0001] Classical antibodies are Y-shaped proteins or immunoglobulins which are heterotetramers formed of two heterodimers, consisting of a light chain portion and a heavy chain portion. The“arms” of the antibody comprise an antigen-binding site and are called a Fab region. Classical antibodies (e.g., those produced by a host immune system) have two identical Fabs and can recognize and bind a specific antigen. The creation of asymmetry in a native antibody structure is a prerequisite for the generation of multispecific binding proteins having two (e.g., bispecific antibodies) or more binding specificities. For example, by separating one or more Fvs on different asymmetric binding arms or Fabs, a bispecific antibody can be made with the flexibility of binding two different antigens simultaneously. Despite these advantages, however, a wide variety of multispecific antibody technologies suffer process and manufacturing problems due to mispairings of various asymmetric heavy and light chains. For example, many of these technologies suffer from the so-called“light-chain problem”, wherein random pairing of the two different light chains with heavy chains generates various combinations of chain pairings other than the desired combination. In some cases, the light-chain problem can be circumvented by the use of a common light chain, which enables binding to both antigens. However, this might not be possible for many antibodies since this format requires de novo antibody generation in transgenic mice or by display technologies. Furthermore, rare antibodies like broadly neutralizing anti-HIV antibodies derived from human patients cannot be adapted to such a format. Accordingly, there remains a need for alternative and creative solutions to the mispairing problem. Series of mutations in the dimer interface have been carefully designed to enable heterodimerization of these antibodies.

SUMMARY OF THE INVENTION

[0001] The present disclosure provides antigen-binding proteins that may optionally comprise a variety of mutations along a dimer interface that enable efficient heterodimerization of the antigen-binding proteins.

[0002] 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 paired with a CL region,

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

[0003] 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 paired with a second CL region.

[0004] In some embodiments, one or both of the first VH and VL pair and the second VH and VL pair comprise opposite 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.

[0005] In some embodiments, one or both CH1 regions comprise a T192E mutation and one or both CL regions comprise N137K and S1 14A mutations.

[0006] In some embodiments, one or both CH1 regions comprise L143Q and S188V mutations and one or both CL regions comprise V133T and S176V mutations.

[0007] In some embodiments, one or both CH1 regions comprise T192E, L143Q and S188V mutations and one or both CL regions comprise N137K, S1 14A, V133T and S176V mutations.

[0008] In some embodiments, one or both CH1 regions comprise a L143E, L143D, L143K, L143R, or L143H mutation and one or both CL regions comprise a S176E, S176D, S176K, S176R, or S176H mutation, wherein the mutation in CH1 is an opposite charge

from the mutation in CL.

[0009] In some embodiments, one or both CH1 regions comprise a 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 an opposite charge from the mutation in CL.

[0010] 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 mutation in VH is an opposite charge from the mutation in VL.

[0011] 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 an opposite charge from the mutation in VL.

[0012] 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.

[0013] In some embodiments, one or both VH regions comprise one or both of 44C and 105C mutations, and one or both VL regions comprise one or both of 100C and 43C mutations.

[0014] In some embodiments, one or both VH regions comprise a 44C mutation and one or both VL regions comprise a 100C mutation.

[0015] In some embodiments, one or both VH regions comprise a 105C mutation and one or both VL regions comprise a 43C mutation.

[0016] In some embodiments, the antigen-binding protein further comprises opposite charged mutations in one or both the CH1/CL pairs.

[0017] In some embodiments, the opposite charged mutations in one or both the CH1/CL pairs are selected from the group consisting of: K221 E 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.

[0018] In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising at least two VL regions respectively paired with at least two VH regions to form at least two antigen-binding sites and at least two CH1 regions respectively paired with two CL regions, wherein at least one CH1/CL pair comprises CH1/CL mutations to facilitate pairing selected from:

(1 ) T192E (CH1 ) mutation and N137K and S1 14A (CL) mutations, and/or

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

(3) T192E, L143Q and S188V (CH1 ) mutations and N137K, S1 14A, V133T and S176V (CL) mutations, and/or

(4) K221 E (CH1 ) mutation and E123K (CL) mutation, and/or

(5) T192E and K221 E (CH1 ) mutation and N137K, S1 14A and E123K (CL) mutations and/or

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

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

(8) wherein when two CH1/CL pairs comprise mutations to facilitate pairing for two different VH/VL pairs, the two CH1/CL pairs do not comprise same mutations, and

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

[0019] In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising at least two VL regions respectively paired with at least two VH regions to form at least two antigen-binding sites and at least two CH1 regions respectively paired with two CL regions,

wherein at least one CH1/CL pair comprises CH1/CL mutations to facilitate pairing selected from the group consisting of one or more of

(1 ) a T192E (CH1 ) mutation and N137K and S1 14A (CL) mutations, and

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

(3) T192E, L143Q and S188V (CH1 ) mutations and N137K, S1 14A, V133T and S176V (CL) mutations,

(4) a K221 E (CH1 ) mutation and a E123K (CL) mutation,

(5) T192E and K221 E (CH1 ) mutations and N137K, S1 14A and E123K (CL) mutations,

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

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

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

(9) K221 E and K228D (CH1 ) mutations and D122K and E123K (CL) mutations, wherein when two CH1/CL pairs comprise mutations to facilitate pairing for two different VH/VL pairs, the two CH1/CL pairs do not comprise same mutations, and

wherein at least one VH/VL pair comprise opposite charged mutations to facilitate pairing, said opposite charged mutations comprising (1 ) a mutated residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutated residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, and

wherein the mutated residue in the VH region has an opposite charge from the mutated residue in the VL region.

[0020] In some embodiments, in the multispecific antigen-binding protein, when at least two different VH/VL pairs comprise a mutation set to facilitate pairing, then the at least two different VH/VL pairs do not comprise the same mutation set.

[0021] In some embodiments, a multispecific antigen-binding protein comprises at least two VL regions respectively paired with at least two VH regions to form at least two antigen-binding sites and at least two CH1 regions respectively paired with two CL regions,

wherein at least one CH1/CL pair comprise CH1/CL mutations to facilitate pairing selected from the group consisting of one or more of:

(1 ) T192E (CH1 ) mutation and N137K and S1 14A (CL) mutations, and/or

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

(3) T192E, L143Q and S188V (CH1 ) mutations and N137K, S1 14A, V133T and S176V (CL) mutations, and/or

(4) K221 E (CH1 ) mutation and E123K (CL) mutation, and/or

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

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

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

and

wherein at least one VH/VL pair comprise opposite charged mutations to facilitate pairing selected from 39E, Q39D, Q39K, Q39R, or Q39H mutation, and Q38E, Q38D, Q38K, Q38R, or Q38H mutation, and wherein the mutation in VH is an opposite charge from the mutation in VL; and when several VH/VL pairs, for which VL or VH are not in the same polypeptide chain, comprise mutations to facilitate pairing for different VH/VL pairs, then each VH/VL pair do not comprise same opposite charged mutations.

[0022] In some embodiments, one or both CH1 regions are operably linked to a heterodimerization domain.

[0023] In some embodiments, the heterodimerization domain comprises a first Fc domain.

[0024] In some embodiments, the first Fc domain heterodimerizes 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.

[0025] In some embodiments, the first CH3 region comprises one or both of S354C and T366W mutations, and the second CH3 region comprises one or more of Y349C, T366S, L368A, and Y407V mutations, wherein the mutations facilitate Fc domain heterodimerization.

