C-terminus, the second heavy chain comprises a second heavy chain variable domain (VH 2), L4, jun domain, and a second dimerization domain from the N-terminus to the C-terminus; or (b)

4) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, a first dimerization domain, L5, and Max domain from the N-terminus to the C-terminus; and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), L3, and Fos domain, and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), L4, jun domain, second dimerization domain, L6, and Myc domain; or (b)

5) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a first light chain variable domain (VL 1), and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a first heavy chain variable domain (VH 1), a first CH1, and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a second light chain variable domain (VL 2), and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain; or (b)

6) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a first light chain variable domain (VL 1), and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a first heavy chain variable domain (VH 1), a first CH1, a first dimerization domain, L5, and WinzipA2 domains; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a second light chain variable domain (VL 2), and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and WinzipB1 domains; or (b)

7) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a second heavy chain variable domain (VH 2), and a second dimerization domain; or (b)

8) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a first heavy chain variable domain (VH 1), a first dimerization domain, L5, and WinzipA2 domains; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a second heavy chain variable domain (VH 2), a second dimerization domain, L6, and WinzipB1 domains; or (b)

9) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, fosW domain, L2, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Myc domain, L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Max domain, L4, a second heavy chain variable domain (VH 2), and a second dimerization domain.

10 The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, fosW domains, L2, a first heavy chain variable domain (VH 1), a first dimerization domain, L5, and WinzipA2 domains; and

The second light chain comprises, from N-terminus to C-terminus, a Myc domain, L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Max domain, L4, a second heavy chain variable domain (VH 2), a second dimerization domain, L6, and WinzipB1 domains.

In some embodiments, wherein

1) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, and a first multimerization domain from the N-terminus to the C-terminus; and

5) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, a first light chain variable domain (VL 1), and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), a first CH1, and a first dimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a cleavage site peptide, a second light chain variable domain (VL 2), and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain; or (b)

6) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, a first light chain variable domain (VL 1), and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), a first CH1, a first dimerization domain, a cleavage site peptide, L5, and WinzipA domains; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a cleavage site peptide, a second light chain variable domain (VL 2), and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, a cleavage site peptide, L6, and WinzipB1 domains; or (b)

7) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a cleavage site peptide, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), and a second dimerization domain; or (b)

8) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), a first dimerization domain, a cleavage site peptide, L5, and WinzipA domains; and

The second light chain comprises from N-terminus to C-terminus a Fos domain, L3, a cleavage site peptide, and a second light chain variable domain (VL 2), and the second heavy chain comprises from N-terminus to C-terminus a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), a second dimerization domain, a cleavage site peptide, L6, and WinzipB1 domains; or (b)

9) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, fosW domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Myc domain, L3, a cleavage site peptide, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Max domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), and a second dimerization domain; or (b)

10 The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, fosW domains, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), a first dimerization domain, a cleavage site peptide, L5, and WinzipA domains; and

The second light chain comprises, from N-terminus to C-terminus, a Myc domain, L3, a cleavage site peptide, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Max domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), a second dimerization domain, a cleavage site peptide, L6, and WinzipB1 domains.

In some embodiments, wherein

The first dimerization domain and the second dimerization domain are joined by a means selected from the group consisting of: handle inlet structure KIH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, ways of increasing flexibility, or combinations thereof;

More preferably, the first and second dimerization domains are bound by a handle entry structure KIH (Knob into Hole), and a protrusion or a pit is created in the interface of the first and second dimerization domains, the protrusion or pit being positioned in the pit or protrusion in the interface to form the handle entry structure; preferably the sequence of the first dimerization domain is as shown in SEQ ID NO. 7 or has at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO. 10 and the sequence of the second dimer is as shown in SEQ ID NO. 8 or has at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO. 810.

In a third aspect the invention discloses an isolated polynucleotide encoding the polypeptide complex of any one of the first aspects or the multispecific antibody of any one of the second aspects.

In a fourth aspect, the invention discloses an isolated vector comprising a polynucleotide according to the third aspect.

In a fifth aspect the invention discloses a host cell comprising an isolated polynucleotide according to the third aspect, or an isolated vector according to 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 the group consisting of a prokaryotic cell, an animal cell, a plant cell, and a fungal cell.

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

In a sixth aspect the present invention discloses a method for preparing a polypeptide complex according to any one of the first aspects or a multispecific antibody according to any one of the second aspects, comprising the steps of:

a) Introducing into a host cell a nucleotide encoding the polypeptide complex of any one of the first aspects or the multispecific antibody of any one of the second aspects;

b) Allowing the host cell to express the antibody.

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

In a seventh aspect the invention discloses a pharmaceutical composition comprising a polypeptide complex according to any of the first aspects or a multispecific antibody according to any of the second aspects, and at least one pharmaceutically acceptable excipient.

In an eighth aspect the invention discloses the use of a polypeptide complex according to any one of the first aspects or a multispecific antibody according to any one of the second aspects in the manufacture of a medicament for immunosuppressive therapy, for treating an autoimmune disease, for treating an inflammatory disease, for treating an infectious disease, for treating allergy or for treating cancer.

Also disclosed is 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 the first aspects or the multispecific antibody of any one of the second aspects.

The invention also discloses a polypeptide complex according to any one of the first aspect or a multispecific antibody according to any one of the second aspect for use in immunosuppressive therapy, in the treatment of autoimmune diseases, in the treatment of inflammatory diseases, in the treatment of infectious diseases, in the treatment of allergies or in the treatment of cancer.

Definition of the definition

The "antibody" of the present invention includes antibodies or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof engineered on the basis of immunoglobulins while retaining the ability to bind antigen; including monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); monovalent or multivalent antibodies are also included. A natural whole antibody comprises two heavy chains and two light chains. Each heavy chain consists of one variable region (VH) and first, second and third constant regions (CH 1, CH2, CH3, respectively), while each light chain consists of one variable region (VL) and one constant region (CL). The antibody is "Y" shaped, the backbone of which is composed of the second and third constant regions of two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure comprises a variable region and a first constant region of one of the heavy chains, which is associated with 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 (CDRs) (CDRs of the light (L) chain comprise LCDR1, LCDR2, LCDR3, CDRs of the heavy (H) chain comprise HCDR1, HCDR2, HCDR 3). Wherein three CDRs are separated by laterally contiguous portions called Framework Regions (FR), which are more highly conserved than the CDRs and form a scaffold-supported hypervariable loop. VH and VL each contain 4 FR, and CDR and FR are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains do not participate in antigen binding, but have multiple effector functions. Antibodies can be classified into several classes according to the amino acid sequence of the heavy chain constant region. Mammalian antibody heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian antibody light chains can be classified as λ or κ. Antibodies can be divided into five main classes or isotypes depending on whether they contain α, δ, ε, γ and μ heavy chains: igA, igD, igE, igG and IgM. Several major classes of antibodies can also be classified into subclasses, such as IgG1 (gamma 1 heavy chain), igG2 (gamma 2 heavy chain), igG3 (gamma 3 heavy chain), igG4 (gamma 4 heavy chain), igA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain), among others. Antibodies of the invention include antibodies with intact constant regions, such as antibodies comprising only CH1, CH1 and CH2, CH1 and CH3, CH2 and CH3, and the like, in addition to antibodies with intact constant regions as described above. In summary, the term "antibody" as used herein includes, but is not limited to, any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific antibody that can bind to a particular antigen, and all such antibodies that exhibit the desired antigen-binding activity are encompassed by the present invention.

The number of different target antigens or different epitopes bound by an antibody is classified into a monospecific antibody, a bispecific antibody, a trispecific antibody, a tetraspecific antibody … …, and a multispecific antibody (which binds to two or more 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 of various structures have been disclosed in the prior art; the IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies can be classified according to the integrity of the IgG molecule; bispecific antibodies that can be classified as bivalent, trivalent, tetravalent, or more multivalent in configuration depending on the number of antigen binding regions; the structure-based bispecific antibody can be classified into a symmetric structure-based bispecific antibody and an asymmetric structure-based bispecific antibody according to whether the structure is symmetric or not.

The term "antigen" or "antigenic molecule" as used herein is used interchangeably and refers to all molecules capable of being specifically bound by an antibody, a bivalent bispecific antibody specifically binding a first antigen and a second antigen, wherein the first antigen and the second antigen may 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 invention), and polysaccharides, etc. Preferably, the antigen is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors. The antigen targeted by the antibody of the present invention is selected from, but not limited to, one of T Cell Receptor (TCR), natural killer group 2D (NKG 2D) receptor, tumor-associated antigen, infectious disease-associated antigen, and autoimmune disease-associated antigen. The antigen specificity may be for any suitable antigen or epitope, for example, an exogenous antigen, an endogenous antigen, a self antigen, a neoantigen, a viral antigen, or a tumor antigen. Exogenous antigens are inhaled, ingested or injected into the body and can be presented by Antigen Presenting Cells (APC) and form MHC class II complexes by endocytosis or phagocytosis. Endogenous antigens can be produced in normal cells due to cellular metabolism, intracellular viral or bacterial infection, which can form MHC class I complexes. Autoantigens (e.g., peptides, DNA or RNA, etc.) are recognized by the immune system of a patient suffering from an autoimmune disease, and under normal conditions, the antigen should not be a target of the immune system. The neoantigen is not present at all in a normal organism but is generated due to a certain disease (e.g., tumor or cancer). In certain embodiments, the antigen is associated with a disease (e.g., tumor or cancer, autoimmune disease, infectious and parasitic disease, cardiovascular disease, neuropathy, neuropsychiatric disorder, injury, inflammation, coagulation disorder). In certain embodiments, the antigen is associated with the immune system (e.g., immune cells, such as B cells, T cells, NK cells, macrophages, etc.).

The terms "specific binding", "selective binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (K _D) of about less than 10 ^-7 M, e.g., about less than 10 ^-8M、10 ^-9M、10 ^-10M、10 ^-11M、10 ^-12 M or less. K _D refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, typically, antibodies of the present disclosure bind antigen with a dissociation equilibrium constant (K _D) of less than about 10 ^-7 M, e.g., less than about 10 ^-8 M or 10 ^-9 M.

