Multispecific binding proteins with mutant Fab domains

文档序号：395287 发布日期：2021-12-14 浏览：36次中文

阅读说明：本技术 具有突变型Fab结构域的多特异性结合蛋白 (Multispecific binding proteins with mutant Fab domains ) 是由 M·阿马拉尔 C·贝尔 I·福肯 G·赫斯勒 S·霍尔珀 J·荣格 C·兰格 W-D· 于 2019-12-23 设计创作，主要内容包括：提供结合蛋白,其包含与VH区配对的VL区和与CL区配对的CH1区,其中所述VL区和所述VH区包含带相反电荷的突变以促进配对,并且其中所述CH1区和所述CL区包含突变以促进配对。还提供结合蛋白,其包含一个或多个半胱氨酸残基,所述半胱氨酸残基被工程化到VH/VL对中以形成一个或多个二硫键。还提供多特异性结合蛋白、编码结合蛋白和多特异性结合蛋白的核酸、表达载体、宿主细胞、药物组合物以及施用本文所述的结合蛋白或多特异性结合蛋白的治疗方法。(Binding proteins are provided comprising a VL region paired with a VH region and a CH1 region paired with a CL region, wherein the VL region and the VH region comprise oppositely charged mutations to facilitate pairing, and wherein the CH1 region and the CL region comprise mutations to facilitate pairing. Also provided are binding proteins comprising one or more cysteine residues engineered into a VH/VL pair to form one or more disulfide bonds. Also provided are multispecific binding proteins, nucleic acids encoding binding proteins and multispecific binding proteins, expression vectors, host cells, pharmaceutical compositions, and therapeutic methods of administering a binding protein or multispecific binding protein described herein.)

1. A multispecific antigen-binding protein comprising at least two VL regions and at least two CH1 regions, the at least two VL regions being paired separately with at least two VH regions to form at least two antigen-binding sites, the at least two CH1 regions being paired separately with two CL regions,

wherein at least one CH1/CL pair comprises a CH1/CL mutation selected from one or more of the following to facilitate pairing:

(1) the T192E (CH1) mutation and the N137K and S114A (CL) mutations, and

(2) the L143Q and S188V (CH1) mutations and the V133T and S176V (CL) mutations, and

(3) the T192E, L143Q and S188V (CH1) mutations and the N137K, S114A, V133T and S176V (CL) mutations,

(4) the K221E (CH1) mutation and the E123K (CL) mutation,

(5) T192E and K221E (CH1) mutations and N137K, S114A and E123K (CL) mutations, (6) L143E, L143D, L143K, L143R or L143H (CH1) mutations and S176E, S176D, S176K, S176R or S176H (CL) mutations,

(7) L124E, L124D, L124K, L124R or L124H (CH1) mutations and V133E, V133D, V133K, V133R or V133H (CL) mutations,

(8) the K228D (CH1) mutation and the D122K (CL) mutation, and

(9) the K221E and K228D (CH1) mutations and the D122K and E123K (CL) mutations,

wherein when two CH1/CL pairs contain a mutation to facilitate pairing of two different VH/VL pairs, the two CH1/CL pairs do not contain the same mutation, and

Wherein at least one VH/VL pair comprises an oppositely charged mutation to facilitate pairing, the oppositely charged mutation comprising (1) a mutant residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutant residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and

wherein a mutated residue in the VH domain has an opposite charge to a mutated residue in the VL domain.

2. A multispecific antigen-binding protein comprising:

a) a first light chain (LC 1)/heavy chain (HC1) pair, the first light chain (LC 1)/heavy chain (HC1) pair comprising:

(1) a first VL region (VL1) that pairs with a first VH region (VH1) to form a first antigen binding site (VL 1);

(2) a first constant heavy chain region 1(CH1-1) operably linked to VH1 and a first constant light chain region (CL1) operably linked to VL 1; and

(3) a first heterodimerization domain (HD 1); and

b) a second light chain (LC 2)/heavy chain (HC2) pair, the second light chain (LC 2)/heavy chain (HC2) pair comprising:

(4) a second VL region (VL2) that pairs with a second VH region (VH2) to form a second antigen binding site;

(5) a second constant heavy chain region 1(CH1-2) operably linked to VH2 and a second constant light chain region (CL2) operably linked to VL 2; and

(6) A second heterodimerization domain (HD 2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of the pair VL1 and VH1 and at least one or both of the pair VL2 and VH2 comprise oppositely charged mutations to facilitate pairing, the oppositely charged mutations comprising (1) a mutant residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutant residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and wherein the mutant residue in the VH region has an opposite charge to the mutant residue in the VL region,

wherein at least one or both of the pair CL1 and CH1-1 and at least one or both of the pair CL2 and CH1-2 comprise mutations to facilitate pairing, and

wherein when both of the pair CL1 and CH1-1 and both of the pair CL2 and CH1-2 comprise mutations to facilitate pairing, the mutations that facilitate pairing in CH1-1 and CL1 are different from the mutations that facilitate pairing in CH1-2 and CL 2.

3. The multispecific antigen-binding protein of claim 1, wherein at least one CH1 region is operably linked to a heterodimerization domain.

4. A multispecific antigen-binding protein comprising at least two polypeptide chains and forming at least two antigen-binding sites, wherein one polypeptide chain comprises a structure represented by the formula:

VL1-L1-VL2-L2-CL [I]

And one polypeptide chain comprises a structure represented by the formula:

VH2-L3-VH1-L4-CH1 [II]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain; and is

L1, L2, L3 and L4 are amino acid linkers,

wherein VH1 is paired with VL1, VH2 is paired with VL2, and CH1 is paired with CL,

wherein said polypeptide of formula I and said polypeptide of formula II form a cross light-heavy chain pair,

wherein one or more cysteine residues are engineered into one or both of the VH1/VL1 pair and the VH2/VL2 pair to form one or more disulfide bonds,

wherein at least one or both of the pair VL1 and VH1 and at least one or both of the pair VL2 and VH2 comprise oppositely charged mutations that facilitate pairing comprising (1) a mutant residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutant residue at Kabat position 38 in the VL region selected from E, D, K, R or H,

Wherein a mutated residue in the VH region has an opposite charge to a mutated residue in the VL region, and

wherein the CH1 and CL domain pair comprises a mutation that facilitates pairing.