[0026] In another aspect, the present disclosure provides a multispecific antibody comprising

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

(2) a first constant heavy chain region 1 (CH1 -1 ) and a first constant light chain region (CL1 ); and

(3) a first heterodimerization domain (HD1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

(4) a second VL region (VL2) paired 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 (CL2) ; and

(6) a second heterodimerization domain (HD2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of VL1 and VH1 pair and of VL2 and VH2 pair comprise opposite charged mutations to facilitate pairing,

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

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

[0027] In another aspect, the present disclosure provides a multispecific antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

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

(3) a first heterodimerization domain (HD1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(5) a second constant heavy chain region 1 (CH1 -2) operatively linked to VH2 and a second constant light chain region (CL2) operatively linked to VL2; and

(6) a second heterodimerization domain (HD2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of VL1 and VH1 pair and of VL2 and VH2 pair comprise opposite charged mutations to facilitate pairing,

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

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

[0028] In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

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

(3) a first heterodimerization domain (HD1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(5) a second constant heavy chain region 1 (CH1 -2) operatively linked to VH2 and a second constant light chain region (CL2) operatively linked to VL2; and

(6) a second heterodimerization domain (HD2);

wherein HD1 and HD2 heterodimerize,

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

from E, D, K, R, or H, and wherein the mutated residue in the VH region has an opposite charge from the mutated residue in the VL region,

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

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

[0029] In some embodiments, CH1 -1 comprises a T192E mutation and CL1 comprises N137K and S1 14A mutations.

[0030] In some embodiments, CH1 -1 comprises L143Q and S188V mutations and CL1 comprises V133T and S176V mutations.

[0031] In some embodiments, CH1 -1 comprises T192E, L143Q and S188V mutations and CL1 comprises N137K, S1 14A, V133T and S176V mutations.

[0032] In some embodiments, CH1 -2 comprises a T192E mutation and CL2 comprises N137K and S1 14A mutations.

[0033] In some embodiments, CH1 -2 comprises L143Q and S188V mutations and CL2 comprises V133T and S176V mutations.

[0034] In some embodiments, CH1 -2 comprises T192E, L143Q and S188V mutations and CL2 comprises N137K, S1 14A, V133T and S176V mutations.

[0035] In some embodiments, one of both of CH1 -1 and CH1 -2 comprises a L143E, L143D, L143K, L143R, or L143H mutation and one or both of CL1 and CL2 comprises a S176E, S176D, S176K, S176R, or S176H mutation, wherein the mutation in one of both of CH1 -1 or CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2.

[0036] In some embodiments, one of both of CH1 -1 and CH1 -2 comprises a 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 of both of CH1 -1 or CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2

[0037] 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 an opposite charge from the mutation in one or both of VL1 and VL2.

[0038] 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 an opposite charge from the mutation in one or both of VL1 and VL2.

[0039] In some embodiments, the multispecific antibody further comprises one or more cysteine residues engineered into the one or both of VH1/VL1 and VH2/VL2 pairs to form one or more disulfide bonds.

[0040] 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.

[0041] In some embodiments, VH1 comprises a 44C mutation and VL1 comprises a 100C mutation.

[0042] In some embodiments, VH1 comprises a 105C mutation and VL1 comprises a 43C mutation.

[0043] In some embodiments, VH2 comprises a 44C mutation and VL2 comprises a 100C.

[0044] In some embodiments, VH2 comprises a 105C mutation and VL2 comprises a 43C mutation.

[0045] In some embodiments, VH1 comprises 39E and 44C mutations and VL1 comprises 38K and 100C mutations.

[0046] In some embodiments, VH1 comprises 39E and 105C mutations and VL1 comprises 38K and 43C mutations.

[0047] In some embodiments, the multispecific antibody further comprises opposite charged mutations in the CH1 -1/CL1 pair.

[0048] In some embodiments, the opposite charged mutations in the CH1 -1/CL1 pair are selected from the group consisting of: K221 E 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.

[0049] In some embodiments, the multispecific antibody further comprises opposite charged mutations in the CH1 -2/CL2 pair.

[0050] In some embodiments, the opposite charged mutations in the CH1 -2/CL2 pair are selected from the group consisting of: K221 E 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.

[0051] In some embodiments, the first and second heterodimerization domains comprise Fc domains.

[0052] In some embodiments, the first heterodimerization domain comprises a first CH3 domain comprising one or both of S354C and T366W mutations, and the second heterodimerization domain comprises a second CH3 domain comprising one or both of Y349C, T366S, L368A, and Y407V mutations, wherein the mutations facilitate Fc domain heterodimerization.

[0053] In some embodiments, the CH1 -1 domain is linked to a first CH2 and first CH3 domain, the CH1 -2 domain is linked to a second CH2 and second CH3 domain, and wherein the first CH2 and CH3 domains and the second CH2 and CH3 domains dimerize to form an Fc domain.

[0054] In some embodiments, the first CH3 domain comprises one or both of S354C and T366W mutations, the second CH3 domain comprises one or more of Y349C, T366S, L368A, and Y407V mutations, and wherein the mutations facilitate Fc domain heterodimerization.

[0055] In another aspect, the present disclosure provides an antigen-binding protein or 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

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

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

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

wherein at least one or both of VL1 and VH1 pair and of VL2 and VH2 pair comprise opposite charged mutations that facilitate pairing, and

wherein the CH1 and CL domain pair comprise mutations that facilitate pairing.

[0056] In some embodiments, VH1 is paired with VL1 , VH2 is paired with VL2, and CH1 is paired with CL.

[0057] 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

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 the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair,

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

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

wherein the CH1 and CL domain pair comprise mutations that facilitate pairing.

[0058] In some embodiments, one or both of VH1 and VH2 comprise VH44C and VH105C mutations.

[0059] In some embodiments, one or both of VL1 and VL2 comprise VL43C and VL100C mutations.

[0060] In some embodiments, one or both of VH1 and VH2 comprise a VH44C mutation and one or both of VL1 and VL2 comprise a VL100C mutation.

[0061] In some embodiments, one or both of VH1 and VH2 comprise a VH105C mutation and one or both of VL1 and VL2 comprise a VL43C mutation.

[0062] In some embodiments, CH1 comprises a T192E mutation and CL comprises N137K and S1 14A mutations.

[0063] In some embodiments, CH1 comprises L143Q and S188V mutations and CL comprises V133T and S176V mutations.

[0064] In some embodiments, CH1 comprises T192E, L143Q and S188V mutations and CL comprises N137K, S1 14A, V133T and S176V mutations.

[0065] In some embodiments, CH1 comprises a L143E, a L143D, a L143K, a L143R, or a L143H mutation, CL comprises a S176E, a S176D, a S176K, a S176R, or a S176H mutation, and wherein the mutation in CH1 is an opposite charge from the mutation in CL.

[0066] In some embodiments, CH1 comprises a L124E, a L124D, a L124K, a L124R, or a L124H mutation, CL comprises a V133E, a V133D, a V133K, a V133R, or a V133H

mutation, and wherein the mutation in CH1 is an opposite charge from the mutation in CL.

[0067] In some embodiments, one or both of VH1 and VH2 comprises a 39E, a 39D, a 39K, a 39R, or a 39H mutation, one or both of VL1 and VL2 comprises a 38E, a 38D, a 38K, a 38R, or a 38H mutation, and wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2.

[0068] In some embodiments, one or both of VH1 and VH2 comprises a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation, one or both of VL1 and VL2 comprises a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, and wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2.

[0069] In some embodiments, CH1 is operatively linked to a dimerization domain.

[0070] In some embodiments, the dimerization domain is an Fc domain comprising a CH2 domain and a CH3 domain.

[0071] In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising four polypeptide chains that form four antigen-binding sites, wherein two polypeptide chains each comprises a structure represented by the formula:

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

and two polypeptide chains each 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

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

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

wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair,

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

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

wherein one or both of the CH1 and CL domain pair comprises mutations that facilitate pairing, and

wherein when at least two CH1/CL pairs comprise mutations to facilitate pairing, then the mutation set in one CH1/CL pair is different from the mutation set in the other CH1/CL pair.

[0072] In another aspect, the present disclosure provides an antigen-binding protein comprising four polypeptide chains that form four antigen-binding sites, wherein two polypeptide chains each comprises a structure represented by the formula:

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

and two polypeptide chains each 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

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

wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair,

wherein VH1 is paired with VL1 to form a first antigen-binding site, VH2 is paired with VL2 to form a second antigen-binding site, and CH1 is paired 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 the one or both of VH1/VL1 and VH2/VL2 pairs to form one or more disulfide bonds,

wherein one or both of the VL1 and VH1 pair and the VL2 and VH2 pair comprise opposite charged mutations that facilitate pairing, and

wherein one or both of the CH1 and CL domain pair comprises mutations that facilitate pairing, and

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

[0073] In some embodiments, one or both of VH1 and VH2 comprise VH44C and VH105C mutations.