The term "Fab fragment" refers to a fragment capable of binding an antigen, which fragment comprises the CH-CH1 structure of the light chain VL-CL and the heavy chain, which fragment can be produced by enzymatic treatment of an antibody.

The term "Fc region" or "Fc fragment" refers to the portion of an antibody that consists of the CH2 constant domain and/or CH3 constant domain of a first heavy chain bound via disulfide bonds to the CH2 constant domain and/or CH3 constant domain of a second heavy chain. The Fc of antibodies is responsible for a number of different effector functions, such as ADCC and CDC, but is not involved in antigen binding.

The term "electrostatic interactions" is a non-covalent interaction and plays an important role in protein folding, stability, flexibility and function, including ionic interactions, hydrogen bonding and halogen bonding. Electrostatic interactions may be formed in the polypeptide, for example between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, trp on the first chain and Arg, val or Thr on the second chain.

The term "hydrophilic interaction" refers to molecules with polar groups that have a great affinity for water and are capable of forming transient bonds with water via hydrogen bonds.

The term "hydrophobic interaction" refers to the phenomenon in which hydrophobic groups aggregate close to each other to avoid boiling water.

The term "leucine zipper" structure is a structural motif found in DNA binding proteins and other proteins, and a leucine zipper is formed when the hydrophobic faces of two alpha-helices (often containing leucine residues) from the same or different polypeptide chains interact to form a loop-to-loop dimer structure. Specifically, the leucine zipper is composed of extended amino acids, the 7 th amino acid of every 7 amino acids is leucine, leucine is a hydrophobic amino acid, arranged on one side of the alpha helix, and all charged amino acid residues are arranged on the other side. When two protein molecules are aligned in parallel, leucine interacts with each other to form a dimer, forming a "zipper" structure. The leucine zipper domain is a peptide that promotes 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-p 1 protein, GCN4 protein/GCN 4 domain, C/EBP protein/C/EBP protein, functional variants of the above proteins/domains. The leucine zipper structure pair described herein may be selected from any of the above groups, but is not limited thereto, and any sequence capable of forming the leucine zipper structure may be used in 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 to SEQ ID NO. 9, said domain being capable of forming a leucine zipper structure with the Fos domain; 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 to SEQ ID NO. 10, said domain being capable of forming a leucine zipper structure with the Jun domain; 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 to SEQ ID NO. 29, said domain being capable of forming a leucine zipper structure with the Jun domain; the Max domain is a sequence shown as SEQ ID NO. 11, or a sequence with at least 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO. 11, said domain being capable of forming a leucine zipper structure with the Myc domain; the Myc domain is a sequence shown as SEQ ID NO. 12, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO. 12, said domain being capable of forming a leucine zipper structure with a Max domain; the WinzipA domain is a sequence as shown in SEQ ID NO. 13, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO. 13, said domain being capable of forming a leucine zipper structure with the WinzipB1 domain; the WinzipB domain is a sequence as set forth in SEQ ID NO. 14, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO. 14, said domain being capable of forming a leucine zipper structure with the WinzipA domain; the ACID-p1 domain is a sequence as shown in SEQ ID NO. 33, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO. 33, said domain being capable of forming a leucine zipper structure with the BASE-p1 domain; the BASE-p1 domain is a sequence as shown in SEQ ID NO. 34, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO. 34, said BASE-p1 domain being capable of forming a leucine zipper structure with the ACID-p1 domain; 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 to SEQ ID NO. 35, said domain being capable of forming a leucine zipper structure with the GCN4 domain; and 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 to SEQ ID NO. 36, said domain being capable of forming a leucine zipper structure with the C/EBP domain.

The term "interface" or "contact interface" as used herein refers to a specific region on the polypeptide that interacts/associates with each other. The interface comprises one or more amino acid residues that interact with corresponding amino acid residues that are contacted or associated when the interaction occurs. The amino acid residues in the interface may or may not be in a contiguous sequence. For example, when the interface is three-dimensional, amino acid residues within the interface may be separated at different positions on the linear sequence.

"Handle entry structure" or "knobs-intos" or "Knob into hole" or "KIH" as used in the present application refers to an interaction between two polypeptides, one having a protuberance (i.e., "knob") due to the presence of an amino acid residue with a bulky side chain (e.g., tyrosine or tryptophan) and the other having a small side chain amino acid residue (e.g., alanine or threonine) to form a cavity/pit (i.e., "hole"), and the protuberance can be placed within the cavity/pit to facilitate the interaction of the two polypeptides to form a heterodimer or complex. Methods for producing polypeptides having knobs-into-holes are well known in the art, for example, as described in U.S. Pat. No. 5,731,168. Illustratively, the Knob sequence is T366W and the Hole sequence is T366S-L368A-Y407V (the numbering sequence is IgG according to the Kabat numbering convention).

The term "operably linked" or "operably linked" refers to the joining of two or more biological sequences such that they are joined in a manner that they function in the intended manner, whether or not a linker (also called a linker, junction sequence) is present. When in a polypeptide, the term means that the polypeptide sequences are linked in such a way that the linked product has the desired 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 having antigen binding activity. The term may also be used for polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), the term indicates that the polynucleotide sequences are linked in a manner that allows for the regulated expression of the polypeptide by the polynucleotide.

The term "linker" or "spacer" or "linker" refers to a convenient or border region where two polypeptide sequences are fused or combined. For example, the linking domain may comprise at least a portion of a C-terminal fragment of a two-character first fusion polypeptide fused to at least a portion of an N-terminal fragment of a leydig second fusion polypeptide with or without additional linkers therebetween. For example, the linker may be enriched in glycine and proline residues, such as a linker having a single or repeated sequence of threonine/serine composition, GGS, GGGGS, TGGGG, SGGGG or tandem repeats thereof (e.g., 2,3, or more repeats).

The term "dimerization domain" refers to a domain capable of promoting association with each other to form a dimer. In some embodiments, the first dimerization domain may be associated with the second dimerization domain. The association may be by any suitable means, such as binding or linking or bonding; for example, via a linker, disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction, or a combination thereof. Exemplary dimerization domains include, but are not limited to, antibody hinge regions, antibody CH2 domains, antibody CH3 domains, and/or other suitable protein monomers or polypeptides capable of dimerizing interactions with each other. The hinge region, CH2 constant domain, CH3 constant domain may be derived from any antibody isotype, e.g., igG1, 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 multispecific pairing. "less prone to mismatches" means that, for example, when polypeptides A1, B2 and B2 are co-expressed, if the amount of desired A1-B1 dimer is greater than the amount of A1-B2 dimer, then preferential pairing between A1-B1 is considered to occur, i.e., mismatches between A1-B2 are not agreed to occur. In some embodiments, in a bispecific/multispecific antibody, a mismatch is not readily occurring between VL1 of the first light chain and VL2 of the second heavy chain, and/or a mismatch is not readily occurring between VL1 of the first heavy chain and VL2 of the second light chain, but rather a preferential pairing between VH1-VL1, and a preferential pairing between VH2-VL 2.

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 specifically limited, the term encompasses polynucleotides containing known natural nucleotide analogs that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implies conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, homologous genes, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

The terms "homology", "identity" refer to sequence similarity between multiple polynucleotides or between two polypeptides. When referring to a nucleic acid or fragment thereof, it means that when optimally aligned with appropriate nucleotide substitutions, insertions or deletions with another nucleic acid (or its complement), there is nucleotide sequence identity in at least about 80%, more preferably at least about 81%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% of the nucleotide bases as calculated using any of the sequence identity calculation programs described below, such as FASTA, BLAST or Gap. When applied to polypeptides, it is intended that when optimally aligned with default Gap or BESTFIT and like procedures, the two peptide sequences have at least 80% sequence identity, more preferably at least 81%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and the different residue positions may differ by amino acid substitutions, deletions or insertions, more preferably 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 (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to the inherent binding affinity of a 1:1 interaction between members of a reactive binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant (K _D), dissociation constant (K _d) or binding 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 for real-time interactive analysis by detecting changes in protein concentration in a biosensor matrix, such as analysis using the Biacore ^TM system.

The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted and which allows expression of the protein. Typically, the construct also comprises appropriate regulatory sequences. Vectors may be used to transform, transduce or transfect host cells such that the genetic elements carried thereby are expressed within 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 a replication origin. The vector may also contain components that assist it in entering the cell, including but not limited to viral particles, liposomes, or protein shells. For example, the carrier comprises: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda or M13 phages, animal viruses, and the like. Animal virus species used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-polyomaviruses (e.g., SV 40). In some embodiments, the vector system comprises mammalian, bacterial, yeast systems, and the like, 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, and the like, as well as other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).

The vector comprising the polynucleotide sequence of the application may be introduced into a host cell for cloning or gene expression, and the term "host cell" as used herein 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 of the application are the prokaryotic cells, animal cells, plant cells and fungal cells described above.

The term "pharmaceutical composition" means a mixture comprising one or more compounds of the present invention or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration of organisms, facilitate the absorption of active ingredients and further exert 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. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antimicrobial or antifungal), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyols, sorbitol, and sodium chloride.

The multispecific antibody of the present invention, wherein the light chain and heavy chain of the first antigen binding portion and/or the second antigen binding portion comprise leucine zipper pair sequences that are effective to reduce mismatches between the light chain and heavy chain, thereby facilitating the formation of heterodimers between the light chain and heavy chain; further, the handle entry structure KIH, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, increased flexibility, or a combination thereof between the heavy chains of the first antigen binding portion and the heavy chains of the second antigen binding portion is effective in reducing mismatch between the heavy chains, particularly when the KIH and leucine zipper structures are used together to provide superior anti-mismatch effects between the heavy chains. The above antibody structure ensures that the antigen binding sequences are expressed and assembled with minimal or little mismatch.