5. A multispecific antigen-binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein each of the two polypeptide chains comprises a structure represented by the formula:

VL1-L1-VL2-L2-CL [I]

and each of the two polypeptide chains comprises a structure represented by the formula:

VH2-L3-VH1-L4-CH1-Fc [II]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

fc comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and is

L1, L2, L3 and L4 are amino acid linkers,

wherein VH1 pairs with VL1 to form a first antigen binding site, VH2 pairs with VL2 to form a second antigen binding site, and CH1 pairs with CL,

wherein said polypeptide of formula I and said polypeptide of formula II form a cross light-heavy chain pair,

Wherein one or more cysteine residues are engineered into one or both of the VH1/VL1 pair and the VH2/VL2 pair to form one or more disulfide bonds,

wherein one or both of the pair VL1 and VH1 and one or both of the pair VL2 and VH2 comprise oppositely charged mutations that facilitate pairing, the oppositely charged mutations comprising (1) a mutated residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutated residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and wherein the mutated residue in the VH region has an opposite charge to the mutated residue in the VL region, and

wherein one or both of the CH1 and CL domain pairs comprise a mutation that facilitates pairing, and

wherein when at least two CH1/CL pairs contain mutations to promote pairing, the set of mutations in one CH1/CL pair is different from the set of mutations in the other CH1/CL pair.

6. A multispecific antigen-binding protein comprising four polypeptide chains forming three antigen-binding sites, wherein

The first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL1 [I]，

the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH 1-1-hinge-CH 2-CH3 [ II ],

The third polypeptide chain comprises a structure represented by the formula:

VH3-CH 1-2-hinge-CH 2-CH3 [ III ], and

the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL2 [IV]，

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL1 is a first immunoglobulin light chain constant domain;

CL2 is a second immunoglobulin light chain constant domain;

CH1-1 is a first immunoglobulin CH1 heavy chain constant domain;

CH1-2 is a second immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

a hinge is an immunoglobulin hinge region connecting the CH1 domain and the CH2 domain; and is

L1, L2, L3 and L4 are amino acid linkers,

wherein said polypeptide of formula I and said polypeptide of formula II form a cross light-heavy chain pair, and

wherein one or more cysteine residues are engineered into one or more of the VH1/VL1 pair, the VH2/VL2 pair and the VH3/VL3 pair to form one or more disulfide bonds,

Wherein one or both of the pair VL1 and VH1 and one or both of the pair VL2 and VH2 comprise oppositely charged mutations that facilitate pairing, the oppositely charged mutations comprising (1) a mutated residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutated residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and wherein the mutated residue in the VH region has an opposite charge to the mutated residue in the VL region,

wherein one or both of the pair CL1 and CH1-1 and one or both of the pair CL2 and CH1-2 comprise mutations that promote pairing, and

wherein the mutations in CH1-1 and CL1 are different from the mutations in CH1-2 and CL2 when both of the CL1 and CH1-1 pairs and both of the CL2 and CH1-2 pairs contain mutations to facilitate pairing.

7. A multispecific antigen-binding protein comprising

a) A first light chain (LC 1)/heavy chain (HC1) pair, the first light chain (LC 1)/heavy chain (HC1) pair comprising:

(1) a first VL region (VL1) that pairs with a first VH region (VH1) to form a first antigen binding site (VL 1);

(2) a first constant heavy chain region 1(CH1-1) operably linked to VH1 and a first constant light chain region (CL1) operably linked to VL1, and

(b) A second light chain (LC 2)/heavy chain (HC2) pair, the second light chain (LC 2)/heavy chain (HC2) pair comprising:

(3) a second VL region (VL2) that pairs with a second VH region (VH2) to form a second antigen binding site; and

(4) a second constant heavy chain region 1(CH1-2) operably linked to VH2 and a second constant light chain region (CL2) operably linked to VL2,

wherein the C-terminus of CH1-1 is operably linked to the N-terminus of VH2,

wherein one or more cysteine residues are engineered into one or both of the VH1/VL1 pair and the VH2/VL2 pair to form one or more disulfide bonds,

wherein one or both of the pair CL1 and CH1-1 and one or both of the pair CL2 and CH1-2 comprise mutations that promote pairing, and

Wherein the mutations in CH1-1 and CL1 are different from the mutations in CH1-2 and CL2 when both of the CL1 and CH1-1 pairs and both of the CL2 and CH1-2 pairs contain mutations to facilitate pairing.

8. The multispecific antigen-binding protein of any one of claims 2-7, wherein CH1 comprises a T192E mutation, and CL comprises N137K and S114A mutations.

9. The multispecific antigen-binding protein of any one of claims 2-8, wherein CH1 comprises L143Q and S188V mutations, and CL comprises V133T and S176V mutations.

10. The multispecific antigen-binding protein of any one of claims 2 to 9, wherein CH1 comprises T192E, L143Q, and S188V mutations, and CL comprises N137K, S114A, V133T, and S176V mutations.

11. The multispecific antigen-binding protein of any one of claims 2-10, wherein CH1 comprises a K221E mutation, and CL comprises an E123K mutation.

12. The multispecific antigen-binding protein of any one of claims 2-11, wherein CH1 comprises the K228D mutation and CL comprises the D122K mutation.

13. The multispecific antigen-binding protein of any one of claims 2 to 12, wherein CH1 comprises K221E and K228D mutations, and CL comprises D122K and E123K mutations.

14. The multispecific antigen-binding protein of any one of claims 2 to 13, wherein CH1 comprises an L143E, L143D, L143K, L143R, or L143H mutation, and CL comprises an S176E, S176D, S176K, S176R, or S176H mutation.

15. The multispecific antigen-binding protein of any one of claims 2-14, wherein CH1 comprises an L124E, L124D, L124K, L124R, or L124H mutation, and CL comprises a V133E, V133D, V133K, V133R, or V133H mutation.

16. The multispecific antigen-binding protein of any one of claims 2 to 15, wherein VH comprises a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, and VL comprises a Q38E, Q38D, Q38K, Q38R, or Q38H mutation.