[0074] In some embodiments, one or both of VL1 and VL2 comprise VL43C and VL100C mutations.

[0075] In some embodiments, one or both of VH1 and VH2 comprise a VH44C mutation and one or both of VL1 and VL2 comprise a VL100C mutation.

[0076] In some embodiments, one or both of VH1 and VH2 comprise a VH105C mutation and one or both of VL1 and VL2 comprise a VL43C mutation.

[0077] In some embodiments, CH1 comprises a T192E mutation and CL comprises N137K and S1 14A mutations.

[0078] In some embodiments, CH1 comprises L143Q and S188V mutations and CL comprises V133T and S176V mutations.

[0079] In some embodiments, CH1 comprises T192E, L143Q and S188V mutations and CL comprises N137K, S1 14A, V133T and S176V mutations.

[0080] In some embodiments, CH1 comprises a L143E, a L143D, a L143K, a L143R, or a L143H mutation, CL comprises a S176E, a S176D, a S176K, a S176R, or a S176H

mutations, and wherein the mutation in CH1 is an opposite charge from the mutation in CL.

[0081] In some embodiments, CH1 comprises a L124E, a L124D, a L124K, a L124R, or a L124H mutation, CL comprises a V133E, a V133D, a V133K, a V133R, or a V133H mutations, and wherein the mutation in CH1 is an opposite charge from the mutation in CL.

[0082] In some embodiments, one or both of VH1 and VH2 comprises a 39E, a 39D, a 39K, a 39R, or a 39H mutation, one or both of VL1 and VL2 comprises a 38E, a 38D, a 38K, a 38R, or a 38H mutation, and wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2.

[0083] In some embodiments, one or both of VH1 and VH2 comprises a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation, one or both of VL1 and VL2 comprises a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, and wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2.

[0084] In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising four polypeptide chains, that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1 -VL1 -L2-CL1 [I],

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

VH 1 -L3-VH2-L4-CH 1 -1 -hinge-CH2-CH3 [II], a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1 -2-hinge-CH2-CH3 [III], and

a 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;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and

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

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

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

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

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

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

[0085] In some embodiments, one or both of VH1 and VH2 comprise VH44C and VH105C mutations.

[0086] In some embodiments, one or both of VL1 and VL2 comprise VL43C and VH100C mutations.

[0087] In some embodiments, one or both of VH1 and VH2 comprise a VH44C mutation and one or both of VL1 and VL2 comprise a VL100C mutation.

[0088] In some embodiments, one or both of VH1 and VH2 comprise a VH105C mutation and one or both of VL1 and VL2 comprise a VL43C mutation.

[0089] In some embodiments, one or both of CH1 -1 and CL1 comprise mutations that facilitate pairing and one or both of CH1 -2 and CL2 comprise mutations that facilitate pairing.

[0090] In some embodiments, CH1 -1 comprises a T192E mutation and CL1 comprises N137K and S1 14A mutations.

[0091] In some embodiments, CH1 -1 comprises L143Q and S188V mutations and CL1 comprises V133T and S176V mutations.

[0092] In some embodiments, CH1 -1 comprises T192E, L143Q and S188V mutations and CL1 comprises N137K, S1 14A, V133T and S176V mutations.

[0093] In some embodiments, CH1 -2 comprises a T192E mutation and CL2 comprises N137K and S1 14A mutations.

[0094] In some embodiments, CH1 -2 comprises L143Q and S188V mutations and CL2 comprises V133T and S176V mutations.

[0095] In some embodiments, CH1 -2 comprises a T192E, L143Q and S188V mutation and CL2 comprises N137K, S1 14A, V133T and S176V mutations.

[0096] In some embodiments, one of both of CH1 -1 and CH1 -2 comprises a L143E, a L143D, a L143K, a L143R, or a L143H mutation, one or both of CL1 and CL2 comprises a S176E, a S176D, a S176K, a S176R, or a S176H mutation, and wherein the mutation in one of both of CH1 -1 or CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2.

[0097] In some embodiments, one of both of CH1 -1 and CH1 -2 comprises a L124E, a L124D, a L124K, a L124R, or a L124H mutation, one or both of CL1 and CL2 comprises a V133E, a V133D, a V133K, a V133R, or a V133H mutation, and wherein the mutation in one of both of CH1 -1 or CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2.

[0098] In some embodiments, one or more of VH1 , VH2, and VH3 comprises a 39E, a 39D, a 39K, a 39R, or a 39H mutation, one or more of VL1 , VL2, and VL3 comprises a 38E, a 38D, a 38K, a 38R, or a 38H mutation, and wherein the mutation in one or more of VH1 , VH2, and VH3 is an opposite charge from the mutation in one or more of VL1 , VL2, and VL3.

[0099] In some embodiments, one or more of VH1 , VH2, and VH3 comprises a Q39E, a

Q39D, a Q39K, a Q39R, or a Q39H mutation, one or more of VL1 , VL2, and VL3 comprises a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, and wherein the mutation in one or more of VH1 , VH2, and VH3 is an opposite charge from the mutation in one or more of VL1 , VL2, and VL3.

[00100] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising:

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

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

b) a second light chain (LC2)/heavy chain (HC2) pair comprising:

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

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

wherein the C terminus of CH1 -1 is operatively linked to the N terminus of VH2, and wherein one or more cysteine residues are engineered into the one or both of VH1/VL1 and VH2/VL2 pairs to form one or more disulfide bonds,

wherein one or both of the VL1 and VH1 pair and the VL2 and VH2 pair comprise opposite charged mutations that facilitate pairing,

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

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

[00101] In some embodiments, CH1 -1 comprises a T192E mutation and CL1 comprises N137K and S1 14A mutations.

[00102] In some embodiments, CH1 -1 comprises L143Q and S188V mutations and CL1 comprises V133T and S176V mutations.

[00103] In some embodiments, CH1 -1 comprises T192E, L143Q and S188V mutations

and CL1 comprises N137K, S1 14A, V133T and S176V mutations.

[00104] In some embodiments, CH1 -2 comprises a T192E mutation and CL2 comprises N137K and S1 14A mutations.

[00105] In some embodiments, CH1 -2 comprises L143Q and S188V mutations and CL2 comprises V133T and S176V mutations.

[00106] In some embodiments, CH1 -2 comprises T192E, L143Q and S188V mutations and CL2 comprises N137K, S1 14A, V133T and S176V mutations.

[00107] In some embodiments, one of both of CH1 -1 and CH1 -2 comprises a L143E, a L143D, a L143K, a L143R, or a L143H mutation, one or both of CL1 and CL2 comprises a S176E, a S176D, a S176K, a S176R, or a S176H mutation, and wherein the mutation in one of both of CH1 -1 or CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2.

[00108] In some embodiments, one of both of CH1 -1 and CH1 -2 comprises a L124E, a L124D, a L124K, a L124R, or a L124H mutation, one or both of CL1 and CL2 comprises a V133E, a V133D, a V133K, a V133R, or a V133H mutation, and wherein the mutation in one of both of CH1 -1 or CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2.

[00109] In some embodiments, one or both of VH1 and VH2 comprises a 39E, a 39D, a 39K, a 39R, or a 39H mutation, one or both of VL1 and VL2 comprises a 38E, a 38D, a 38K, a 38R, or a 38H mutation, and wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2.

[00110] In some embodiments, one or both of VH1 and VH2 comprises a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation, one or both of VL1 and VL2 comprises a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, and wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2.

[00111] In some embodiments, the present disclosure provides a multispecific antigen binding protein comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising:

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

(2) a first constant heavy chain region 1 (CH1 -1 ) operatively linked to VH1 and a first constant light chain region (CL1 ) operatively linked to VL1 , and b) a second light chain (LC2)/heavy chain (HC2) pair comprising:

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

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

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

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

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

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

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

[00112] In some embodiments, the multispecific antibody further comprises one or more cysteine residues engineered into the one or both of VH1/VL1 and VH2/VL2 pairs to form one or more disulfide bonds.