Drawings

Fig. 1-1: schematic structural diagram of antibody AmF1

Fig. 1-2: schematic structural diagram of antibody AmF2

Fig. 1-3: schematic structural diagram of antibody AmF3

Fig. 1-4: schematic structural diagram of antibody AmF4

Fig. 1-5: schematic structural diagram of antibody AmF5

Fig. 2-1: SEC-HPLC detection result of antibody AmF1

Fig. 2-2: SEC-HPLC detection result of antibody AmF2

Fig. 2-3: SEC-HPLC detection result of antibody AmF3

Fig. 2-4: SEC-HPLC detection result of antibody AmF5

Fig. 3: cell killing activity assay results for antibody AmF2, horizontal axis: log (antibody concentration, ng/Ml), vertical axis: killing efficiency (%)

Fig. 4: schematic of antibody BmF structure

Fig. 5: SEC-HPLC detection results of antibody mAb-T

Fig. 6: SEC-HPLC detection result of antibody BmF

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Examples

The present invention illustratively 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 multi-specific antibody platform technology according to the concepts of the present invention. Examples 1-7 are bispecific antibody-related experiments capable of simultaneously targeting DLL3 and CD3, and examples 8-12 are bispecific antibodies capable of simultaneously targeting TROP2 and PRLR and targeted TROP2 monoclonal antibody-related experiments.

Target introduction

DLL3: notch signaling pathways are widely found in a variety of animals, are highly conserved in evolution, and regulate the differentiation of neuroendocrine and epithelial cells upon embryonic development through interactions between adjacent cells. Mammals have 4 Notch receptors (Notch 1, notch2, notch3, notch 4), 5 Notch ligands (DLL 1, DLL3, DLL4, jagged2, and Jagged 2). DLL3 (Delta-like protein 3) is one of the 5 ligands of Notch, DLL3 is typically expressed in the developing central nervous system and plays a key role in somite generation. DLL3 can regulate the differentiation, growth and proliferation processes of cells and indirectly promote or inhibit the occurrence of tumors by inhibiting Notch signal channels. DLL3 is specifically highly expressed on the surface of SCLC (SMALL CELL lung cancer) cells as a ligand of the Notch pathway. DLL3 plays an important role in tumor initiation and epithelial-mesenchymal transition, invasion and metastasis of SCLC.

CD3: t cell co-receptors are protein complexes consisting of four distinct chains (the cd3γ chain, the cd3δ chain, and two cd3ε chains). These four chains associate with molecules known as T Cell Receptors (TCRs) and zeta chains to generate activation signals in T lymphocytes. TCR, zeta chain and CD3 molecules constitute TCR complexes in which TCR as subunit is used to recognize and bind antigen, and CD3 as subunit is used to transfer and carry antigen stimuli to the signaling pathway, and ultimately modulate T cell activity. 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 the elimination of pathogenic organisms and the control of tumor growth through a broad range of cytotoxic effects. Mouse monoclonal antibodies specific for human CD3, such as OKT3 (Kung et al, science,206:347-9 (1979)), are the first generation of CD3 antibodies developed for use in therapy.

TROP2: human trophoblast cell surface antigen 2 (human trophoblast cell surface antigen, TROP2) is a cell surface glycoprotein encoded by the TACSTD gene. Numerous clinical studies and literature reports indicate that TROP2 is overexpressed in a variety of cancers of epithelial origin, such as gastric, lung, large intestine, ovarian, breast, prostate, pancreatic, liver, esophageal (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;andWu et al.(2013)Head&Neck.35:1373–1378). In contrast, TROP2 is expressed little or no in adult normal tissues (Cubas et al. (2009) Biochimica et Biophysica acta.1796:309-314 (Zhang et al. (1997)

Science 276:1268-1272), cells limited to the epithelial region had a small expression, and the expression level was also lower than in cancer, indicating that TROP2 was associated with tumor formation. Overexpression of TROP2 in tumor tissue is closely related to poor prognosis (Wang et al (2008) Mol CANCER THER 7:280-285) and metastasis of cancer cells in patients, while affecting overall survival in patients. Thus, TROP2 has become an attractive target in tumor molecular targeted therapies.

PRLR: the prolactin receptor (PRLR) is capable of interacting with prolactin binding. PRLR is a single transmembrane receptor belonging to the superfamily of class I cytokine receptors. Binding of prolactin to PRLR results in receptor dimerization and intracellular signaling. Signaling through PRLR is associated with various processes such as those involved in breast development, lactation, reproduction and immunomodulation. In addition, 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 variable region sequence for DLL3 of the present invention is as follows:

VL amino acid sequence of anti-DLL 3 antibody (VLDLL 3)

VH amino acid sequence of anti-DLL 3 antibody (VHDLL)

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

VL amino acid sequence of anti-CD 3 antibody (VLCD 3)

VH amino acid sequence of anti-CD 3 antibody (VHCD)

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

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

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

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

VL amino acid sequence of anti-TROP 2 antibody (VLTROP 2)

VH amino acid sequence of anti-TROP 2 antibody (VHTROP 2)

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

CL constant Domain

CH1 constant domain

First dimerization Domain

Remarks: in the sequence, the single underlined part is the Hinge region of Hinge, the non-underlined part is the Fc region, and the bolded part is Knob, specifically T366W (Knob).

Second dimerization domain

Remarks: in the sequence, the single underlined part is the Hinge region of Hinge, the non-underlined part is the Fc region, and the bolded part is Hole, specifically, T366S-L368A-Y407V (Hole).

First light chain sequences of AmF1 and AmF2

First heavy chain sequences of AmF1 and 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 sequences of AmF3 and 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

A 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

An exemplary bispecific antibody of the invention comprises a first antigen binding moiety and a second antigen binding moiety, wherein the first antigen binding moiety targets the first antigen DLL3 and the second antigen binding moiety targets the second antigen CD3.

According to the bispecific antibody platform technology of the present invention, the bispecific antibody molecule DLL3 xcd 3 of the present invention is designed as follows:

Antibody AmF1: the C-terminal of the light chain variable domain VLDLL (sequence shown as SEQ ID NO: 1) of the monoclonal antibody targeting DLL3 is linked to the N-terminal of the Jun domain (sequence shown as SEQ ID NO: 9) via a first linker L1 (GGGGS), and the C-terminal of the heavy chain variable domain VHDLL (sequence shown as SEQ ID NO: 2) of the monoclonal antibody targeting DLL3 is linked to the N-terminal of the Fos domain (sequence shown as SEQ ID NO: 10) via a second linker L2 (GGGGS); the C-terminus of the light chain variable domain VLCD3 (sequence shown as SEQ ID NO: 3) of the CD 3-targeting monoclonal antibody is linked to the N-terminus of the Fos domain (sequence shown as SEQ ID NO: 10) via a third linker L3 (GGGGS), and the C-terminus of the heavy chain variable domain VHCD (sequence shown as SEQ ID NO: 4) of the CD 3-targeting monoclonal antibody is linked to the Jun domain (sequence shown as SEQ ID NO: 9) via a fourth linker L4 (GGGGS). The first and second antigen binding portions further comprise a first dimerization domain (SEQ ID NO: 7) and a second dimerization domain (SEQ ID NO: 8), the first and second dimerization domains comprising a hinge region, a CH2 constant domain and a CH3 constant domain; and the C-terminal end of the CH3 constant domain of the first dimerization domain is connected with the max domain (the sequence is shown as SEQ ID NO: 11) through a fifth connector L5 (GGS), the C-terminal end of the CH3 constant domain of the second dimerization domain is connected with the Myc domain (the sequence is shown as SEQ ID NO: 12) through a sixth connector L6 (GGS), the structure shown as the attached figure 1-1 is formed, the structure is named as AmF1, the first light chain sequence of the AmF1 is shown as SEQ ID NO:15, the first heavy chain sequence is shown as SEQ ID NO:16, the second light chain sequence of the AmF1 is shown as SEQ ID NO:17, and the second heavy chain sequence is shown as SEQ ID NO: 18.

Antibody AmF2: the C-terminal of the light chain variable domain VLDLL (sequence shown as SEQ ID NO: 1) of the monoclonal antibody targeting DLL3 is linked to the N-terminal of the Jun domain (sequence shown as SEQ ID NO: 9) via a first linker L1 (GGGGS), and the C-terminal of the heavy chain variable domain VHDLL (sequence shown as SEQ ID NO: 2) of the monoclonal antibody targeting DLL3 is linked to the N-terminal of the Fos domain (sequence shown as SEQ ID NO: 10) via a second linker L2 (GGGGS); the C-terminus of the light chain variable domain VLCD3 (SEQ ID NO: 3) of the CD 3-targeting monoclonal antibody is linked to the N-terminus of the CL constant domain (SEQ ID NO: 5), and the C-terminus of the heavy chain variable domain VHCD (SEQ ID NO: 4) of the CD 3-targeting monoclonal antibody is linked to the N-terminus of the CH1 constant domain (SEQ ID NO: 6); the first and second antigen binding portions further comprise a first dimerization domain (SEQ ID NO: 7) and a second dimerization domain (SEQ ID NO: 8), the first and second dimerization domains comprising a hinge region, a CH2 domain and a CH3 domain; and the C end of the CH3 domain of the first dimerization domain is connected with the max domain (the sequence is shown as SEQ ID NO: 11) through a fifth connector L5 (GGS), the C end of the CH3 domain of the second dimerization domain is connected with the Myc domain (the sequence is shown as SEQ ID NO: 12) through a sixth connector L6 (GGS), the structure shown as the attached figure 1-2 is formed, the structure is named as AmF2, the first light chain sequence of the AmF2 is shown as SEQ ID NO:15, the first heavy chain sequence is shown as SEQ ID NO:16, the second light chain sequence of the AmF2 is shown as SEQ ID NO:19, and the second heavy chain sequence is shown as SEQ ID NO: 20.