17. The multispecific antigen-binding protein of any one of claims 2 to 16, wherein at least one CH1/CL pair comprises a CH1/CL mutation selected from one or more of:

(1) the T192E (CH1) mutation and the N137K and S114A (CL) mutations,

(2) the L143Q and S188V (CH1) mutations and the V133T and S176V (CL) mutations,

(3) the T192E, L143Q and S188V (CH1) mutations and the N137K, S114A, V133T and S176V (CL) mutations,

(4) the K221E (CH1) mutation and the E123K (CL) mutation,

(5) the T192E and K221E (CH1) mutations and the N137K, S114A and E123K (CL) mutations,

(6) L143E, L143D, L143K, L143R or L143H (CH1) mutations and S176E, S176D, S176K, S176R or S176H (CL) mutations,

(7) L124E, L124D, L124K, L124R or L124H (CH1) mutations and V133E, V133D, V133K, V133R or V133H (CL) mutations,

(8) the K228D (CH1) mutation and the D122K (CL) mutation, and

(9) the K221E and K228D (CH1) mutations and the D122K and E123K (CL) mutations,

wherein when at least two CH1/CL pairs comprise a set of mutations to facilitate pairing of two different VH/VL pairs, then the at least two CH1/CL pairs do not comprise the same mutation, and

wherein at least one VH/VL pair comprises an oppositely charged mutation to facilitate pairing, the oppositely charged mutation comprising (1) a mutated residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutated residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and wherein the mutated residue in the VH region has an opposite charge to the mutated residue in the VL region.

18. An antigen binding protein comprising:

an antigen binding domain; and

a constant heavy chain CH1 region paired with a constant light chain CL region,

wherein the antigen binding domain selectively binds to a target antigen, and wherein the CH1 region and the CL region comprise one or both of:

a) L143E, L143D, L143K, L143R or L143H mutations in the CH1 region and S176E, S176D, S176K, S176R or S176H mutations in the CL region; and

b) the L124E, L124D, L124K, L124R or L124H mutations in the CH1 region and the V133E, V133D, V133K, V133R or V133H mutations in the CL region,

wherein the mutant residue in the CH1 region has an opposite charge to the mutant residue in the CL region.

19. The antigen binding protein of claim 18, further comprising a CH1/CL mutation selected from one or more of the following to facilitate pairing:

(1) the T192E (CH1) mutation and the N137K and S114A (CL) mutations,

(2) the L143Q and S188V (CH1) mutations and the V133T and S176V (CL) mutations,

(3) the T192E, L143Q and S188V (CH1) mutations and the N137K, S114A, V133T and S176V (CL) mutations,

(4) the K221E (CH1) mutation and the E123K (CL) mutation,

(5) the K228D (CH1) mutation and the D122K (CL) mutation, and

(6) the K221E and K228D (CH1) mutations and the D122K and E123K (CL) mutations,

wherein when two CH1/CL pairs comprise a mutation to facilitate pairing of two different VH/VL pairs, the two CH1/CL pairs do not comprise the same mutation.

20. The antigen binding protein of claim 18 or 19, further comprising at least one VH/VL pair comprising an oppositely charged mutation to facilitate pairing, the oppositely charged mutation comprising (1) a mutated residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutated residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and wherein the mutated residue in the VH region has an opposite charge to the mutated residue in the VL region.

21. An antigen binding protein comprising:

an antigen binding domain; and

a constant heavy chain CH1 region paired with a constant light chain CL region,

wherein the antigen binding domain selectively binds to a target antigen, and wherein the CH1 region and the CL region comprise one or both of:

a) L143E, L143D, L143K, L143R or L143H mutations in the CH1 region and S176E, S176D, S176K, S176R or S176H mutations in the CL region; and

b) the K221E and K228D mutations in the CH1 region and the D122K and E123K mutations in the CL region,

wherein the mutant residue in the CH1 region has an opposite charge to the mutant residue in the CL region.

22. The antigen binding protein of claim 21, further comprising at least one VH/VL pair comprising oppositely charged mutations to facilitate pairing, the oppositely charged mutations comprising (1) a mutant residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutant residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and wherein the mutant residue in the VH region has an opposite charge to the mutant residue in the VL region.

23. The multispecific antigen-binding protein of any one of claims 1 to 22, further comprising one or more cysteine residues engineered into one or several VH/VL pairs to form one or more disulfide bonds.

24. The multispecific antigen-binding protein of claim 23, wherein one or both VH regions comprise one or both of the 44C and 105C mutations and one or both VL regions comprise one or both of the 100C and 43C mutations.

25. A kit comprising one or more isolated nucleic acid molecules comprising one or more nucleotide sequences encoding the multispecific antigen-binding protein of any one of claims 1-24.

26. A kit comprising one or more expression vectors comprising one or more nucleic acid molecules according to claim 25.

27. An isolated host cell comprising one or more nucleic acid molecules according to claim 25 or one or more expression vectors according to claim 26.

28. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the multispecific antigen-binding protein of any one of claims 1-24.

29. A method of treating a disorder in which antigenic activity is detrimental, comprising administering to a subject in need thereof an effective amount of a multispecific antigen-binding protein of any one of claims 1-24.

30. The multispecific antigen-binding protein or antigen-binding protein of any one of claims 1 to 24, comprising three HCDRs per VH region and three LCDRs per VL region, and further comprising binding specificity for one or more target antigens or one or more target epitopes.

Background

Classical antibodies are Y-proteins or immunoglobulins, which are heterotetramers formed from two heterodimers consisting of a light chain portion and a heavy chain portion. The "arm" of an antibody contains the antigen binding site and is referred to as the Fab region. Classical antibodies (e.g., those produced by the host immune system) have two identical fabs and can recognize and bind to a particular antigen. The generation of asymmetry in the native antibody structure is a prerequisite for the production of multispecific binding proteins with two (e.g., bispecific antibodies) or more binding specificities. For example, bispecific antibodies can be made flexible to bind two different antigens simultaneously by separating one or more fvs on different asymmetric binding arms or fabs. However, despite these advantages, many multispecific antibody technologies have process and manufacturing problems due to the fact that each is not paired incorrectly with a weighed chain and a light chain. For example, many of these techniques have a so-called "light chain problem," in which random pairing of two different light chains with a heavy chain results in various chain pairing combinations in addition to the desired combination. In some cases, light chain problems can be avoided by using a common light chain that enables binding to both antigens. However, this may not be feasible for many antibodies, as this format requires de novo antibody production in transgenic mice or by display technology. Furthermore, rare antibodies such as broadly neutralizing anti-HIV antibodies derived from human patients cannot adapt to such formats. Thus, there remains a need for an alternative and inventive solution to the problem of wrong pairing. A series of mutations in the dimer interface have been carefully designed to allow these antibodies to heterodimerize.