[00113] In some embodiments, one or both of VH1 and VH2 comprises a 44C and a 105C mutation and one or both of VL1 and VL2 comprises a 100C and a 43C mutation.

[00114] In some embodiments, VH1 comprises a 44C mutation and VL1 comprises a 100C mutation.

[00115] In some embodiments, VH1 comprises a 105C mutation and VL1 comprises a

43C mutation.

[00116] In some embodiments, VH2 comprises a 44C mutation and VL2 comprises a 100C mutation.

[00117] In some embodiments, VH2 comprises a 105C mutation and VL2 comprises a 43C mutation.

[00118] In some embodiments, VH1 comprises 39E and 44C mutations and VL1 comprises 38K and 100C mutations.

[00119] In some embodiments, VH1 comprises 39E and 105C mutations and VL1 comprises 38K and 43C mutations.

[00120] In some embodiments, the multispecific antibody further comprises opposite charged mutations in the CH1 -1/CL1 pair.

[00121] In some embodiments, the opposite charged mutations in the CH1 -1/CL1 pair are selected from the group consisting of: K221 E 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.

[00122] In some embodiments, the multispecific antibody further comprises opposite charged mutations in the CH1 -2/CL2 pair.

[00123] In some embodiments, the opposite charged mutations in the CH1 -2/CL2 pair are selected from the group consisting of: K221 E 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.

[00124] In some embodiments, the CH1 -1 domain is linked to a first CH2 domain and 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.

[00125] In some embodiments, the first CH3 domain comprises one or both of S354C and T366W mutations and the second CH3 domain comprises one or both of Y349C, T366S, L368A, and Y407V mutations to facilitate Fc domain heterodimerization.

[00126] In some embodiments, the C terminus of CH1 -1 is operatively linked to the N terminus of VH2 via a peptide linker.

[00127] In some embodiments, the peptide linker comprises a (GGGGS)n (SEQ ID NO: X) linker, wherein n is any integer from 1 to 5.

[00128] In some embodiments, the peptide linker comprises all or part of the sequence of a hinge region of one or more immunoglobulin(s) selected from IgA, IgG, and IgD.

[00129] In some embodiments, the peptide linker comprises the following sequence: EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: X).

[00130] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

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

(3) a first heterodimerization domain (HD1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(5) a second constant heavy chain region 1 (CH1 -2) operatively linked to VH2 and a second constant light chain region (CL2) operatively linked to VL2; and

(6) a second heterodimerization domain (HD2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of VH1 and VH2 comprise a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation and one or both of VL1 and VL2 comprise a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2 to facilitate pairing, and wherein at least one or both of CH1 -1 and CH1 -2 comprise one or more of a T192E, a L143Q, and a S188V mutation and at least one or both of CL1 and CL2 comprise one or more of a N137K, a S1 14A, a V133T, and a S176V mutation.

[00131] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

(2) a first constant heavy chain region 1 (CH1 -1 ) and a first constant light chain region (CL1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(4) a second constant heavy chain region 1 (CH1 -2) and a second constant light chain region (CL2);

wherein at least one or both of VH1 and VH2 comprise a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation and one or both of VL1 and VL2 comprise a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2 to facilitate pairing, and wherein at least one or both of CH1 -1 and CH1 -2 comprise one or more of a T192E, a L143Q, and a S188V mutation and at least one or both of CL1 and CL2 comprise one or more of a N137K, a S1 14A, a V133T, and a S176V mutation.

[00132] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

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

(3) a first heterodimerization domain (HD1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(5) a second constant heavy chain region 1 (CH1 -2) operatively linked to VH2 and a second constant light chain region (CL2) operatively linked to VL2; and

(6) a second heterodimerization domain (HD2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of CH1 -1 and CH1 -2 comprise a K221 E, a K221 D, a K221 K, a K221 R, or a K221 H mutation and one or both of CL1 and CL2 comprise a E123E, a E123D, a E123K, a E123R, or a E123H mutation, wherein the mutation in one or both of CH1 -1 and CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2 to facilitate pairing, and

wherein at least one or both of CH1 -1 and CH1 -2 comprise one or more of a T192E, a L143Q, and a S188V mutation and at least one or both of CL1 and CL2 comprise one or more of a N137K, a S1 14A, a V133T, and a S176V mutation.

[00133] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

(2) a first constant heavy chain region 1 (CH1 -1 ) and a first constant light chain region (CL1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(4) a second constant heavy chain region 1 (CH1 -2) and a second constant light chain region (CL2);

wherein at least one or both of CH1 -1 and CH1 -2 comprise a K221 E, a K221 D, a K221 K, a K221 R, or a K221 H mutation and one or both of CL1 and CL2 comprise a E123E, a E123D, a E123K, a E123R, or a E123H mutation, wherein the mutation in one or both of CH1 -1 and CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2 to facilitate pairing, and

wherein at least one or both of CH1 -1 and CH1 -2 comprise one or more of a T192E, a L143Q, and a S188V mutation and at least one or both of CL1 and CL2 comprise one or more of a N137K, a S1 14A, a V133T, and a S176V mutation.

[00134] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

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

(3) a first heterodimerization domain (HD1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(5) a second constant heavy chain region 1 (CH1 -2) operatively linked to VH2 and a second constant light chain region (CL2) operatively linked to VL2; and

(6) a second heterodimerization domain (HD2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of VH1 and VH2 comprise a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation and one or both of VL1 and VL2 comprise a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2 to facilitate pairing, wherein at least one or both of CH1 -1 and CH1 -2 comprise a K221 E, a K221 D, a K221 K, a K221 R, or a K221 H mutation and one or both of CL1 and CL2 comprise a E123E, a E123D, a E123K, a E123R, or a E123H mutation, wherein the mutation in one or both of CH1 -1 and CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2 to facilitate pairing, and

wherein at least one or both of CH1 -1 and CH1 -2 comprise one or more of a T192E, a L143Q, and a S188V mutation and at least one or both of CL1 and CL2 comprise one or more of a N137K, a S1 14A, a V133T, and a S176V mutation.

[00135] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

(2) a first constant heavy chain region 1 (CH1 -1 ) and a first constant light chain region (CL1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(4) a second constant heavy chain region 1 (CH1 -2) and a second constant light chain region (CL2);

wherein at least one or both of VH1 and VH2 comprise a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation and one or both of VL1 and VL2 comprise a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2 to facilitate pairing, wherein at least one or both of CH1 -1 and CH1 -2 comprise a K221 E, a K221 D, a K221 K, a K221 R, or a K221 H mutation and one or both of CL1 and CL2 comprise a E123E, a E123D, a E123K, a E123R, or a E123H mutation, wherein the mutation in one or both of CH1 -1 and CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2 to facilitate pairing, and

wherein at least one or both of CH1 -1 and CH1 -2 comprise one or more of a T192E, a L143Q, and a S188V mutation and at least one or both of CL1 and CL2 comprise one or more of a N137K, a S1 14A, a V133T, and a S176V mutation.

[00136] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

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

(3) a first heterodimerization domain (HD1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(5) a second constant heavy chain region 1 (CH1 -2) operatively linked to VH2 and a second constant light chain region (CL2) operatively linked to VL2; and

(6) a second heterodimerization domain (HD2);

wherein HD1 and HD2 heterodimerize,

wherein at least one or both of VH1 and VH2 comprise a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation and one or both of VL1 and VL2 comprise a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2 to facilitate pairing, and wherein at least one or both of CH1 -1 and CH1 -2 comprise one or more of a T192E, a L143Q, and a S188V mutation and at least one or both of CL1 and CL2 comprise one or more of a N137K, a S1 14A, a V133T, and a S176V mutation, and

wherein at least one or both of VH1 and VH2 comprise a 44C mutation and one or both of VL1 and VL2 comprise a 100C mutation.