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

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

Antibody AmF5: the structure is shown in FIGS. 1-5 and is designated AmF5, and the difference between the structure and AmF3 is that the second pairing domain is FosW domain (sequence shown as SEQ ID NO: 29) rather than Fos domain, and the third pairing domain X3 is myc domain (sequence shown as SEQ ID NO: 12) and the fourth pairing domain is max domain (sequence shown as SEQ ID NO: 11). The first light chain sequence of the AmF5 is shown as SEQ ID NO. 21, the first heavy chain sequence is shown as SEQ ID NO. 30, the second light chain sequence of the AmF5 is shown as SEQ ID NO. 31, and the second heavy chain sequence is shown as SEQ ID NO. 32.

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

Gene synthesis

Genes expressing the antibody sequences against DLL3 and CD3 in example 1 were constructed, codon optimized, and submitted to the gene synthesis company for total gene synthesis.

PCGS3 expression vector construction

Using a seamless cloning procedure, the heavy and light chains of the first antigen binding portion were cloned into the multiple cloning site of the pCGS vector, and the heavy and light chains of the second antigen binding portion were also cloned into the multiple cloning site of the pCGS vector, with the heavy chain cloned into BstBI and PacI cleavage sites and the light chain cloned into HindIII and XhoI cleavage sites. The specific method comprises the following steps:

The enzyme-cleaved components in Table 1 were mixed well and the resulting reaction solution incubated in a 37℃metal bath for 3h; the digested products were recovered using a general agarose gel DNA recovery kit (Tiangen Biochemical technologies (Beijing) Co., ltd.).

Table 1 heavy chain double cleavage System

T4 DNA ligase (NEB Co., USA) was used to ligate the recovered DNA with pCGS vector for cleavage, the specific ligation reaction system was shown in Table 2, the ligation reaction components were mixed, and the well-mixed reaction solution was placed in a metal bath at 16℃overnight.

TABLE 2 ligation reaction System

Transformation and inoculation were performed as follows: mu.L of the above-mentioned ligation product was added to 50. Mu.L of Trans 5. Alpha. Competent cells (Beijing full gold Biotechnology Co., ltd.), mixed well and placed on ice for 30min, then placed in a constant temperature water bath at 42℃for heat shock for 90s, then placed on ice for 1min, finally added with 250. Mu.L of LB medium, and cultured for 1h at 37℃under shaking at 220rpm, 100. Mu.L of LB medium was applied to ampicillin ⁺ (amp+, tiangen Biochemical technology (Beijing Co., ltd.) and cultured overnight at 37 ℃.

Selecting a monoclonal from each LB culture plate, respectively placing the monoclonal into 1.5mL EP tubes, adding 300 mu L of Amp+ LB culture medium into each tube, and carrying out shake culture for 8 hours at 37 ℃ and 220 rpm; culturing thallus PCR, electrophoresis, and sequencing bacterial liquid with correct band.

Plasmid DNA was extracted as follows: after sequencing the correct bacterial solution and amplifying by shaking, plasmid DNA was extracted using a plasmid miniprep kit (Beijing full gold Biotechnology Co., ltd.).

Light chain double cleavage and ligation were continued, wherein the light chain double cleavage system is shown in table 3:

TABLE 3 light chain double cleavage System

The rest steps are the same as above.

The constructed plasmids were extracted from endotoxin-free plasmid large extraction kit (Tiangen Biochemical Co., ltd.) and named p-AmF1, p-AmF2, p-AmF3, p-AmF4 and p-AmF5, and the concentration and purity of the plasmid DNA were measured by a NanoDrop ultraviolet spectrophotometer (Thermo FISHER SCIENTIFIC Co., USA) for use.

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

Bispecific antibodies p-AmF1, p-AmF2, p-AmF3, p-AmF4 and p-AmF5 were expressed as follows:

HEK-293 cells (Martensitic kai R Biotechnology Co., ltd.) are placed in an 8% CO ₂ constant temperature shaking table (Shanghai-He Sci Co., ltd.) and subjected to constant temperature shaking culture at 37 ℃ and a rotation speed of 125rpm, and the cell density and the survival rate are determined by counting, and cells with the growth in an exponential phase (density of about 4 to 6X 106/ml) and the survival rate of more than 98% are used for transfection;

two 15mL sterile centrifuge tubes were prepared, 5mL KPM (supplemental manufacturer information) and 100 μg sterile plasmid DNA were added to one of the tubes, gently swirled and mixed; taking the other branch, adding 5mL KPM and 500 mu L TA-293 (supplementary manufacturer information) transfection reagent, blowing and mixing uniformly;

transferring all the liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and blowing and uniformly mixing; standing for 10 minutes at room temperature to prepare a plasmid-carrier compound;

Taking out cells from the constant temperature shaking table, adding the prepared plasmid-carrier compound while shaking, and placing the cells back into the CO ₂ constant temperature shaking table for culturing; 600. Mu.L of 293 cell protein expression enhancer (KE-293, make-up manufacturer) and transient transfection nutrition additive (KT-feed 50X, martensitic kai R Biotechnology Co., ltd.) were added 24 hours after transfection to increase the product expression level; on day 5 post transfection, cell supernatants were harvested.

Example 4: purification and concentration of bispecific antibodies AmF1, amF2, amF3, amF4, amF5

Purification of antibodies

The antibodies were purified using an AKTA purifier chromatograph, specifically, protein a affinity chromatography column.

Preparing Protein A affinity chromatographic column buffer (balance buffer: 9.5mM sodium dihydrogen phosphate, 40.5mM disodium hydrogen phosphate, 200mM sodium chloride, pH 7.0; elution buffer: 100mM glycine, 100mM sodium chloride, pH 3.0; neutralization buffer: 1M Tris, pH 8.0), and filtering with 0.45 μm filter membrane; centrifuging the sample, taking supernatant, and filtering with a 0.45 μm filter membrane; cleaning and preparing the pipeline; installing a Protein A column, preparing to load a sample after the column is balanced, and zeroing UV; loading, wherein no bubbles enter the pipeline during the loading period; the breakthrough peak was again washed with equilibration buffer until the UV value dropped to baseline level; adjusting the flow rate to 5.0mL/min, eluting the protein from the column by using an elution buffer and collecting the protein; finally, the pH of the collection solution was adjusted to about 7.0 using a neutralization buffer.

Ultrafiltration concentration

Collecting the purified product in a 50mL centrifuge tube, adding the product into an ultrafiltration tube in batches, and centrifuging the product to 1mL under the conditions of the rotating speed of 3,800rpm and the temperature of 4 ℃; after all proteins pass through the ultrafiltration tube, filling 1xPBS in the ultrafiltration tube for replacement, centrifuging to 1mL at the rotation speed of 3,800rpm and the temperature of 4 ℃, and repeating for 3 times; collecting the liquid on the ultrafilter tube, packaging protein, and storing in a refrigerator at-80deg.C.

Example 5: bispecific antibodies AmF1, amF2, amF3, amF5 purity assay

BCA (biquinolinecarboxylic acid) quantitative protein concentration

Protein concentration of the ultrafiltration concentrated product obtained in example 4 was measured with BCA assay kit (Shanghai bi yun biotechnology limited).

High performance liquid chromatography (SEC-HPLC) for detecting antibody purity

Antibody purity was measured as follows: the mobile phase (50 mM PBS (phosphate buffer) +150mM NaCl, pH 6.8) was prepared and filtered with a 0.45um filter membrane, sonicated; filtering the ultrafiltered protein by using a needle type filter; inserting an inner cannula into a glass penicillin bottle, and adding 200 mu L of a sample to be detected; the sample is placed in an HPLC sample tank, the HPLC working software is opened, the system is flushed with equilibration buffer, the baseline change is observed, and when the system has reached an equilibrium state, the procedure is set, and the sample detection is started. Detection conditions: the flow rate is 0.5mL/min; the time is 30min, the sample injection amount is 20 mu L, the column temperature is 25 ℃, and the detection wavelength is 214nm;280nm; isocratic elution; and after the sample detection is finished, analyzing the result.

Example 6: bispecific antibodies AmF1, amF2, amF3, amF5 purity assay results

BCA quantitative protein concentration results (see Table 4)

Table 4 BCA quantitative protein concentration results

As can be seen from the results of the quantitative protein concentration in BCA, the antibody of the present invention has a high supernatant expression level.

High performance liquid chromatography (SEC-HPLC) detection results

After the sample has been tested by SEC-HPLC, the SEC-HPLC results of the antibodies AmF1, amF2, amF3, amF5 are shown in FIGS. 2-1,2-2,2-3,2-4, in particular in Table 5:

TABLE 5 SEC HPLC detection results

ND refers to not measured.

As can be seen from the above SEC-HPLC detection results, the purity of the antibodies obtained in the present invention was very high.

Example 7: cell killing activity assay for bispecific antibody AmF2

Cell killing activity assay for CD3/DLL3 was performed using a DLL3 positive SHP-77 cell line (human small cell lung carcinoma cell, ATCC) as follows:

Activated T cells were co-cultured with a SHP-77GFP & luc cell line (SHP-77 cell line overexpressing the Green Fluorescent Protein (GFP) gene and the luciferase (luciferase) gene), T cells and cancer cell efficiency target ratio (E: t=5:1). Wherein the SHP-77GFP & luc cell number is 2x10 ⁴ cells/well, the T cell number is 1x10 ⁵ cells/well, the co-culture system is 200. Mu.l, and the co-culture is carried out for 24h. The antibody concentration is set to 1000ng/ml,100ng/ml,10ng/ml and 1ng/ml, co-culture groups (without antibody) and co-culture groups (with antibody), cancer cell groups alone (with or without antibody), T cell groups alone, three compound wells are set in each group, and 96-U type plates are selected as culture containers. The specific operation is as follows:

8ml of 200ng/ml antibody solution was prepared using R10F (RPMI-1640 medium containing 10% FBS (Gibco) wherein the RPMI-1640 medium was purchased from ATCC), and after sterilization filtration through a 0.22. Mu.M filter, 500. Mu.l of 20ng/ml antibody solution (50ul+450 ul R10F) was obtained by gradient dilution, 500. Mu.l of 2ng/ml antibody solution;

The corresponding number of SHP-77luc & GFP, the concentration of 2x10 ^{^5} cells/ml and the concentration of R10F culture medium are re-suspended by using different concentrations of antibody liquid, the concentration of T cells is 1x10 ^{^6} cells/ml, 100 mu l T cells and cancer cells are respectively added to each hole of a co-culture group, 100 mu L of cancer cells and 100 mu l R F are added to the separate culture groups, and finally, the mixture is uniformly mixed by a row gun.