Disclosure of Invention

The present disclosure provides antigen binding proteins, which may optionally comprise a plurality of mutations along the dimer interface that allow the antigen binding protein to effectively heterodimerize.

In one aspect, the present disclosure provides an antigen binding protein comprising

A VL region paired with a VH region to form an antigen binding site; and

a CH1 region, the CH1 region being paired with a CL region,

wherein the VL region and the VH region comprise oppositely charged mutations to facilitate pairing, and wherein the CH1 region and the CL region comprise mutations to facilitate pairing.

In some embodiments, the antigen binding protein further comprises a second VL region paired with a second VH region to form a second antigen binding site; and a second CH1 region, the second CH1 region paired with a second CL region.

In some embodiments, one or both of the first VH and VL pair and the second VH and VL pair comprise oppositely charged mutations to facilitate pairing, and one or both of the first CH1 and CL pair and the second CH1 and CL pair comprise mutations to facilitate pairing.

In some embodiments, one or both CH1 regions comprise the T192E mutation and one or both CL regions comprise the N137K and S114A mutations.

In some embodiments, one or both CH1 regions comprise the L143Q and S188V mutations, and one or both CL regions comprise the V133T and S176V mutations.

In some embodiments, one or both CH1 regions comprise T192E, L143Q, and S188V mutations, and one or both CL regions comprise N137K, S114A, V133T, and S176V mutations.

In some embodiments, one or both CH1 regions comprise an L143E, L143D, L143K, L143R, or L143H mutation, and one or both CL regions comprise an S176E, S176D, S176K, S176R, or S176H mutation, wherein the mutation in CH1 is oppositely charged to the mutation in CL.

In some embodiments, one or both CH1 regions comprise an L124E, L124D, L124K, L124R, or L124H mutation, and one or both CL regions comprise a V133E, V133D, V133K, V133R, or V133H mutation, wherein the mutation in CH1 is oppositely charged to the mutation in CL.

In some embodiments, one or both VH regions comprise a 39E, 39D, 39K, 39R, or 39H mutation, and one or both VL regions comprise a 38E, 38D, 38K, 38R, or Q38H mutation, wherein the mutations in VH are oppositely charged to the mutations in VL.

In some embodiments, one or both VH regions comprise a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, and one or both VL regions comprise a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, wherein the mutation in VH is oppositely charged to the mutation in VL.

In some embodiments, the antigen binding protein further comprises one or more cysteine residues engineered into one or both of the VH/VL pairs to form one or more disulfide bonds.

In some embodiments, one or both VH regions comprise one or both of the 44C and 105C mutations, and one or both VL regions comprise one or both of the 100C and 43C mutations.

In some embodiments, one or both VH regions comprise the 44C mutation, and one or both VL regions comprise the 100C mutation.

In some embodiments, one or both VH regions comprise the 105C mutation, and one or both VL regions comprise the 43C mutation.

In some embodiments, the antigen binding protein further comprises oppositely charged mutations in one or both CH1/CL pairs.

In some embodiments, the oppositely charged mutations in one or both of the CH1/CL pairs are selected from: K221E in the CH1 region and E123K in the CL region; K228D in the CH1 region and D122K in the CL region; L145E in the CH1 region and S176K in the CL region; and L128E in the CH1 region and V133K in the CL region.

In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising at least two VL regions that are paired separately with at least two VH regions to form at least two antigen-binding sites, and at least two CH1 regions that are paired separately with two CL regions, wherein at least one CH1/CL pair comprises a CH1/CL mutation selected from:

(1) The T192E (CH1) mutation and the N137K and S114A (CL) mutations, and/or

(2) The L143Q and S188V (CH1) mutations and the V133T and S176V (CL) mutations, and/or

(3) T192E, L143Q and S188V (CH1) mutations and N137K, S114A, V133T and S176V (CL) mutations, and/or

(4) The K221E (CH1) mutation and the E123K (CL) mutation, and/or

(5) The T192E and K221E (CH1) mutations and the N137K, S114A and E123K (CL) mutations, and/or

(6) L143E, L143D, L143K, L143R or L143H (CH1) mutations and S176E, S176D, S176K, S176R or S176H (CL) mutations, and/or

(7) L124E, L124D, L124K, L124R or L124H (CH1) mutations and V133E, V133D, V133K, V133R or V133H (CL) mutations, and

(8) wherein when two CH1/CL pairs comprise a mutation to facilitate pairing of two different VH/VL pairs, the two CH1/CL pairs do not comprise the same mutation,

and is

Wherein at least one VH/VL pair comprises an oppositely charged mutation to facilitate pairing, the oppositely charged mutation comprising (1) a mutated residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutated residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and wherein the mutated residue in the VH region has an opposite charge to the mutated residue in the VL region.

wherein at least one CH1/CL pair comprises a CH1/CL mutation selected from one or more of the following to facilitate pairing:

(1) the T192E (CH1) mutation and the N137K and S114A (CL) mutations, and

(2) the L143Q and S188V (CH1) mutations and the V133T and S176V (CL) mutations, and

(3) the T192E, L143Q and S188V (CH1) mutations and the N137K, S114A, V133T and S176V (CL) mutations,

(4) the K221E (CH1) mutation and the E123K (CL) mutation,

(5) the T192E and K221E (CH1) mutations and the N137K, S114A and E123K (CL) mutations,

(6) L143E, L143D, L143K, L143R or L143H (CH1) mutations and S176E, S176D, S176K, S176R or S176H (CL) mutations,

(7) L124E, L124D, L124K, L124R or L124H (CH1) mutations and V133E, V133D, V133K, V133R or V133H (CL) mutations,

(8) the K228D (CH1) mutation and the D122K (CL) mutation, and

(9) the K221E and K228D (CH1) mutations and the D122K and E123K (CL) mutations,

wherein when two CH1/CL pairs contain a mutation to facilitate pairing of two different VH/VL pairs, the two CH1/CL pairs do not contain the same mutation, and

wherein a mutated residue in the VH domain has an opposite charge to a mutated residue in the VL domain.