[00137] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

(2) a first constant heavy chain region 1 (CH1 -1 ) and a first constant light chain region (CL1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(4) a second constant heavy chain region 1 (CH1 -2) and a second constant light chain region (CL2);

wherein at least one or both of VH1 and VH2 comprise a Q39E, a Q39D, a Q39K, a Q39R, or a Q39H mutation and one or both of VL1 and VL2 comprise a Q38E, a Q38D, a Q38K, a Q38R, or a Q38H mutation, wherein the mutation in one or both of VH1 and VH2 is an opposite charge from the mutation in one or both of VL1 and VL2 to facilitate pairing, and wherein at least one or both of CH1 -1 and CH1 -2 comprise one or more of a T192E, a L143Q, and a S188V mutation and at least one or both of CL1 and CL2 comprise one or more of a N137K, a S1 14A, a V133T, and a S176V mutation, and

wherein at least one or both of VH1 and VH2 comprise a 44C mutation and one or both of VL1 and VL2 comprise a 100C mutation.

[00138] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

(2) a first constant heavy chain region 1 (CH1 -1 ) and a first constant light chain region (CL1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(4) a second constant heavy chain region 1 (CH1 -2) and a second constant light chain region (CL2);

wherein at least one or both of CH1 -1 and CH1 -2 comprises a L143E, a L143D, a L143K, a L143R, or a L143H mutation and at least one or both of CL1 and CL2 comprises a S176E, a S176D, a S176K, a S176R, or a S176H mutation, and wherein the mutation in one of both of CH1 -1 or CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2.

[00139] In another aspect, the present disclosure provides a multispecific antigen-binding protein or antibody comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising

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

(2) a first constant heavy chain region 1 (CH1 -1 ) and a first constant light chain region (CL1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising

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

(4) a second constant heavy chain region 1 (CH1 -2) and a second constant light chain region (CL2);

wherein at least one or both of CH1 -1 and CH1 -2 comprises a L124E, a L124D, a L124K, a L124R, or a L124H mutation and at least one or both of CL1 and CL2 comprises a V133E, a V133D, a V133K, a V133R, or a V133H mutation, and wherein the mutation in one of both of CH1 -1 or CH1 -2 is an opposite charge from the mutation in one or both of CL1 and CL2.

[00140] In some embodiments, CH1 -1 further comprises a T192E mutation and CL1 comprises N137K and S1 14A mutations.

[00141] In some embodiments, CH1 -2 comprises a T192E mutation and CL2 comprises N137K and S1 14A mutations.

[00142] 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 an opposite charge from the mutation in one or both of VL1 and VL2.

[00143] In some embodiments, the multispecific antibody further comprises one or more cysteine residues engineered into the one or both of VH1/VL1 and VH2/VL2 pairs to form one or more disulfide bonds.

[00144] 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.

[00145] In some embodiments, VH1 comprises a 44C mutation and VL1 comprises a 100C mutation.

[00146] In some embodiments, VH1 comprises a 105C mutation and VL1 comprises a 43C mutation.

[00147] In some embodiments, VH2 comprises a 44C mutation and VL2 comprises a 100C.

[00148] In some embodiments, VH2 comprises a 105C mutation and VL2 comprises a 43C mutation.

[00149] In some embodiments, VH1 comprises 39E and 44C mutations and VL1

comprises 38K and 100C mutations.

[00150] In some embodiments, VH1 comprises 39E and 105C mutations and VL1 comprises 38K and 43C mutations.

[00151] In some embodiments, the multispecific antibody further comprises opposite charged mutations in one or both of the CH1 -1/CL1 pair and the CH1 -2/CL2 pair.

[00152] In some embodiments, the opposite charged mutations in the one or both of the CH1 -1/CL1 pair and the CH1 -2/CL2 pair comprise K221 E in one or both of the CH1 -1 region and CH2-2 region and E123K in one or both of the CL1 region and CL2 region.

[00153] In some embodiments, the CH1 -1 domain is operatively linked to a first Fc domain comprising a first CH2 and first CH3 domain, and the CH1 -2 domain is operatively linked to a second Fc domain comprising a second CH2 and second CH3 domain, and wherein the first Fc domain and the second Fc domain dimerize.

[00154] In some embodiments, the first CH3 domain comprises one or both of S354C and T366W mutations, the second CH3 domain comprises one or more of Y349C, T366S, L368A, and Y407V mutations, and wherein the mutations facilitate Fc domain heterodimerization.

[00155] In another aspect, the present disclosure provides 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 CL region comprise one or both of:

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

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

wherein the mutated residue in the CH1 region has an opposite charge from the mutated residue in the CL region.

[00156] In another aspect, the present disclosure provides a binding protein comprising a protein binding domain; and

a CH1 region paired with a CL region,

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

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

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

wherein the mutation in the CH1 region is an opposite charge from the mutation in the CL region.

[00157] In another aspect, the present disclosure provides an antigen-binding protein comprising

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 CL region comprise one or both of:

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

b) a K221 E and a K228D mutation in the CH1 region and a D122K and a E123K mutation in the CL region,

[00158] wherein the mutated residue in the CH1 region has an opposite charge from the mutated residue in the CL region.

[00159] In some embodiments, the binding protein further comprises a K221 E mutation in the CH1 region and a E123K mutation in the CL region.

[00160] In some embodiments, the present disclosure provides a multispecific antigen binding protein comprising a first Fab, said first Fab comprising CH1 -1 , VH1 , CL1 and VL1 domains, and a second Fab, said second Fab comprising CH1 -2, VH2, CL2 and VL2 domains, wherein first Fab and second Fab are selected from one of the following alternatives :

i. A first Fab comprises 143R mutation in CH1 -1 , 39K mutation in VH1 , 176E mutation in CL1 , 38E mutation in VL1 and a second Fab comprises 143E mutation in CH1 -2, 39E mutation in VH2, 176R mutation in CL2 and 38K mutation in VL2; ii. A first Fab comprises 143K mutation in CH1 -1 , 39K mutation in VH1 , 176E mutation in CL1 , 38E mutation in VL1 and a second Fab comprises 143E mutation

in CH1 -2, 39E mutation in VH2, 176K mutation in CL2 and 38K mutation in VL2; iii. A first Fab comprises 143H mutation in CH1 -1 , 39K mutation in VH1 , 176E mutation in CL1 , 38E mutation in VL1 and a second Fab comprises 143E mutation in CH1 -2, 39E mutation in VH2, 176H mutation in CL2 and 38K mutation in VL2; iv. A first Fab comprises 143R mutation in CH1 -1 , 39K mutation in VH1 , 176D mutation in CL1 , 38E mutation in VL1 and a second Fab comprises 143D mutation in CH1 -2, 39E mutation in VH2, 176R mutation in CL2 and 38K mutation in VL2; v. A first Fab comprises 143K mutation in CH1 -1 , 39K mutation in VH1 , 176D mutation in CL1 , 38E mutation in VL1 and a second Fab comprises 143D mutation in CH1 -2, 39E mutation in VH2, 176K mutation in CL2 and 38K mutation in VL2; vi. A first Fab comprises 143H mutation in CH1 -1 , 39K mutation in VH1 , 176D mutation in CL1 , 38E mutation in VL1 and a second Fab comprises 143D mutation in CH1 -2, 39E mutation in VH2, 176H mutation in CL2 and 38K mutation in VL2.

[00161] In some embodiments, the CH1 region is operably linked to a heterodimerization domain.

[00162] In some embodiments, the heterodimerization domain comprises a first Fc domain.

[00163] In some embodiments, the first Fc domain heterodimerizes 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.

[00164] In some embodiments, the first CH3 region comprises one or both of S354C and T366W mutations, and the second CH3 region comprises one or more of Y349C, T366S, L368A, and Y407V mutations, wherein the mutations facilitate Fc domain heterodimerization.

[00165] In some embodiments, the antigen-binding protein further comprises at least one VH/VL pair comprising opposite charged mutations to facilitate pairing, said opposite charged mutations comprising (1 ) a mutated residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutated residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, and wherein the mutated residue in the VH region has an opposite charge from the mutated residue in the VL region.