After co-culturing for 24 hours, detecting by a substrate detection method, detecting the killing rate, centrifuging for 5 minutes by 500g, absorbing 100ul of supernatant (the supernatant which can remain below 80ul in a hole) by a pipetting gun, adding a substrate (Biyun Tian good number: RG 055M) of an One-Lumi ^TM firefly luciferase reporter gene detection kit for recovering room temperature, 100 ul/hole, uniformly mixing by a pipetting device, cracking for 5 minutes at room temperature, transferring all lysates into a black 96-well plate, and detecting by an enzyme-labeled instrument, and then calculating the killing rate.

GRAPHPAD PRISM 5.0.0 data were analyzed and organized and killing%1-co-cultured group luminescence/individual cancer cell luminescence%100%

The experimental results are shown in fig. 3 and table 6, and the results show that the bispecific antibody AmF2 provided by the invention has a strong killing effect on the DLL3 positive SHP-77 cells. Further, the antibody assembly platform can effectively prevent the double-specificity antibody from being mismatched, and the produced multi-specificity antibody has stronger specificity.

TABLE 6 killing of DLL3 positive SHP-77 cells by bispecific antibody AmF2

Example 8: design of bispecific antibody molecule TROP2 XPRR and monoclonal antibody molecule TROP2

An exemplary bispecific antibody of the invention comprises a first antigen binding moiety and a second antigen binding moiety, wherein the first antigen binding moiety targets a first antigen TROP2 and the second antigen binding moiety targets a 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 of the second light chain variable domain VLPRLR (sequence shown as SEQ ID NO: 37) of the PRLR-targeting monoclonal antibody is linked to the N-terminus of the Fos domain (sequence shown as SEQ ID NO: 10) via a first linker L1 (GGGGS), and the C-terminus of the second heavy chain variable domain VHPRLR (sequence shown as SEQ ID NO: 38) of the PRLR-targeting monoclonal antibody is linked to the N-terminus of the Jun domain (sequence shown as SEQ ID NO: 9) via a second linker L2 (GGGGS); the first light chain variable domain VLTROP (sequence shown as SEQ ID NO: 39) and the first heavy chain variable domain VHTROP2 (sequence shown as SEQ ID NO: 40) of a TROP 2-targeting monoclonal antibody form the structure shown in FIG. 4, designated as BmF. The first light chain sequence is shown as SEQ ID NO. 41, the first heavy chain sequence is shown as SEQ ID NO. 42, the second light chain sequence is shown as SEQ ID NO. 43, the second heavy chain sequence is shown as SEQ ID NO. 44, and the amino acids T366S-L368A-Y407V (Hole) on the first heavy chain sequence and the 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 was designated mAb-T, the light chain amino acid sequence was SEQ ID NO. 45, and the heavy chain amino acid sequence was SEQ ID NO. 46.

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

Gene synthesis

The sequences expressing bispecific antibody BmF and monoclonal antibody mAb-T of example 7 were constructed and subjected to codon optimization and then submitted to total gene synthesis by Nanjing Style, a gene synthesis company.

PCGS3 expression vector construction

For bispecific antibodies BmF:

Using a seamless cloning procedure, the heavy and light chains of the first antigen binding portion were cloned into the multiple cloning site of the pCGS vector, and the heavy and light chains of the second antigen binding portion were also cloned into the multiple cloning site of the pCGS vector, with the heavy chain cloned into BstBI and PacI cleavage sites and the light chain cloned into HindIII and XhoI cleavage sites.

The specific method comprises the following steps:

The enzyme-cleaved components in Table 8 were mixed well and the resulting reaction solution incubated in a 37℃metal bath for 3h; the digested products were recovered using a general agarose gel DNA recovery kit (Tiangen Biochemical technologies (Beijing) Co., ltd.).

TABLE 7 heavy chain double cleavage System

T4 DNA ligase (NEB Co., USA) ligated DNA recovered product with pCGS vector cleavage recovered product, specific ligation reaction system was shown in Table 8, ligation reaction components were mixed, and the well-mixed reaction solution was placed in a metal bath at 16℃overnight.

Table 8 connection reaction System

Transformation and inoculation were performed as follows: mu.L of the above-mentioned ligation product was added to 50. Mu.L of Trans 5. Alpha. Competent cells (Beijing full gold Biotechnology Co., ltd.), mixed well on ice for 30min, then placed in a constant temperature water bath at 42℃for heat shock for 90s, then placed on ice for 1min, finally 250. Mu.L of LB medium was added, and shaking culture was performed at 37℃and 220rpm for 1h, 100. Mu.L of LB medium (Luria-Bertani medium, broth medium) coated with ampicillin+ (amp+, tiangen Biochemical Co., ltd.) was used, and culture was performed overnight at 37 ℃.

Selecting a monoclonal from each LB culture plate, respectively placing the monoclonal into 1.5mL EP tubes, adding 300 mu LAmp + LB culture medium into each tube, and carrying out shake culture for 8 hours at 37 ℃ and 220 rpm; culturing thallus PCR, electrophoresis, and sequencing bacterial liquid with correct band. Plasmid DNA was extracted as follows: after sequencing the correct bacterial solution and amplifying by shaking, plasmid DNA was extracted using a plasmid miniprep kit (Beijing full gold Biotechnology Co., ltd.).

Light chain double cleavage and ligation were continued, wherein the light chain double cleavage system is shown in table 9:

Table 9 light chain double cleavage System

The rest steps are the same as above.

The constructed plasmid was extracted with endotoxin-free plasmid big extraction kit (DP 117, tiangen Biochemical technology Co., ltd.) and named p-BmF, and the concentration and purity of plasmid DNA were measured with a Nanodrop ultraviolet spectrophotometer (Thermo FISHER SCIENTIFIC Co., USA) for use.

The mAb-T sequence of the monoclonal antibody is constructed into pCGS expression vectors according to the same method as above, and the constructed plasmid is named p-mAb-T.

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

Bispecific antibody p-BmF and monoclonal antibody p-mAb-T were expressed separately as follows:

293f cells (Zhuhai kai R. Biotechnology Co., ltd.) were cultured using KOP293 cell medium (Zhuhai kai R., K03252) in a 5% CO ₂ shaker at 37℃under a rotation speed of 100-130 rpm and a humidity of 75% or higher. The day before transfection, 293f cells in the logarithmic growth phase, well grown, were passaged to 2X10 ⁶/mL, incubated overnight on a shaker (110 rpm,37 ℃,5% CO 2) and the next day for transfection. TA-293 (293 cell suspension chemical transfection reagent) is preheated at normal temperature before transfection, KPM (serum-free cell transfection buffer solution) is used for measuring cell density and activity rate, the density is 4X 10 ⁶/mL, and the activity rate is more than 97%. Transfection was performed according to the guidelines for the use of the KOP293 transient transfection protein expression system. The expression supernatants were harvested 5 days after transfection by centrifugation.

Example 11: purification of bispecific antibodies BmF and monoclonal antibodies mAb-T

The expression supernatants of bispecific antibody p-BmF and monoclonal antibody p-mAb-T were purified by the following steps: the antibody with Fc domain was obtained from the expression supernatant by filtration through a 0.45uM filter membrane using a ProteinA affinity column. Preparing a ProteinA affinity chromatography column buffer, wherein the balance buffer is 9.5mM sodium dihydrogen phosphate, 40.5mM disodium hydrogen phosphate and the pH value is 7.4; the elution buffer was 0.1M glycine, pH 3.0. The eluted antibody was replaced with PBS buffer. Purified bispecific antibody BmF and monoclonal antibody mAb-T are obtained.

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

The purified bispecific antibody BmF and monoclonal antibody mAb-T were subjected to high performance liquid chromatography (SEC-HPLC) detection: the mobile phase (50 mM PBS (phosphate buffer) +150mM NaCl, pH 6.8) was prepared and filtered through a 0.45um filter, sonicated; filtering the ultrafiltered protein by using a needle type filter; inserting an inner cannula into a glass penicillin bottle, and adding 200 mu L of a sample to be detected; the sample is placed in an HPLC sample tank, the HPLC working software is opened, the system is flushed with equilibration buffer, the baseline change is observed, and when the system has reached an equilibrium state, the procedure is set, and the sample detection is started. Detection conditions: the flow rate is 0.5mL/min; the time is 30min, the sample injection amount is 20 mu L, the column temperature is 25 ℃, and the detection wavelength is 214nm;280nm; isocratic elution; and after the sample detection is finished, analyzing the result.

From the results of fig. 5 and 6, it is calculated that: monoclonal antibody mAb-T was 96% pure (see FIG. 5) and bispecific antibody BmF was 94% pure (see FIG. 6). From the above results, it can be seen that the purity of the antibodies obtained in the present invention is relatively high, and the bispecific antibodies BmF of the present invention are effective in preventing 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 for antigen

The binding affinities of bispecific antibody BmF, monoclonal antibody mAb-T to TROP2 and PRLR, respectively, were measured using a surface plasmon resonance biosensor. Affinity analysis the analysis was performed by SPR using the instrument Biacore, T200 (purchased from GE HEALTHCARE); mobile phase (GE, BR-1006-69). All reagents and materials can be purchased from GE unless otherwise indicated.

Table 10 instrument and reagent specific information

Experimental procedure

Human Fab capture molecules were covalently coupled to CM5 biosensing chips (Cat.#BR-1000-12, GE) to affinity capture antibodies to be tested, as described in the instructions of the human Fab capture kit (Cat.#28-9583-25, GE). The antigen used was human TROP2-his (CAT #10428-H08H-100,Sino biological) antigen or human PRLR antigen (available from Baipusais, cat# PRR-H52 Ha); bispecific antibody BmF and monoclonal antibody mAb-T are analytes and their affinity for TROP2 or PRLR is detected. After analyte gradient dilution (100 nM,50nM,25nM,10nM,5 nM), the following was performed:

Capture antibody: the antibody was captured at a flow rate of 10. Mu.l/min for 60s.