In some embodiments, in the multispecific antigen-binding protein, when at least two different VH/VL pairs comprise a mutant set to facilitate pairing, then the at least two different VH/VL pairs do not comprise the same mutant set.

In some embodiments, the multispecific antigen-binding protein comprises at least two VL regions that are paired separately with at least two VH regions to form at least two antigen-binding sites, and at least two CH1 regions that are paired separately with two CL regions,

wherein at least one CH1/CL pair comprises a CH1/CL mutation selected from one or more of the following to facilitate pairing:

(1) the T192E (CH1) mutation and the N137K and S114A (CL) mutations, and/or

(2) The L143Q and S188V (CH1) mutations and the V133T and S176V (CL) mutations, and/or

(3) T192E, L143Q and S188V (CH1) mutations and N137K, S114A, V133T and S176V (CL) mutations, and/or

(4) The K221E (CH1) mutation and the E123K (CL) mutation, and/or

(5) L143E, L143D, L143K, L143R or L143H (CH1) mutations and S176E, S176D, S176K, S176R or S176H (CL) mutations, and/or

(6) L124E, L124D, L124K, L124R or L124H (CH1) mutations and V133E, V133D, V133K, V133R or V133H (CL) mutations, and

(7) when two CH1/CL pairs contain mutations to facilitate the pairing of two different VH/VL pairs, the two CH1/CL pairs do not contain the same mutations,

and is

Wherein at least one VH/VL pair comprises oppositely charged mutations selected from: 39E, Q39D, Q39K, Q39R or Q39H mutations and Q38E, Q38D, Q38K, Q38R or Q38H mutations, and wherein the mutations in VH are oppositely charged to the mutations in VL; and when several VH/VL pairs (where VL or VH are not in the same polypeptide chain) contain mutations to facilitate pairing of different VH/VL pairs, then each VH/VL pair does not contain the same oppositely charged mutation.

In some embodiments, one or both CH1 regions are operably linked to a heterodimerization domain.

In some embodiments, the heterodimerization domain comprises a first Fc domain.

In some embodiments, the first Fc domain is heterodimerized with a second Fc domain, and wherein the first Fc domain comprises a first CH3 region and the second Fc domain comprises a second CH3 region.

In some embodiments, the first CH3 region comprises one or both of the S354C and T366W mutations and the second CH3 region comprises one or more of the Y349C, T366S, L368A, and Y407V mutations, wherein the mutations promote Fc domain heterodimerization.

In another aspect, the disclosure provides a multispecific antibody comprising

a) A first light chain (LC 1)/heavy chain (HC1) pair, said first light chain (LC 1)/heavy chain (HC1) pair comprising

(1) A first VL region (VL1) that pairs with a first VH region (VH1) to form a first antigen binding site (VL 1);

(2) a first constant heavy chain region 1(CH1-1) and a first constant light chain region (CL 1); and

(3) a first heterodimerization domain (HD 1); and

b) a second light chain (LC 2)/heavy chain (HC2) pair comprising

(4) A second VL region (VL2) that pairs with a second VH region (VH2) to form a second antigen binding site;

(5) a second constant heavy chain region 1(CH1-2) and a second constant light chain region (CL 2); and

(6) A second heterodimerization domain (HD 2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of the VL1 and VH1 pairs and at least one or both of the VL2 and VH2 pairs comprise oppositely charged mutations to facilitate pairing,

wherein at least one or both of the pair CL1 and CH1-1 and at least one or both of the pair CL2 and CH1-2 comprise mutations to facilitate pairing, and

In another aspect, the present disclosure provides a multispecific antibody comprising:

a) a first light chain (LC 1)/heavy chain (HC1) pair, said first light chain (LC 1)/heavy chain (HC1) pair comprising

(1) A first VL region (VL1) that pairs with a first VH region (VH1) to form a first antigen binding site (VL 1);

(2) a first constant heavy chain region 1(CH1-1) operably linked to VH1 and a first constant light chain region (CL1) operably linked to VL 1; and

(3) a first heterodimerization domain (HD 1); and

b) a second light chain (LC 2)/heavy chain (HC2) pair comprising

(4) A second VL region (VL2) that pairs with a second VH region (VH2) to form a second antigen binding site;

(5) a second constant heavy chain region 1(CH1-2) operably linked to VH2 and a second constant light chain region (CL2) operably linked to VL 2; and

(6) a second heterodimerization domain (HD 2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of the VL1 and VH1 pairs and at least one or both of the VL2 and VH2 pairs comprise oppositely charged mutations to facilitate pairing,

wherein at least one or both of the pair CL1 and CH1-1 and at least one or both of the pair CL2 and CH1-2 comprise mutations to facilitate pairing, and

In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising:

a) a first light chain (LC 1)/heavy chain (HC1) pair, said first light chain (LC 1)/heavy chain (HC1) pair comprising

(1) A first VL region (VL1) that pairs with a first VH region (VH1) to form a first antigen binding site (VL 1);

(2) A first constant heavy chain region 1(CH1-1) operably linked to VH1 and a first constant light chain region (CL1) operably linked to VL 1; and

(3) a first heterodimerization domain (HD 1); and

b) a second light chain (LC 2)/heavy chain (HC2) pair comprising

(4) A second VL region (VL2) that pairs with a second VH region (VH2) to form a second antigen binding site;

(5) a second constant heavy chain region 1(CH1-2) operably linked to VH2 and a second constant light chain region (CL2) operably linked to VL 2; and

(6) a second heterodimerization domain (HD 2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of the pair CL1 and CH1-1 and at least one or both of the pair CL2 and CH1-2 comprise mutations to facilitate pairing, and

Wherein when both of the pair CL1 and CH1-1 and both of the pair CL2 and CH1-2 comprise mutations to facilitate pairing, the mutations that facilitate pairing in CH1-1 and CL1 are different from the mutations that facilitate pairing in CH1-2 and CL 2.