[00166] In some embodiments, a multispecific antigen-binding protein or antigen-

binding protein comprises three HCDRs for each VH region and three LCDRs for each VL region, and further comprises binding specificity to one or more target antigens or one or more target epitopes. In some embodiments, the HCDRs, LCDRs and/or antigen are disclosed herein. In some embodiments, the HCDRs, LCDRs and/or antigen are known in the art. In some embodiments, the HCDRs, LCDRs and/or antigen have been newly identified or discovered.

[00167] In some embodiments, the present disclosure provides 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 CL region comprise one or both of:

a) a L143E, a L143D, a L143K, a L143R, or a L143H mutation in the CH1 region and a S176E, a S176D, a S176K, a S176R, or a S176H mutation in the CL region; and b) a L124E, a L124D, a L124K, a L124R, or a L124H mutation in the CH1 region and a V133E, a V133D, a V133K, a V133R, or a V133H mutation in the CL region, wherein the mutated residue in the CH1 region has an opposite charge from the mutated residue in the CL region.

[00168] In some embodiments, the antigen-binding protein further comprises CH1/CL mutations to facilitate pairing selected from the group consisting of one or more of:

(1 ) a T192E (CH1 ) mutation and N137K and S1 14A (CL) mutations,

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

(3) T192E, L143Q and S188V (CH1 ) mutations and N137K, S1 14A, V133T and S176V (CL) mutations,

(4) a K221 E (CH1 ) mutation and a E123K (CL) mutation,

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

(6) K221 E and K228D (CH1 ) mutations and D122K and E123K (CL) mutations, wherein when two CH1/CL pairs comprise mutations to facilitate pairing for two different VH/VL pairs, the two CH1/CL pairs do not comprise the same mutations.

[00169] In some embodiments, the CH1 region is operably linked to a Fc domain.

[00170] In some embodiments, an isolated nucleic acid molecule comprising a nucleotide sequence encoding the multispecific antibody or antigen-binding protein, is provided. In some embodiments, a kit comprising one or more isolated nucleic acid molecules comprising one or more nucleotide sequences encoding the multispecific antigen-binding protein or antigen-binding protein, is provided.

[00171] In some embodiments, an expression vector comprising the nucleic acid molecule is provided. In some embodiments, a kit comprising one or more expression vectors comprising one or more of the nucleic acid molecules is provided.

[00172] In some embodiments, an isolated host cell comprising the one or more nucleic acid molecules or the one or more expression vectors is provided. In some embodiments, an isolated host cell comprising the kit of nucleic acid molecules or the kit of expression vectors is provided.

[00173] In some embodiments, the host cell is a mammalian cell or an insect cell.

[00174] In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the multispecific antibody or antigen-binding protein is provided.

[00175] In some embodiments, a method of treating a disorder in which antigen activity is detrimental, the method comprising administering to a subject in need thereof an effective amount of a multispecific antibody or antigen-binding protein is provided.

[00176] In some embodiments, a polynucleotide encoding a multispecific antibody or antigen-binding protein is provided.

[00177] In some embodiments, a host cell expressing a multispecific antibody or antigen binding protein is provided.

[00178] In some embodiments, a method of producing a multispecific antibody or antigen binding protein comprising culturing the host cell under conditions such that a multispecific antibody or antigen-binding protein is expressed is provided. In some embodiments, a multispecific antibody or antigen-binding protein for use as a medicament is provided.

[00179] The summary of the disclosure described above is non-limiting and other features and advantages of the disclosed antigen-binding proteins and methods will be apparent from the following brief description of the drawings, detailed description of the disclosure, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00180] FIG. 1A- FIG. 1C schematically depict cross-over dual variable (CODV) antigen binding protein formats with several different mutations to enhance heterodimerization. FIG. 1A depicts CH1/kappa mutations Ό” on CODV-Fab (CH1 : L143Q, S188V; Ck: V133T, S176V) and“D” on Fab2 (CH1 : T192E; Ck: N137K, S1 14A). FIG. 1 B depicts mutations on CODV-Fab (VH39E/VL38K + O) and Fab2 (VH39K/VL38E + D). FIG. 1C depicts mutations on CODV-Fab ([VH39E/VL38K + O] + [VH44Cys/VL100Cys] or [VH 105Cys/VL43Cys]) and Fab2 ([VH39K/VL38E + D] + [VH44Cys/VL100Cys] or [VH105Cys/VL43Cys]).

[00181] FIG. 2A - FIG. 2G graphically depict the results for Table 5. FIG. 2A depicts results for the WT CODV antibody. FIG. 2B depicts results for a CODV antibody with only the CH1/Ck mutations. FIG. 2C depicts results for a CH1/Ck and charge mutation (CM) combination. FIG. 2D depicts results for the disulfide-stabilized (ds) only CODV antibody. FIG. 2E depicts results for a CH1/Ck and ds mutation combination. FIG. 2F depicts results for a CM and ds mutation combination. FIG. 2G depicts results for the combination of all three mutation sets, CH1/Ck, CM, and ds stabilized, in a CODV antibody.

[00182] FIG. 3A - FIG. 3D schematically depict tandem Fabs antibody formats in open (FIG. 3A, FIG. 3B) and closed (FIG. 3C, FIG. 3D) configurations. FIG. 3A shows open configuration with CH1 /kappa mutations“D” on Fab1 (CH1 : T192E; Ck: N137K, S1 14A) and “O” on Fab2 (CH1 : L143Q, S188V; Ck: V133T, S176V. FIG. 3B shows open configuration with mutations on Fab1 (VH39K/VL38E + D) and Fab2 (VH39E/VL38K + O). FIG. 3C shows closed configuration with CH1 /kappa mutations D on Fab1 (CH1 : T192E; Ck: N137K, S1 14A) and O on Fab2 (CH1 : L143Q, S188V; Ck: V133T, S176V). FIG. 3D shows closed configuration with mutations on Fab1 (VH39K/VL38E + D) and Fab2 (VH39E/VL38K + O).

[00183] FIG. 4 depicts the results of SEC and HIC analysis for tandem Fabs in the open configuration in WT, CH1/Ck, and CH1/Ck + CM formats.

[00184] FIG. 5A - FIG. 5D depict the results of HIC analysis, yield, and binding affinity for an anti-CD40 x anti-PD-L1 tandem Fab antibody in the open and closed configurations. FIG. 5A shows a closed configuration with CH1/Ck mutations only. FIG. 5B shows a closed configuration with Fab domain swap with CH1/Ck mutations only. FIG. 5C shows a closed configuration with CH1/Ck and CM mutations. FIG. 5D shows an open configuration with CH1/Ck and CM mutations.

[00185] FIG. 6A - FIG. 6C depict the results of HIC analysis, yield, and binding affinity for an anti-PD-1 x anti-OX40 tandem Fab antibody in the open and closed configurations. FIG. 6A shows a closed configuration with CH1/Ck mutations only. FIG. 6B shows an open configuration with CH1/Ck mutations only. FIG. 6C shows an open configuration with CH1/Ck and CM mutations.

[00186] FIG. 7A- FIG. 7B depict purity results for an anti-4-1 BB x anti-PD-L1 tandem Fab antibody (FIG. 7A) and an anti-4-1 BB x anti-PD-1 tandem Fab antibody (FIG. 7B). Both antibodies are in the open configuration with CH1/Ck and CM mutations.

[00187] FIG. 8A- FIG. 8B schematically depict Y shaped bispecific antibody formats with several different mutations to enhance heterodimerization. FIG. 8A shows CH1 /kappa mutations Ό” on Fab1 (CH1 : L143Q, S188V; Ck: V133T, S176V) and“D” on Fab2 (CH1 : T192E; Ck: N137K, S1 14A). FIG. 8B shows mutations on Fab1 (VH39E/VL38K + O) and Fab2 (VH39K/VL38E + D).

[00188] FIG. 9A - FIG. 9C depicts purity results for an anti-PD-1 x anti-OX40 antibody. NanoDSF (differential scanning fluorimetry) was used to measure thermal stability of the antibodies for T onset. HIC was used to measure purity. Wild-type antibody is shown in FIG. 9 A. CH1/Ck mutations (Fab1 = CH1 : L143Q, S188V; Ck: V133T, S176V) and (Fab2= CH1 : T192E; Ck: N137K, S1 14A) are shown in FIG. 9B. CH1/Ck and CM mutations (Fab1 = CH1 : L143Q, S188V; Ck: V133T, S176V; VH39E/VL38K) and (Fab2= CH1 : T192E; Ck: N137K, S1 14A; VH39K/VL38E) are shown in FIG. 9C.

[00189] FIG. 10A - FIG. 10L depicts SEC and HIC profiles for various anti-PD-1 x anti-0X40 antibodies with several different combinations of mutations. The specific mutations are recited in Table 9 below.

[00190] FIG. 11 A - FIG. 11 E depicts chain mispairing data for various Y-shaped antibodies. FIG. 11 A depicts mispairing data for anti-PD-1 x anti-GITR antibodies. FIG. 11 B depicts mispairing data for anti-TNF x anti-GITR antibodies. FIG. 11C depicts mispairing data for anti-TNF x anti-OX40 antibodies. FIG. 11 D depicts mispairing data for anti-CD40 x anti-PD-L1 antibodies. FIG. 11 E depicts mispairing data for anti-CD3 x anti-CD123 antibodies. The specific mutations and biophysical characterization data are recited in Table 11 below.

[00191] FIG. 12 depicts a cytotoxic assay of human panT cells against THP-1 target cells co-incubated with bispecific CD3 x CD123-hlgG1 -LALA molecules. T effector cells and CFSE-labeled THP-1 target cells were seeded in an effector to target ratio of 10:1 and co-incubated with serial dilutions of respective bispecific molecules (10nM - 0 nM) for 20h at 37°C. Dead cells were stained with 7-AAD and measured by flow cytometry. Cytotoxic activity was calculated based on percentage of dead THP-1 target cells (7-AAD/CFSE double positive). Data show dead target cells [%] against concentration of bispecific molecules [pM] as mean of two representative healthy donors.

DETAILED DESCRIPTION OF THE DISCLOSURE

I. DEFINITIONS

[00192] So that the disclosure may be more readily understood, selected terms are defined below.

[00193] Sequence position numbers used herein refer to Kabat numbering (Kabat, E.A. et al. , Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication no 91 -3242, pp 662,680,689, 1991 ).

[00194] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids; unnatural amino acids such as a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins described herein. Examples of unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, c-N,N,N-trimethyllysine, c-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, u-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention. Naturally occurring residues may be divided into classes based on common side chain properties (see Table 1 ).

Table 1 - Amino acid residues by classes.

[00195] Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid residues. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

[00196] As used herein, the term“mutation” or“mutated” refers to an alteration of the amino acid sequence by deletion, insertion and/or substitution of one or more amino acids. In particular, it refers to a substitution. A mutation is introduced with respect to a given sequence, e.g., the amino acid sequence of a VL1 and/or VH1 pair that specifically recognizes a first antigen.

[00197] As used herein, a“T192E (CH1 ) mutation” refers to the substitution of a threonine (T) residue for a glutamic acid (E) residue, in the immunoglobulin CH1 heavy chain constant domain of an antigen-binding protein, at the Kabat position 192.

[00198] As used herein, a“mutation set” refers to a group of different mutations present in a sequence.

[00199] As used herein, the term“variant” refers to an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence it is derived from. The determination of percent identity between two sequences is accomplished using the mathematical algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Alternatively, a variant can also be defined as having up to 20, 15, 10, 5, 4, 3, 2, or 1 amino acid substitutions, in particular conservative amino acid substitutions. Conservative substitutions are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company). An overview of physical and chemical properties of amino acids is given in Table 1 above. In a particular embodiment, conservative substitutions are substitutions made with amino acids having at least one property

according to Table 1 in common (i.e., of column 1 and/or 2). The term“variant” also includes fragments. A fragment has an N-terminal and/or C-terminal deletion of up to 20, 15, 10, 5, 4, 3, 2, or 1 amino acid(s) in total. In addition or alternatively, the variant may be modified, for example by N-terminal and/or C-terminal amino acid additions of up to 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 amino acid(s) in total.

[00200] As used herein, the term“antigen” or“target antigen” or“antigen target” refers to a molecule or a portion of a molecule (e.g., epitope) that is capable of being specifically bound by a binding protein described herein, and additionally is capable of being used in an animal to produce antibodies capable of specific binding to an epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[00201] As used herein, the term“epitope” refers to any determinant, e.g., a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or by an antigen-binding fragment of an antibody or by a binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is < 10 8 M, when the equilibrium dissociation constant is < T9 M, or when the dissociation constant is < 10 10 M.

[00202] As used herein, the term“antigen-binding protein” or“binding protein” or “binding polypeptide” refers to a polypeptide (e.g., an antibody or fragment thereof) that contains at least one binding site which is responsible for selectively binding to a target antigen of interest (e.g., a human antigen). Exemplary binding sites include, but are not limited to, an antibody variable domain, a ligand binding site of a receptor, or a receptor binding site of a ligand. In certain aspects, the binding polypeptides comprise multiple (e.g., two, three, four, or more) binding sites. In certain aspects, the binding protein is not a therapeutic enzyme.

[00203] As used herein a“heterodimerization domain” or“HD” refers to a subunit of a bispecific, trispecific or a multispecific binding protein that facilitates, directs or forces the correct assembly of light chains and their cognate heavy chains to result in the desired protein while preventing mispairing of the respective light or heavy chains.

[00204] As used herein, a“multispecific” binding protein is a binding protein that binds two or more antigens, and/or two or more different epitopes. A multispecific binding protein that binds two antigens, and/or two different epitopes, is also referred to herein as a“bispecific” binding protein. A multispecific binding protein that binds three antigens, and/or three different epitopes, is also referred to herein as a“trispecific” binding protein.

[00205] As used herein, the term“heterodimerizing Fc” or“functional fragment of a heterodimerizing Fc” refers to a mutant form of the constant domain, e.g., the CH2-CH3 or CH2-CH3-CH4, that is mutated with regard to a naturally occurring Fc part in that it no longer forms homodimers but forms a heterodimer with a correspondingly mutated Fc part. Thus, the term refers to one part of the two chains that form a heterodimer. Several of such pairs are known in the art and comprise, e.g., knob-in-hole (KIH) variants or EV-RWT variants.

[00206] Ridgeway and coworkers generated a CH3 interface favoring heterodimeric assembly by replacing small side chains on one CH3 interface with larger side chains to create a knob and replacing large side chains on the other CH3 domain with smaller side chains to generate a hole. Testing such variants demonstrated a preferential heterodimerization. The original knobs-into-holes mutations were further extended to identify further suitable combinations by phage display which were used to generate bispecific IgG antibodies testing additional substitutions allowing for disulfide bond formation. The knobs-in-hole variants are described further in U.S. Patent No. 5,732,168 and U.S. Patent No. 8,216,805, which are herein incorporated by reference. Accordingly, in an embodiment, the CH3 domain of one Fc domain or heterodimerization domain contains the mutations Y349C, T366S, L368A, and Y407V, and the CH3 domain of another FC domain or heterodimerization domain contains the mutations S354C and T366W (amino acid position being indicated by reference to an lgG1 sequence).

[00207] As used herein, the term“homodimerization domain” refers to a domain mediating the homodimerization of to like domains, e.g., two heavy chains. Heavy chain pairing is mediated by the last domain of the constant region, i.e. , CH3 in IgG molecules, which forms high-affinity homodimer complexes (KD of approximately 10 pM). Further interactions reside in the hinge region responsible for covalent linkage of two heavy chains, which form after heavy chain assembly. Interaction in a CH3 homodimer involves approximately 16 residues at the CH3-CH3 interface as shown for human y1 CH3 with patch formed by 6 residues (T366, L368, F405, Y407 and K409) at the center of the interface strongly contributing to stability. Homodimerization domains include, but are not limited to, Fc regions and effector modified variants thereof and fragments of either, CH2 domains or fragments thereof, CH3 domains or fragments thereof, CH4 domains or fragments or the like.

[00208] Naturally-occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length“light” chain (typically having a molecular weight of about 25 kDa) and one full-length“heavy” chain (typically having a molecular weight of about 50-70 kDa). The terms “heavy chain” and “light chain,” as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 1 10 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain IgG immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CH1 , CH2, and CH3), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.

[00209] In some embodiments, the multispecific antigen-binding proteins of the disclosure comprise one or more VH domains from any one of the VH domain sequences recited in Tables 2, 3, and 4. In some embodiments, the multispecific antigen-binding

proteins of the disclosure comprise one or more VL domains from any one of the VL domain sequences recited in Tables 2, 3, and 4. In some embodiments, the multispecific antigen-binding proteins of the disclosure comprise one or more VH domains from any one of the VH domain sequences recited in Tables 2, 3, and 4, paired with one or more VL domains from any one of the VL domain sequences recited in Tables 2, 3, and 4.

[00210] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, lgG1 , lgG2, lgG3, and lgG4. IgM has subclasses including, but not limited to, lgM1 and lgM2. IgA is similarly subdivided into subclasses including, but not limited to, lgA1 and lgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a“J” region of about 12 or more amino acids, with the heavy chain also including a“D” region of about 10 more amino acids. See, e.g., Fundamental Immunology (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4.

CLAIMS

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

wherein at least one CH1/CL pair comprises CH1/CL mutations to facilitate pairing selected from the group consisting of one or more of

(1 ) a T192E (CH1 ) mutation and N137K and S1 14A (CL) mutations, and

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

(3) T192E, L143Q and S188V (CH1 ) mutations and N137K, S1 14A, V133T and S176V (CL) mutations,

(4) a K221 E (CH1 ) mutation and a E123K (CL) mutation,

(5) T192E and K221 E (CH1 ) mutations and N137K, S1 14A and E123K (CL) mutations,

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

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

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

(9) K221 E and K228D (CH1 ) mutations and D122K and E123K (CL) mutations, wherein when two CH1/CL pairs comprise mutations to facilitate pairing for two different VH/VL pairs, the two CH1/CL pairs do not comprise same mutations, and

wherein at least one VH/VL pair comprise opposite charged mutations to facilitate pairing, said opposite charged mutations comprising (1 ) a mutated residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutated residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, and

wherein the mutated residue in the VH region has an opposite charge from the mutated residue in the VL region.

2. A multispecific antigen-binding protein comprising:

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising:

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

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

(3) a first heterodimerization domain (HD1 ); and

b) a second light chain (LC2)/heavy chain (HC2) pair comprising:

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

(5) a second constant heavy chain region 1 (CH1 -2) operatively linked to VH2 and a second constant light chain region (CL2) operatively linked to VL2; and

(6) a second heterodimerization domain (HD2);

wherein HD1 and HD2 heterodimerize,

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

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

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

3. A multispecific antigen-binding protein of claims 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

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 the polypeptide of formula I and the polypeptide of formula II form a cross over light chain-heavy chain pair,

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

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

wherein the mutated residue in the VH region has an opposite charge from the mutated residue in the VL region, and

wherein the CH1 and CL domain pair comprise mutations that facilitate pairing.

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

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

and two polypeptide chains each 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

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

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

wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair,

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

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

wherein one or both of the CH1 and CL domain pair comprises mutations that facilitate pairing, and

wherein when at least two CH1/CL pairs comprise mutations to facilitate pairing, then the mutation set in one CH1/CL pair is different from the mutation set in the other CH1/CL pair.

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

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

VL2-L1 -VL1 -L2-CL1 [I],

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

VH 1 -L3-VH2-L4-CH 1 -1 -hinge-CH2-CH3 [II], a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1 -2-hinge-CH2-CH3 [III], and

a 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;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and

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

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

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

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

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

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

7. A multispecific antigen-binding protein comprising

a) a first light chain (LC1 )/heavy chain (HC1 ) pair comprising:

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

(2) a first constant heavy chain region 1 (CH1 -1 ) operatively linked to VH1 and a first constant light chain region (CL1 ) operatively linked to VL1 , and b) a second light chain (LC2)/heavy chain (HC2) pair comprising:

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

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

wherein the C terminus of CH1 -1 is operatively linked to the N terminus of VH2, wherein one or more cysteine residues are engineered into the one or both of VH1/VL1 and VH2/VL2 pairs to form one or more disulfide bonds,

wherein one or both of the VL1 and VH1 pair and the VL2 and VH2 pair comprise opposite charged mutations that facilitate pairing, said opposite charged mutations

comprising (1 ) a mutated residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutated residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, and wherein the mutated residue in the VH region has an opposite charge from the mutated residue in the VL region,

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

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

8. A multispecific antigen-binding protein of any of claims 2 to 7, wherein CH1 comprises a T192E mutation and CL comprises N137K and S1 14A mutations.

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

10. A multispecific antigen-binding protein of any of claims 2 to 9, wherein CH1 comprises T192E, L143Q and S188V mutations and CL comprises N137K, S1 14A, V133T and S176V mutations.

1 1. A multispecific antigen-binding protein of any of claims 2 to 10, wherein CH1 comprises a K221 E mutation and CL comprises a E123K mutation.

12. A multispecific antigen-binding protein of any of claims 2 to 1 1 , wherein CH1 comprises a K228D mutation and CL comprises a D122K mutation.

13. A multispecific antigen-binding protein of any of claims 2 to 12, wherein CH1 comprises K221 E and K228D mutations and CL comprises D122K and E123K mutations.

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

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

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

17. A multispecific antigen-binding protein of any of claims 2 to 16, wherein at least one CH1/CL pair comprises CH1/CL mutations to facilitate pairing selected from the group consisting of one or more of:

(1 ) a T192E (CH1 ) mutation and N137K and S1 14A (CL) mutations,

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

(3) T192E, L143Q and S188V (CH1 ) mutations and N137K, S1 14A, V133T and S176V (CL) mutations,

(4) a K221 E (CH1 ) mutation and a E123K (CL) mutation,

(5) T192E and K221 E (CH1 ) mutations and N137K, S1 14A and E123K (CL) mutations,

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

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

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

(9) K221 E and K228D (CH1 ) mutations and D122K and E123K (CL) mutations, wherein when at least two CH1/CL pairs comprise a mutation set to facilitate pairing for two different VH/VL pairs, then the at least two CH1/CL pairs do not comprise same mutations, and

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

a) a L143E, a L143D, a L143K, a L143R, or a L143H mutation in the CH1 region and a S176E, a S176D, a S176K, a S176R, or a S176H mutation in the CL region; and b) a L124E, a L124D, a L124K, a L124R, or a L124H mutation in the CH1 region and a V133E, a V133D, a V133K, a V133R, or a V133H mutation in the CL region, wherein the mutated residue in the CH1 region has an opposite charge from the mutated residue in the CL region.

19. The antigen-binding protein of claim 18, further comprising CH1/CL mutations to facilitate pairing selected from the group consisting of one or more of:

(1 ) a T192E (CH1 ) mutation and N137K and S1 14A (CL) mutations,

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

(3) T192E, L143Q and S188V (CH1 ) mutations and N137K, S1 14A, V133T and S176V (CL) mutations,

(4) a K221 E (CH1 ) mutation and a E123K (CL) mutation,

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

(6) K221 E and K228D (CH1 ) mutations and D122K and E123K (CL) mutations, wherein when two CH1/CL pairs comprise mutations to facilitate pairing for two different VH/VL pairs, the two CH1/CL pairs do not comprise same mutations.

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

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

b) a K221 E and a K228D mutation in the CH1 region and a D122K and a E123K mutation in the CL region,

wherein the mutated residue in the CH1 region has an opposite charge from the mutated residue in the CL region.

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

23. A multispecific antigen-binding protein of any 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. A multispecific antigen-binding protein of claim 23, wherein one or both VH regions comprise one or both of 44C and 105C mutations, and one or both VL regions comprise one or both of 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 the one or more nucleic acid molecules of claim 25.

27. An isolated host cell comprising the one or more nucleic acid molecules of claim 25 or the one or more expression vectors of 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 antigen activity is detrimental, the method comprising administering to a subject in need thereof an effective amount of a multispecific antigen-binding protein of any one claims 1 -24.

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