Analysis: after analyte gradient dilution (100 nM,50nM,25nM,10nM,5 nM), the antigen was bound for 120s at a flow rate of 30. Mu.l/min followed by dissociation for 900s at a flow rate of 30. Mu.l/min.

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

Treatment results: and waiting for the end of the experiment, and opening the analysis software to process the result.

Experimental results

TABLE 11 affinity results of bispecific antibody BmF and monoclonal antibody mAb-T for antigen

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

The experimental results show that: the antibody produced by the antibody assembly platform can effectively prevent the mismatch and assembly between the light chain and the heavy chain and between the heavy chain and the heavy chain.

The present invention has been illustrated by the above-described embodiments, but it should be understood that the above-described embodiments are for purposes of illustration and description only and are not intended to limit the invention to the embodiments described. In addition, those skilled in the art will appreciate that the present invention is not limited to the embodiments described above, and that many variations and modifications are possible in light of the teachings of the invention, which variations and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

A polypeptide complex comprising a first antigen binding portion comprising:

A first polypeptide comprising a first light chain variable domain from N-terminus to C-terminus (VL 1) and a first pairing domain from N-terminus to C-terminus (X1), said VL1 being operably linked to X1, and

A second polypeptide comprising a first heavy chain variable domain from N-terminus to C-terminus (VH 1) and a second pairing domain from N-terminus to C-terminus (X2), said VH1 being operably linked to X2, wherein,

The first antigen binding portion specifically binds to a first antigen, and the X1 and X2 are capable of forming a leucine zipper structure.
The polypeptide complex of claim 1, wherein,

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

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

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

The N-terminus of VH1 is operably linked to the C-terminus of X2; or (b)

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

The N-terminus of VH1 is operably linked to the C-terminus of X2, and the C-terminus of VH1 is further operably linked to the N-terminus of the second domain comprising the first CH1; or (b)

4) The C-terminus of VL1 is operably linked to the N-terminus of a first domain comprising a first CL, the C-terminus of the first domain being operably linked to the N-terminus of X1, and

The C-terminus of VH1 is operably linked to the N-terminus of a second domain comprising the first CH1, which is operably linked to the N-terminus of X2.
The polypeptide complex according to claim 1 or 2, wherein,

X1 comprises a 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 domain, X2 comprises a domain capable of forming a leucine zipper with said 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 domain; or (b)

X2 comprises a 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 domain, and X1 comprises a domain capable of forming a leucine zipper with said 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 domain.
The polypeptide complex according to any one of claim 1 to 3, wherein,

X1 comprises a Jun domain and X2 comprises a Fos domain;

X1 comprises a Fos domain and X2 comprises a Jun domain;

x1 comprises a Jun domain and X2 comprises a FosW domain;

x1 comprises FosW domains and X2 comprises Jun domains;

x1 comprises a Myc domain and X2 comprises a Max domain;

X1 comprises a Max domain and X2 comprises a Myc domain;

X1 comprises WinzipA domains and X2 comprises WinzipB1 domains;

x1 comprises WinzipB1 domains and X2 comprises WinzipA2 domains;

x1 comprises an ACID-p1 domain and X2 comprises a BASE-p1 domain;

X1 comprises a BASE-p1 domain and X2 comprises an ACID-p1 domain;

X1 comprises a GCN4 domain and X2 comprises a GCN4 domain; or (b)

X1 comprises a C/EBP domain and X2 comprises a C/EBP domain.
The polypeptide complex according to claim 3 or 4, wherein,

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 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 to SEQ ID NO. 10; and/or

The FosW domain is a sequence as set forth in SEQ ID NO. 29, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to 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 to SEQ ID NO. 11; and/or

The Myc domain is 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; and/or

The WinzipA domain is a sequence as set forth in SEQ ID NO. 13, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO. 13; and/or

The WinzipB domain is a sequence as set forth in SEQ ID NO. 14, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO. 14; and/or

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

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

The GCN4 domain is the sequence shown as SEQ ID NO. 35, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to 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 to SEQ ID NO. 36.
The polypeptide complex of any one of claims 1-5, wherein VL1 is operably linked to X1 via a first linker L1, VH1 is operably linked to X2 via a second linker L2, L1 and L2 may or may not be present, and when L1 and L2 are present, L1 and L2 are each independently selected from the following polypeptide fragments: (G _xS) _y、(T _xG) _y, and (S _xG) _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 of any one of claims 1-6, wherein the first antigen is selected from the group consisting of: foreign antigens, endogenous antigens, autoantigens, neoantigens, viral antigens and tumor antigens.
A multi-specific polypeptide complex comprising:

A first polypeptide complex which is a polypeptide complex according to any one of claims 1 to 7, which specifically binds to a first antigen, and

A second polypeptide complex comprising a second antigen binding portion that specifically binds to a second antigen,

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

Preferably, the second polypeptide complex comprises a second heavy chain variable domain (VH 2) from N-terminus to C-terminus and a second light chain variable domain (VL 2) from N-terminus to C-terminus, the VH1 of the first polypeptide complex and VL2 of the second polypeptide complex, and/or a mismatch is not likely to occur between VH2 of the second polypeptide complex and VL1 of the first polypeptide complex; preferably, the multispecific polypeptide complex is a bispecific polypeptide complex; preferably, VH2 and VL2 of the second antigen binding portion form a second antigen binding site that specifically binds to a second antigen.
The multi-specific polypeptide complex of claim 8, wherein the second antigen-binding portion of the second polypeptide complex comprises:

A third polypeptide comprising the VL2, the VL2 operably linked to a third domain comprising a second CL or a third mating domain (X3), and

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

The X3 and X4 can form a leucine zipper structure.
The multi-specific polypeptide complex of claim 9, wherein,

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

The C-terminal of VH2 is operably connected to the N-terminal of X4; or (b)

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

The N-terminus of VH2 is operably linked to the C-terminus of X4; or (b)

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

The C-terminal of VH2 is operably linked to the N-terminal of the fourth domain; or (b)

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

The N-terminus of VH2 is operably linked to the C-terminus of X4, and the C-terminus of VH2 is further operably linked to the N-terminus of the fourth domain; or (b)

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

The C-terminus of VH2 is operably linked to the N-terminus of the fourth domain, which is operably linked to the N-terminus of X4.
The multi-specific polypeptide complex of any one of claims 8-10, further comprising a first dimerization domain and the second polypeptide complex further comprises a second dimerization domain, the first dimerization domain and the second dimerization domain being associated.
The multi-specific polypeptide complex of claim 11, wherein the C-terminus of the first dimerization domain is operably linked to the N-terminus of the fifth mating domain (X5) and the C-terminus of the second dimerization domain is operably linked to the N-terminus of the sixth mating domain (X6), wherein

The X5 and X6 form dimers, preferably the X5 and X6 form leucine zipper structures.
The multi-specific polypeptide complex of 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 N-terminus of the second dimerization domain; or (b)

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 operably linked to the N-terminus of the second dimerization domain; or (b)

The C-terminus of the second domain of the first polypeptide complex is operably linked to the N-terminus of the first multimerization domain, and the C-terminus of the fourth domain of the second polypeptide complex is operably linked to the N-terminus of the second multimerization domain; or (b)

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

preferably, the first and second dimerization domains are joined by a means selected from the group consisting of: an antibody hinge region or portion thereof, a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, a hydrophobic-hydrophilic interaction, or a combination thereof;

More preferably, the first and second dimerization domains are bound by: the antibody hinge region or portion thereof binds, more preferably the hinge region or portion thereof of IgG1, igG2, igG3 or IgG 4.
The multi-specific polypeptide complex of claim 12 or 13, wherein 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, the first CH2, first CH3, second CH2, and second CH3 are each independently derived from IgG1, igG2, igG3, or IgG4.
The multi-specific polypeptide complex of any one of claims 12-14, wherein the first and second dimerization domains are not identical and bind in a manner that prevents homodimerization and/or facilitates heterodimerization;

Preferably, the first and second dimerization domains are joined by a means selected from the group consisting of: knob-into-hole, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and ways of increasing flexibility;

Further preferably, the first dimerization domain and the second dimerization domain are bound by a knob-intoo-hole manner.
The multi-specific polypeptide complex of any one of claim 8-15, wherein,

X3 and X5 each independently comprise a Jun domain, fos domain, fosW domain, myc domain, max domain, winzipA2 domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain, X4 and X6 each independently comprise a domain capable of forming a leucine zipper structure with said 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 domain; or (b)

X4 and X6 each independently comprise a Jun domain, fos domain, fosW domain, myc domain, max domain, winzipA2 domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain, and X3 and X5 each independently comprise a domain capable of forming a leucine zipper with said 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 domain.
The multi-specific polypeptide complex of any one of claim 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 FosW domains, and X4 and X6 each independently comprise Jun domains;

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 WinzipA domain, and X4 and X6 each independently comprise WinzipB domain;

x3 and X5 each independently comprise WinzipB domain, and X4 and X6 each independently comprise WinzipA 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 (b)

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, winzipA domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain and C/EBP domain are as defined in claim 5.
The multi-specific polypeptide complex of any one of claims 8-16, wherein the VL2 is operably linked to X3 via a third linker L3, the VH2 is operably linked to X4 via a fourth linker L4, L3 and L4 are each independently present or absent, and when L3 and L4 are present, L3 and L4 are each independently selected from the following polypeptide fragments: (G _xS) _y、(T _xG) _y, and (S _xG) _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, L3 and L4 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG.
The polypeptide complex of any one of claims 1-18, wherein the polypeptide complex is a domain engineered antibody or antigen binding fragment thereof.
A multispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein,

The first antigen binding portion comprises:

a first light chain comprising a first light chain variable domain (VL 1), said VL1 being operably linked to a first pairing domain (X1), and