In some embodiments, CH1-1 comprises the T192E mutation, and CL1 comprises the N137K and S114A mutations.

In some embodiments, CH1-1 comprises L143Q and S188V mutations, and CL1 comprises V133T and S176V mutations.

In some embodiments, CH1-1 comprises T192E, L143Q, and S188V mutations, and CL1 comprises N137K, S114A, V133T, and S176V mutations.

In some embodiments, CH1-2 comprises the T192E mutation, and CL2 comprises the N137K and S114A mutations.

In some embodiments, CH1-2 comprises L143Q and S188V mutations, and CL2 comprises V133T and S176V mutations.

In some embodiments, CH1-2 comprises T192E, L143Q, and S188V mutations, and CL2 comprises N137K, S114A, V133T, and S176V mutations.

In some embodiments, one or both of CH1-1 and CH1-2 comprises an L143E, L143D, L143K, L143R, or L143H mutation, and one or both of CL1 and CL2 comprises an S176E, S176D, S176K, S176R, or S176H mutation, wherein the mutation in one or both of CH1-1 or CH1-2 is oppositely charged than the mutation in one or both of CL1 and CL 2.

In some embodiments, one or both of CH1-1 and CH1-2 comprises an L124E, L124D, L124K, L124R, or L124H mutation, and one or both of CL1 and CL2 comprises a V133E, V133D, V133K, V133R, or V133H mutation, wherein the mutation in one or both of CH1-1 or CH1-2 is oppositely charged than the mutation in one or both of CL1 and CL 2.

In some embodiments, one or both of VH1 and VH2 comprises a 39E, 39D, 39K, 39R, or 39H mutation, and one or both of VL1 and VL2 comprises a 38E, 38D, 38K, 38R, or 38H mutation, wherein the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL 2.

In some embodiments, one or both of VH1 and VH2 comprises a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, and one or both of VL1 and VL2 comprises a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, wherein the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL 2.

In some embodiments, the multispecific antibody further comprises one or more cysteine residues engineered into one or both of the VH1/VL1 and VH2/VL2 pairs to form one or more disulfide bonds.

In some embodiments, one or both of VH1 and VH2 comprises 44C and 105C mutations, and one or both of VL1 and VL2 comprises 100C and 43C mutations.

In some embodiments, VH1 comprises the 44C mutation, and VL1 comprises the 100C mutation.

In some embodiments, VH1 comprises the 105C mutation and VL1 comprises the 43C mutation.

In some embodiments, VH2 comprises the 44C mutation, and VL2 comprises 100C.

In some embodiments, VH2 comprises the 105C mutation and VL2 comprises the 43C mutation.

In some embodiments, VH1 comprises 39E and 44C mutations, and VL1 comprises 38K and 100C mutations.

In some embodiments, VH1 comprises the 39E and 105C mutations and VL1 comprises the 38K and 43C mutations.

In some embodiments, the multispecific antibody further comprises an oppositely charged mutation in the CH1-1/CL1 pair.

In some embodiments, the oppositely charged mutations in the CH1-1/CL1 pair are selected from: K221E in the CH1-1 region and E123K in the CL1 region; K228D in the CH1-1 region and D122K in the CL1 region; L145E in the CH1-1 region and S176K in the CL1 region; and L128E in the CH1-1 region and V133K in the CL1 region.

In some embodiments, the multispecific antibody further comprises an oppositely charged mutation in the CH1-2/CL2 pair.

In some embodiments, the oppositely charged mutations in the CH1-2/CL2 pair are selected from: K221E in the CH1-2 region and E123K in the CL2 region; K228D in the CH1-2 region and D122K in the CL2 region; L145E in the CH1-2 region and S176K in the CL2 region; and L128E in the CH1-2 region and V133K in the CL2 region.

In some embodiments, the first heterodimerization domain and the second heterodimerization domain comprise Fc domains.

In some embodiments, the first heterodimerization domain comprises a first CH3 domain, the first CH3 domain comprises one or both of the S354C and T366W mutations, and the second heterodimerization domain comprises a second CH3 domain, the second CH3 domain comprises one or both of the Y349C, T366S, L368A, and Y407V mutations, wherein the mutations promote Fc domain heterodimerization.

In some embodiments, the CH1-1 domain is linked to a first CH2 domain and a first CH3 domain, the CH1-2 domain is linked to a second CH2 domain and a second CH3 domain, and wherein the first CH2 and CH3 domains and the second CH2 and CH3 domains dimerize to form an Fc domain.

In some embodiments, the first CH3 domain comprises one or both of the S354C and T366W mutations, the second CH3 domain comprises one or more of the Y349C, T366S, L368A, and Y407V mutations, and wherein the mutations promote heterodimerization of Fc domains.

In another aspect, the present disclosure provides an antigen binding protein or multispecific antigen binding protein comprising at least two polypeptide chains and forming at least two antigen binding sites, wherein one polypeptide chain comprises a structure represented by the formula:

VL1-L1-VL2-L2-CL[I]

and one polypeptide chain comprises a structure represented by the formula:

VH2-L3-VH1-L4-CH1[II]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain; and is

L1, L2, L3 and L4 are amino acid linkers,

wherein said polypeptide of formula I and said polypeptide of formula II form a cross light-heavy chain pair,

wherein one or more cysteine residues are engineered into one or both of the VH1/VL1 pair and the VH2/VL2 pair to form one or more disulfide bonds,

Wherein at least one or both of the pair VL1 and VH1 and at least one or both of the pair VL2 and VH2 comprise oppositely charged mutations that promote pairing, and

wherein the CH1 and CL domain pair comprises a mutation that facilitates pairing.

In some embodiments, VH1 is paired with VL1, VH2 is paired with VL2, and CH1 is paired with CL.

In some embodiments, the present disclosure provides a multispecific antigen-binding protein comprising at least two polypeptide chains and forming at least two antigen-binding sites, wherein one polypeptide chain comprises a structure represented by the formula:

VL1-L1-VL2-L2-CL[I]

and one polypeptide chain comprises a structure represented by the formula:

VH2-L3-VH1-L4-CH1[II]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain; and is

L1, L2, L3 and L4 are amino acid linkers,

wherein VH1 is paired with VL1, VH2 is paired with VL2, and CH1 is paired with CL,

wherein said polypeptide of formula I and said polypeptide of formula II form a cross light-heavy chain pair,

Wherein one or more cysteine residues are engineered into one or both of the VH1/VL1 pair and the VH2/VL2 pair to form one or more disulfide bonds,

wherein at least one or both of the pair VL1 and VH1 and at least one or both of the pair VL2 and VH2 comprise oppositely charged mutations that facilitate pairing, the oppositely charged mutations comprising (1) a mutant residue at Kabat position 39 in the VH region selected from E, D, K, R or H, and (2) a mutant residue at Kabat position 38 in the VL region selected from E, D, K, R or H, and wherein the mutant residue in the VH region has an opposite charge to the mutant residue in the VL region, and

wherein the CH1 and CL domain pair comprises a mutation that facilitates pairing.

In some embodiments, one or both of VH1 and VH2 comprises VH44C and VH105C mutations.

In some embodiments, one or both of VL1 and VL2 comprises VL43C and VL100C mutations.

In some embodiments, one or both of VH1 and VH2 comprises a VH44C mutation, and one or both of VL1 and VL2 comprises a VL100C mutation.

In some embodiments, one or both of VH1 and VH2 comprises a VH105C mutation, and one or both of VL1 and VL2 comprises a VL43C mutation.

In some embodiments, CH1 comprises the T192E mutation and CL comprises the N137K and S114A mutations.

In some embodiments, CH1 comprises L143Q and S188V mutations, and CL comprises V133T and S176V mutations.

In some embodiments, CH1 comprises T192E, L143Q, and S188V mutations, and CL comprises N137K, S114A, V133T, and S176V mutations.

In some embodiments, CH1 comprises an L143E, L143D, L143K, L143R, or L143H mutation, CL comprises an S176E, S176D, S176K, S176R, or S176H mutation, and wherein the mutation in CH1 is oppositely charged to the mutation in CL.

In some embodiments, CH1 comprises an L124E, L124D, L124K, L124R, or L124H mutation, CL comprises a V133E, V133D, V133K, V133R, or V133H mutation, and wherein the mutation in CH1 is oppositely charged to the mutation in CL.

In some embodiments, one or both of VH1 and VH2 comprises a 39E, 39D, 39K, 39R, or 39H mutation, one or both of VL1 and VL2 comprises a 38E, 38D, 38K, 38R, or 38H mutation, and wherein the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL 2.

In some embodiments, one or both of VH1 and VH2 comprises a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, one or both of VL1 and VL2 comprises a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, and wherein the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL 2.

In some embodiments, CH1 is operably linked to a dimerization domain.

In some embodiments, the dimerization domain is an Fc domain comprising a CH2 domain and a CH3 domain.

In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein each of the two polypeptide chains comprises a structure represented by the formula:

VL1-L1-VL2-L2-CL[I]

and each of the two polypeptide chains comprises a structure represented by the formula:

VH2-L3-VH1-L4-CH1-Fc[II]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

Fc comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and is

L1, L2, L3 and L4 are amino acid linkers,

wherein VH1 pairs with VL1 to form a first antigen binding site, VH2 pairs with VL2 to form a second antigen binding site, and CH1 pairs with CL,

wherein said polypeptide of formula I and said polypeptide of formula II form a cross light-heavy chain pair,

wherein one or more cysteine residues are engineered into one or both of the VH1/VL1 pair and the VH2/VL2 pair to form one or more disulfide bonds,

wherein one or both of the CH1 and CL domain pairs comprise a mutation that facilitates pairing, and

wherein when at least two CH1/CL pairs contain mutations to promote pairing, the set of mutations in one CH1/CL pair is different from the set of mutations in the other CH1/CL pair.

In another aspect, the present disclosure provides an antigen binding protein comprising four polypeptide chains forming four antigen binding sites, wherein each of the two polypeptide chains comprises a structure represented by the formula:

VL1-L1-VL2-L2-CL[I]

and each of the two polypeptide chains comprises a structure represented by the formula:

VH2-L3-VH1-L4-CH1-Fc[II]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

fc comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and is

L1, L2, L3 and L4 are amino acid linkers,

wherein said polypeptide of formula I and said polypeptide of formula II form a cross light-heavy chain pair,

wherein VH1 pairs with VL1 to form a first antigen-binding site, VH2 pairs with VL2 to form a second antigen-binding site, and CH1 pairs with CL, and more particularly, the VH1/VL1 pair comprises a first antigen-binding specificity and the VH2/VL2 pair comprises a second antigen-binding specificity,

wherein one or more cysteine residues are engineered into one or both of the VH1/VL1 pair and the VH2/VL2 pair to form one or more disulfide bonds,

Wherein one or both of the pair VL1 and VH1 and one or both of the pair VL2 and VH2 comprise oppositely charged mutations that promote pairing, and

wherein one or both of the CH1 and CL domain pairs comprise a mutation that facilitates pairing, and

wherein when both CH1 and CL pairs contain mutations to promote pairing, the mutations in one CH1 and CL pair are different from the mutations in the other CH1 and CL pair to promote pairing.

In some embodiments, one or both of VH1 and VH2 comprises VH44C and VH105C mutations.

In some embodiments, one or both of VL1 and VL2 comprises VL43C and VL100C mutations.

In some embodiments, one or both of VH1 and VH2 comprises a VH44C mutation, and one or both of VL1 and VL2 comprises a VL100C mutation.

In some embodiments, one or both of VH1 and VH2 comprises a VH105C mutation, and one or both of VL1 and VL2 comprises a VL43C mutation.

In some embodiments, CH1 comprises the T192E mutation and CL comprises the N137K and S114A mutations.

In some embodiments, CH1 comprises L143Q and S188V mutations, and CL comprises V133T and S176V mutations.