A first heavy chain comprising a first heavy chain variable domain (VH 1), said VH1 being operably linked to a second pairing domain (X2), wherein,

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

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

The first antigen and the second antigen are different, 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, between the second heavy chain and the first light chain are not likely to occur;

preferably, the multispecific antibody is a bispecific antibody.
The antibody of claim 20, wherein,

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

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

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

The N-terminus of VH1 is operably linked to the C-terminus of X2; or (b)

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

The N-terminus of VH1 is operably linked to the C-terminus of X2, and the C-terminus of VH1 is operably linked to the N-terminus of CH 1; or (b)

4) The C-terminus of VL1 is operably linked to the N-terminus of a first domain comprising a first CL, the C-terminus of the first domain being operably linked to the N-terminus of X1, and

The C-terminus of VH1 is operably linked to the N-terminus of a second domain comprising the first CH1, which is operably linked to the N-terminus of X2.
The antibody according to claim 20 or 21, wherein,

The second light chain comprises a second light chain variable domain (VL 2), the VL2 being operably linked to a second CL, the second heavy chain comprising a second heavy chain variable domain (VH 2), the VH2 being operably linked to a second CH1; or (b)

The second light chain comprises a second light chain variable domain (VL 2), the VL2 is operably linked to a third mating domain (X3), the second heavy chain comprises a second heavy chain variable domain (VH 2), the VH2 is operably linked to a fourth mating domain (X4), the X3 and X4 are capable of forming a leucine zipper structure.
The antibody of claim 22, wherein,

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

The C-terminal of VH2 is operably connected to the N-terminal of X4; or (b)

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

The N-terminus of VH2 is operably linked to the C-terminus of X4; or (b)

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

The C-terminal of VH2 is operably connected to the N-terminal of CH 1; or (b)

4) The N-terminus of VL2 is operably linked to the C-terminus of X3, the C-terminus of VL2 is further operably linked to the N-terminus of a second CL, and

The N-terminus of VH2 is operably linked to the C-terminus of X4, and the C-terminus of VH2 is further operably linked to the N-terminus of CH 1; or (b)

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

The C-terminal of VH2 is operably linked to the N-terminal of CH1, and the C-terminal of CH1 is operably linked to the N-terminal of X4.
The antibody of any one of claim 20 to 23, wherein,

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

The second light chain comprises a second light chain variable domain (VL 2) and a second CL from the N-terminus to the C-terminus, and the second heavy chain comprises a second heavy chain variable domain (VH 2) and a constant domain (CH 1) from the N-terminus to the C-terminus; or (b)

2) The first light chain comprises a first light chain variable domain (VL 1) from the N-terminus to the C-terminus, and a first pairing domain (X1), and the first heavy chain comprises a first heavy chain variable domain (VH 1) from the N-terminus to the C-terminus, and a second pairing domain (X2); and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), and a third pairing domain (X3), and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), and a fourth pairing domain (X4); or (b)

3) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), a first light chain variable domain (VL 1) and a second CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), a first heavy chain variable domain (VH 1) and a constant domain (CH 1); and

The second light chain comprises from N-terminus to C-terminus a third pairing domain (X3), a second light chain variable domain (VL 2) and a second CL, and the second heavy chain comprises from N-terminus to C-terminus a fourth pairing domain (X4), a second heavy chain variable domain (VH 2) and a constant domain (CH 1); or (b)

4) The first light chain comprises a first pairing domain (X1) from the N-terminus to the C-terminus, and a first light chain variable domain (VL 1), and the first heavy chain comprises a second pairing domain (X2) from the N-terminus to the C-terminus, and a first heavy chain variable domain (VH 1); and

The second light chain comprises a third pairing domain (X3) from the N-terminus to the C-terminus, and a second light chain variable domain (VL 2), and the second heavy chain comprises a fourth pairing domain (X4) from the N-terminus to the C-terminus, and a second heavy chain variable domain (VH 2).
The antibody of any one of claims 20-24, wherein the first antigen binding portion further comprises a first dimerization domain and the second antigen binding portion further comprises a second dimerization domain, the first dimerization domain and the second dimerization domain binding.
The antibody of any one of claims 25, wherein the C-terminus of the first dimerization domain is operably linked to the N-terminus of the fifth mating domain (X5) and the C-terminus of the second dimerization domain is operably linked to the N-terminus of the sixth mating 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; or (b)

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 N-terminus of the second dimerization domain; or (b)

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

Preferably, the first and second dimerization domains are joined by a means selected from the group consisting of: an antibody hinge region or portion thereof, a linker, disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, hydrophobic-hydrophilic interactions, and combinations thereof;

more preferably, the first and second dimerization domains are bound by: the antibody hinge region or portion thereof, more preferably the hinge region or portion thereof of IgG1, igG2, igG3 or IgG 4.
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 CH3;

Preferably, the antibodies first CH2, first CH3, second CH2 and second CH3 are each independently derived from IgG1, igG2, igG3 or IgG4.
The antibody of any one of claims 24-28, wherein the first and second dimerization domains are not identical and bind in a manner that prevents homodimerization and/or favors heterodimerization.

Preferably, the first and second dimerization domains are joined by a means selected from the group consisting of: knob-into-hole, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or ways of increasing flexibility.
The antibody of any one of claims 20-29, wherein

1) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), and a first mating domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), a second mating domain (X2), and a first multimerization domain; and

The second light chain comprises a second light chain variable domain (VL 2) and a second CL from the N-terminus to the C-terminus, the second heavy chain comprises a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain from the N-terminus to the C-terminus; or (b)

2) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), and a first pairing domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), a second pairing domain (X2), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises from N-terminus to C-terminus a second light chain variable domain (VL 2) and a constant domain (CL), the second heavy chain comprises from N-terminus to C-terminus a second heavy chain variable domain (VH 2), a constant domain (CH 1), a second dimerization domain, and a sixth pairing domain (X6); or (b)

3) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), and a first mating domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), a second mating domain (X2), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), and a third mating domain (X3), and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), a fourth mating domain (X4), and a second dimerization domain; or (b)

4) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), and a first pairing domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), a second pairing domain (X2), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), and a third pairing domain (X3), the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), a fourth pairing domain (X4), a second dimerization domain, and a sixth pairing domain (X6); or (b)

5) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), a first light chain variable domain (VL 1) and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), a first heavy chain variable domain (VH 1), a first CH1, and a first dimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), a second light chain variable domain (VL 2) and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain; or (b)

6) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), a first light chain variable domain (VL 1) and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), a first heavy chain variable domain (VH 1), a first CH1, a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), a second light chain variable domain (VL 2) and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, and a sixth pairing domain (X6); or (b)

7) The first light chain comprises a first pairing domain (X1) from the N-terminus to the C-terminus, and a first light chain variable domain (VL 1), the first heavy chain comprises a second pairing domain (X2), a first heavy chain variable domain (VH 1), and a first multimerization domain from the N-terminus to the C-terminus; and

The second light chain comprises a third pairing domain (X3) from the N-terminus to the C-terminus, and a second light chain variable domain (VL 2), and the second heavy chain comprises a fourth pairing domain (X4), a second heavy chain variable domain (VH 2), and a second dimerization domain from the N-terminus to the C-terminus.

8) The first light chain comprises from N-terminus to C-terminus a first pairing domain (X1), and a first light chain variable domain (VL 1), and the first heavy chain comprises from N-terminus to C-terminus a second pairing domain (X2), a first heavy chain variable domain (VH 1), a first dimerization domain, and a fifth pairing domain (X5); and

The second light chain comprises a third pairing domain (X3) from the N-terminus to the C-terminus, and a second light chain variable domain (VL 2), and the second heavy chain comprises a fourth pairing domain (X4), a second heavy chain variable domain (VH 2), a second dimerization domain, and a sixth pairing domain (X6) from the N-terminus to the C-terminus.
The antibody of any one of claim 20 to 30, wherein,

X1, X3 and X5 each independently comprise a Jun domain, fos domain, fosW domain, myc domain, max domain, winzipA2 domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain, X2, X4 or X6 each independently comprise a domain capable of forming a leucine zipper with said 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 domain; or (b)

X2, X4 and X6 each independently comprise a Jun domain, fos domain, fosW domain, myc domain, max domain, winzipA2 domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain or C/EBP domain, and X1, X3 or X5 each independently comprise a domain capable of forming a leucine zipper with said 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 domain.
The antibody of any one of claim 20 to 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 FosW domains, and X2, X4 and X6 each independently comprise Jun domains;

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 WinzipA domain, X2, X4 and X6 each independently comprise WinzipB domain;

X1, X3 and X5 each independently comprise WinzipB1 domains, and X2, X4 and X6 each independently comprise WinzipA domains;

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 (b)

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, winzipA domain, winzipB domain, ACID-p1 domain, BASE-p1 domain, GCN4 domain and C/EBP domain are 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, the VL2 is operably linked to X3 through a third linker L3, the VH2 is operably linked to X4 through a fourth linker L4, the C-terminus of the first dimerization domain is linked to X5 through a fifth linker L5, the C-terminus of the second dimerization domain is linked to X6 through a sixth linker L6, each independently present or absent, and when L1-L6 are optionally present, each L1-L6 is independently selected from the following polypeptide fragments: (G _xS) _y、(T _xG) _y, and (S _xG) _y, wherein x is an integer of 1, 2, 3, 4, or 5 for each occurrence, and y is an integer of 1, 2, 3, 4, 5, or 6 for each occurrence;

preferably, each of L1-L6 is independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG;

Further preferably, L1-L4 are each independently linked via their C-terminus to a cleavage site peptide selected from the group consisting of factor Xa, thrombin, enterokinase, and SUMO, and L5-L6 are each independently linked via their N-terminus to a cleavage site peptide.
The antibody of any one of claim 20 to 33, wherein,

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

The second light chain comprises a second light chain variable domain (VL 2) and a constant domain (CL) from the N-terminus to the C-terminus, the second heavy chain comprises a second heavy chain variable domain (VH 2), a constant domain (CH 1), and a second dimerization domain from the N-terminus to the C-terminus; or (b)

2) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), L1, and a first pairing domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), 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 (VL 2) and a second CL from the N-terminus to the C-terminus, the second heavy chain comprising a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and a sixth pairing domain (X6); or (b)

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

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), L3, and a third pairing domain (X3), and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), L4, a fourth pairing domain (X4), and a second dimerization domain; or (b)

4) The first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL 1), L1, and a first pairing domain (X1), and the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH 1), L2, a second pairing domain (X2), a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), L3, and a third pairing domain (X3), and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), L4, a fourth pairing domain (X4), a second dimerization domain, L6, and a sixth pairing domain (X6); or (b)

5) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), L1, a first light chain variable domain (VL 1) and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), L2, a first heavy chain variable domain (VH 1), a first CH1, and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), L3, a second light chain variable domain (VL 2) and a second CL, the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain; or (b)

6) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), L1, a first light chain variable domain (VL 1) and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), L2, a first heavy chain variable domain (VH 1), a first CH1, a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), L3, a second light chain variable domain (VL 2) and a second CL, the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and a sixth pairing domain (X6); or (b)

7) The first light chain comprises, from N-terminus to C-terminus, a first pairing domain (X1), L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a second pairing domain (X2), L2, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises a third pairing domain (X3), L3, and a second light chain variable domain (VL 2) from the N-terminus to the C-terminus, and the second heavy chain comprises a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH 2), and a second dimerization domain from the N-terminus to the C-terminus.

8) The first light chain comprises from N-terminus to C-terminus a first pairing domain (X1), L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises from N-terminus to C-terminus a second pairing domain (X2), L2, a first heavy chain variable domain (VH 1), a first dimerization domain, L5, and a fifth pairing domain (X5); and

The second light chain comprises, from N-terminus to C-terminus, a third pairing domain (X3), L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a fourth pairing domain (X4), L4, a second heavy chain variable domain (VH 2), a second dimerization domain, L6, and a sixth pairing domain (X6).
The antibody of any one of claim 20 to 34, wherein,

1) The first light chain comprises a first light chain variable domain (VL 1), L1, and Fos domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, jun domain, and a first multimerization domain from the N-terminus to the C-terminus; and

The second light chain comprises a second light chain variable domain (VL 2) and a second CL from the N-terminus to the C-terminus, the second heavy chain comprises a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain from the N-terminus to the C-terminus; or (b)

2) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, a first dimerization domain, L5, and Max domain from the N-terminus to the C-terminus; and

The second light chain comprises a second light chain variable domain (VL 2) and a second CL from the N-terminus to the C-terminus, the second heavy chain comprising a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and a Myc domain; or (b)

3) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, and a first multimerization domain from the N-terminus to the C-terminus; and

The second light chain comprises a second light chain variable domain (VL 2), L3, and Fos domain from the N-terminus to the C-terminus, the second heavy chain comprises a second heavy chain variable domain (VH 2), L4, jun domain, and a second dimerization domain from the N-terminus to the C-terminus; or (b)

4) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, a first dimerization domain, L5, and Max domain from the N-terminus to the C-terminus; and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), L3, and Fos domain, and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), L4, jun domain, second dimerization domain, L6, and Myc domain; or (b)

5) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a first light chain variable domain (VL 1), and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a first heavy chain variable domain (VH 1), a first CH1, and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a second light chain variable domain (VL 2), and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain; or (b)

6) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a first light chain variable domain (VL 1), and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a first heavy chain variable domain (VH 1), a first CH1, a first dimerization domain, L5, and WinzipA2 domains; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a second light chain variable domain (VL 2), and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and WinzipB1 domains; or (b)

7) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a second heavy chain variable domain (VH 2), and a second dimerization domain; or (b)

8) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a first heavy chain variable domain (VH 1), a first dimerization domain, L5, and WinzipA2 domains; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a second heavy chain variable domain (VH 2), a second dimerization domain, L6, and WinzipB1 domains; or (b)

9) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, fosW domain, L2, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Myc domain, L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Max domain, L4, a second heavy chain variable domain (VH 2), and a second dimerization domain.

10 The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, fosW domains, L2, a first heavy chain variable domain (VH 1), a first dimerization domain, L5, and WinzipA2 domains; and

The second light chain comprises, from N-terminus to C-terminus, a Myc domain, L3, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Max domain, L4, a second heavy chain variable domain (VH 2), a second dimerization domain, L6, and WinzipB1 domains.
The antibody of any one of claim 20 to 35, wherein,

1) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, and a first multimerization domain from the N-terminus to the C-terminus; and

The second light chain comprises a second light chain variable domain (VL 2) and a constant domain (CL) from the N-terminus to the C-terminus, the second heavy chain comprising a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain from the N-terminus to the C-terminus; or (b)

2) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, a first dimerization domain, L5, and Max domain from the N-terminus to the C-terminus; and

The second light chain comprises a second light chain variable domain (VL 2) and a second CL from the N-terminus to the C-terminus, the second heavy chain comprising a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, L6, and a Myc domain; or (b)

3) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, and a first multimerization domain from the N-terminus to the C-terminus; and

The second light chain comprises a second light chain variable domain (VL 2), L3, and Fos domain from the N-terminus to the C-terminus, the second heavy chain comprises a second heavy chain variable domain (VH 2), L4, jun domain, and a second dimerization domain from the N-terminus to the C-terminus; or (b)

4) The first light chain comprises a first light chain variable domain (VL 1), L1, and Jun domain from the N-terminus to the C-terminus, and the first heavy chain comprises a first heavy chain variable domain (VH 1), L2, fos domain, a first dimerization domain, L5, and Max domain from the N-terminus to the C-terminus; and

The second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain (VL 2), L3, and Fos domain, and the second heavy chain comprises, from N-terminus to C-terminus, a second heavy chain variable domain (VH 2), L4, jun domain, second dimerization domain, L6, and Myc domain; or (b)

5) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, a first light chain variable domain (VL 1), and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), a first CH1, and a first dimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a cleavage site peptide, a second light chain variable domain (VL 2), and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), a second CH1, and a second dimerization domain; or (b)

6) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, a first light chain variable domain (VL 1), and a first CL, and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), a first CH1, a first dimerization domain, a cleavage site peptide, L5, and WinzipA domains; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a cleavage site peptide, a second light chain variable domain (VL 2), and a second CL, and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), a second CH1, a second dimerization domain, a cleavage site peptide, L6, and WinzipB1 domains; or (b)

7) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Fos domain, L3, a cleavage site peptide, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), and a second dimerization domain; or (b)

8) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, a Fos domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), a first dimerization domain, a cleavage site peptide, L5, and WinzipA domains; and

The second light chain comprises from N-terminus to C-terminus a Fos domain, L3, a cleavage site peptide, and a second light chain variable domain (VL 2), and the second heavy chain comprises from N-terminus to C-terminus a Jun domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), a second dimerization domain, a cleavage site peptide, L6, and WinzipB1 domains; or (b)

9) The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, fosW domain, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), and a first multimerization domain; and

The second light chain comprises, from N-terminus to C-terminus, a Myc domain, L3, a cleavage site peptide, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Max domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), and a second dimerization domain; or (b)

10 The first light chain comprises, from N-terminus to C-terminus, a Jun domain, L1, a cleavage site peptide, and a first light chain variable domain (VL 1), and the first heavy chain comprises, from N-terminus to C-terminus, fosW domains, L2, a cleavage site peptide, a first heavy chain variable domain (VH 1), a first dimerization domain, a cleavage site peptide, L5, and WinzipA domains; and

The second light chain comprises, from N-terminus to C-terminus, a Myc domain, L3, a cleavage site peptide, and a second light chain variable domain (VL 2), and the second heavy chain comprises, from N-terminus to C-terminus, a Max domain, L4, a cleavage site peptide, a second heavy chain variable domain (VH 2), a second dimerization domain, a cleavage site peptide, L6, and WinzipB1 domains.
The antibody of any one of claim 20 to 36, wherein,

The first dimerization domain and the second dimerization domain are joined by a means selected from the group consisting of: handle inlet structure KIH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, ways of increasing flexibility, or combinations thereof;

More preferably, the first and second dimerization domains are bound by a handle entry structure KIH (Knob into Hole), and a protrusion or a pit is created in the interface of the first and second dimerization domains, the protrusion or pit being positioned in the pit or protrusion in the interface to form the handle entry structure; preferably the sequence of the first dimerization domain is as shown in SEQ ID NO. 7 or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO. 7 and the sequence of the second dimer 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.
A host cell according to claim 40, which is a prokaryotic cell, preferably E.coli.
A host cell according to claim 40, which is a eukaryotic cell, preferably one selected from the group consisting of a prokaryotic cell, an animal cell, a plant cell and a fungal cell.
The host cell according to claim 40, wherein the cell is selected from one of CHO cells, COS cells and yeast cells.
A method for preparing the polypeptide complex of any one of claims 1-19 or the multispecific antibody of any one of claims 20-37, comprising the steps of:

a) Introducing into a host cell a nucleotide encoding the nucleotide of claim 37;

b) Allowing the host cell to express the antibody.
The method of claim 44, further comprising purifying the antibody.
A pharmaceutical composition comprising the polypeptide complex of any one of claims 1-19 or the multispecific antibody of any one of claims 20-37 and at least one pharmaceutically acceptable excipient.
Use of the polypeptide complex of any one of claims 1-20 or the multispecific antibody of any one of claims 20-37 in the manufacture of a medicament for immunosuppressive therapy, for treating an autoimmune disease, for treating an inflammatory disease, for treating an infectious disease, for treating allergy, or for treating 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 of any one of claims 1-19 or the multispecific antibody of any one of claims 20-37 for use in immunosuppressive therapy, in the treatment of an autoimmune disease, in the treatment of an inflammatory disease, in the treatment of an infectious disease, in the treatment of allergy or in the treatment of cancer.