In some embodiments, CH1 comprises T192E, L143Q, and S188V mutations, and CL comprises N137K, S114A, V133T, and S176V mutations.

In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising four polypeptide chains forming three antigen-binding sites, wherein

The first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL1[I]，

the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH 1-1-hinge-CH 2-CH3[ II ],

the third polypeptide chain comprises a structure represented by the formula:

VH3-CH 1-2-hinge-CH 2-CH3[ III ], and

the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL2[IV]，

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL1 is a first immunoglobulin light chain constant domain;

CL2 is a second immunoglobulin light chain constant domain;

CH1-1 is a first immunoglobulin CH1 heavy chain constant domain;

CH1-2 is a second immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

A hinge is an immunoglobulin hinge region connecting the CH1 domain and the CH2 domain; and L1, L2, L3 and L4 are amino acid linkers,

wherein said polypeptide of formula I and said polypeptide of formula II form a cross light-heavy chain pair, and

wherein one or more cysteine residues are engineered into one or more of the VH1/VL1 pair, the VH2/VL2 pair and the VH3/VL3 pair to form one or more disulfide bonds,

wherein one or both of the pair CL1 and CH1-1 and one or both of the pair CL2 and CH1-2 comprise mutations that promote pairing, and

In some embodiments, one or both of VH1 and VH2 comprises VH44C and VH105C mutations.

In some embodiments, one or both of VL1 and VL2 comprises VL43C and VH100C mutations.

In some embodiments, one or both of VH1 and VH2 comprises a VH44C mutation, and one or both of VL1 and VL2 comprises a VL100C mutation.

In some embodiments, one or both of VH1 and VH2 comprises a VH105C mutation, and one or both of VL1 and VL2 comprises a VL43C mutation.

In some embodiments, one or both of CH1-1 and CL1 comprise a mutation that facilitates pairing and one or both of CH1-2 and CL2 comprise a mutation that facilitates pairing.

In some embodiments, CH1-1 comprises the T192E mutation, and CL1 comprises the N137K and S114A mutations.

In some embodiments, CH1-1 comprises L143Q and S188V mutations, and CL1 comprises V133T and S176V mutations.

In some embodiments, CH1-1 comprises T192E, L143Q, and S188V mutations, and CL1 comprises N137K, S114A, V133T, and S176V mutations.

In some embodiments, CH1-2 comprises the T192E mutation, and CL2 comprises the N137K and S114A mutations.

In some embodiments, CH1-2 comprises L143Q and S188V mutations, and CL2 comprises V133T and S176V mutations.

In some embodiments, CH1-2 comprises T192E, L143Q, and S188V mutations, and CL2 comprises N137K, S114A, V133T, and S176V mutations.

In some embodiments, one or both of CH1-1 and CH1-2 comprises an L143E, L143D, L143K, L143R, or L143H mutation, one or both of CL1 and CL2 comprises an S176E, S176D, S176K, S176R, or S176H mutation, and wherein the mutation in one or both of CH1-1 or CH1-2 is oppositely charged to the mutation in one or both of CL1 and CL 2.

In some embodiments, one or both of CH1-1 and CH1-2 comprises an L124E, L124D, L124K, L124R, or L124H mutation, one or both of CL1 and CL2 comprises a V133E, V133D, V133K, V133R, or V133H mutation, and wherein the mutation in one or both of CH1-1 or CH1-2 is oppositely charged than the mutation in one or both of CL1 and CL 2.

In some embodiments, one or more of VH1, VH2, and VH3 comprises a 39E, 39D, 39K, 39R, or 39H mutation, one or more of VL1, VL2, and VL3 comprises a 38E, 38D, 38K, 38R, or 38H mutation, and wherein the mutation in one or more of VH1, VH2, and VH3 is oppositely charged to the mutation in one or more of VL1, VL2, and VL 3.

In some embodiments, one or more of VH1, VH2, and VH3 comprises a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, one or more of VL1, VL2, and VL3 comprises a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, and wherein the mutation in one or more of VH1, VH2, and VH3 is oppositely charged to the mutation in one or more of VL1, VL2, and VL 3.

In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC 1)/heavy chain (HC1) pair, the first light chain (LC 1)/heavy chain (HC1) pair comprising:

(1) a first VL region (VL1) that pairs with a first VH region (VH1) to form a first antigen binding site (VL 1);

(2) a first constant heavy chain region 1(CH1-1) operably linked to VH1 and a first constant light chain region (CL1) operably linked to VL1, and

b) a second light chain (LC 2)/heavy chain (HC2) pair, the second light chain (LC 2)/heavy chain (HC2) pair comprising:

(3) a second VL region (VL2) that pairs with a second VH region (VH2) to form a second antigen binding site; and

(4) a second constant heavy chain region 1(CH1-2) operably linked to VH2 and a second constant light chain region (CL2) operably linked to VL2,

Wherein the C-terminus of CH1-1 is operably linked to the N-terminus of VH2, and

wherein one or more cysteine residues are engineered into one or both of the VH1/VL1 pair and the VH2/VL2 pair to form one or more disulfide bonds,

wherein one or both of the VL1 and VH1 pairs and one or both of the VL2 and VH2 pairs comprise oppositely charged mutations that promote pairing,

wherein one or both of the pair CL1 and CH1-1 and one or both of the pair CL2 and CH1-2 comprise mutations that promote pairing, and

In some embodiments, CH1-1 comprises the T192E mutation, and CL1 comprises the N137K and S114A mutations.

In some embodiments, CH1-1 comprises L143Q and S188V mutations, and CL1 comprises V133T and S176V mutations.

In some embodiments, CH1-1 comprises T192E, L143Q, and S188V mutations, and CL1 comprises N137K, S114A, V133T, and S176V mutations.

In some embodiments, CH1-2 comprises the T192E mutation, and CL2 comprises the N137K and S114A mutations.

In some embodiments, CH1-2 comprises L143Q and S188V mutations, and CL2 comprises V133T and S176V mutations.

In some embodiments, CH1-2 comprises T192E, L143Q, and S188V mutations, and CL2 comprises N137K, S114A, V133T, and S176V mutations.

In some embodiments, the present disclosure provides a multispecific antigen-binding protein comprising: