Multispecific binding proteins containing a mutant fab domain

Abstract

Binding proteins are provided that include a VL region paired with a VH region and a CH1 region paired with a CL region, wherein the VL and VH regions contain oppositely charged mutations to promote pairing, and the CH1 and CL regions contain mutations to promote pairing. Also provided are binding proteins that include one or more cysteine ​​residues incorporated into the VH / VL pair to form one or more disulfide bonds. Multispecific binding proteins, nucleic acids encoding the binding proteins and multispecific binding proteins, expression vectors, host cells, pharmaceutical compositions, and therapeutic methods of administering the binding proteins or multispecific binding proteins described herein are also provided.

Description

[Technical Field]

[0001] Related applications This application claims priority to European Application No. 18306843.6 filed on 24 December 2018 and EP Application No. 19305812.0 filed on 21 June 2019, the contents of which are incorporated herein by reference for all purposes. [Background technology]

[0002] Classical antibodies are Y-shaped proteins or immunoglobulins, which are heterotetramers formed from two heterodimers consisting of a light chain and a heavy chain. The "arms" of the antibody contain the antigen-binding site and are called Fab regions. Classical antibodies (e.g., antibodies produced by the host immune system) have two identical Fab regions and can recognize and bind to specific antigens. The generation of asymmetry in native antibody structures is a prerequisite for the creation of multispecific binding proteins with two (e.g., bispecific antibodies) or more binding specificities. For example, by separating one or more Fv on different asymmetric binding arms or Fab regions, it is possible to create bispecific antibodies that have the flexibility to bind two different antigens simultaneously. However, despite these advantages, a wide variety of multispecific antibody technologies suffer from process and manufacturing problems due to various asymmetric heavy and light chain mispairings. For example, many of these technologies suffer from the so-called "light chain problem," where random pairing of two different light and heavy chains produces chain pairings of various combinations other than the desired combination. In some cases, the light chain problem can be avoided by using a common light chain that allows binding to both antigens. However, this format may not be feasible for many antibodies because it requires the generation or display of de novo antibodies in transgenic mice. Furthermore, rare antibodies, such as broadly neutralizing anti-HIV antibodies derived from human patients, cannot be adapted to such a format. Therefore, alternative and creative solutions to the mispairing problem are still needed. A series of mutations at the dimer interface have been carefully designed to enable heterodimerization of these antibodies. [Overview of the project] [Problems that the invention aims to solve]

[0003] This disclosure provides an antigen-binding protein that may optionally contain various mutations along the dimer interface, which may enable efficient heterodimerization of the antigen-binding protein. [Means for solving the problem]

[0004] In one aspect, this disclosure is as follows: The VL region paired with the VH region for forming an antigen-binding site; and CH1 region opposite the CL region We provide antigen-binding proteins that include, The VL and VH regions contain opposite-charged mutations to promote pairing, while the CH1 and CL regions contain mutations to promote pairing.

[0005] 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.

[0006] In some embodiments, one or both of the first VH and VL pairs and the second VH and VL pairs include an anti-charge mutation to promote pairing, and one or both of the first CH1 and CL pairs and the second CH1 and CL pairs include a mutation to promote pairing.

[0007] In some embodiments, one or both CH1 regions contain the T192E mutation, and one or both CL regions contain the N137K and S114A mutations.

[0008] In some embodiments, one or both CH1 regions contain L143Q and S188V mutations, and one or both CL regions contain V133T and S176V mutations.

[0009] In some embodiments, one or both CH1 regions contain T192E, L143Q, and S188V mutations, and one or both CL regions contain N137K, S114A, V133T, and S176V mutations.

[0010] In some embodiments, one or both CH1 regions contain L143E, L143D, L143K, L143R, or L143H mutations, and one or both CL regions contain S176E, S176D, S176K, S176R, or S176H mutations, where the mutations in CH1 are oppositely charged to the mutations in CL.

[0011] In some embodiments, one or both CH1 regions contain L124E, L124D, L124K, L124R, or L124H mutations, and one or both CL regions contain V133E, V133D, V133K, V133R, or V133H mutations, where the mutations in CH1 are oppositely charged to the mutations in CL.

[0012] In some embodiments, one or both VH regions contain 39E, 39D, 39K, 39R, or 39H mutations, and one or both VL regions contain 38E, 38D, 38K, 38R, or Q38H mutations, where the mutations in VH are oppositely charged to the mutations in VL.

[0013] In some embodiments, one or both VH regions contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and one or both VL regions contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations, where the mutations in VH are oppositely charged to the mutations in VL.

[0014] In some embodiments, the antigen-binding protein further comprises one or more cysteine ​​residues incorporated into one or both of the VH / VL pair to form one or more disulfide bonds.

[0015] In some embodiments, one or both VH regions contain one or both of the 44C and 105C mutations, and one or both VL regions contain one or both of the 100C and 43C mutations.

[0016] In some embodiments, one or both of the VH regions contain a 44C mutation, and one or both of the VL regions contain a 100C mutation.

[0017] In some embodiments, one or both of the VH regions contain a 105C mutation, and one or both of the VL regions contain a 43C mutation.

[0018] In some embodiments, the antigen-binding protein further contains charge-reversal mutations in one or both of the CH1 / CL pairs.

[0019] In some embodiments, the charge-reversal mutations in one or both of the CH1 / CL pairs are selected from the group consisting of K221E in the CH1 region and E123K in the CL region; K228D in the CH1 region and D122K in the CL region; L145E in the CH1 region and S176K in the CL region; and L128E in the CH1 region and V133K in the CL region.

[0020] In another aspect, the present disclosure provides a multispecific antigen-binding protein comprising at least two VH regions for forming at least two antigen-binding sites and at least two VL regions paired with the at least two VH regions respectively, and at least two CH1 regions paired with the two CL regions respectively, wherein at least one CH1 / CL pair is as follows: (1) a T192E (CH1) mutation and N137K and S114A (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, S114A, V133T and S176V (CL) mutations, and / or (4) a K221E (CH1) mutation and an E123K (CL) mutation, and / or (5) T192E and K221E(CH1) mutations, as well as N137K, S114A and E123K(CL) mutations, and / or (6) L143E, L143D, L143K, L143R or L143H(CH1) mutations, and S176E, S176D, S176K, S176R or S176H(CL) mutations, and / or (7) L124E, L124D, L124K, L124R or L124H(CH1) mutations, and V133E, V133D, V133K, V133R or V133H(CL) mutations, and (8) If two CH1 / CL pairs contain mutations that facilitate pairing of two different VH / VL pairs, then the two CH1 / CL pairs do not contain the same mutations. Includes CH1 / CL mutations to promote pairing selected from, At least one VH / VL pair includes an opposite-charged mutation to facilitate pairing, the opposite-charged mutation comprising (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, wherein the mutant residue in the VH region has the opposite charge to the mutant residue in the VL region.

[0021] In another embodiment, the present disclosure provides a multispecific antigen-binding protein comprising at least two VL regions paired with at least two VH regions to form at least two antigen-binding sites, and at least two CH regions paired with two CL regions, respectively. At least one CH1 / CL pair is: (1) T192E(CH1) mutation, as well as N137K and S114A(CL) mutations, (2) L143Q and S188V(CH1) mutations, and V133T and S176V(CL) mutations, (3) T192E, L143Q and S188V(CH1) mutations, as well as N137K, S114A, V133T and S176V(CL) mutations, (4) K221E(CH1) mutation and E123K(CL) mutation, (5) T192E and K221E(CH1) mutations, as well as N137K, S114A and E123K(CL) mutations, (6) L143E, L143D, L143K, L143R or L143H(CH1) mutations, and S176E, S176D, S176K, S176R or S176H(CL) mutations, (7) L124E, L124D, L124K, L124R or L124H(CH1) mutations, and V133E, V133D, V133K, V133R or V133H(CL) mutations, (8) K228D(CH1) mutation and D122K(CL) mutation, and (9) K221E and K228D(CH1) mutations, and D122K and E123K(CL) mutations Includes CH1 / CL mutations to promote pairing selected from one or more of the following groups, If two CH1 / CL pairs contain mutations that facilitate pairing for two different VH / VL pairs, then the two CH1 / CL pairs do not contain the same mutations. At least one VH / VL pair contains an anti-charged mutation to facilitate pairing, the anti-charged mutation comprising (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H. Mutant residues in the VH region have the opposite charge to those in the VL region.

[0022] In some embodiments, if a multispecific antigen-binding protein contains at least two different sets of mutations to facilitate pairing, then at least two different VH / VL pairs do not contain the same set of mutations.

[0023] In some embodiments, the multispecific antigen-binding protein comprises at least two VL regions paired with at least two VH regions to form at least two antigen-binding sites, and at least two CH1 regions paired with at least two CL regions, At least one CH1 / CL pair is: (1) T192E(CH1) mutations, as well as N137K and S114A(CL) mutations, and / or (2) L143Q and S188V(CH1) mutations, as well as V133T and S176V(CL) mutations, and / or (3) T192E, L143Q and S188V(CH1) mutations, as well as N137K, S114A, V133T and S176V(CL) mutations, and / or (4) K221E(CH1) mutations and E123K(CL) mutations, and / or (5) L143E, L143D, L143K, L143R or L143H(CH1) mutations, and S176E, S176D, S176K, S176R or S176H(CL) mutations, and / or (6) L124E, L124D, L124K, L124R or L124H(CH1) mutations, and V133E, V133D, V133K, V133R or V133H(CL) mutations, and (7) If two CH1 / CL pairs contain mutations that facilitate pairing of two different VH / VL pairs, then the two CH1 / CL pairs do not contain the same mutations. Includes CH1 / CL mutations to promote pairing selected from one or more of the following groups, At least one VH / VL pair contains an anti-charged mutation to facilitate pairing selected from the 39E, Q39D, Q39K, Q39R, or Q39H mutations and the Q38E, Q38D, Q38K, Q38R, or Q38H mutations, wherein the mutation in VH is anti-charged compared to the mutation in VL; if several VH / VL pairs contain mutations to facilitate pairing of different VH / VL pairs where VL or VH are not in the same polypeptide chain, each VH / VL pair does not contain the same anti-charged mutation.

[0024] In some embodiments, one or both CH1 regions are operably linked to a heterodimerization domain.

[0025] In some embodiments, the heterodimerization domain includes a first Fc domain.

[0026] In some embodiments, the first Fc domain heterodimerizes with the second Fc domain, the first Fc domain includes a first CH3 region, and the second Fc domain includes a second CH3 region.

[0027] In some embodiments, the first CH3 region contains one or both of the S354C and T366W mutations, and the second CH3 region contains one or more of the Y349C, T366S, L368A, and Y407V mutations, the mutations promoting Fc domain heterodimerization.

[0028] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) First steady heavy chain region 1 (CH1-1) and first steady light chain region (CL1); and (3) First heterodimerization domain (HD1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) Second steady heavy chain region 1 (CH1-2) and second steady light chain region (CL2); and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including We provide multispecific antibodies including, HD1 and HD2 heterodimerize, At least one or both of the VL1 and VH1 pairs, and the VL2 and VH2 pairs, contain opposite-charged mutations to facilitate pairing. At least one or both of the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations to promote pairing. If both the CL1 and CH1-1 pair, and the CL2 and CH1-2 pair, contain mutations that promote pairing, then the mutations in CH1-1 and CL1 that promote pairing are different from the mutations in CH1-2 and CL2 that promote pairing.

[0029] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including We provide multispecific antibodies including, HD1 and HD2 heterodimerize, At least one or both of the VL1 and VH1 pairs, and the VL2 and VH2 pairs, contain opposite-charged mutations to facilitate pairing. At least one or both of the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations to promote pairing. If both the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations that promote pairing, then the mutations in CH1-1 and CL1 that promote pairing are different from the mutations in CH1-2 and CL2 that promote pairing.

[0030] In another aspect, this disclosure is: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including We provide a multispecific antigen-binding protein containing, HD1 and HD2 heterodimerize, At least one or both of the VL1 and VH1 pairs, and the VL2 and VH2 pairs, include an opposite-charged mutation to facilitate pairing, wherein the opposite-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, wherein the mutant residue in the VH region has the opposite charge to the mutant residue in the VL region. At least one or both of the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations that promote pairing. If both the CL1 and CH1-1 pair, and the CL2 and CH1-2 pair, contain mutations that promote pairing, then the mutations in CH1-1 and CL1 that promote pairing are different from the mutations in CH1-2 and CL2 that promote pairing.

[0031] In some embodiments, CH1-1 includes the T192E mutation, and CL1 includes the N137K and S114A mutations.

[0032] In some embodiments, CH1-1 includes L143Q and S188V mutations, and CL1 includes V133T and S176V mutations.

[0033] In some embodiments, CH1-1 includes T192E, L143Q, and S188V mutations, and CL1 includes N137K, S114A, V133T, and S176V mutations.

[0034] In some embodiments, CH1-2 includes the T192E mutation, and CL2 includes the N137K and S114A mutations.

[0035] In some embodiments, CH1-2 includes L143Q and S188V mutations, and CL2 includes V133T and S176V mutations.

[0036] In some embodiments, CH1-2 includes T192E, L143Q, and S188V mutations, and CL2 includes N137K, S114A, V133T, and S176V mutations.

[0037] In some embodiments, one or both of CH1-1 and CH1-2 contain L143E, L143D, L143K, L143R, or L143H mutations, one or both of CL1 and CL2 contain S176E, S176D, S176K, S176R, or S176H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0038] In some embodiments, one or both of CH1-1 and CH1-2 contain L124E, L124D, L124K, L124R, or L124H mutations, one or both of CL1 and CL2 contain V133E, V133D, V133K, V133R, or V133H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0039] In some embodiments, one or both of VH1 and VH2 include a 39E, 39D, 39K, 39R, or 39H mutation, and one or both of VL1 and VL2 include a 38E, 38D, 38K, 38R, or 38H mutation, and the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL2.

[0040] In some embodiments, one or both of VH1 and VH2 include Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and one or both of VL1 and VL2 include Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in one or both of VH1 and VH2 are oppositely charged to the mutations in one or both of VL1 and VL2.

[0041] In some embodiments, the multispecific antibody further comprises one or more cysteine ​​residues incorporated into one or both of the VH1 / VL1 and VH2 / VL2 pairs to form one or more disulfide bonds.

[0042] In some embodiments, one or both of VH1 and VH2 include 44C and 105C mutations, and one or both of VL1 and VL2 include 100C and 43C mutations.

[0043] In some embodiments, VH1 includes the 44C mutation, and VL1 includes the 100C mutation.

[0044] In some embodiments, VH1 includes the 105C mutation, and VL1 includes the 43C mutation.

[0045] In some embodiments, VH2 includes the 44C mutation, and VL2 includes the 100C mutation.

[0046] In some embodiments, VH2 includes the 105C mutation, and VL2 includes the 43C mutation.

[0047] In some embodiments, VH1 includes 39E and 44C mutations, and VL1 includes 38K and 100C mutations.

[0048] In some embodiments, VH1 includes 39E and 105C mutations, and VL1 includes 38K and 43C mutations.

[0049] In some embodiments, the multispecific antibody further includes an anti-charge mutation in the CH1-1 / CL1 pair.

[0050] In some embodiments, the opposite charge mutation in the CH1-1 / CL1 pair is selected from the group consisting of K221E in the CH1-1 region and E123K in the CL1 region; K228D in the CH1-1 region and D122K in the CL1 region; L145E in the CH1-1 region and S176K in the CL1 region; and L128E in the CH1-1 region and V133K in the CL1 region.

[0051] In some embodiments, the multispecific antibody further includes opposite charge mutations in the CH1-2 / CL2 pair.

[0052] In some embodiments, the opposite charge mutations in the CH1-2 / CL2 pair are selected from the group consisting of K221E in the CH1-2 region and E123K in the CL2 region; K228D in the CH1-2 region and D122K in the CL2 region; L145E in the CH1-2 region and S176K in the CL2 region; and L128E in the CH1-2 region and V133K in the CL2 region.

[0053] In some embodiments, the first and second heterodimerization domains include an Fc domain.

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

[0055] In some embodiments, the CH1-1 domain is linked to the first CH2 and first CH3 domains, the CH1-2 domain is linked to the second CH2 and second CH3 domains, and the first CH2 and CH3 domains, as well as the second CH2 and CH3 domains, dimerize to form an Fc domain.

[0056] In some embodiments, the first CH3 domain contains one or both of the S354C and T366W mutations, and the second CH3 domain contains one or more of the Y349C, T366S, L368A, and Y407V mutations, the mutations promoting heterodimerization of the Fc domain.

[0057] In another aspect, the 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, One polypeptide chain has the formula: VL1-L1-VL2-L2-CL[I] The structure includes the form represented by , and one polypeptide chain is given by formula: VH2-L3-VH1-L4-CH1[II] Includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; and L1, L2, L3, and L4 are amino acid linkers. The polypeptides of formula I and formula II form crossed light-heavy chain pairs. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 and VH2 / VL2 pairs to form one or more disulfide bonds. At least one or both of the VL1 and VH1 pairs, and the VL2 and VH2 pairs, contain opposite-charged mutations to facilitate pairing. The CH1 and CL domain pairs contain mutations that facilitate pairing.

[0058] In some embodiments, VH1 is paired with VL1, VH2 is paired with VL2, and CH1 is paired with CL.

[0059] In some embodiments, the disclosure provides a multispecific antigen-binding protein comprising at least two polypeptide chains and forming at least two antigen-binding sites, one polypeptide chain having the formula: VL1-L1-VL2-L2-CL[I] The structure includes the form represented by , and one polypeptide chain is given by formula: VH2-L3-VH1-L4-CH1[II] Includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; and L1, L2, L3, and L4 are amino acid linkers. VH1 pairs with VL1, VH2 pairs with VL2, and CH1 pairs with CL. The polypeptides of formula I and formula II form crossed light-heavy chain pairs. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds. At least one or both of the VL1 and VH1 pairs, and the VL2 and VH2 pairs, contain an opposite-charged mutation to facilitate pairing, wherein the opposite-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, the mutant residue in the VH region having the opposite charge to the mutant residue in the VL region. The CH1 and CL domain pairs contain mutations that facilitate pairing.

[0060] In some embodiments, one or both of VH1 and VH2 include VH44C and VH105C mutations.

[0061] In some embodiments, one or both of VL1 and VL2 include the VL43C and VL100C mutations.

[0062] In some embodiments, one or both of VH1 and VH2 contain the VH44C mutation, and one or both of VL1 and VL2 contain the VL100C mutation.

[0063] In some embodiments, one or both of VH1 and VH2 contain the VH105C mutation, and one or both of VL1 and VL2 contain the VL43C mutation.

[0064] In some embodiments, CH1 includes the T192E mutation, and CL includes the N137K and S114A mutations.

[0065] In some embodiments, CH1 includes L143Q and S188V mutations, and CL includes V133T and S176V mutations.

[0066] In some embodiments, CH1 includes T192E, L143Q, and S188V mutations, and CL includes N137K, S114A, V133T, and S176V mutations.

[0067] In some embodiments, CH1 includes L143E, L143D, L143K, L143R, or L143H mutations, and CL includes S176E, S176D, S176K, S176R, or S176H mutations, where the mutations in CH1 are oppositely charged to the mutations in CL.

[0068] In some embodiments, CH1 includes L124E, L124D, L124K, L124R, or L124H mutations, and CL includes V133E, V133D, V133K, V133R, or V133H mutations, where the mutations in CH1 are oppositely charged to the mutations in CL.

[0069] In some embodiments, one or both of VH1 and VH2 include a 39E, 39D, 39K, 39R, or 39H mutation, and one or both of VL1 and VL2 include a 38E, 38D, 38K, 38R, or 38H mutation, and the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL2.

[0070] In some embodiments, one or both of VH1 and VH2 include Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and one or both of VL1 and VL2 include Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in one or both of VH1 and VH2 are oppositely charged to the mutations in one or both of VL1 and VL2.

[0071] In some embodiments, CH1 is operably linked to the dimerization domain.

[0072] In some embodiments, the dimerization domain is an Fc domain containing a CH2 domain and a CH3 domain.

[0073] In another aspect, the disclosure provides a multispecific antigen-binding protein comprising four polypeptide chains forming four antigen-binding sites, where two polypeptide chains each have the formula: VL1-L1-VL2-L2-CL[I] The structure includes the form represented by , and the two polypeptide chains are each represented by formula: VH2-L3-VH1-L4-CH1-Fc[II] Includes a structure represented by, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; Fc includes an immunoglobulin hinge region and constant domains of the CH2 and CH3 immunoglobulin heavy chains; L1, L2, L3, and L4 are amino acid linkers. VH1 pairs with VL1 to form the first antigen-binding site, VH2 pairs with VL2 to form the second antigen-binding site, and CH1 pairs with CL. The polypeptides of formula I and formula II form crossed light-heavy chain pairs. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 and VH2 / VL2 pairs to form one or more disulfide bonds. One or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs contain an opposite-charged mutation that promotes pairing, and the opposite-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, wherein the mutant residue in the VH region has the opposite charge to the mutant residue in the VL region. One or both of the CH1 and CL domain pairs contain mutations to facilitate pairing. If at least two CH1 / CL pairs contain mutations that facilitate pairing, the set of mutations in one CH1 / CL pair is different from the set of mutations in the other CH1 / CL pair.

[0074] In another embodiment, the disclosure provides an antigen-binding protein comprising four polypeptide chains forming four antigen-binding sites, where two polypeptide chains each have the formula: VL1-L1-VL2-L2-CL[I] The structure includes the form represented by the formula, and the two polypeptide chains are each: VH2-L3-VH1-L4-CH1-Fc[II] Includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; Fc comprises an immunoglobulin hinge region, as well as the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, and L4 are amino acid linkers. The polypeptides of formula I and formula II form crossed light-heavy chain pairs. VH1 pairs with VL1 to form a first antigen-binding site, VH2 pairs with VL2 to form a second antigen-binding site, CH1 pairs with CL, more specifically the VH1 / VL1 pair has a first antigen-binding specificity, and the VH2 / VL2 pair has a second antigen-binding specificity. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds. One or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs contain opposite-charged mutations to facilitate pairing. One or both of the CH1 and CL domain pairs contain mutations to facilitate pairing. If both CH1 and CL pairs contain mutations that promote pairing, the mutations in one CH1 and CL pair are different from the mutations in the other CH1 and CL pair that promote pairing.

[0075] In some embodiments, one or both of VH1 and VH2 include VH44C and VH105C mutations.

[0076] In some embodiments, one or both of VL1 and VL2 include the VL43C and VL100C mutations.

[0077] In some embodiments, one or both of VH1 and VH2 contain the VH44C mutation, and one or both of VL1 and VL2 contain the VL100C mutation.

[0078] In some embodiments, one or both of VH1 and VH2 contain the VH105C mutation, and one or both of VL1 and VL2 contain the VL43C mutation.

[0079] In some embodiments, CH1 includes the T192E mutation, and CL includes the N137K and S114A mutations.

[0080] In some embodiments, CH1 includes L143Q and S188V mutations, and CL includes V133T and S176V mutations.

[0081] In some embodiments, CH1 includes T192E, L143Q, and S188V mutations, and CL includes N137K, S114A, V133T, and S176V mutations.

[0082] In some embodiments, CH1 includes L143E, L143D, L143K, L143R, or L143H mutations, and CL includes S176E, S176D, S176K, S176R, or S176H mutations, where the mutations in CH1 are oppositely charged to the mutations in CL.

[0083] In some embodiments, CH1 includes L124E, L124D, L124K, L124R, or L124H mutations, and CL includes V133E, V133D, V133K, V133R, or V133H mutations, where the mutations in CH1 are oppositely charged to the mutations in CL.

[0084] In some embodiments, one or both of VH1 and VH2 contain 39E, 39D, 39K, 39R, or 39H mutations, and one or both of VL1 and VL2 contain 38E, 38D, 38K, 38R, or 38H mutations, and the mutations in one or both of VH1 and VH2 are oppositely charged to the mutations in one or both of VL1 and VL2.

[0085] In some embodiments, one or both of VH1 and VH2 contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and one or both of VL1 and VL2 contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in one or both of VH1 and VH2 are oppositely charged to the mutations in one or both of VL1 and VL2.

[0086] In another aspect, the disclosure provides a multispecific antigen-binding protein comprising four polypeptide chains that form three antigen-binding sites. The first polypeptide chain is given by formula: VL2-L1-VL1-L2-CL1[I] Includes a structure represented by, The second polypeptide chain is given by formula: VH1-L3-VH2-L4-CH1-1-Hinge-CH2-CH3[II] Includes a structure represented by, The third polypeptide chain is given by formula: VH3-CH1-2-Hinge-CH2-CH3[III] Includes a structure represented by, The fourth polypeptide chain is given by formula: VL3-CL2[IV] Includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; CL1 is the first constant domain of the immunoglobulin light chain; CL2 is the second constant domain of the immunoglobulin light chain; CH1-1 is the first constant domain of the immunoglobulin CH1 heavy chain; CH1-2 is the second constant domain of the immunoglobulin CH1 heavy chain; CH2 is the constant domain of the immunoglobulin CH2 double chain; CH3 is the constant domain of the immunoglobulin CH3 heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 domain and the CH2 domain. L1, L2, L3, and L4 are amino acid linkers. The polypeptides of formula I and formula II form crossed light-heavy chain pairs. One or more cysteine ​​residues are incorporated into one or more of the VH1 / VL1 pairs, VH2 / VL2 pairs, and VH3 / VL3 pairs to form one or more disulfide bonds. One or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs contain an anti-charged mutation to facilitate pairing, the anti-charged mutation comprising (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H. Mutant residues in the VH region have the opposite charge to those in the VL region. One or both of the CL1 and CH1-1 pairs and the CL2 and CH1-2 pairs contain mutations to promote pairing. If both the CL1 and CH1-1 pair, and the CL2 and CH1-2 pair, contain mutations that promote pairing, the mutations in CH1-1 and CL1 are different from the mutations in CH1-2 and CL2.

[0087] In some embodiments, one or both of VH1 and VH2 include VH44C and VH105C mutations.

[0088] In some embodiments, one or both of VL1 and VL2 include the VL43C and VH100C mutations.

[0089] In some embodiments, one or both of VH1 and VH2 contain the VH44C mutation, and one or both of VL1 and VL2 contain the VL100C mutation.

[0090] In some embodiments, one or both of VH1 and VH2 contain the VH105C mutation, and one or both of VL1 and VL2 contain the VL43C mutation.

[0091] In some embodiments, one or both of CH1-1 and CL1 include mutations to promote pairing, and one or both of CH1-2 and CL2 include mutations to promote pairing.

[0092] In some embodiments, CH1-1 includes the T192E mutation, and CL1 includes the N137K and S114A mutations.

[0093] In some embodiments, CH1-1 includes L143Q and S188V mutations, and CL1 includes V133T and S176V mutations.

[0094] In some embodiments, CH1-1 includes T192E, L143Q, and S188V mutations, and CL1 includes N137K, S114A, V133T, and S176V mutations.

[0095] In some embodiments, CH1-2 includes the T192E mutation, and CL2 includes the N137K and S114A mutations.

[0096] In some embodiments, CH1-2 includes L143Q and S188V mutations, and CL2 includes V133T and S176V mutations.

[0097] In some embodiments, CH1-2 includes T192E, L143Q, and S188V mutations, and CL2 includes N137K, S114A, V133T, and S176V mutations.

[0098] In some embodiments, one or both of CH1-1 and CH1-2 contain L143E, L143D, L143K, L143R, or L143H mutations, one or both of CL1 and CL2 contain S176E, S176D, S176K, S176R, or S176H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0099] In some embodiments, one or both of CH1-1 and CH1-2 contain L124E, L124D, L124K, L124R, or L124H mutations, one or both of CL1 and CL2 contain V133E, V133D, V133K, V133R, or V133H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0100] In some embodiments, one or more of VH1, VH2, and VH3 include a 39E, 39D, 39K, 39R, or 39H mutation, one or more of VL1, VL2, and VL3 include a 38E, 38D, 38K, 38R, or 38H mutation, and the mutation in one or more of VH1, VH2, and VH3 is oppositely charged to the mutation in one or more of VL1, VL2, and VL3.

[0101] In some embodiments, one or more of VH1, VH2, and VH3 include Q39E, Q39D, Q39K, Q39R, or Q39H mutations, one or more of VL1, VL2, and VL3 include Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in one or more of VH1, VH2, and VH3 are oppositely charged to the mutations in one or more of VL1, VL2, and VL3.

[0102] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1 The first light chain (LC1) / heavy chain (HC1) pair including; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2 The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, The C-terminus of CH1-1 is operably connected to the N-terminus of VH2. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds. One or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs contain opposite-charged mutations to facilitate pairing. One or both of the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations to promote pairing. If both the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations that promote pairing, then the mutations in CH1-1 and CL1 are different from the mutations in CH1-2 and CL2.

[0103] In some embodiments, CH1-1 includes the T192E mutation, and CL1 includes the N137K and S114A mutations.

[0104] In some embodiments, CH1-1 includes L143Q and S188V mutations, and CL1 includes V133T and S176V mutations.

[0105] In some embodiments, CH1-1 includes T192E, L143Q, and S188V mutations, and CL1 includes N137K, S114A, V133T, and S176V mutations.

[0106] In some embodiments, CH1-2 includes the T192E mutation, and CL2 includes the N137K and S114A mutations.

[0107] In some embodiments, CH1-2 includes L143Q and S188V mutations, and CL2 includes V133T and S176V mutations.

[0108] In some embodiments, CH1-2 includes T192E, L143Q, and S188V mutations, and CL2 includes N137K, S114A, V133T, and S176V mutations.

[0109] In some embodiments, one or both of CH1-1 and CH1-2 contain L143E, L143D, L143K, L143R, or L143H mutations, one or both of CL1 and CL2 contain S176E, S176D, S176K, S176R, or S176H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0110] In some embodiments, one or both of CH1-1 and CH1-2 contain L124E, L124D, L124K, L124R, or L124H mutations, one or both of CL1 and CL2 contain V133E, V133D, V133K, V133R, or V133H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0111] In some embodiments, one or both of VH1 and VH2 include a 39E, 39D, 39K, 39R, or 39H mutation, and one or both of VL1 and VL2 include a 38E, 38D, 38K, 38R, or 38H mutation, and the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL2.

[0112] In some embodiments, one or both of VH1 and VH2 include Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and one or both of VL1 and VL2 include Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in one or both of VH1 and VH2 are oppositely charged to the mutations in one or both of VL1 and VL2.

[0113] In some embodiments, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1 The first light chain (LC1) / heavy chain (HC1) pair including; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2 The second light chain (LC2) / heavy chain (HC2) pair including We provide a multispecific antigen-binding protein containing, The C-terminus of CH1-1 is operably connected to the N-terminus of VH2. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds. One or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs contain an anti-charged mutation to facilitate pairing, the anti-charged mutation comprising (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H. Mutant residues in the VH region have the opposite charge to those in the VL region. One or both of the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations to promote pairing. If both the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations that promote pairing, then the mutations in CH1-1 and CL1 are different from the mutations in CH1-2 and CL2.

[0114] In some embodiments, the multispecific antibody further comprises one or more cysteine ​​residues incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds.

[0115] In some embodiments, one or both of VH1 and VH2 include 44C and 105C mutations, and one or both of VL1 and VL2 include 100C and 43C mutations.

[0116] In some embodiments, VH1 includes the 44C mutation, and VL1 includes the 100C mutation.

[0117] In some embodiments, VH1 includes the 105C mutation, and VL1 includes the 43C mutation.

[0118] In some embodiments, VH2 includes the 44C mutation, and VL2 includes the 100C mutation.

[0119] In some embodiments, VH2 includes the 105C mutation, and VL2 includes the 43C mutation.

[0120] In some embodiments, VH1 includes 39E and 44C mutations, and VL1 includes 38K and 100C mutations.

[0121] In some embodiments, VH1 includes 39E and 105C mutations, and VL1 includes 38K and 43C mutations.

[0122] In some embodiments, the multispecific antibody further includes an anti-charge mutation in the CH1-1 / CL1 pair.

[0123] In some embodiments, the opposite charge mutation in the CH1-1 / CL1 pair is selected from the group consisting of K221E in the CH1-1 region and E123K in the CL1 region; K228D in the CH1-1 region and D122K in the CL1 region; L145E in the CH1-1 region and S176K in the CL1 region; and L128E in the CH1-1 region and V133K in the CL1 region.

[0124] In some embodiments, the multispecific antibody further includes opposite charge mutations in the CH1-2 / CL2 pair.

[0125] In some embodiments, the opposite charge mutations in the CH1-2 / CL2 pair are selected from the group consisting of K221E in the CH1-2 region and E123K in the CL2 region; K228D in the CH1-2 region and D122K in the CL2 region; L145E in the CH1-2 region and S176K in the CL2 region; and L128E in the CH1-2 region and V133K in the CL2 region.

[0126] In some embodiments, the CH1-1 domain is linked to a first CH2 domain and a first CH3 domain, the CH1-2 domain is linked to a second CH2 domain and a second CH3 domain, and the first CH2 domain and CH3 domain, as well as the second CH2 domain and CH3 domain, dimerize to form an Fc domain.

[0127] In some embodiments, the first CH3 domain includes one or both of the S354C and T366W mutations, and the second CH3 domain includes one or both of the Y349C, T366S, L368A, and Y407V mutations to promote Fc domain heterodimerization.

[0128] In some embodiments, the C-terminus of CH1-1 is functionally linked to the N-terminus of VH2 via a peptide linker.

[0129] In some embodiments, the peptide linker is (GGGGS) n (Sequence array X) contains the linker, where n is any integer from 1 to 5.

[0130] In some embodiments, the peptide linker includes all or part of a sequence of hinge regions of one or more immunoglobulins selected from IgA, IgG, and IgD.

[0131] In some embodiments, the peptide linker has the following sequence: EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG(Sequence ID X) Includes.

[0132] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, HD1 and HD2 heterodimerize, At least one or both of VH1 and VH2 contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and one or both of VL1 and VL2 contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in one or both of VH1 and VH2 are oppositely charged to the mutations in one or both of VL1 and VL2 that promote pairing. At least one or both of CH1-1 and CH1-2 contain one or more of the T192E, L143Q, and S188V mutations, and at least one or both of CL1 and CL2 contain one or more of the N137K, S114A, V133T, and S176V mutations.

[0133] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) First steady heavy chain region 1 (CH1-1) and first steady light chain region (CL1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) Second steady heavy chain region 1 (CH1-2) and second steady light chain region (CL2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, At least one or both of VH1 and VH2 contain a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, and one or both of VL1 and VL2 contain a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, and the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL2 in order to facilitate pairing. At least one or both of CH1-1 and CH1-2 contain one or more of the T192E, L143Q, and S188V mutations, and at least one or both of CL1 and CL2 contain one or more of the N137K, S114A, V133T, and S176V mutations.

[0134] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, HD1 and HD2 heterodimerize, At least one or both of CH1-1 and CH1-2 contain a K221E, K221D, K221K, K221R, or K221H mutation, and one or both of CL1 and CL2 contain an E123E, E123D, E123K, E123R, or E123H mutation, and the mutation in one or both of CH1-1 and CH1-2 is oppositely charged to the mutation in one or both of CL1 and CL2 in order to facilitate pairing. At least one or both of CH1-1 and CH1-2 contain one or more of the T192E, L143Q, and S188V mutations, and at least one or both of CL1 and CL2 contain one or more of the N137K, S114A, V133T, and S176V mutations.

[0135] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) First steady heavy chain region 1 (CH1-1) and first steady light chain region (CL1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) Second steady heavy chain region 1 (CH1-2) and second steady light chain region (CL2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, At least one or both of CH1-1 and CH1-2 contain a K221E, K221D, K221K, K221R, or K221H mutation, and one or both of CL1 and CL2 contain an E123E, E123D, E123K, E123R, or E123H mutation, and the mutation in one or both of CH1-1 and CH1-2 is oppositely charged to the mutation in one or both of CL1 and CL2 in order to facilitate pairing. At least one or both of CH1-1 and CH1-2 contain one or more of the T192E, L143Q, and S188V mutations, and at least one or both of CL1 and CL2 contain one or more of the N137K, S114A, V133T, and S176V mutations.

[0136] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, HD1 and HD2 heterodimerize, At least one or both of VH1 and VH2 contain a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, and one or both of VL1 and VL2 contain a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, and the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL2 in order to facilitate pairing. At least one or both of CH1-1 and CH1-2 contain a K221E, K221D, K221K, K221R, or K221H mutation, and one or both of CL1 and CL2 contain an E123E, E123D, E123K, E123R, or E123H mutation, and the mutation in one or both of CH1-1 and CH1-2 is oppositely charged to the mutation in one or both of CL1 and CL2 in order to facilitate pairing. At least one or both of CH1-1 and CH1-2 contain one or more of the T192E, L143Q, and S188V mutations, and at least one or both of CL1 and CL2 contain one or more of the N137K, S114A, V133T, and S176V mutations.

[0137] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) First steady heavy chain region 1 (CH1-1) and first steady light chain region (CL1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) Second steady heavy chain region 1 (CH1-2) and second steady light chain region (CL2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, At least one or both of VH1 and VH2 contain a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, and one or both of VL1 and VL2 contain a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, and the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL2 in order to facilitate pairing. At least one or both of CH1-1 and CH1-2 contain a K221E, K221D, K221K, K221R, or K221H mutation, and one or both of CL1 and CL2 contain an E123E, E123D, E123K, E123R, or E123H mutation, and the mutation in one or both of CH1-1 and CH1-2 is oppositely charged to the mutation in one or both of CL1 and CL2 in order to facilitate pairing. At least one or both of CH1-1 and CH1-2 contain one or more of the T192E, L143Q, and S188V mutations, and at least one or both of CL1 and CL2 contain one or more of the N137K, S114A, V133T, and S176V mutations.

[0138] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, HD1 and HD2 heterodimerize, At least one or both of VH1 and VH2 contain a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, and one or both of VL1 and VL2 contain a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, and the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL2 in order to facilitate pairing. CH1-1 and CH1-2, at least one or both, contain one or more of the T192E, L143Q, and S188V mutations, and CL1 and CL2, at least one or both, contain one or more of the N137K, S114A, V133T, and S176V mutations. At least one or both of VH1 and VH2 contain the 44C mutation, and one or both of VL1 and VL2 contain the 100C mutation.

[0139] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) First steady heavy chain region 1 (CH1-1) and first steady light chain region (CL1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) Second steady heavy chain region 1 (CH1-2) and second steady light chain region (CL2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, At least one or both of VH1 and VH2 contain a Q39E, Q39D, Q39K, Q39R, or Q39H mutation, and one or both of VL1 and VL2 contain a Q38E, Q38D, Q38K, Q38R, or Q38H mutation, and the mutation in one or both of VH1 and VH2 is oppositely charged to the mutation in one or both of VL1 and VL2 in order to facilitate pairing. CH1-1 and CH1-2, at least one or both, contain one or more of the T192E, L143Q, and S188V mutations, and CL1 and CL2, at least one or both, contain one or more of the N137K, S114A, V133T, and S176V mutations. At least one or both of VH1 and VH2 contain the 44C mutation, and one or both of VL1 and VL2 contain the 100C mutation.

[0140] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) First steady heavy chain region 1 (CH1-1) and first steady light chain region (CL1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) Second steady heavy chain region 1 (CH1-2) and second steady light chain region (CL2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, At least one or both of CH1-1 and CH1-2 contain the L143E, L143D, L143K, L143R, or L143H mutation, at least one or both of CL1 and CL2 contain the S176E, S176D, S176K, S176R, or S176H mutation, and the mutation in one or both of CH1-1 or CH1-2 is oppositely charged to the mutation in one or both of CL1 and CL2.

[0141] In another aspect, this disclosure is as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) First steady heavy chain region 1 (CH1-1) and first steady light chain region (CL1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) Second steady heavy chain region 1 (CH1-2) and second steady light chain region (CL2) The second light chain (LC2) / heavy chain (HC2) pair including The present invention provides a multispecific antigen-binding protein or antibody containing, At least one or both of CH1-1 and CH1-2 contain L124E, L124D, L124K, L124R, or L124H mutations, at least one or both of CL1 and CL2 contain V133E, V133D, V133K, V133R, or V133H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0142] In some embodiments, CH1-1 further includes the T192E mutation, and CL1 includes the N137K and S114A mutations.

[0143] In some embodiments, CH1-2 includes the T192E mutation, and CL2 includes the N137K and S114A mutations.

[0144] In some embodiments, one or both of VH1 and VH2 include Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and one or both of VL1 and VL2 include Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in one or both of VH1 and VH2 are oppositely charged to the mutations in one or both of VL1 and VL2.

[0145] In some embodiments, the multispecific antibody further comprises one or more cysteine ​​residues incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds.

[0146] In some embodiments, one or both of VH1 and VH2 include 44C and 105C mutations, and one or both of VL1 and VL2 include 100C and 43C mutations.

[0147] In some embodiments, VH1 includes the 44C mutation, and VL1 includes the 100C mutation.

[0148] In some embodiments, VH1 includes the 105C mutation, and VL1 includes the 43C mutation.

[0149] In some embodiments, VH2 includes the 44C mutation, and VL2 includes the 100C mutation.

[0150] In some embodiments, VH2 includes the 105C mutation, and VL2 includes the 43C mutation.

[0151] In some embodiments, VH1 includes 39E and 44C mutations, and VL1 includes 38K and 100C mutations.

[0152] In some embodiments, VH1 includes 39E and 105C mutations, and VL1 includes 38K and 43C mutations.

[0153] In some embodiments, the multispecific antibody further comprises opposite-charge mutations in one or both of the CH1-1 / CL1 pair and the CH1-2 / CL2 pair.

[0154] In some embodiments, the opposite charge mutations in one or both of the CH1-1 / CL1 pair and the CH1-2 / CL2 pair include K221E in one or both of the CH1-1 region and the CH2-2 region, and E123K in one or both of the CL1 region and the CL2 region.

[0155] In some embodiments, the CH1-1 domain is operably linked to a first Fc domain including a first CH2 domain and a first CH3 domain, and the CH1-2 domain is operably linked to a second Fc domain including a second CH2 domain and a second CH3 domain, and the first and second Fc domains dimerize.

[0156] In some embodiments, the first CH3 domain contains one or both of the S354C and T366W mutations, and the second CH3 domain contains one or more of the Y349C, T366S, L368A, and Y407V mutations, the mutations promoting heterodimerization of the Fc domain.

[0157] In another aspect, this disclosure is: Antigen-binding domain and constant heavy chain CH1 region paired with constant light chain CL region We provide antigen-binding proteins that include, The antigen-binding domain selectively binds to the target antigen, and the CH1 and CL regions are as follows: a) L143E, L143D, L143K, L143R, or L143H mutations in the CH1 region, and S176E, S176D, S176K, S176R, or S176H mutations in the CL region; and b) L124E, L124D, L124K, L124R, or L124H mutations in the CH1 region, and V133E, V133D, V133K, V133R, or V133H mutations in the CL region. Including one or both of the following: Mutant residues in the CH1 region have the opposite charge to those in the CL region.

[0158] In another aspect, this disclosure is: Protein-binding domain; and CH1 region opposite the CL region Provides a binding protein containing, The protein-binding domain selectively binds to the target antigen, and the CH1 and CL regions are as follows: a) L143E, L143D, L143K, L143R, or L143H mutations in the CH1 region, and S176E, S176D, S176K, S176R, or S176H mutations in the CL region; and b) L124E, L124D, L124K, L124R, or L124H mutations in the CH1 region, and V133E, V133D, V133K, V133R, or V133H mutations in the CL region, It includes one or both of the above, and mutations in the CH1 region are oppositely charged to mutations in the CL region.

[0159] In another aspect, this disclosure is: Steady-state light chain CL region paired with steady-state heavy chain CH1 region We provide antigen-binding proteins that include, The antigen-binding domain selectively binds to the target antigen, and the CH1 and CL regions are as follows: a) L143E, L143D, L143K, L143R, or L143H mutations in the CH1 region, and S176E, S176D, S176K, S176R, or S176H mutations in the CL region; and b) K221E and K228D mutations in the CH1 region, and D122K and E123K mutations in the CL region Including one or both of the following: Mutant residues in the CH1 region have the opposite charge to mutant residues in the CL region.

[0160] In some embodiments, the binding protein further includes a K221E mutation in the CH1 region and an E123K mutation in the CL region.

[0161] In some embodiments, the Disclosure provides a multispecific antigen-binding protein comprising a first Fab and a second Fab, wherein the first Fab comprises CH1-1, VH1, CL1 and VL1 domains, and the second Fab comprises CH1-2, VH2, CL2 and VL2 domains, and the first and second Fabs are selected from one of the following options: i. The first Fab includes a 143R mutation in CH1-1, a 39K mutation in VH1, a 176E mutation in CL1, and a 38E mutation in VL1; the second Fab includes a 143E mutation in CH1-2, a 39E mutation in VH2, a 176R mutation in CL2, and a 38K mutation in VL2; ii. The first Fab includes a 143K mutation in CH1-1, a 39K mutation in VH1, a 176E mutation in CL1, and a 38E mutation in VL1; the second Fab includes a 143E mutation in CH1-2, a 39E mutation in VH2, a 176K mutation in CL2, and a 38K mutation in VL2; iii. The first Fab includes the 143H mutation in CH1-1, the 39K mutation in VH1, the 176E mutation in CL1, and the 38E mutation in VL1; the second Fab includes the 143E mutation in CH1-2, the 39E mutation in VH2, the 176H mutation in CL2, and the 38K mutation in VL2; iv. The first Fab includes the 143R mutation in CH1-1, the 39K mutation in VH1, the 176D mutation in CL1, and the 38E mutation in VL1; the second Fab includes the 143D mutation in CH1-2, the 39E mutation in VH2, the 176R mutation in CL2, and the 38K mutation in VL2; v. The first Fab includes a 143K mutation in CH1-1, a 39K mutation in VH1, a 176D mutation in CL1, and a 38E mutation in VL1; the second Fab includes a 143D mutation in CH1-2, a 39E mutation in VH2, a 176K mutation in CL2, and a 38K mutation in VL2; vi. The first Fab includes a 143H mutation in CH1-1, a 39K mutation in VH1, a 176D mutation in CL1, and a 38E mutation in VL1, while the second Fab includes a 143D mutation in CH1-2, a 39E mutation in VH2, a 176H mutation in CL2, and a 38K mutation in VL2.

[0162] In some embodiments, the CH1 region is operably linked to the heterodimerization domain.

[0163] In some embodiments, the heterodimerizing domain includes a first Fc domain.

[0164] In some embodiments, the first Fc domain heterodimerizes with the second Fc domain, the first Fc domain includes a first CH3 region, and the second Fc domain includes a second CH3 region.

[0165] In some embodiments, the first CH3 region contains one or both of the S354C and T366W mutations, and the second CH3 region contains one or more of the Y349C, T366S, L368A, and Y407V mutations, the mutations promoting Fc domain heterodimerization.

[0166] In some embodiments, the antigen-binding protein further comprises at least one VH / VL pair including an opposite-charged mutation to facilitate pairing, wherein the opposite-charged mutation comprises (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, the mutant residue in the VH region having the opposite charge to the mutant residue in the VL region.

[0167] In some embodiments, the 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 antigens are disclosed herein. In some embodiments, the HCDRs, LCDRs and / or antigens are known in the art. In some embodiments, the HCDRs, LCDRs and / or antigens are newly identified or discovered.

[0168] In some embodiments, this disclosure is, antigen-binding domain; and Steady-state light chain CL region paired with steady-state heavy chain CH1 region We provide antigen-binding proteins that include, The antigen-binding domain selectively binds to the target antigen, and the CH1 and CL regions are as follows: a) L143E, L143D, L143K, L143R, or L143H mutations in the CH1 region, and S176E, S176D, S176K, S176R, or S176H mutations in the CL region; and b) L124E, L124D, L124K, L124R, or L124H mutations in the CH1 region, and V133E, V133D, V133K, V133R, or V133H mutations in the CL region. Including one or both of the following: Mutant residues in the CH1 region have the opposite charge to those in the CL region.

[0169] In some embodiments, the antigen-binding protein is as follows: (1) T192E (CH1) mutation, and N137K and S114A (CL) mutations, (2) L143Q and S188V (CH1) mutations, and V133T and S176V (CL) mutations, (3) T192E, L143Q and S188V (CH1) mutations, and N137K, S114A, V133T and S176V (CL) mutations, (4) K221E (CH1) mutation and E123K (CL) mutation, (5) K228D (CH1) mutation and D122K (CL) mutation, and (6) K221E and K228D (CH1) mutations, and D122K and E123K (CL) mutations further comprises a CH1 / CL mutation for promoting pairing selected from the group consisting of one or more of the following, When two CH1 / CL pairs contain mutations for promoting pairing for two different VH / VL pairs, the two CH1 / CL pairs do not contain the same mutation.

[0170] In some embodiments, the CH1 region is operably linked to the Fc domain.

[0171] In some embodiments, an isolated nucleic acid molecule comprising a nucleotide sequence encoding a 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 a multispecific antigen-binding protein or antigen-binding protein is provided.

[0172] In some embodiments, an expression vector comprising a nucleic acid molecule is provided. In some embodiments, a kit comprising one or more expression vectors comprising one or more nucleic acid molecules is provided.

[0173] In some embodiments, isolated host cells containing one or more nucleic acid molecules or one or more expression vectors are provided. In some embodiments, isolated host cells containing a kit of nucleic acid molecules or a kit of expression vectors are provided.

[0174] In some embodiments, the host cell is a mammalian cell or an insect cell.

[0175] In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a multispecific antibody or antigen-binding protein.

[0176] In some embodiments, a method is provided for treating a disorder in which antigenic activity is harmful, comprising administering an effective amount of a multispecific antibody or antigen-binding protein to a subject in need thereof.

[0177] In some embodiments, polynucleotides encoding multispecific antibodies or antigen-binding proteins are provided.

[0178] In some embodiments, host cells expressing multispecific antibodies or antigen-binding proteins are provided.

[0179] In some embodiments, a method is provided for producing a multispecific antibody or antigen-binding protein, comprising culturing host cells under conditions such that the multispecific antibody or antigen-binding protein is expressed. In some embodiments, a multispecific antibody or antigen-binding protein for use as a drug is provided.

[0180] The above-mentioned summary of the disclosure 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, the detailed description of this disclosure, and the claims. [Brief explanation of the drawing]

[0181] [Figure 1]Figures 1A and 1C schematically illustrate cross-bivariate (CODV) antigen-binding protein formats with several different mutations to promote heterodimerization. Figure 1A shows the CH1 / kappa mutations of "O" on CODV-Fab(CH1:L143Q, S188V;Ck:V133T, S176V) and "Δ" on Fab2(CH1:T192E;Ck:N137K, S114A). Figure 1B shows the mutations on CODV-Fab(VH39E / VL38K+O) and Fab2(VH39K / VL38E+Δ). Figure 1C shows mutations on CODV-Fab ([VH39E / VL38K+O]+[VH44Cys / VL100Cys] or [VH105Cys / VL43Cys]) and Fab2 ([VH39K / VL38E+Δ]+[VH44Cys / VL100Cys] or [VH105Cys / VL43Cys]). [Figure 2-1] Figures 2A to 2G are diagrammatic illustrations of the results in Table 5. Figure 2A shows the results for WT CODV antibodies. Figure 2B shows the results for CODV antibodies containing only the CH1 / Ck mutation. Figure 2C shows the results for the combination of CH1 / Ck and charged mutation (CM). Figure 2D shows the results for CODV antibodies containing only disulfide-stabilized (ds). Figure 2E shows the results for the combination of CH1 / Ck and ds mutation. Figure 2F shows the results for the combination of CM and ds mutation. Figure 2G shows the results for the combinations of the three mutation sets in CODV antibodies: CH1 / Ck, CM, and ds stabilization. [Figure 2-2] Continuation of Figure 2-1. [Figure 2-3] Continuation of Figure 2-2. [Figure 3-1]Figures 3A to 3D schematically show the tandem Fabs antibody format in open configurations (Figures 3A and 3B) and closed configurations (Figures 3C and 3D). Figure 3A shows the open configuration with the CH1 / kappa mutation "Δ" (CH1:T192E;Ck:N137K, S114A) on Fab1 and the CH1 / kappa mutation "O" (CH1:L143Q, S188V;Ck:V133T, S176V) on Fab2. Figure 3B shows the open configuration with the mutations on Fab1 (VH39K / VL38E+Δ) and Fab2 (VH39E / VL38K+O). Figure 3C shows the closed configuration with the Ch1 / kappa mutation Δ (CH1:T192E;Ck:N137K, S114A) on Fab1 and the CH1 / kappa mutation O (CH1:L143Q, S188V;Ck:V133T, S176V) on Fab2. Figure 3D shows the closed configuration with the mutation (VH39K / VL38E+Δ) on Fab1 and the mutation (VH39E / VL38K+O) on Fab2. [Figure 3-2] Continuation of Figure 3-1. [Figure 4] Figure 4 shows the results of SEC and HIC analysis of tandem Fab in open configurations in WT, CH1 / Ck, and CH1 / Ck+CM formats. [Figure 5] Figures 5A to 5D show the results of HIC analysis, yield, and binding affinity for anti-CD40 × anti-PD-L1 tandem Fab antibodies in open and closed configurations. Figure 5A shows the closed configuration with only the CH1 / Ck mutation. Figure 5B shows the closed configuration with Fab domain exchange with only the CH1 / Ck mutation. Figure 5C shows the closed configuration with both the CH1 / Ck and CM mutations. Figure 5D shows the open configuration with both the CH1 / Ck and CM mutations. [Figure 6]Figures 6A to 6C show the results of HIC analysis, yield, and binding affinity for anti-PD-1 × anti-OX40 tandem Fab antibodies in open and closed configurations. Figure 6A shows the closed configuration with only the CH1 / Ck mutation. Figure 6B shows the open configuration with only the CH1 / Ck mutation. Figure 6C shows the open configuration with both the CH1 / Ck and CM mutations. [Figure 7] Figures 7A and 7B show the purity results for anti-4-1BB × anti-PD-L1 tandem Fab antibody (Figure 7A) and anti-4-1BB × anti-PD-1 tandem Fab antibody (Figure 7B). Both antibodies are in an open configuration with CH1 / Ck and CM mutations. [Figure 8] Figures 8A and 8B schematically illustrate Y-shaped bispecific antibody formats with several different mutations to enhance heterodimerization. Figure 8A shows the CH1 / kappa mutation "O" (CH1:L143Q, S188V; Ck:V133T, S176V) on Fab1 and the CH1 / kappa mutation "Δ" (CH1:T192E; Ck:N137K, S114A) on Fab2. Figure 8B shows the mutation on Fab1 (VH39E / VL38K+O) and the mutation on Fab2 (VH39K / VL38E+Δ). [Figure 9-1] Figures 9A to 9C show the purity results for anti-PD-1 × anti-OX40 antibodies. The thermal stability of the antibody against T initiation was measured using NanoDSF (differential scanning fluorescence measurement). Purity was measured using HIC. The wild-type antibody is shown in Figure 9A. CH1 / Ck mutations (Fab1=CH1:L143Q, S188V;Ck:V133T, S176V) and (Fab2=CH1:T192E;Ck:N137K, S114A) are shown in Figure 9B. CH1 / Ck and CM mutations (Fab1=CH1:L143Q, S188V;Ck:V133T, S176V;VH39E / VL38K) and (Fab2=CH1:T192E;Ck:N137K, S114A;VH39K / VL38E) are shown in Figure 9C. [Figure 9-2] Continuation of Figure 9-1. [Figure 10-1]Figures 10A to 10L are diagrams showing SEC and HIC profiles for various anti-PD-1 × anti-OX40 antibodies having several different combinations of mutations. Specific mutations are listed in Table 9 below. [Figure 10-2] Continuation of Figure 10-1. [Figure 10-3] Continuation of Figure 10-2. [Figure 10-4] Continuation of Figure 10-3. [Figure 11A] Figures 11A to 11E are diagrams showing chain mispairing data for various Y-shaped antibodies. Figure 11A shows mispairing data for an anti-PD-1 × anti-GITR antibody. [Figure 11B] Figures 11A to 11E are diagrams showing chain mispairing data for various Y-shaped antibodies. Figure 11B shows mispairing data for an anti-TNF × anti-GITR antibody. [Figure 11C] Figures 11A to 11E are diagrams showing chain mispairing data for various Y-shaped antibodies. Figure 11C shows mispairing data for an anti-TNF × anti-OX40 antibody. [Figure 11D] Figures 11A to 11E are diagrams showing chain mispairing data for various Y-shaped antibodies. Figure 11D shows mispairing data for an anti-CD40 × anti-PD-L1 antibody. [Figure 11E] Figures 11A to 11E are diagrams showing chain mispairing data for various Y-shaped antibodies. Figure 11E shows mispairing data for an anti-CD3 × anti-CD123 antibody. Specific mutations and biophysical characterization data are listed in Table 11 below. [Figure 12]Figure 12 shows a cytotoxic assay of human pan-T cells against THP-1 target cells co-incubated with the bispecific CD3×CD123-hIgG1-LALA molecule. Effector cells and CFSE-labeled THP-1 target cells were seeded in an effector-to-target ratio of 10:1 and co-incubated for 20 hours at 37°C with serial dilutions of the respective bispecific molecules (10 nM to 0 nM). Dead cells were stained with 7-AAD and measured by flow cytometry. Cytotoxic activity was calculated based on the percentage of dead THP-1 target cells (7-AAD / CFSE bi-positive). Data are shown as the mean of two representative healthy donors, with dead target cells [%] relative to the concentration [pM] of the bispecific molecule. [Modes for carrying out the invention]

[0182] I. Definition To facilitate easier understanding of the disclosure, the selected terms are defined below.

[0183] The sequence position numbers used herein refer to Kabat numbering (Kabat, EA et al., Sequences of proteins of immunological interest. 5th ed. - US Department of Health and Human Services, NIH publications 91-3242, pp. 662, 680, 689, 1991).

[0184] Where used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids); unnatural amino acids, e.g., α-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, γ-carboxyglutamic acid, cN,N,N-trimethyllysine, cN-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, uN-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In polypeptide notation used herein, the left-handed direction is towards the amino terminus and the right-handed direction is towards the carboxyl terminus, following standard usage and convention. Naturally occurring residues can be classified into classes based on common side-chain properties (see Table 1).

[0185] [Table 1]

[0186] Conservative amino acid substitutions can involve the exchange of one member of one of these classes with another member of the same class. Conservative amino acid substitutions can include amino acid residues that do not exist naturally and are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimes and other forms of the return or inversion of amino acid residues. Non-conservative substitutions can involve the exchange of one member of one of these classes with a member of another class.

[0187] As used herein, the terms “mutation” or “mutant” refer to an alteration of an amino acid sequence by the deletion, insertion, and / or substitution of one or more amino acids. In particular, this refers to substitution. A mutation is introduced into a given sequence, for example, the amino acid sequence of a VL1 and / or VH1 pair that specifically recognizes a first antigen.

[0188] As used herein, “T192E(CH1) mutation” refers to the substitution of a threonine (T) residue for a glutamic acid (E) residue in the constant domain of the immunoglobulin CH1 heavy chain of an antigen-binding protein at Kabat position 192.

[0189] As used herein, a “mutation set” refers to a group of different mutations present in a sequence.

[0190] As used herein, the term “mutant” 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 from which it is derived. The determination of the percentage of identity between two sequences is achieved using the mathematical algorithm described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 5873–5877, 1993. Such algorithms are incorporated into the BLASTN and BLASTP programs described in Altschul et al., (1990) J. Mol. Biol. Vol. 215, pp. 403–410. To obtain gap alignment for comparison purposes, Gapped BLAST is used as described in Altschul et al., (1997) Nucleic Acids Res. Vol. 25, pp. 3389–3402. When using the BLAST program and the Gapped BLAST program, the default parameters of each program 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, particularly conserved amino acid substitutions. Conservative substitutions are well known in the art (see, for example, Creighton (1984) Proteins. WH Freeman and Company). A summary of the physical and chemical properties of amino acids is shown in Table 1 above. In certain embodiments, a conservative substitution is a substitution made to an amino acid having at least one property according to Table 1 (i.e., in columns 1 and / or 2). The term “variant” also includes fragments. A fragment has N-terminal and / or C-terminal deletions with a total of up to 20, 15, 10, 5, 4, 3, 2, or 1 amino acid(s). Furthermore, or alternatively, the variants may be modified by the addition of N-terminal and / or C-terminal amino acids of up to 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 amino acid in total.

[0191] As used herein, the terms “antigen,” “target antigen,” or “antigen target” mean a molecule or part of a molecule (e.g., an epitope) that can be specifically bound by the binding proteins described herein and can also be used in animals to produce an antibody that can specifically bind to the epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by the binding protein, the binding protein may compete with an intact antibody that recognizes the target antigen.

[0192] As used herein, the term “epitope” refers to any determinant that can specifically bind to an immunoglobulin or T cell receptor, such as a polypeptide determinant. In certain embodiments, an epitope determinant comprises a chemically active surface grouping of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural properties and / or specific charge properties. An epitope is a region of an antigen that is bound by an antibody or an antigen-binding fragment of an antibody, or by a binding protein. In certain embodiments, a binding protein is said to bind specifically to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, the binding protein has an equilibrium dissociation constant of 10 -8 If M is less than 10 -9 If M is less than 10 -10 It is said that when the molecule is less than M, it specifically binds to the antigen.

[0193] As used herein, the terms “antigen-binding protein” or “binding protein” or “binding polypeptide” refer to a polypeptide (e.g., an antibody or a fragment thereof) that includes at least one binding site involved in the selective binding of a target antigen of interest (e.g., a human antigen). Examples of 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 embodiments, a binding polypeptide includes multiple (e.g., two, three, four, or more) binding sites. In certain embodiments, a binding protein is not a therapeutic enzyme.

[0194] As used herein, “heterodimizing domain” or “HD” refers to a subunit of a bispecific, tripspecific, or multispecificity binding protein that facilitates, directs, or forces the correct assembly of light chains and their homologous heavy chains to yield a desired protein, while preventing mispairing of the respective light chains or heavy chains.

[0195] As used herein, a “multispecific” binding protein is a binding protein that binds to two or more antigens and / or two or more different epitopes. A multispecific binding protein that binds to two antigens and / or two different epitopes is also referred herein to as a “bispecific” binding protein. A multispecific binding protein that binds to three antigens and / or three different epitopes is also referred herein to as a “triplespecific” binding protein.

[0196] As used herein, the terms “heterodimerated Fc” or “functional fragment of heterodimerized Fc” refer to mutant forms of a constant domain, such as the CH2-CH3 or CH2-CH3-CH4 mutant forms that no longer form a homodimer but form a heterodimer with respect to the naturally occurring Fc moiety. Thus, the term refers to one of the two strands that form the heterodimer. Some of these sites are known in the art and include, for example, the knob-in-hole (KIH) mutant or the EV-RWT mutant.

[0197] Ridgeway and collaborators generated CH3 interfaces favorable to heterodimer assembly by substituting a smaller side chain on one CH3 interface with a larger side chain to create a knob, and by substituting a larger side chain on the other CH3 domain with a smaller side chain to generate a pore. Testing of such mutants demonstrated selective heterodimerization. Further extensions of the original knob-in-hole mutation were used to identify more suitable combinations by phage display, which was used to generate bispecific IgG antibodies to test for further substitutions that would enable disulfide bond formation. Knob-in-hole variants are further described in U.S. Patents 5,732,168 and 8,216,805, which are incorporated herein by reference. Therefore, in one embodiment, the CH3 domain of one Fc domain or heterodimer domain contains mutants Y349C, T366S, L368A, and Y407V, and the CH3 domain of another FC domain or heterodimer domain contains mutants S354C and T366W (amino acid positions are indicated by reference to the IgG1 sequence).

[0198] As used herein, the term “homodimerizing domain” refers to a similar domain, such as the domain that mediates homodimerization to two heavy chains. Heavy chain pairing is mediated by the last domain of the constant region, i.e., CH3 in the IgG molecule, resulting in a high-affinity homodimer complex (approximately 10 pM K). D) forms. Further interactions exist in the hinge region involved in the covalent bond between the two heavy chains formed after heavy chain assembly. In the CH3 homodimer, as shown for human γ1 CH3, approximately 16 residues are involved at the CH3 interface, with 6 residues (T366, L368, F405, Y407, and K409) forming a patch at the center of the interface, which strongly contributes to stability. Homodimerization domains include, but are not limited to, the Fc region and its effector-modified variants or fragments thereof, as well as the CH2 domain or fragments thereof, the CH3 domain or fragments thereof, the CH4 domain or fragments thereof, etc.

[0199] Naturally occurring antibodies typically contain tetramers. Such tetramers typically consist of two identical polypeptide chain pairs, each pair comprising one full-length "light chain" (typically with a molecular weight of approximately 25 kDa) and one full-length "heavy chain" (typically with a molecular weight of approximately 50–70 kDa). The terms "heavy chain" and "light chain," as used herein, refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity to a target antigen. The amino-terminal portions of each light and heavy chain typically contain a variable domain of approximately 100–110 or more amino acids involved in antigen recognition. The carboxyl-terminal portions of each chain typically define a constant domain involved in effector function. Therefore, in naturally occurring antibodies, full-length heavy-chain IgG immunoglobulin polypeptides contain a variable domain (VH) and three constant domains (CH1, CH2, and CH3), with the VH domain at the amino terminus of the polypeptide and the CH3 domain at the carboxyl terminus, while full-length light-chain immunoglobulin polypeptides contain a variable domain (VL) and a constant domain (CL), with the VL domain at the amino terminus of the polypeptide and the CL domain at the carboxyl terminus.

[0200] In some embodiments, the multispecific antigen-binding proteins of this disclosure include one or more VH domains from any one of the VH domain sequences listed in Tables 2, 3, and 4. In some embodiments, the multispecific antigen-binding proteins of this disclosure include one or more VL domains from any one of the VL domain sequences listed in Tables 2, 3, and 4. In some embodiments, the multispecific antigen-binding proteins of this disclosure include one or more VH domains from any one of the VH domain sequences listed in Tables 2, 3, and 4, and are paired with one or more VL domains from any one of the VL domain sequences listed in Tables 2, 3, and 4.

[0201] 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, defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, but is not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, but is not limited to IgM1 and IgM2. IgA is similarly subdivided into subclasses, but is not limited to IgA1 and IgA2. Within full-length light and heavy chains, variable and constant domains are typically linked by "J" regions of approximately 12 or more amino acids, and heavy chains also contain "D" regions of approximately 10 or more amino acids. See, for example, Basic Immunology (Paul, W., ed., Raven Press, 2nd edition, 1989), which is incorporated by reference as a whole for all purposes. The variable regions of each light / heavy chain pair typically form antigen-binding sites. The variable domains of naturally occurring antibodies typically exhibit the same general structure of a relatively conserved framework region (FR), connected by three hypervariable regions, also known as complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework region, potentially enabling binding to specific epitopes. From the amino terminus to the carboxyl terminus, both the light and heavy chain variable domains typically contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0202] As used herein, the term “CDR set” refers to a group of three CDRs that reside within a single variable region capable of binding to an antigen. The precise boundaries of these CDRs are defined differently by different systems. The Kabat system, described by Kabat et al. in *SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST* (National Institutes of Health, Bethesda, MD (1987) and (1991)), not only provides obvious residue numbers applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs. These CDRs are called Kabat CDRs. Chothia and collaborators (Chothia and Lesk, 1987, J. Affol. Biol. Vol. 196: pp. 901-917; Chothia et al., 1989, Nature Vol. 342: pp. 877-83) found that certain sub-parts within Kabat CDs adopt nearly identical peptide backbone structures despite significant diversity at the amino acid sequence level. These sub-parts are named L1, L2, and L3, or H1, H2, and H3, where "L" and "H" indicate the light chain and heavy chain regions, respectively. These regions are called Kabat CDRs. A CDR with a boundary that overlaps with the CDR is called a Chothia CDR. Other boundaries defining CDRs that overlap with the Kabat CDR are described by Padlan, 1995, FASEB J. 9: pp. 133-39; MacCallum, 1996, J. Mol. Biol. 262 (No. 5): pp. 732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27: pp. 55-77. Further other CDR boundary definitions cannot strictly adhere to one of the systems described herein, but nevertheless, in light of predictions or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding, they are shortened or extended but overlap with the Kabat CDR.The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia. Identification of predictive CDRs using amino acid sequences is well known in the art, for example, in Martin, AC., "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Vol. 2, edited by Kontermann R. and Dikel S., Springer-Verlag, Berlin, pp. 33-51 (2010). The sequences of CDRs can also be identified by examining the amino acid sequences of the heavy chain variable domains and / or light chain variable domains and determining regions of sequence hypervariability by other conventional methods, e.g., by comparing them with known amino acid sequences of other heavy chain variable and light chain variable regions. Numbered sequences can be aligned visually or by using an alignment program, such as one of the CLUSTAL program suites, as described in Thompson, 1994, Nucleic Acids Res. Vol. 22: pp. 4673-80. Molecular models are conventionally used to accurately depict the framework and CDR region, and therefore to correct sequence-based assignments.

[0203] In some embodiments, the CDR / FR of the immunoglobulin light chain or heavy chain is determined based on the IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, Vol. 27 (No. 1): pp. 55-77; website: imgt.org).

[0204] In some embodiments, the multispecific antigen-binding proteins of this disclosure include three variable heavy chain CDRs (HCDRs) from any one of the variable heavy chain sequences listed in Tables 2, 3, and 4. In some embodiments, the multispecific antigen-binding proteins of this disclosure include three variable light chain CDRs (LCDRs) from any one of the variable light chain sequences listed in Tables 2, 3, and 4. In some embodiments, the multispecific antigen-binding proteins of this disclosure include three HCFRs from any one of the variable heavy chain sequences listed in Tables 2, 3, and 4, as well as three LCFRs from any one of the variable light chain sequences listed in Tables 2, 3, and 4. The CDR sequences from the heavy chain and light chain variable sequences of Tables 2, 3, and 4 are readily determined using methods recognized by those skilled in the art for identifying CDR sequences.

[0205] The term "Fc," as used herein, refers to a molecule comprising a sequence of non-antigen-binding fragments resulting from the digestion of an antibody or produced by other means, whether in monomeric or polymeric form, and may include a hinge region. The original immunoglobulin source of natural Fc is typically of human origin and may be any of the immunoglobulins, but in exemplary embodiments, IgG1 and IgG2 are used. Fc molecules consist of monomeric polypeptides that can be linked into dimeric or polymeric forms by covalent (i.e., disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between monomeric subunits of a natural Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of Fc is the disulfide-bonded dimer resulting from the papain digestion of IgG. As used herein, the term "natural Fc" generally refers to monomeric, dimeric, and polymeric forms.

[0206] A Fab fragment typically comprises one light chain and one heavy chain with VH and CH1 domains, and the VH-CH1 heavy chain portion of an F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a Fab fragment may also include one light chain containing two variable domains separated by an amino acid linker and a CL domain, and one heavy chain containing two variable domains separated by an amino acid linker and a CH1 domain.

[0207] A F(ab') fragment typically consists of one light chain and a portion of one heavy chain containing more constant regions (between the CH1 and CH2 domains), thereby allowing an interchain disulfide bond to form between the two heavy chains to create an F(ab')2 molecule.

[0208] As used herein, the term “binding protein” refers to a molecule that does not exist in nature (or is recombinant, engineered, or substituted) that specifically binds to at least one target antigen.

[0209] As used herein, the term "Tm" refers to the melting temperature of binding proteins, antigen-binding proteins, and antibodies, and is an important parameter for the thermal stability of antigen-binding proteins. Generally, Tm refers to the thermal stability of the Fv fragment, i.e., the variable region heavy and light chain (VH / VL). Tm can be measured by differential scanning calorimetry (DSC) or differential scanning fluorescence (DSF).

[0210] One embodiment of the present disclosure provides a binding protein having biological and immunological specificity to 1 to 4 target antigens and / or specificity to 1 to 4 target epitopes. Another embodiment of the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a binding protein. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a binding protein. Yet another embodiment of the present disclosure provides a host cell that expresses such a binding protein (i.e., comprising a nucleic acid molecule or vector encoding a polypeptide chain that forms such a binding protein).

[0211] As used herein, the term “linker” refers to a sequence of 0 to 100 consecutive amino acid residues. Linkers may or may not be present, and may be identical or different. Linkers contained in a protein or polypeptide may all have the same amino acid sequence or may have different amino acid sequences.

[0212] In some embodiments, the peptide linker includes the following sequence:EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (Sequence ID X).

[0213] In some embodiments, the term “linker” refers to 1 to 15 consecutive amino acid residues. Typically, linkers provide flexibility and spatial separation between two amino acids or between two polypeptide domains. Linkers can be inserted between VH, VL, CH, and / or CL domains to provide sufficient flexibility and mobility to light and heavy chain domains, depending on the molecular format, for example, to fold into a bivariate variable-region immunoglobulin. Linkers are typically inserted at the amino sequence level, in transitions between variable domains, between a variable domain and a knockout domain, or between a variable domain and a constant domain, respectively. Interdomain transitions can be identified because the size of immunoglobulin domains is well understood. The precise location of domain transitions can be determined by arranging peptide stretches that do not form secondary structure elements, such as beta-sheets or alpha-helices, as indicated by experimental data or determined by modeling or secondary structure prediction techniques. In certain exemplary embodiments, linkers can be inserted between Fab domains to produce tandem Fab antibodies. In certain embodiments, the linker is inserted between the N-terminus of the VH domain of the first Fab and the C-terminus of the CH1 domain of the second Fab.

[0214] The identity and sequence of amino acid residues in a linker can vary depending on the type of secondary structural element(s) required to achieve it within the linker. For example, glycine, serine, and alanine are suitable for linkers with maximum flexibility. Certain combinations of glycine, proline, threonine, and serine are more rigid and useful when an elongated linker is desired. Any amino acid residue can be considered a linker in combination with other amino acid residues to construct a larger peptide linker, if required depending on the desired properties.

[0215] In some embodiments, the linker includes a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide having four glycine residues (Gly-Gly-Gly-Gly; Sequence ID x); a peptide having five glycine residues (Gly-Gly-Gly-Gly-Gly; Sequence ID x); a peptide having six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; Sequence ID x); a peptide having seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; Sequence ID x); and a peptide having eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; Sequence ID x).

[0216] In some embodiments, the linker contains small amino acids such as Gly, Ala, or Ser.

[0217] In some embodiments, the linker includes Gly(G) and Ser(S), or GS, GGS, GGGS, or GGGGS. In some embodiments, the linker includes (Gly-Gly-Gly-Gly-Ser)2 (i.e., (GGGGS)2). In some embodiments, the linker includes (Gly-Gly-Gly-Gly-Ser)3 (i.e., (GGGGS)3).

[0218] In some embodiments, the linker includes Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser (SEQ ID NO: x), and peptide Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x).

[0219] In some embodiments, the linker comprises a single Ser residue; a single Val residue; a dipeptide selected from Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; or a polypeptide selected from Thr-Lys-Gly-Pro-Ser (sequence ID x), Thr-Val-Ala-Ala-Pro (sequence ID x), Gln-Pro-Lys-Ala-Ala (sequence ID x), Gln-Arg-Ile-Glu-Gly (sequence ID x); Ala-Ser-Thr-Lys-Gly-Pro-Ser (sequence ID x), Arg-Thr-Val-Ala-Ala-Pro-Ser (sequence ID x), Gly-Gln-Pro-Lys-Ala-Ala-Pro (sequence ID x), His-Ile-Asp-Ser-Pro-Asn-Lys (sequence ID x), and Asp-Lys-Thr-His-Thr (sequence ID x).

[0220] In some embodiments, two tandem Fabs are connected via a (Gly-Gly-Gly-Gly-Ser)2 linker.

[0221] In some embodiments having a CODV-Fab moiety where L1 and L2 are on the light chain and L3 and L4 are on the heavy chain, L1 has a length of 3 to 12 amino acid residues, L2 has a length of 3 to 14 amino acid residues, L3 has a length of 1 to 8 amino acid residues, and L4 has a length of 1 to 3 amino acid residues. In some embodiments, L1 has a length of 5 to 10 amino acid residues, L2 has a length of 5 to 8 amino acid residues, L3 has a length of 1 to 5 amino acid residues, and L4 has a length of 1 to 2 amino acid residues. In some embodiments, L1 has a length of 7 amino acid residues, L2 has a length of 5 amino acid residues, L3 has a length of 1 amino acid residue, and L4 has a length of 2 amino acid residues. In some embodiments, L1 has a length of 10 amino acid residues, L2 has a length of 10 amino acid residues, L3 has a length of 0 amino acid residues, and L4 has a length of 0 amino acid residues. In some embodiments, L1, L2, L3, and L4 each have a length independently selected from 0 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids), and at least two of the linkers have a length of 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In some embodiments, L1, L2, L3, and L4 are Asp-Lys-Thr-His-Thr (sequence number x). In some embodiments, the linker contains the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (sequence number x). In some embodiments, L1 contains the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (sequence number x). In some embodiments, L1 includes the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (sequence number x), L2 includes the sequence Thr-Lys-Gly-Pro-Ser-Arg (sequence number x), L3 includes the sequence Ser, and L4 includes the sequence Arg-Thr. In some embodiments, L3 includes the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (sequence number x).In some embodiments, L1 includes the sequence Ser, L2 includes the sequence Arg-Thr, L3 includes the sequence Gly-Gln-Pro-Lys-Ala-Pro (sequence number x), and L4 includes the sequence Thr-Lys-Gly-Pro-Ser-Arg (sequence number x).

[0222] In some embodiments, L1, L2, L3, and L4 are each independently (Gly-Gly-Gly-Gly-Ser) nIncludes sequences selected from (n is an integer from 0 to 5; sequence number x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(sequence number x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(sequence number x), Ser, Arg-Thr, Thr-Lys-Gly-Pro-Ser(sequence number x), Gly-Gln-Pro-Lys-Ala-Ala-Pro(sequence number x), and Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly(sequence number x). In some embodiments, L1 contains the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (sequence code x), L2 contains the sequence Thr-Lys-Gly-Pro-Ser (sequence code x), L3 contains the sequence Ser, and L4 contains the sequence Arg-Thr. In some embodiments, L1 contains the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (sequence code x), L2 contains the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (sequence code x), L3 has an amino acid length of 0, and L4 has an amino acid length of 0. In some embodiments, L1 contains the sequence Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (sequence number x), L2 contains the sequence Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (sequence number x), L3 has an amino acid length of 0, and L4 has an amino acid length of 0. In some embodiments, L1 contains the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (sequence number x), L2 has an amino acid length of 0, L3 contains the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (sequence number x), and L4 has an amino acid length of 0. In some embodiments, L1 and L2 have an amino acid length of 0, and L3 and L4 are (Gly-Gly-Gly-Gly-Ser) nThe sequence includes (SEQ ID NO: x) (where n is an integer from 0 to 5; SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser(SEQ ID NO: x), Ser, Arg-Thr, Thr-Lys-Gly-Pro-Ser(SEQ ID NO: x), Gly-Gln-Pro-Lys-Ala-Ala-Pro(SEQ ID NO: x), and Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly(SEQ ID NO: x). In some embodiments, L3 and L4 are 0 amino acid lengths, and L1 and L2 are (Gly-Gly-Gly-Gly-Ser) n Includes sequences independently selected from (n is an integer between 0 and 5; sequence number x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(sequence number x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(sequence number x), Ser, Arg-Thr, Thr-Lys-Gly-Pro-Ser(sequence number x), Gly-Gln-Pro-Lys-Ala-Ala-Pro(sequence number x), and Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly(sequence number x).

[0223] In some embodiments, the linker(s) include sequences derived from naturally occurring sequences at the junction between the antibody variable domain and the antibody constant domain (e.g., those described in International Publication No. 2012 / 135345, incorporated by reference). For example, in some embodiments, the linker includes sequences found in the transition between endogenous VH and the CH1 domain, or between endogenous VL and the CL domain (e.g., κ or λ). In some embodiments, the linker includes sequences found in the transition between endogenous human VH and the CH1 domain, or between endogenous human VL and the CL domain (e.g., human κ or λ).

[0224] The examples listed above are not intended to limit the scope of the disclosure in any way, and linkers containing randomly selected amino acids from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline are suitable for use in the binding proteins described herein. For further descriptions of linker sequences, see, for example, International Publication Nos. 2012 / 135345 and International Publication Nos. 2017 / 180913, incorporated by reference.

[0225] As used herein, the term “valence” refers to the number of binding sites on a binding protein, epitope, antigen-binding protein, or antibody. For example, the term “monovalent binding protein” refers to a binding protein having one antigen-binding site. The term “bivalent binding protein” refers to a binding protein having two binding sites. The term “trivalent binding protein” refers to a binding protein having three binding sites. The term “tetravalent binding protein” refers to a binding protein having four binding sites. In certain embodiments, a bivalent binding protein can bind to one antigen target. In other embodiments, a bivalent binding protein can bind to two different antigen targets. In certain embodiments, a trivalent binding protein can bind to one antigen target, i.e., it is monospecific. In other embodiments, a trivalent binding protein can bind to two different antigen targets, i.e., it is bispecific. In other embodiments, a trivalent binding protein can bind to three different antigen targets, i.e., it is triplicate. In certain embodiments, a tetravalent binding protein can bind to one antigen target, i.e., it is monospecific. In other embodiments, the tetravalent conjugate protein can bind to two different antigen targets, i.e., it is bispecific. In other embodiments, the tetravalent conjugate protein can bind to three different antigen targets, i.e., it is triplicate. In other embodiments, the tetravalent conjugate protein can bind to four different antigen targets, i.e., it is quadruplicate.

[0226] As used herein, the term "specificity" refers to the number of binding specificities of a binding protein, epitope, antigen-binding protein, or antibody. For example, the term "single-specific binding protein" refers to a binding protein that specifically binds to one antigen target. The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. The term "triple-specific binding protein" refers to a binding protein that specifically binds to three different antigen targets. The term "quadrispecific binding protein" refers to a binding protein that specifically binds to four different antigen targets, etc.

[0227] As used herein, the term “selective recognition site” refers to a modification in a binding protein that enables selective recognition by affinity reagents that bind to that site. Examples of selective recognition sites include the binding site of protein A on the Fc portion of immunoglobulins.

[0228] As used herein, the term “affinity reagent” refers to a reagent containing a ligand that is immobilized on a matrix and specifically binds to the surface grouping of molecules, such as amino acids or sugar side chains, and typically possesses specific three-dimensional structural and specific charge properties. Affinity reagents are tools of affinity chromatography, and purification is possible through specific interactions between ligands and products. “Protein L” is an example of an affinity reagent, referring to recombinant protein L that is immobilized on a matrix and forms a ligand that has affinity for a subset of variable domains of the immunoglobulin kappa light chain. Such a matrix may be a resin. Another example of an affinity reagent is “KappaSelect,” which refers to a recombinant 13kDa single-chain antibody derived from camelids that is immobilized on a matrix and forms a ligand that has affinity for the constant domain of the human immunoglobulin kappa light chain. Another example of an affinity reagent is protein A. Protein A is a 42kDa surface protein naturally found in the cell wall of the bacterium Staphylococcus aureus. Crystallographic refinement has shown that the primary binding site for protein A is located on the Fc region between the CH2 and CH3 domains. Furthermore, protein A has been shown to bind to human IgG molecules containing the IgG F(ab')2 fragment derived from the human VH3 gene family. Protein A can bind with strong affinity to the Fc portion of certain immunoglobulins.

[0229] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures the real-time binding interaction between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing an analyte (binding protein on a biosensor matrix) and presenting a ligand (target antigen). As used herein, the term "KD" refers to the dissociation constant of the interaction between a specific binding protein and a target antigen.

[0230] As used herein, the term "specifically binds" refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with a Kd of at least about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 M or higher, and / or the ability to bind to an epitope with an affinity at least 2-fold higher than its affinity for a non-specific antigen. The binding affinity of an antigen for a binding protein or antibody can be determined by surface plasmon resonance (SPR) using a BIAcore device.

[0231] As used herein, the term “nucleic acid” refers to macromolecules, oligomeric macromolecules, or large biomolecules essential to all known forms of life. Nucleic acids, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made up of monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biomolecular chains that are wrapped around each other, forming a double helix. DNA chains are also known as polynucleotides, which are made up of nucleotides. Each nucleotide consists of a nitrogen-containing nucleic acid base, as well as a monosaccharide called deoxyribose or ribose and a phosphate group. Naturally occurring nucleic acid bases include guanine (G), adenine (A), thymine (T), uracil (U), or cytosine (C). Nucleotides are linked to each other in a chain by a covalent bond between the sugar of one nucleotide and the phosphate of the next nucleotide, resulting in alternating sugar-phosphate backbones. When the sugar is deoxyribose, the polymer is DNA. When the sugar is ribose, the polymer is RNA. Typically, polynucleotides are formed via phosphodiester bonds between individual nucleotide monomers.

[0232] As used herein, the term “polynucleotide” refers to a single-stranded or double-stranded nucleic acid polymer having a length of at least 10 nucleotides. It is understood that nucleotides comprising a polynucleotide may be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications, e.g., bromlysine; ribose modifications, e.g., arabinosides and 2',3'-dideoxyribose; and internucleotide bond modifications, e.g., phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladetes, and phosphoramidates. The term “polynucleotide” includes, in particular, DNA in single-stranded and double-stranded forms.

[0233] "Isolated polynucleotide" means a polynucleotide or some combination thereof of genomic, cDNA, or synthetic origin that (1) is not bound to all or part of naturally occurring polynucleotides; (2) is bound to polynucleotides that are not naturally linked; or (3) is not naturally present as part of a larger sequence.

[0234] An "isolated polypeptide" is defined as (1) it does not contain at least some other polypeptides that are normally found, (2) it is substantially free of other polypeptides from the same source, e.g., from the same species, (3) it is expressed by cells from a different species, (4) it is isolated from at least about 50% of the polynucleotides, lipids, carbohydrates or other substances to which it is bound in nature, (5) the "isolated polypeptide" is not bound (covalently or noncovalently) to any of the polypeptides that are bound in nature, (6) it is operably bound (covalently or noncovalently) to polypeptides that are not bound in nature, or (7) it does not exist in nature. Such isolated polypeptides are encoded by synthetic genomic DNA, cDNA, mRNA or other RNA, or any combination thereof. In exemplary embodiments, the isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, preventive, research or otherwise).

[0235] As used herein, the term “expression vector” also refers to an expression construct and typically refers to a plasmid or virus designed for protein expression in cells. The term “vector” refers to a protein or polynucleotide or mixture thereof that can or will introduce a protein and / or nucleic acid contained in a vector into a cell. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, viruses, or artificial chromosomes. In particular, vectors are used to transport a gene product of interest, such as exogenous or heterologous DNA, into a suitable host cell. A vector may contain a “replicon” polynucleotide sequence that facilitates the autonomous replication of the vector in the host cell. Exogenous DNA is defined as heterologous DNA that is not naturally found in a host cell and, for example, replicates a vector molecule, codes for a selection marker or a screenable marker, or codes for a transgene. Once inside a host cell, a vector can replicate independently of or concurrently with the host chromosomal DNA, and several copies of the vector and its inserted DNA can be produced. Furthermore, a vector may also contain necessary elements that enable the transcription of the inserted DNA into an mRNA molecule, or otherwise cause the inserted DNA to be replicated into multiple copies of the RNA. A vector may also contain “expression control sequences” that regulate the expression of the gene of interest. Typically, expression control sequences are polypeptides or polynucleotides, e.g., promoters, enhancers, silencers, insulators, or repressors, but are not limited to these. In a vector containing more than one polynucleotide encoding one or more gene products of interest, expression is controlled collectively or separately by one or more expression control sequences. More specifically, each polynucleotide contained on the vector may be controlled by a separate expression control sequence, or all polynucleotides contained on the vector may be controlled by a single expression control sequence. Polynucleotides contained on a single vector controlled by a single expression control sequence may form an open reading frame.Some expression vectors further include sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and / or enable the translation of the mRNA into protein molecules. Therefore, many molecules of mRNA and polypeptide encoded by the inserted DNA are rapidly synthesized.

[0236] As used herein, the term “host cell” refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to a specific target cell but also to the offspring of such cells. Such offspring may not actually be identical to the parent cell, as certain modifications may occur in the next generation either by mutation or environmental influence, but such cells are included within the scope of the term “host cell” as used herein. A wide range of host cell expression systems, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems), can be used to express the binding protein. An example of a suitable bacterial expression vector is pUC19. To recombinantly express the binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chain of the binding protein so that the polypeptide chain of the binding protein is expressed within the host cell, and in exemplary embodiments, the host cell may be secreted into the culture medium from which the binding protein is recovered.

[0237] As used herein, the term “pharmaceutical composition” means a compound or composition that, when appropriately administered to a subject, for example, a human subject, can induce a desired therapeutic effect.

[0238] The terms “pharmaceutically acceptable carrier” or “physiologically acceptable carrier,” as used herein, refer to one or more pharmaceutical materials suitable for achieving or enhancing the delivery of a binding protein.

[0239] The terms “effective dose” and “therapeutic dose” refer to the amount or dosage sufficient to produce the desired therapeutic effect when used in reference to a pharmaceutical composition containing one or more binding proteins (e.g., an antibody or its antigen-binding fragment). More specifically, the therapeutic dose is the amount of binding protein (e.g., an antibody or its antigen-binding fragment) sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated for a given period of time. The effective dose can vary depending on the specific antibody-like binding protein being used and also on various factors and conditions related to the patient being treated and the severity of the disorder. For example, when a binding protein or multispecific binding protein is administered in vivo, factors such as the patient's age, weight, and health, as well as dose-response curves and toxicity data obtained from preclinical animal studies, are considered among these factors. Determining the effective dose or therapeutic dose of a given pharmaceutical composition is within the scope of the skills of those skilled in the art.

[0240] As used herein, the term “method for generating a binding protein” means a method for recombining protein expression using techniques well known in the art.

[0241] II. CH1 / CL mutations that promote pairing In certain embodiments, mutations are induced at the CH1-CL interface to promote specific pairings and prevent CH1 / CL mispairings. Such mutations are described below and further described in International Publication 2013 / 005194, each incorporated herein by reference, and in Golay et al., (2016) J.Immunol. 196, pp. 3199–3211.

[0242] The first set of mutations is made for a pair of interacting polar interfacial amino acids within the CH1 and CL domains. Such polar amino acids can be replaced with a pair of neutral and salt-forming amino acids. In certain embodiments, the first set of mutations may include a T192E CH1 mutation and an N137K,S114A CL kappa mutation. The T192E CH1 mutation and the N137K CL kappa mutation form a salt bridge, which may enhance the specificity of association, but undesirable pairings should be avoided due to a lack of steric and charge complementarity between the wild-type and mutant CH1 and CL kappa domains. Furthermore, the S114A mutation on the CL kappa domain is made to avoid steric collisions with larger lysine side chains. The CH1 T192E and CL N137K / S114A mutation set may instead be called the "CR3" mutation set.

[0243] A second set of mutations is created for pairs of interacting hydrophobic and polar interface amino acid residues in the CH1 and CL kappa domains. A single mutation may constitute a switch from hydrophobic to polar interactions. In certain embodiments, the second set of mutations may include L143Q, S188V CH1 mutations and V133T, S176V CL kappa mutations. The L134Q CH1 mutation and the V133T CL kappa mutation constitute a switch from hydrophobic to polar interactions. The simultaneous S188V CH1 mutation and the S176V CL kappa mutation constitute a switch from polar to hydrophobic interactions. The exchange of polar / hydrophobic properties of the interface interaction is expected to maintain the affinity between the mutant CH1 domain and the CL kappa domain without changing it, while reducing their respective affinities to other wild-type corresponding CH1 and CL kappa domains, and thus prevent mispairing due to undesirable interactions occurring in mismatched (mutant / wild-type) domains. The CH1 L143Q / S188V and CL V133T / S176V mutation sets may instead be called the "MUT4" mutation set.

[0244] The third and fourth sets of mutations are "knob-into-hole" mutations. More specifically, the third set of mutations (KH1) produces the L124A, L143E CH1 mutation and the V133W CL kappa mutation. The fourth set of mutations (KH2) produces the V190A CH1 mutation and the L135W, N137A CL kappa mutation. The first, second, third, and fourth sets of mutations are described in more detail in International Publication No. 2013 / 005194A1.

[0245] Fifth and sixth sets of mutations can be created to exchange the static charge of the CH1 and CL kappa domains. In certain embodiments, the fifth set of mutations may include the K221E CH1 mutation and the E123K CL kappa mutation. The fifth set of mutations is instead referred to as the "K221E / E123K opposite charge" mutation set or the "NN1" mutation set. In certain embodiments, the sixth set of mutations may include the K228D CH1 mutation and the D122K CL kappa mutation. The sixth set of mutations is instead referred to as the "K228D / D122K opposite charge" mutation set or the "NN2" mutation set. The fifth and sixth sets of mutations are described in more detail in International Publication No. 2007 / 147901A1. In certain embodiments, the "K221E / E123K opposite charge" mutation set and the "K228D / D122K opposite charge" mutation set can be combined. Therefore, this combination may include K221E and K228D CH1 mutant pairs, as well as E123K and D122K CL kappa mutant pairs. Mutations in the combined set are instead called the "K221E:K228D / E123K:D122K opposite charge" mutant set or the "NN3" mutant set.

[0246] Seventh and eighth sets of mutations can be created to exchange the static charge of the CH1 and CL kappa domains. In certain embodiments, the seventh set of mutations may include L143E, L143D, L143R, L143K, or L143H CH1 mutations and S176E, S176D, S176R, S176k, or S176H CL kappa mutations, provided that the CH1 mutation has the opposite charge to the CL kappa mutation. The seventh set of mutations is alternatively referred to as the “L143 / S176 opposite charge” mutation set. In certain embodiments, the CH1 L143E or L143D mutation pairs with the CL S176R or S176K mutation and is referred to as the “CM3” mutation set. In certain embodiments, the CH1 L143R or L143K mutation pairs with the CL S176E or S176D mutation and is referred to as the “CM4” mutation set. In certain embodiments, the eighth set of mutations may include L124E, L124D, L124R, L124K, or L124H CH1 mutations, and V133E, V133D, V133R, V133K, or V133H CL Kappa mutations, provided that the CH1 mutation has the opposite charge to the CL Kappa mutation. The eighth set of mutations is alternatively referred to as the “L124 / V133 opposite charge” mutation set. In certain embodiments, the CH1 L124E or L124D mutation pairs with the CL V133R or V133K mutation and is referred to as the “CM5” mutation set. In certain embodiments, the CH1 L124R or L124K mutation pairs with the CL V133E or V133D mutation and is referred to as the “CM6” mutation set.

[0247] In further embodiments, one or more of the above-described mutations may be combined with each other and / or with the mutations listed below. For example, the CH1 domain of the first Fab may contain the T192E, K221E mutation, and the CL kappa domain of the first Fab may contain the E123K, N137K, S114A mutation. The CH1 domain of the second Fab may further contain the L143Q, S188V mutation, and the CL kappa domain of the second Fab may contain the V133T, S176V mutation. Alternatively, the second Fab may be wild-type.

[0248] The sequence position numbers used herein for the CH1 and CL kappa domains refer to Kabat numbering (Kabat, EA et al., Sequences of proteins of immunological interest. 5th ed. - US Department of Health and Human Services, NIH publications 91-3242, pp. 662, 680, 689, 1991).

[0249] III. VH / VL opposite-charged mutations to promote pairing In certain embodiments, mutations are induced at the VH and VL interface to promote specific pairings and prevent VH / VL mispairings. In certain embodiments, mutations made in the VH and VL domains introduce oppositely charged amino acid residues to promote heterodimerization via electrostatic interactions.

[0250] In certain embodiments, a pair of opposite-charged mutations may include a mutation in the VH domain at Kabat position 39 and a mutation in the VL domain at Kabat position 38. Provided that the mutation introduced at VL Kabat position 38 is oppositely charged, any known positively or negatively charged residue can be introduced at VH Kabat position 39. For example, one possible mutation pair includes a VH K39 mutant residue (introducing a positive charge) and a VL E38 mutant residue (introducing a negative charge). Alternatively, a reversion mutation set can be constructed, introducing a VH E39 mutant residue (introducing a negative charge) and a VL K38 mutant residue (introducing a positive charge). In certain embodiments, the VH domain may contain a residue mutated to E, D, K, R, or H at Kabat position 39, and the VL domain may contain a residue mutated to E, D, K, R, or H at Kabat position 38, provided that the mutant residue in the VH domain is oppositely charged to the residue mutated in the VL domain. For example, if the VH domain contains a mutant residue E or D at Kabat position 39, the VL domain contains a mutant residue K, R, or H at Kabat position 38. Alternatively, if the VH domain contains a mutant residue K, R, or H at Kabat position 39, the VL domain contains a mutant residue E or D at Kabat position 38. In certain embodiments where two VH / VL pairs are not present in the same polypeptide chain, if one VH / VL pair contains a positive charge (e.g., E or D) at VH39 and a negative charge (e.g., K, R, or H) at VL38, another VH / VL pair may contain a negative charge (e.g., K, R, or H) at VH39 and a positive charge (e.g., E or D) at VL38. For example, if the first VH / VL pair contains VH39K and VL38E mutations, the second VH / VL pair may contain VH39E and VL38K mutations. The counter-charged mutations in this set can instead be called the "VH39 / VL38 counter-charged" mutation set. These counter-charged mutations are described in more detail by Tan et al., Biophysical Journal, Vol. 75, pp. 1473-1482, 1998.

[0251] In certain embodiments, a pair of opposite-charged mutations may include a Q39 mutation in the VH domain and a Q38 mutation in the VL domain. Any known positively or negatively charged residue can be introduced at the VH Q39 position, provided that the mutation introduced at the VL Q38 position has the opposite charge. For example, one possible mutation pair includes a VH Q39K mutation (introducing a positive charge) and a VL Q38E mutation (introducing a negative charge). Alternatively, a revertant mutation set can be constructed that introduces a VH Q39E mutation (introducing a negative charge) and a VL Q38K mutation (introducing a positive charge). In certain embodiments, the VH domain may include Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and the VL domain may include Q38E, Q38D, Q38K, Q38R, or Q38H mutations, provided that the mutation in the VH domain has the opposite charge to the mutation in the VL domain.

[0252] In further embodiments, one or more of the above-described mutations can be combined with each other and / or with the mutations described below.

[0253] IV. VH / VL disulfide-stabilized mutation In certain embodiments, mutations can be introduced at the VH and VL interfaces to improve the stability between the VH and VL interfaces. A specific series of amino acid mutations in the VH and VL domains can improve stability through the introduction of unnatural cysteine ​​residues that form disulfide bridges.

[0254] A first set of disulfide-stabilizing mutations can be generated for amino acid residues in the VH and VL domains. In certain embodiments, the first set of disulfide-stabilizing mutations may include a 44C mutation in the VH domain and a 100C mutation in the VL domain. The first set of disulfide-stabilizing mutations is alternatively referred to as the “VH44C / VL100C” mutation set. The first set of disulfide-stabilizing mutations is described in further detail in Reiter et al., Nature Biotechnology, Vol. 14, pp. 1239–1245, 1996, which is incorporated herein by reference for all purposes.

[0255] A second set of disulfide-stabilizing mutations can be generated for amino acid residues in the VH and VL domains. In certain embodiments, the second set of disulfide-stabilizing mutations may include a 105C mutation in the VH domain and a 43C mutation in the VL domain. The second set of disulfide-stabilizing mutations is alternatively referred to as the "VH105C / VL43C" mutation set. The second set of disulfide-stabilizing mutations is described in further detail in U.S. Patent No. 9,527,927, which is incorporated herein by reference for all purposes.

[0256] V. Combinations of antibody mutations In certain embodiments, the combination of mutation sets is located at the CH1 and CL kappa interface and / or the VH and VL interface, which can further promote pairing and improve stability.

[0257] In certain embodiments, the first Fab domain in the antibody may, in combination with the VH / VL opposite-charged mutant set, contain one or both of the CR3 and MUT4 mutant sets. In further embodiments, the second Fab domain in the antibody may, in combination with the VH / VL opposite-charged mutant set, contain one or both of the CR3 and MUT4 mutant sets.

[0258] In certain embodiments, the first Fab domain may contain one or both of the CR3 and MUT4 mutation sets in combination with the VH / VL opposite-charged mutation set and the disulfide-stabilized mutation set. In further embodiments, the second Fab domain in the antibody may contain one or both of the CR3 and MUT4 mutation sets in combination with the VH / VL opposite-charged mutation set and the disulfide-stabilized mutation set.

[0259] Any of the above embodiments may further include a set of oppositely charged mutations at the CH1 / CL interface. For example, the first Fab may include a K221E CH1 mutation and an E123K CL kappa mutation. The second Fab may include a K221E CH1 mutation and an E123K CL kappa mutation.

[0260] VI. Antibody format and mutation set therein Any of the above-described mutant sets and combinations thereof can be applied to the multispecific antigen-binding proteins described herein.

[0261] Crossover dual variables In certain embodiments, “crossover dual variable” or “CODV” refers to an antigen-binding domain comprising at least two polypeptide chains that specifically bind to at least one target antigen or at least one target epitope and form at least two antigen-binding sites, wherein at least one polypeptide chain has the formula: VL1-L1-VL2-L2-CL [I] The structure includes the form represented by , and at least one polypeptide chain is of the formula: VH2-L3-VH1-L4-CH1 [II] Includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; L1, L2, L3, and L4 are amino acid linkers. The polypeptides of formula I and formula II form crossed light-chain-heavy-chain pairs.

[0262] In certain embodiments, the CODV antigen-binding domain comprises four polypeptide chains that specifically bind to at least one target antigen or at least one target epitope, forming four antigen-binding sites, two polypeptide chains each having the formula: VL1-L1-VL2-L2-CL [I] The structure includes the form represented by the formula, and the two polypeptide chains are each: VH2-L3-VH1-L4-CH1-Fc [II] Includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; Fc is the immunoglobulin hinge region, as well as the constant domains of the CH2 and CH3 immunoglobulin heavy chains; L1, L2, L3, and L4 are amino acid linkers. The polypeptides of formula I and formula II form crossed light-heavy chain pairs. The VH1 / VL1 pair contains a primary antigen-binding specificity, and the VH2 / VL2 pair contains a secondary antigen-binding specificity.

[0263] In certain embodiments, the antigen-binding protein described herein is a trispecific and / or trivalent antigen-binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more different antigen targets, wherein the first polypeptide chain has the formula: VL2-L1-VL1-L2-CL [I] Includes a structure represented by, The second polypeptide chain is given by formula: VH1-L3-VH2-L4-CH1-Hinge-CH2-CH3 [II] The structure includes the form represented by , and the third polypeptide chain is given by formula: VH3-CH1-Hinge-CH2-CH3 [III] The structure includes the form represented by , and the fourth polypeptide chain is given by formula: VL3-CL [IV] Includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; CH2 is the constant domain of the immunoglobulin CH2 double chain; CH3 is the constant domain of the immunoglobulin CH3 heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 domain and the CH2 domain; and L1, L2, L3, and L4 are amino acid linkers. The polypeptides of formula I and formula II form crossed light-chain-heavy-chain pairs.

[0264] In certain embodiments, the first and second polypeptide chains have a cross-orientation that forms two different antigen-binding sites. In some embodiments, VH1 and VL1 form a binding pair to form a first antigen-binding site. In some embodiments, VH2 and VL2 form a binding pair to form a second antigen-binding site. In some embodiments, the third and fourth polypeptides form a third antigen-binding site. In some embodiments, VH3 and VL3 form a binding pair to form a third antigen-binding site.

[0265] Such an antigen-binding protein contains at least three antigen-binding sites. It is at least a trivalent antigen-binding molecule. In a particular embodiment, it specifically binds to one antigen target, i.e., it is a single-specific antigen-binding molecule. In another embodiment, it specifically binds to two different antigen targets, i.e., it is a bispecific antigen-binding molecule. In yet another embodiment, it specifically binds to three different antigen targets, i.e., it is a triplicate antigen-binding molecule.

[0266] The examples listed above are not intended to limit the scope of the present invention in any way, and linkers comprising amino acids randomly selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline are shown to be suitable for antibody-like binding proteins as described herein.

[0267] Further details regarding the CODV antibody format, various substitutions of the CODV antibody format, and linkers are further described in International Publication Nos. 2012 / 135345A1 and International Publication Nos. 2017 / 180913A2, which are incorporated herein by reference in their entirety.

[0268] Tandem Fab In certain embodiments, “tandem Fab” refers to an antigen-binding protein in which the C-terminus of one CH1 region of the first Fab domain is operably ligated to the N-terminus of the VH region of the second Fab domain. In certain embodiments, the tandem fab antibody may be tetravalent and monospecific (each of the four Fabs binds to the same antigen). In certain embodiments, the tandem fab antibody may be tetravalent and bispecific (two of the four Fabs bind to the first antigen or epitope, while the other two Fabs bind to the second antigen or epitope).

[0269] A tandem fab is operably linked to any known peptide linker in the art used to link two or more antigen-binding domains. In certain embodiments, the peptide linker is a Gly-Ser linker, i.e., a linker containing only glycine amino acids(s) and serine amino acids(s). In certain embodiments, the peptide linker is (Gly-Gly-Gly-Gly-Ser) n The linker is (sequence number x), where n is any integer from 1 to 5. In certain embodiments, the peptide linker is (Gly-Gly-Gly-Gly-Ser)3(sequence number x) linker. In certain embodiments, the peptide linker is (Gly-Gly-Gly-Gly-Ser)2(sequence number x) linker.

[0270] Alternatively, or in combination with the Gly-Ser linker described above, the peptide linker may contain all or part of the sequence of the hinge region of one or more immunoglobulins selected from IgA, IgG, and IgD. The sequences of the hinge regions of human IgG, IgA, and IgD are shown below: IgA1 (Sequence ID X): Val-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser IgA2 (Sequence ID X): Val-Pro-Pro-Pro-Pro-Pro IgD (Sequence ID X): Glu-Ser-Pro-Lys-Ala-Gln-Ala-Ser-Ser-Val-Pro-Thr-Ala-Gln-Pro-Gln-Ala-Glu-Gly-Ser-Leu-Ala-Lys-Ala-Thr-Thr-Ala-Pro-Ala -Thr-Thr-Arg-Asn-Thr-Gly-Arg-Gly-Gly-Glu-Glu-Lys-Lys-Lys-Glu-Lys-Glu-Lys-Glu-Glu-Gln-Glu-Glu-Arg-Glu-Thr-Lys-Thr-Pro IgG1 (Sequence Number X): Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys-Pro IgAG2 (Sequence ID X): Glu-Arg-Lys-Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys-Pro IgG3: Glu-Leu-Lys-Thr-Pro-Leu-Gly-Asp-Thr-Thr-His-Thr-Cys-Pro-Arg-Cys-Pro(Sequence ID X) followed by 0 or 1 to 4 repetitions of Glu-Pro-Lys-Ser-Cys-Asp-Thr-Pro-Pro-Pro-Cys-Pro-Arg-Cys-Pro(Sequence ID X) IgG4: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro(Sequence ID X)

[0271] The peptide linker described above may contain all or part of the sequence of the hinge region of a single immunoglobulin. In this case, the immunoglobulin may belong to the same isotype and subclass as the immunoglobulin from which the adjacent CH1 domain originates, or to a different isotype or subclass.

[0272] Alternatively, the peptide linker may include all or part of the hinge region sequences of at least two immunoglobulins of different isotypes or subclasses. In this case, the N-terminal portion of the peptide linker directly following the CH1 domain may consist of all or part of the hinge region of an immunoglobulin belonging to the same isotype and subclass as the immunoglobulin from which the CH1 domain originates. Optionally, the peptide linker may further include sequences of 2 to 15 or 5 to 10 N-terminal amino acids of the CH2 domain of the immunoglobulin.

[0273] In certain embodiments, sequences derived from natural hinge regions can be used. In other embodiments, point mutations can be used to replace one or more cysteine ​​residues in these sequences, particularly natural IgG1, IgG2, or IgG3 hinge sequences, with alanine or serine to avoid undesirable intra-chain or inter-chain disulfide bonds.

[0274] A non-limiting example of a peptide linker that can be used in the antigen-binding protein of this disclosure is a peptide linker having the following sequence: Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-Glu-Leu-Leu-Gly-Gly-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Gly-Gly (SEQ ID NO: x). The peptide linker consists of the full-length sequence of the human IgG1 hinge, followed by the nine N-terminal amino acids of human IgG1 CH2 (Ala-Pro-Glu-Leu-Leu-Gly-Gly-Pro-Ser, SEQ ID NO: x), followed by a portion of the sequence of the human IgA1 hinge (Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser, SEQ ID NO: x), and a dipeptide GG, which is conferred to the linker to provide supplemental flexibility. In certain embodiments, the peptide linker Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-Glu-Leu-Leu-Gly-Gly-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Gly-Gly(Sequence ID X) may have one or more cysteine ​​residues substituted to eliminate disulfide bond formation. In certain embodiments, the peptide linker comprises the following sequence:Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Ala-Pro-Glu-Leu-Leu-Gly-Gly-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Gly-Gly(Sequence ID X). The hinge-derived peptide linker is further described in International Publication No. 2013 / 005194A2, which is incorporated herein by reference in its entirety.

[0275] VII. Exemplary antibody mutations and antibody format combinations Any of the antibody mutations described above can be combined with any of the antibody formats described above.

[0276] In certain embodiments, the antigen-binding protein of this disclosure may comprise a CODV antibody format having one or more of the CR3 mutant set, the MUT4 mutant set, the L143 / S176 countercharged mutant set, and the L124 / V133 countercharged mutant set. The antigen-binding protein may further comprise one or more VH / VL countercharged mutant sets. One or more VH / VL countercharged mutant sets include, but are not limited to, the VH39 / VL38 countercharged mutant set. The antigen-binding protein may further comprise one or more VH / VL disulfide-stabilized mutant sets. One or more VH / VL disulfide mutant sets include, but are not limited to, the VH44C / VL100C mutant set and the VH105C / VL43C mutant set. The antigen-binding protein may further comprise one or more CH1 / CL countercharged mutant sets. One or more CH1 / CL opposite-charged mutation sets may include, but are not limited to, K221E / E123K opposite-charged mutation sets.

[0277] In certain embodiments, the antigen-binding protein of the present disclosure comprises two polypeptide chains forming two antigen-binding sites, one polypeptide chain having the formula: VL1-L1-VL2-L2-CL[I] It has a structure represented by the formula: VH2-L3-VH1-L4-CH1[II] It has a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; and L1, L2, L3, and L4 are amino acid linkers. The polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair. VH1 and VH2, or one or both, contain the VH44C mutation, and VL1 and VL2, or one or both, contain the VL100C mutation, forming a disulfide bond. VH1 and VH2, or either one or both, contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and VL1 and VL2, or either one or both, contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations. Mutations in one or both of VH1 and VH2 are oppositely charged to mutations in one or both of VL1 and VL2. The CH1 domain contains one or more of the T192E, L143Q, and S188V mutations, and the CL domain contains one or more of the N137K, S114A, V133T, and S176V mutations.

[0278] In certain embodiments, the antigen-binding protein of this disclosure may comprise a tandem Fab antibody format having one or more of the following: a CR3 mutation set, a MUT4 mutation set, an L143 / S176 countercharged mutation set, and an L124 / V133 countercharged mutation set. The antigen-binding protein may further comprise one or more VH / VL countercharged mutation sets. One or more VH / VL countercharged mutation sets include, but are not limited to, a VH39 / VL38 countercharged mutation set. The antigen-binding protein may further comprise one or more VH / VL disulfide-stabilized mutation sets. One or more VH / VL disulfide mutation sets include, but are not limited to, a VH44C / VL100C mutation set and a VH105C / VL43C mutation set. The antigen-binding protein may further comprise one or more CH1 / CL countercharged mutation sets. One or more CH1 / CL opposite-charged mutant sets may include, but are not limited to, the K221E / E123K opposite-charged mutant set.

[0279] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1 The first light chain (LC1) / heavy chain (HC1) pair including; and b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2 The second light chain (LC2) / heavy chain (HC2) pair including It can include, The C-terminus of CH1-1 is operably connected to the N-terminus of VH2. VH1 and VH2, or both, contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and VL1 and VL2, or both, contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in VH1 and VH2, or both, are oppositely charged to the mutations in VL1 and VL2, or both, One or both of the CH1-1 and CH1-2 domains contain one or more of the T192E, L143Q, and S188V mutations, and one or both of the CL1 and CL2 domains contain one or more of the N137K, S114A, V133T, and S176V mutations. Mutations in CH1-1 and CL1 that promote pairing are different from mutations in CH1-2 and CL2 that promote pairing.

[0280] In certain embodiments, the antigen-binding protein of this disclosure may include a conventional Y-shaped antibody format having one or more CR3 mutant sets, MUT4 mutant sets, L143 / S176 countercharged mutant sets, and L124 / V133 countercharged mutant sets. The antigen-binding protein may further include one or more VH / VL countercharged mutant sets. One or more VH / VL countercharged mutant sets include, but are not limited to, the VH39 / VL38 countercharged mutant set. The antigen-binding protein may further include one or more VH / VL disulfide-stabilized mutant sets. One or more VH / VL disulfide-stabilized mutant sets include, but are not limited to, the VH44C / VL100C mutant set and the VH105C / VL43C mutant set. The antigen-binding protein may further include one or more CH1 / CL countercharged mutant sets. One or more CH1 / CL opposite-charged mutant sets may include, but are not limited to, the K221E / E123K opposite-charged mutant set.

[0281] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, VH1 and VH2, or both, contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and VL1 and VL2, or both, contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in VH1 and VH2, or both, are oppositely charged to the mutations in VL1 and VL2, or both, The CH1-1 domain and / or the CH1-2 domain contain one or more of the T192E, L143Q, and S188V mutations, and the CL1 and / or the CL2 domain contain one or more of the N137K, S114A, V133T, and S176V mutations. Mutations in CH1-1 and CL1 that promote pairing are different from mutations in CH1-2 and CL2 that promote pairing.

[0282] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, VH1 and VH2, or both, contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and VL1 and VL2, or both, contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in VH1 and VH2, or both, are oppositely charged to the mutations in VL1 and VL2, or both, The CH1-1 domain and / or the CH1-2 domain contain one or more of the T192E, L143Q, and S188V mutations, and the CL1 and / or the CL2 domain contain one or more of the N137K, S114A, V133T, and S176V mutations. Mutations in CH1-1 and CL1 that promote pairing differ from mutations in CH1-2 and CL2 that promote pairing. The CH1-1 and CH1-2 domains, or both, further contain the K221E mutation, and the CL1 and CL2 domains, or both, further contain the E123K mutation.

[0283] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, VH1 and VH2, or both, contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and VL1 and VL2, or both, contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in VH1 and VH2, or both, are oppositely charged to the mutations in VL1 and VL2, or both, One or both of the CH1-1 and CH1-2 domains contain L143E, L143D, L143K, L143R, or L143H mutations, one or both of the CL1 and CL2 domains contain S176E, S176D, S176K, S176R, or S176H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0284] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, VH1 and VH2, or both, contain Q39E, Q39D, Q39K, Q39R, or Q39H mutations, and VL1 and VL2, or both, contain Q38E, Q38D, Q38K, Q38R, or Q38H mutations, and the mutations in VH1 and VH2, or both, are oppositely charged to the mutations in VL1 and VL2, or both, One or both of the CH1-1 and CH1-2 domains contain L124E, L124D, L124K, L124R, or L124H mutations, one or both of the CL1 and CL2 domains contain V133E, V133D, V133K, V133R, or V133H mutations, and the mutations in one or both of CH1-1 or CH1-2 are oppositely charged to the mutations in one or both of CL1 and CL2.

[0285] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143E or L143D mutations, and the CL1 domain contains S176R or S176K mutations.

[0286] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143R or L143K mutations, and the CL1 domain contains S176E or S176D mutations.

[0287] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD2) The first light chain (LC1) / heavy chain (HD1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation.

[0288] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143E or L143D mutations, and the CL1 domain contains S176R or S176K mutations. One or both of the CH1-1 and CH1-2 domains contain the K221E mutation, and one or both of the CL1 and CL2 domains contain the E123K mutation.

[0289] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143R or L143K mutations, and the CL1 domain contains S176E or S176D mutations. One or both of the CH1-1 and CH1-2 domains contain the K221E mutation, and one or both of the CL1 and CL2 domains contain the E123K mutation.

[0290] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain the K221E mutation, and one or both of the CL1 and CL2 domains contain the E123K mutation.

[0291] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) Including the first light chain (LC1) / heavy chain (HC1) pair; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143E or L143D mutations, and the CL1 domain contains S176R or S176K mutations. One or both of the CH1-1 and CH1-2 domains contain the K228D mutation, and one or both of the CL1 and CL2 domains contain the D122K mutation.

[0292] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143R or L143K mutations, and the CL1 domain contains S176E or S176D mutations. One or both of the CH1-1 and CH1-2 domains contain the K228D mutation, and one or both of the CL1 and CL2 domains contain the D122K mutation.

[0293] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain the K228D mutation, and one or both of the CL1 and CL2 domains contain the D122K mutation.

[0294] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143E or L143D mutations, and the CL1 domain contains S176R or S176K mutations. One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations.

[0295] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143R or L143K mutations, and the CL1 domain contains S176E or S176D mutations. One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations.

[0296] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations.

[0297] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143E or L143D mutations, and the CL1 domain contains S176R or S176K mutations. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation.

[0298] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143R or L143K mutations, and the CL1 domain contains S176E or S176D mutations. The VH1 domain contains the VH39E mutation, and the VL1 domain contains the VL38K mutation.

[0299] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation.

[0300] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation. The VH2 domain contains the VH39E mutation, and the VL2 domain contains the VL38K mutation.

[0301] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143E or L143D mutations, and the CL1 domain contains S176R or S176K mutations. One or both of the CH1-1 and CH1-2 domains contain the K221E mutation, and one or both of the CL1 and CL2 domains contain the E123K mutation. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation.

[0302] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143R or L143K mutations, and the CL1 domain contains S176E or S176D mutations. One or both of the CH1-1 and CH1-2 domains contain the K221E mutation, and one or both of the CL1 and CL2 domains contain the E123K mutation. The VH1 domain contains the VH39E mutation, and the VL1 domain contains the VL38K mutation.

[0303] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain the K221E mutation, and one or both of the CL1 and CL2 domains contain the E123K mutation, The VH2 domain contains the VH39E mutation, and the VL2 domain contains the VL38K mutation.

[0304] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1); The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain the K221E mutation, and one or both of the CL1 and CL2 domains contain the E123K mutation. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation. The VH2 domain contains the VH39E mutation, and the VL2 domain contains the VL38K mutation.

[0305] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143E or L143D mutations, and the CL1 domain contains S176R or S176K mutations. One or both of the CH1-1 and CH1-2 domains contain the K228D mutation, and one or both of the CL1 and CL2 domains contain the D122K mutation. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation.

[0306] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143R or L143K mutations, and the CL1 domain contains S176E or S176D mutations. One or both of the CH1-1 and CH1-2 domains contain the K228D mutation, and one or both of the CL1 and CL2 domains contain the D122K mutation. The VH1 domain contains the VH39E mutation, and the VL1 domain contains the VL38K mutation.

[0307] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain the K228D mutation, and one or both of the CL1 and CL2 domains contain the D122K mutation. The VH2 domain contains the VH39E mutation, and the VL2 domain contains the VL38K mutation.

[0308] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) A first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain the K228D mutation, and one or both of the CL1 and CL2 domains contain the D122K mutation. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation. The VH2 domain contains the VH39E mutation, and the VL2 domain contains the VL38K mutation.

[0309] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143E or L143D mutations, and the CL1 domain contains S176R or S176K mutations. One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation.

[0310] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains L143R or L143K mutations, and the CL1 domain contains S176E or S176D mutations. The VH1 domain contains the VH39E mutation, and the VL1 domain contains the VL38K mutation. One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations.

[0311] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations. The VH2 domain contains the VH39E mutation, and the VL2 domain contains the VL38K mutation.

[0312] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, The CH1-1 domain contains an L143E or L143D mutation, the CH1-2 domain contains an L143R or L143K mutation, the CL1 domain contains an S176R or S176K mutation, and the CL2 domain contains an S176E or S176D mutation. One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation. The VH2 domain contains the VH39E mutation, and the VL2 domain contains the VL38K mutation.

[0313] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation.

[0314] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations. The VH1 domain contains the VH39E mutation, and the VL1 domain contains the VL38K mutation.

[0315] In certain embodiments, the multispecific antibodies of this disclosure are as follows: a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) The first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) to form a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including It can include, HD1 and HD2 heterodimerize, One or both of the CH1-1 and CH1-2 domains contain K221E and K228D mutations, and one or both of the CL1 and CL2 domains contain D122K and E123K mutations. The VH1 domain contains the VH39K mutation, and the VL1 domain contains the VL38E mutation. The VH2 domain contains the VH39E mutation, and the VL2 domain contains the VL38K mutation.

[0316] VIII. Pharmaceutical Formulations / Pharmaceutical Compositions In certain embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antigen-binding protein as described herein. Some embodiments include a pharmaceutical composition comprising a therapeutically effective amount of any one of the binding proteins described herein, or a binding protein-drug conjugate, mixed with a pharmaceutically or physiologically acceptable formulation selected for suitability with the mode of administration.

[0317] Acceptable formulation materials are typically non-toxic to the recipient at the dosage and concentration used.

[0318] In some embodiments, the pharmaceutical composition may include, for example, formulation materials for modifying, maintaining, or preventing the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition.Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), fillers (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavorings, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), and preservatives. Examples include benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., Pluronics; PEG; sorbitan esters; polysorbates, e.g., polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol, or tyroxapal), stability enhancers (e.g., sucrose or sorbitol), isotonic enhancers (e.g., alkali metal halides, e.g., sodium chloride or potassium chloride, or mannitol sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (e.g., REMINGTON'S, incorporated herein by reference for all purposes). See PHARMACEUTICAL SCIENCES (18th edition, edited by Argennaro, Mack Publishing Company, 1990) and subsequent editions.

[0319] In some embodiments, the optimal pharmaceutical composition is determined by those skilled in the art, depending, for example, on the intended route of administration, the delivery format, and the desired dosage. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0320] In some embodiments, the primary vehicle or carrier in the pharmaceutical composition may be either naturally aqueous or non-aqueous. For example, a suitable vehicle or carrier for injection may be water, saline, or artificial cerebrospinal fluid, and other common substances may be supplemented in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at approximately pH 7.0–8.5, or acetate buffer at approximately pH 4.0–5.5, which may further include sorbitol or suitable substitutions. In one embodiment of the present disclosure, the binding protein composition may be prepared for storage by mixing a selected composition having a desired degree of purity with any formulation in the form of a lyophilized cake or aqueous solution. Furthermore, the binding protein may be formulated as a lyophilized agent using a suitable excipient, such as sucrose.

[0321] In some embodiments, the pharmaceutical compositions of this disclosure may be selected for parenteral or subcutaneous delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the gastrointestinal tract, for example, orally. The preparation of such pharmaceutically acceptable compositions is within the scope of the art.

[0322] In some embodiments, the formulation components are present at an acceptable concentration at the administration site. For example, buffers are used to maintain the composition at or slightly below the physiological pH, typically within a pH range of about 5 to about 8.

[0323] When parenteral administration is intended, the therapeutic composition for use may be in the form of a parenterally acceptable aqueous solution that is pyrogenically free and contains the desired binding protein in a pharmaceutically acceptable vehicle. A vehicle particularly suitable for parenteral injection is sterile distilled water, in which the binding protein is formulated as a properly preserved sterile isotonic solution. Yet another preparation may involve the formulation of the desired molecule using the drug, e.g., microspheres for injection, bioerosive particles, polymer compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, which then provide controlled or sustained release of the product delivered via depot injection. Hyaluronic acid may also be used, which may have the effect of promoting duration in circulation. Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0324] In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the binding protein can be formulated as a dry powder for inhalation. The binding protein inhalation solution can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be sprayed.

[0325] Furthermore, it is intended that certain formulations may be administered orally. In one embodiment of this disclosure, the multispecific binding protein administered in this manner may be formulated with or without carriers conventionally used in solid drug formulations, such as tablets and capsules. For example, capsules may be designed to release the active portion of the formulation at the gastrointestinal tract, when bioavailability is maximized and pre-systemic degradation is minimized. Further substances may be included to enhance the absorption of the binding protein. Diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0326] Another pharmaceutical composition may involve an effective amount of multispecific binding protein in a mixture with a non-toxic excipient suitable for the manufacture of tablets. The solution can be prepared in unit dose form by dissolving the tablets in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents, e.g., calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders, e.g., starch, gelatin or acacia; or lubricants, e.g., magnesium stearate, stearic acid or talc.

[0327] Further pharmaceutical compositions of this disclosure will be apparent to those skilled in the art and include formulations with binding proteins in sustained-release or controlled-release formulations. Various other sustained-release or controlled-release means, such as liposome carriers, bio-erosive microparticles or porous beads, and techniques for formulating depot injections are also known to those skilled in the art. Further examples of sustained-release preparations include semipermeable polymer matrices in the form of articles, such as films or microcapsules. Sustained-release matrices may include copolymers of polyesters, hydrogels, polylactides, L-glutamic acid and gamma-ethyl-L-glutamic acid, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0328] In some embodiments, the pharmaceutical composition is to be used for in vivo administration and is typically sterile. This can be achieved by filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Furthermore, parenteral compositions are generally placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be penetrated by a subcutaneous needle.

[0329] Once a pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as an anhydrous or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form requiring reconstitution before administration (e.g., lyophilized).

[0330] The disclosure also encompasses kits for generating single-dose units. Each kit may include both a first container having a dried multispecific binding protein and a second container having an aqueous formulation. The scope of the disclosure also includes kits comprising single and multi-chamber pre-filled syringes (e.g., liquid syringes and rio-syringes).

[0331] The therapeutically effective amount of binding protein in a pharmaceutical composition depends, for example, on the therapeutic background and purpose. Therefore, those skilled in the art will understand that the appropriate dose level for treatment varies in part depending on the delivered molecule, the indication in which the binding protein is used, the route of administration, and the patient's size (body weight, body surface, or organ size) and condition (age and overall health). Accordingly, clinicians can determine the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0332] The frequency of administration depends on the pharmacokinetic parameters of the binding protein in the formulation used. Typically, clinicians administer the composition until the desired effect is achieved. Therefore, the composition can be administered as a single dose, as two or more doses (which may or may not contain the same amount as the desired molecule), over time, or as a continuous infusion via an implantable device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of routine tasks performed by those skilled in the art. The appropriate dosage can be confirmed by using appropriate dose-response data.

[0333] The route of administration of the pharmaceutical composition shall follow known methods, such as oral administration; injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by a continuous-release system; or by an implantable device. If desired, the composition may be administered by bolus injection, by continuous infusion, or by an implantable device.

[0334] In some embodiments, the composition can also be administered topically via implantation of a membrane, sponge, or other suitable material in which the desired molecule is absorbed or encapsulated. When an implantation device is used, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be carried out via diffusion, sustained-release bolus, or continuous administration.

[0335] VIII. Treatment / Usage Another aspect of this disclosure is multispecific antibodies and / or antigen-binding proteins described herein for use as pharmaceuticals.

[0336] In certain embodiments, a method is provided for treating a disease in which antigenic activity is harmful, comprising administering an effective amount of the antigen-binding protein described herein to a subject in need thereof.

[0337] The binding protein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantification of one or more target antigens. The binding protein binds to one or more target antigens with an affinity suitable for the assay method used.

[0338] For diagnostic purposes, in some embodiments, the binding protein can be labeled with a detectable moiety. The detectable moiety may be any substance that can directly or indirectly generate a detectable signal. For example, the detectable moiety may be a radioactive isotope, e.g., 3 H, 14 C,32 P, 35 S, 125 I, 99 Tc, 111 In or 67 Ga; fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, rhodamine, or luciferin; or enzymes, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0339] Binding proteins are also useful for in vivo imaging. Binding proteins labeled at a detectable region can be administered to animals, for example, in the bloodstream, and the presence and location of the labeled antibody in the host can be assayed. Binding proteins can be labeled at any region detectable in animals, whether or not by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0340] For clinical or research purposes, in some embodiments, the binding protein is conjugated to a cytotoxic agent. Various antibodies conjugated to cytotoxic agents (i.e., antibody-drug conjugates) are used to target cytotoxic payloads to specific tumor cells. Cytotoxic agents and linkers that conjugate drugs to antibodies are known in the art; see, for example, Parslow, AC et al., (2016) Biomedicines Vol. 4: p. 14, and Kalim, M. et al., (2017) Drug Des. Devel. Ther. Vol. 11: pp. 2265-2276.

[0341] This disclosure also refers to a kit comprising binding proteins and other reagents useful for detecting target antigen levels in biological samples. Such reagents may include detectable labels, blocking serums, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and / or host cell as described herein. In some embodiments, the kit comprises a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and IV solution bags. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold a composition, either by itself or in combination with another composition effective for treating, preventing, and / or diagnosing a condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a subcutaneous needle). In some embodiments, the label or packaging insert indicates that the composition is used to prevent, diagnose, and / or treat a selected condition. Alternatively, the manufactured article or kit may further include a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0342] In some embodiments, the binding proteins of this disclosure are administered to patients who require them to treat or prevent cancer. In some embodiments, this disclosure relates to a method for preventing and / or treating proliferative disorders or disorders (e.g., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins or related pharmaceutical compositions described herein. In some embodiments, the patient is human.

[0343] In some embodiments, at least one binding protein is administered in combination with one or more anticancer therapies (e.g., any anticancer chemotherapy agent or therapy known in the art). In some embodiments, at least one binding protein is administered before one or more anticancer therapies. In some embodiments, at least one binding protein is administered concurrently with one or more anticancer therapies. In some embodiments, at least one binding protein is administered after one or more anticancer therapies. [Examples]

[0344] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and that these may vary. It should also be understood that the terms used herein are used only to describe specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In certain embodiments, terms used herein are defined as those found in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," edited by Leuenberger, HGW, Nagel, B., and Kolb, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Unless otherwise defined herein, scientific and technical terms used herein have the meaning as commonly understood by those skilled in the art. In any case of potential ambiguity, the definitions provided herein take precedence over any dictionary or foreign definitions. Unless otherwise specified by the context, singular terms include plurals, and plural terms include singulars. The use of "or" means "and / or" unless otherwise specified. The use of the term "including," as well as other forms such as "includes" and "included," is not limited. Where used herein, unless otherwise specified, the singular forms "a," "an," and "the" include plural references. Thus, for example, a reference to "protein" includes multiple protein molecules.

[0345] Furthermore, unless otherwise specified, the experiments described herein utilize conventional molecular and cell biological and immunological techniques within the scope of those skilled in the art. Such techniques are well known to skilled practitioners and are adequately described in the literature. For example, see Ausubel et al., *Current Protocols in Molecular Biology*, John Wiley & Sons, Inc., NY, NY (1987–2008), including appendices, *Molecular Cloning: A Laboratory Manual* (4th edition); and MR. Green, J. Sambrook, and Harlow et al., *Antibodies: A Laboratory Manual*, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0346] This specification contains references to several documents. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing in this specification should be construed as admitting that the present invention does not have prior rights to such disclosures by prior art.

[0347] The elements of the present invention are described below. These elements are described in specific embodiments, however, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and specific embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed elements. Furthermore, unless otherwise indicated in the context, all substitutions and combinations of the elements described in this application should be deemed to be disclosed by the specification of this application.

[0348] In general, the nomenclature used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, as described herein, is well known and commonly used in the art. The methods and techniques provided herein are generally carried out in accordance with conventional methods well known in the art, and, unless otherwise indicated, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in the art or as described herein. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry, as well as their experimental procedures and techniques, as described herein, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and delivery, and for patient treatment.

[0349] Fab assemblies are driven by VH / VL and CH1 / CL domain interactions. Various multispecific constructs with different structures containing different Fab interface mutations were fabricated. The ability of combined mutation pairs within the CH1 / CL and VH / VL interfaces to direct correct pairing was investigated and described in the following examples. [Examples]

[0350] General expression and purification scheme Expression of bispecific and tripspecific molecules HEK293-FS cells, grown in F17 serum-free suspension culture medium (Invitrogen), were transfected with plasmids encoding the indicated light and heavy chains using polyethyleneimine transfection reagents. After culturing at 37°C for 7 days, the cells were removed by centrifugation, and the supernatant was passed through a 0.22 μm filter to remove particles.

[0351] For purification, the antibody was captured on a MabSelect SuRe column (catalog number: 11-0034-93, GE Healthcare), eluted with 0.1M citrate buffer pH 3.0, and directly desalted using a HiPrep 26 / 10 desalting column (catalog number: 17-05087-02, GE Healthcare). After final purification (polish) of the protein by size exclusion chromatography (SEC) using a Superdex 200 26 / 60 (GE) and a final ultrafiltration concentration step, the protein was used for further characterization.

[0352] Analytical size exclusion chromatography (SEC) Analytical SEC was performed at 25°C using a BioSECcurity instrument (PSS Polymer) with a TSKgel SuperSW3000 column (4.6 mm × 300 mm) and a TSKgel SuperSW HPLC guard column (Tosoh Bioscience). Analysis was performed at a flow rate of 0.25 μm / min using 250 mM NaCl and 100 mM Na-phosphate pH 6.7, and detection was performed at 280 nm and 260 nm. Static light scattering was detected at 436 nm. 5 μl of protein sample (1 mg / ml) was applied to the column. Data evaluation was performed using WinGPC software v8.1 (PSS Polymer). For molecular weight estimation, the SEC column was calibrated with protein standards in the molecular weight range of 6.5–670 kDa.

[0353] Hydrophobic interaction chromatography (HIC) for analysis Analysis was performed using an LC10 HPLC system (Shimadzu) at 25°C with a TSKgel Ether-5PW 10 μm, 2 × 75 mm (Tosoh Bioscience) column. Analysis was performed at a flow rate of 0.1 ml / min, and detection was performed at 280 nm. 5 μg of undiluted protein sample was applied to the column. Gradient elution was performed from 0 to 30 minutes (0% to 100% B), followed by re-equilibrium at 100% B for 10 minutes and then 15 minutes. Buffer A consisted of 1.5 M ammonium sulfate and 25 mM sodium phosphate, pH 7.0. Buffer B consisted of 25 mM sodium phosphate, pH 7.0. Data evaluation was performed using LabSolutions software v5.85 (Shimadzu).

[0354] Mass spectrometry (MS) Protein integrity was analyzed by LC-MS. Protein samples were deglycosylated with 12.5 μg of protein diluted to 0.5 mg / ml in D-PBS buffer treated with 0.5 μl of PNGaseF (glycerol-free, New England Biolabs) at 37°C for 15 hours. LC-MS analysis was performed using a 6540UHD high-precision mass Q-TOF LC / MS instrument (Agilent). Reverse-phase (RP) chromatography was performed at 180 μL / min using a Poroshell 300SB-C8, 5 μm, 75 × 0.5 mm (Agilent) guard column. Elutions were LC water, 0.1% formic acid (A) and 90% acetonitrile, 10% LC water, 0.1% formic acid (B). 2 μg of protein was injected into the column and eluted over 13 minutes using a linear gradient from 0% to 100% B. Data analysis was performed using MassHunter software B.06 (Agilent). Molecular weight was calculated based on the protein's amino acid sequence using GPMAW software version 9.13a2 (Lighthouse Data).

[0355] Surface plasmon resonance (SPR) Antigen binding to antibody constructs was measured using surface plasmon resonance (SPR) on a BIAcore 3000 instrument (GE Healthcare) with HBS-EP buffer (GE Healthcare). Anti-human Fc capture antibody (Human Antibody Capture Kit, GE Life Sciences) was immobilized via primary amine groups (11000 RU) on a research-grade CM5 chip (GE Life Sciences) using standard procedure. The ligand was captured at a flow rate of 10 μl / min using a adjusted RU value, resulting in a maximum analyte binding of 30 RU. The tested antibody construct was used as the analyte and injected at a flow rate of 30 μl / min at a concentration of 100 nM for 240 seconds, with a dissociation time of 300 seconds. Binding kinetics measurements were performed using the capture antibody by injecting 2-fold series dilutions of the analyte from 3 nM to 100 nM. The chip surface was regenerated by injecting the regeneration buffer provided with the capture kit for 2 minutes. Sensograms were dual-referenced on the blank chip surface and the HBS-EP buffer blank. Data analysis was performed using BIA evaluation software v4.1.

[0356] Differential scanning fluorescence (DSF) measurement Melting point (Tm) data were measured using differential scanning fluorescence (DSF). The sample was diluted in D-PBS buffer (Invitrogen) to a final concentration of 0.2 μg / μl, containing a 4× concentrated solution of SYPRO-Orange dye (Invitrogen, 5000x stock in DMSO) in D-PBS in a white semi-skirted 96-well plate (BIORAD). All measurements were performed in duplication using a MyiQ2 real-time PCR instrument (BIORAD). The negative first derivative curve (-d(RFU) / dT) of the melting curve was generated using iQ5 software v2.1 (BIORAD). The data was then exported to Excel for Tm determination and graphical visualization.

[0357] The sequences for the selected tandem Fab antibody, triplicate CODV antibody, and bispecific Y-type antibody are listed in Tables 2, 3, and 4 below.

[0358] Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21

[0359] Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Table 29

[0360] Table 30 Table 31 Table 32 Table 33 Table 34 Table 35 Table 36 Table 37 Table 38 Table 39 Table 40 Table 41 Table 42 Table 43 Table 44 Table 45 Table 46 Table 47 Table 48 Table 49 Table 50 Table 51 Table 52 Table 53 Table 54 Table 55 Table 56 Table 57 Table 58 Table 59 Table 60 Table 61 Table 62 Table 63 Table 64 Table 65 Table 66 Table 67 Table 68 Table 69 Table 70 Table 71 Table 72 Table 73 Table 74 Table 75 Table 76 Table 77 Table 78 Table 79 Table 80 Table 81 Table 82 Table 83 Table 84 Table 85 Table 86 Table 87 Table 88 [Table 89] [Table 90] [Table 91] [Examples]

[0361] Heterodimerization enabling mutations in the CODV format Expression, yield, and homogeneity were investigated for three different mutant formats of the CODV antibody. The first format contained a CODV antibody with a knob-in-hole mutation in the Fc domain, accompanied by a CH1 / kappa MUT4 mutation (CH1:L143Q, S188V; Ck:V133T, S176V) in CODV-Fab and a CH1 / kappa CR3 mutation (CH1:T192E; Ck:N137K, S114A) in Fab2 (Figure 1A). The second format contained a CODV antibody with a knob-in-hole mutation in the Fc domain, accompanied by the MUT4 / CR3 mutation from the first format, combined with the electrostatic mutation VH39E / VL38K in both CODV-Fab and Fab2 (Figure 1B). The third format included a CODV antibody with a knob-in-hole mutation in the Fc domain, combined with the disulfide-stabilizing mutation VH44C / VL100C on CODV-Fab, along with the MUT4 / CR3 mutation and the electrostatic mutation of the second format (Figure 1C).

[0362] Table 4 outlines the electrostatic and disulfide bond formation mutations in the variable region. Table 5 outlines the CH1 / CL kappa mutations. A detailed description of the expression and purification scheme is provided in Example 1. Figures 2A to 2G show graphs of data from various CODV antibody formats tested.

[0363] Table 5 below summarizes the characterization results of various CODV antibody formats tested with the ([OX40×PD1]+GITR) trispecific CODV antibody. CH1 / Ck mutations: CODV-Fab (CH1:L143Q, S188V; Ck:V133T, S176V) and Fab (CH1:T192E; Ck:N137K, S114A). CM (charged mutations): CODV-Fab (VH39E / VL38K) and Fab2 (VH39K / VL38E). ds (disulfide bond stabilization mutations): VH44Cys / VL100Cys. As shown in Table 5 below, the introduction of mutations within the CH1 / CL interface increased the onset melting point (Tm onset) and demonstrated increased thermal stability. Further incorporation of VH / VL modifications significantly increased correct pairing, as shown by HIC and MS (see Figures 2A–2G). The combination of CH1 / CL kappa mutation sets (CR3 and MUT4) and disulfide-stabilizing mutations showed promising results, exhibiting high HIC monomer content and increased thermal stability. Combinations of CH1 / CL kappa mutation sets (CR3 and MUT4), disulfide-stabilizing mutations, and counter-charged mutations also showed promising results.

[0364] [Table 92] [Examples]

[0365] Heterodimization enables mutations in the tandem Fab format. Two conformations of the tandem Fab antibody were investigated. Each conformation was further tested for efficient expression, purification, and homogeneity. The first format consists of an open conformation of tandem Fab in which a Y-shaped antibody is bound to another Fab fragment via a flexible linker on the VH domain (see Figures 3A and 3B). The second tandem Fab consists of a closed conformation of tandem Fab in which the linker in the aforementioned VH domain can form a disulfide bond (see Figures 3C and 3D).

[0366] Two mutants were explored for efficient expression, purification, and homogeneity, one for open tandem Fab and the other for closed tandem Fab conformations. The first mutant consisted of an open fab with only MUT4 and CR3 mutations (Figure 3A). The second mutant consisted of an open fab with MUT4 and CR3 mutations combined with counter-charged mutations (Figure 3B).

[0367] Two mutants were explored for efficient expression, purification, and homogeneity for each closed tandem Fab conformation. The first mutant consisted of a closed fab with only MUT4 and CR3 mutations (Figure 3C). The second mutant consisted of a closed fab with MUT4 and CR3 mutations combined with opposite-charged mutations (Figure 3D).

[0368] As shown in Table 6 below, the combination of CH1 / CL and VH / VL interface mutations increased the targeted HIC profile compared to the case where only CH1 / CL modification was introduced. Therefore, combinatorial mutations reduced the amount of mispaired species and increased the amount of correctly paired molecules (targeted HIC peak). The intensity of the pairing result depended on the antibody sequence and domain alignment.

[0369] [Table 93] [Table 94] [Table 95]

[0370] Open-conceptual tandem Fabs containing only the MUT4 / CR3 mutation yielded approximately 68% correct pairing. However, the introduction of the opposite-charged mutation was observed to increase the yield and homogeneity of correct polypeptides from 68% to 96% (Figure 4).

[0371] Open and closed configurations were compared in parallel with anti-CD40 × anti-PD-L1 antibody. As can be seen in Figures 5A–5D, the closed configuration yielded lower yield and purity compared to the open configuration, as measured by HIC. The addition of counter-charged mutations combined with CH1 / CL kappa mutations (CR3 / MUT4) also improved purity. These effects were supported by anti-PD-1 × anti-OX40 antibody, as shown in Figures 6A–6C. The open configuration yielded higher yield and purity. Further improvements in yield and purity were achieved by including counter-charged mutations. [Examples]

[0372] Y-shaped antibody mutation Orthogonal Fab design in bispecific antibodies with a Y-shaped structure was achieved by introducing knob-into-hole mutations (knob: S354C, T366W; hole: Y349C, T366S, L368A, Y407V) into the CH3 domain of each heavy chain. The two different Fab arms contained the mutations described in Figures 8A and 8B, as well as the mutations shown in Tables 7-9 below. The mutations in Figure 8A are the CH1 / kappa MUT4 mutation in Fab1 (CH1: L143Q, S188V; Ck: V133T, S176V) and the CH1 / kappa CR3 mutation in Fab2 (CH1: T192E; Ck: N137K, S114A). The mutations in Figure 8B are the MUT4 / CR3 mutations in Figure 8A, combined with the electrostatic mutation VH39E / VL38K in both Fab1 and Fab2.

[0373] Anti-PD-1 × anti-OX40 antibodies with various mutations were tested. Table 7 below provides an overview of the expression and biophysical characterization of the PD-1 × OX40 bispecific antibody variants altered by their Fab interface mutations. Modifications of CH1 / CL alone or VH / VL alone were not effective in preventing mispairing. The proposed combination of CH1 / CL mutation and VH39 / VL38 interface mutation significantly affected guided pairing when determined by HIC and MS (see Figures 9A–9C).

[0374] [Table 96] [Table 97]

[0375] Anti-CD40×anti-PD-L1 antibodies with various mutations were tested. Table 8 below provides an overview of the expression and biophysical characterization of mutants of the CD40×PD-L1 bispecific antibody that differ in their Fab interface mutations. Modifications of CH1 / CL alone or VH / VL alone were not effective in preventing mispairing. Combinations of CH1 / CL mutations and VH39 / VL38 interface mutations significantly affected guided pairing when determined by HIC and MS.

[0376] [Table 98] [Table 99]

[0377] Anti-PD-1 × anti-OX40 antibodies with different mutations than those of the previously tested anti-PD-1 × anti-OX40 antibodies were examined. Table 9 below provides an overview of the expression and biophysical characterization of variants of PD1 × OX40 bispecific antibodies with diverse Fab interface mutations. As shown in Table 9, CH1 / CL mutations at L143 / S176 or L124 / V133, combined with VH / VL interface mutations at the VH38 / VL39 position, significantly increased the amount of the correct pair species, as observed by HIC profiling and validated by MS analysis (Figures 9A–9C and 10A–10L).

[0378] [Table 100]

[0379] The antigen binding levels in the SPR data in Tables 7-9 are reported relative to the captured antibody level (RU antigen / RU Fc capture) to facilitate comparison between constructs. The SPR data in Tables 7-9 show that all tested antibody constructs bound to their respective antigens at comparable binding levels. [Examples]

[0380] General expression and purification schemes for Examples 6 and 7 Analytical size exclusion chromatography (SEC) Analytical SEC was performed at 25°C using a BioSECcurity instrument (PSS Polymer) equipped with an AdvanceBio 300 column (4.6 mm × 300 mm) and an AdvanceBio 300 guard column (Agilent Technologies). Analysis was performed at a flow rate of 0.5 ml / min using 2 × concentrated D-PBS buffer (Thermo Fisher Scientific), and detection was performed at 280 nm. 10 μl of protein sample (1 mg / ml) was applied to the column. Data evaluation was performed using WinGPC software v8.1 (PSS Polymer). To estimate molecular weight, the SEC column was calibrated with a protein calibration standard mix (Agilent Technologies).

[0381] Hydrophobic interaction chromatography (HIC) for analysis Analytical HIC was performed at 25°C using an LC10 HPLC system (Shimadzu) or a Vanquish HPLC system (Thermo Fisher Scientific) equipped with a TSKgel Butyl-NPR column (2.5 μm, 4.6 × 35 mm) (Tosoh Bioscience). Analysis was performed at a flow rate of 1 ml / min, and detection was performed at 280 nm. 5 μg of undiluted protein sample was applied to the column. Gradient elution was performed from 15%B to 85%B for 7 minutes, followed by 1 minute to 100%B, then 1 minute to 15%B, and finally equilibrated at 15%B for 3 minutes. Buffer A consisted of 1.5 M ammonium sulfate and 25 mM sodium phosphate pH 7.0. Buffer B consisted of 25 mM sodium phosphate pH 7.0. Data evaluation was performed using either LabSolutions software v5.85 (Shimadzu) or Chromeleon 7 software (Thermo Fisher Scientific).

[0382] Mass spectrometry (MS) The protein integrity and potential mispairing of heterodimer constructs were analyzed by LC-mass spectrometry (LC-MS). Protein samples were deglycosylated with 12.5 μg of protein diluted to 0.17 mg / ml in LC-MS grade water (Thermo Scientific) treated with 0.5 μl of PNGase F (glycerol-free, New England Biolabs) at 37°C for 16 hours. LC-MS analysis was performed using a Thermo Fisher Orbitrap Lumos LC / MS instrument. Reverse-phase (RP) chromatography was performed at 300 μL / min using a MabPac RP HPLC column, analytical 4 μm particle size, 2.1 × 100 mm (Thermo Scientific). Elutions were LC water, 0.1% formic acid (A), and 90% acetonitrile, 10% LC water, 0.1% formic acid (B). 2 μg of protein was injected into the column and eluted over 12 minutes using a linear gradient from 0% to 95% B. Data analysis was performed using Expressionist software 13.0.3 (Genedata). Molecular weight was calculated based on the protein's amino acid sequence using GPMAW software version 10.32b1 (Lighthouse data).

[0383] Surface plasmon resonance (SPR) Antigen binding to antibody constructs was measured using surface plasmon resonance (SPR) with a BIAcore T200 instrument (GE Healthcare) using HBS-EP+ buffer (GE Healthcare). To evaluate the relative binding levels (Rmax%) of antibodies against each antigen, the antibodies were captured by anti-Fc affinity capture on a sensor chip. This assay used recombinant human antigens (PD-L1-His (9049-B7-100, R&D Systems), CD40-His (10774-H08H, Sino Biological), TNFα (130-094-022, Miltenyi), GITR-His (in-house prepared), PD1-His (8986-PD-100, R&D Systems), CD3εδ-FLAG-His (#CT038-H2508H, Sino Biological), and human CD123 (#301-R3 / CF, R&D Systems)). Anti-human Fc capture antibodies (Human Antibody Capture Kit, GE Life Sciences) were immobilized via primary amine groups (11000RU) on research-grade CM5 chips (GE Life Sciences) using standard procedures. Antibodies were captured at a flow rate of 10 μl / min using adjusted RU values, resulting in maximum analyte binding of 10–30 RU. Antigens were used as analytes, injected at either 400 nM or 100 nM concentrations for CD3εδ-FLAG-His, or at 100 nM concentration for all other antigens used. Antigens were injected for 240 seconds at a flow rate of 30 μL / min with a dissociation time of 300 seconds. The chip surface was regenerated by injecting the regeneration buffer provided with the capture kit for 2 minutes. Sensegments were double-referenced on a blank chip surface and an HBS-EP buffer blank. Data analysis and binding level determination were performed using Biacore 8K Evaluation software v1.11.7442 (GE Healthcare). Rmax% values ​​were calculated using the maximum binding level divided by the theoretical Rmax value. The Rmax value was calculated from the capture level R, binding stoichiometry N, and the molecular weights of the antibody Mw(Ab) and antigen Mw(Ag), with Rmax = R capture × N × (Mw(Ag) / Mw(Ab)).

[0384] Nano-differential scanning fluorescence (nanoDSF) The protein denaturation onset temperature (T onset) and melting point (Tm) were measured using nano-differential scanning fluorescence (nanoDSF). The sample was diluted to a final concentration of 0.5 μg / μl in formulation buffer and double-loaded into a nanoDSF capillary (Nanotemper Technologies). All measurements were performed using a Prometheus NT.plex nanoDSF device (Nanotemper Technologies). The heating rate was 1 °C / min from 20 °C to 95 °C. Data were recorded using PR.ThermControl Software v2.3.1 (Nanotemper Technologies) and analyzed using PR.Stability Analysis Software v1.0.3 (Nanotemper Technologies). [Examples]

[0385] Further Y-shaped antibody mutations Further bispecific antibodies with Y-shaped structures were constructed and tested with different mutant sets. Specifically, anti-PD-1 × anti-OX40 antibodies were used with the mutants listed in Table 10 below. The data below shows that all Y-shaped antibodies with Fab mutations reduced mispairing compared to wild-type controls. Antibodies with the VH39 / VL38 opposite-charged mutant set, the L143 / S176 opposite-charged mutant set, and the K221E:K228D / E123K:D122K opposite-charged mutant set (protein ID Y61) showed excellent results, and no mispairing was detected.

[0386] [Table 101] [Examples]

[0387] Further heterodimerization enabling mutations in the Y-shaped antibody format Further Y-shaped antibodies with different heterodimerization mutation sets were tested. Table 11 below lists the biophysical characterization data for each Y-shaped antibody tested. As shown in Table 11 and Figures 11A-11E, “CM1” refers to the VH39K / VL38E mutation pair, “CM2” refers to the VH39E / VL38K mutation pair, “CM3” refers to the CH1 L143E or L143D / CL S176R or S176K mutation pair, “CM4” refers to the CH1 L143R or L143K / CL S176E or S176D mutation pair, and “NN3” refers to the CH1 K221E and K228D / CL D122K and E123K mutation pairs.

[0388] [Table 102]

[0389] As shown in Table 11 and Figures 11A-11E, the various heterodimerizing mutations used each reduce mispairing compared to the wild-type antibody. In the case of the anti-TNF × anti-OX40 antibody (Y49), which is known to be free from mispairing in its wild-type configuration, the inclusion of the heterodimerizing mutation did not negatively affect mispairing (Figure 11C).

[0390] In addition to the biophysical characterization described above, the T cell-inducing (engager) antibody CD3×CD123 was effective in cell-based cytotoxicity assays. The bispecific antibody molecule was analyzed in cytotoxicity assays using primary human T cells. Human peripheral mononuclear cells from healthy donor blood were isolated in Leucosep-Tubes (Greiner Bio-One, #227290) using 15 ml of Histopaque (Sigma-Aldrich, #10771) and centrifugation at 1000×g for 10 minutes. The isolated PBMCs were washed twice in autoMACS rinsing buffer (Miltenyi Biotec, #130-091-222) supplemented with 5% MACS BSA stock solution (Miltenyi Biotec, #130-091-370). Primary human T cells were isolated from human PBMCs using MACSpro separator (Miltenyi Biotec) and Pan T cell isolation kit (Miltenyi Biotec, #130-096-535) according to the manufacturer's protocol. The isolated human T cells were placed in RPMI GlutaMAX I medium (Gibco, #72400) supplemented with 10% FCS HI (Gibco, #10082-147) in 5 × 10⁶ units. 6 The cells were resuspended at a concentration of cells / mL. Prior to the cytotoxicity assay, THP-1 target cells (ADCC TIB-202) were stained with 1 μM CFSE (Invitrogen, #C1157) for 15 minutes at 37°C. The cells were washed twice in RPMI+GlutaMAX I medium and centrifuged at 400×g for 5 minutes. The THP-1 target cells were then placed in RPMI medium supplemented with 10% FCS HI in 5×10⁶ units. 5The cells were resuspended at a concentration of cells / mL. CFSE-labeled THP-1 cells and human pan-T cells were mixed in an effector-to-target ratio of 10:1 and seeded in a 96-well assay plate (Greiner BioOne, #650185) at a total volume of 100 μl / well. Bispecific antibody molecules were added to the cells in 11 dilution series starting from 10 nM to 0 nM (1:6 dilution) at a volume of 5 μL / well, and incubated for 20 hours at 37°C and 5% CO2. After incubation, cells were stained with 5 μg / ml 7-AAD (Invitrogen, #A1310) for 30 minutes at 4°C. To determine cytotoxicity, dead cells were measured by gating CFSE / 7-AAD bipositive THP-1 cells on an LSRII flow cytometer (BD), and the EC50 value was determined using Xlfit software.

[0391] As shown in Figure 12, all three CD3×CD123 bispecific antibodies exhibited comparable potent activity. The presence of heterodimerization mutations at Y56 and Y57 did not negatively affect activity, while reducing chain mispairing.

Claims

1. A multispecific antigen-binding protein comprising at least two VL regions paired with at least two VH regions for forming at least two antigen-binding sites, and at least two CH1 regions paired with at least two CL regions, At least one CH1 / CL pair is: (1) T192E(CH1) mutation, and N137K and S114A(CL) mutation, (2) L143Q and S188V(CH1) mutations, and V133T and S176V(CL) mutations, (3) T192E, L143Q and S188V(CH1) mutations, and N137K, S114A, V133T and S176V(CL) mutations, (4) K221E(CH1) mutation and E123K(CL) mutation, (5) T192E and K221E (CH1) mutations, as well as N137K, S114A and E123K (CL) mutations, (6) L143E, L143D, L143K, L143R or L143H(CH1) mutations, and S176E, S176D, S176K, S176R or S176H(CL) mutations, where the L143 mutation has the opposite charge to the S176 mutation, and (7) K221E and K228D (CH1) mutations, and D122K and E123K (CL) mutations A group consisting of one or more of the following, comprising a CH1 / CL mutation to promote pairing, If two CH1 / CL pairs contain mutations that facilitate pairing for two different VH / VL pairs, the two CH1 / CL pairs do not contain the same mutation. At least one VH / VL pair includes an anti-charged mutation to facilitate pairing, the anti-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H. Mutant residues in the VH region have the opposite charge to mutant residues in the VL region. and, Mutations are numbered by Kabat. The multispecific antigen-binding protein.

2. It is a multispecific antigen-binding protein, a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1; and (3) First heterodimerization domain (HD1) A first light chain (LC1) / heavy chain (HC1) pair including; and b) (4) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; (5) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2; and (6) Second heterodimerization domain (HD2) The second light chain (LC2) / heavy chain (HC2) pair including Includes, HD1 and HD2 heterodimerize, At least one or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs include an opposite-charged mutation to facilitate pairing, wherein the opposite-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, wherein the mutant residue in the VH region has the opposite charge to the mutant residue in the VL region. At least one or both of the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations that promote pairing. If both the CL1 and CH1-1 pair, and the CL2 and CH1-2 pair, contain mutations that promote pairing, the mutations in CH1-1 and CL1 that promote pairing are different from the mutations in CH1-2 and CL2 that promote pairing. Here, (1) CH1-1 contains the T192E mutation, CL1 contains the N137K and S114A mutations, and / or CH1-2 contains the T192E mutation, CL2 contains the N137K and S114A mutations; (2) CH1-1 contains L143Q and S188V mutations, CL1 contains V133T and S176V mutations, and / or CH1-2 contains L143Q and S188V mutations, CL2 contains V133T and S176V mutations; (3) CH1-1 contains T192E, L143Q and S188V mutations, CL1 contains N137K, S114A, V133T and S176V mutations, and / or CH1-2 contains T192E, L143Q and S188V mutations, CL2 contains N137K, S114A, V133T and S176V mutations; (4) CH1-1 contains the K221E mutation, CL1 contains the E123K mutation, and / or CH1-2 contains the K221E mutation, CL2 contains the E123K mutation; (5) CH1-1 contains T192E and K221E mutations, CL1 contains N137K, S114A and E123K mutations, and / or CH1-2 contains T192E and K221E mutations, CL2 contains N137K, S114A and E123K mutations; (6) CH1-1 contains K221E and K228D mutations, CL1 contains D122K and E123K mutations, and / or CH1-2 contains K221E and K22 CL2 contains the 8D mutation, and CL2 contains the D122K and E123K mutations; and / or, (7) CH1-1 contains L143E, L143D, L143K, L143R or L143H mutations, CL1 contains S176E, S176D, S176K, S176R or S176H mutations, where the L143 mutation has the opposite charge to the S176 mutation, and / or CH1-2 contains L143E, L143D, L143K, L143R or L143H mutations, CL2 contains S176E, S176D, S176K, S176R or S176H mutations, where the L143 mutation has the opposite charge to the S176 mutation, and, Mutations are numbered by Kabat. The multispecific antigen-binding protein.

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

4. A multispecific antigen-binding protein comprising at least two polypeptide chains and forming at least two antigen-binding sites, wherein one polypeptide chain has the formula: VL1-L1-VL2-L2-CL[I] The structure includes the form represented by , and one polypeptide chain is given by formula: VH2-L3-VH1-L4-CH1 [II] Includes a structure represented by During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; and L1, L2, L3, and L4 are amino acid linkers. VH1 pairs with VL1, VH2 pairs with VL2, CH1 pairs with CL, The polypeptide of formula I and the polypeptide of formula II form crossed light chain-heavy chain pairs. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds. At least one or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs include an anti-charged mutation to promote pairing, wherein the anti-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H. Mutant residues in the VH region have the opposite charge to mutant residues in the VL region. The CH1 and CL domain pairs contain mutations to facilitate pairing. Here, (1) CH1 contains the T192E mutation, and CL contains the N137K and S114A mutations; (2) CH1 contains L143Q and S188V mutations, and CL contains V133T and S176V mutations; (3) CH1 contains T192E, L143Q and S188V mutations, and CL contains N137K, S114A, V133T and S176V mutations; (4) CH1 contains the K221E mutation, and CL contains the E123K mutation; (5) CH1 contains T192E and K221E mutations, and CL contains N137K, S114A and E123K mutations; (6) CH1 contains K221E and K228D mutations, CL contains D122K and E123K mutations; and / or, (7) CH1 contains L143E, L143D, L143K, L143R, or L143H mutations, CL contains S176E, S176D, S176K, S176R, or S176H mutations, where the L143 mutation has the opposite charge to the S176 mutation, and Mutations are numbered by Kabat. The multispecific antigen-binding protein.

5. A multispecific antigen-binding protein comprising four polypeptide chains that form four antigen-binding sites, wherein two polypeptide chains each have the formula: VL1-L1-VL2-L2-CL[I] The structure includes the form represented by the formula, and the two polypeptide chains are each: VH2-L3-VH1-L4-CH1-Fc[II] It includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; Fc comprises an immunoglobulin hinge region and the constant domains of the CH2 and CH3 immunoglobulin heavy chains; and L1, L2, L3, and L4 are amino acid linkers. VH1 pairs with VL1 to form a first antigen-binding site, VH2 pairs with VL2 to form a second antigen-binding site, and CH1 pairs with CL. The polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds. One or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs include an anti-charged mutation to promote pairing, wherein the anti-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H. Mutant residues in the VH region have the opposite charge to those in the VL region, and One or both of the CH1 and CL domain pairs contain mutations to facilitate pairing. If at least two CH1 / CL pairs contain mutations to facilitate pairing, the set of mutations in one CH1 / CL pair is different from the set of mutations in the other CH1 / CL pair. Here, (1) CH1 contains the T192E mutation, and CL contains the N137K and S114A mutations; (2) CH1 contains L143Q and S188V mutations, and CL contains V133T and S176V mutations; (3) CH1 contains T192E, L143Q and S188V mutations, and CL contains N137K, S114A, V133T and S176V mutations; (4) CH1 contains the K221E mutation, and CL contains the E123K mutation; (5) CH1 contains T192E and K221E mutations, CL is N137K, S11 Includes 4A and E123K mutations; (6) CH1 contains K221E and K228D mutations, CL contains D122K and E123K mutations; and / or, (7) CH1 contains L143E, L143D, L143K, L143R, or L143H mutations, CL contains S176E, S176D, S176K, S176R, or S176H mutations, where the L143 mutation has the opposite charge to the S176 mutation, and Mutations are numbered by Kabat. The multispecific antigen-binding protein.

6. A multispecific antigen-binding protein comprising four polypeptide chains that form three antigen-binding sites, The first polypeptide chain is given by formula: VL2-L1-VL1-L2-CL1 [I], It includes a structure represented by, The second polypeptide chain is given by formula: VH1-L3-VH2-L4-CH1-1-Hinge-CH2-CH3[II] It includes a structure represented by, The third polypeptide chain is given by formula: VH3-CH1-2-Hinge-CH2-CH3[III] It includes a structure represented by, The fourth polypeptide chain is given by formula: VL3-CL2[IV] It includes a structure represented by, During the ceremony, VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; CL1 is the first constant domain of the immunoglobulin light chain; CL2 is the second constant domain of the immunoglobulin light chain; CH1-1 is the first immunoglobulin CH1 heavy chain constant domain; CH1-2 is the second constant domain of the immunoglobulin CH1 heavy chain; CH2 is the constant domain of the immunoglobulin CH2 heavy chain; CH3 is the constant domain of the immunoglobulin CH3 heavy chain; The hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3, and L4 are amino acid linkers. VH1 pairs with VL1, VH2 pairs with VL2, VH3 pairs with VL3, and CH1-1 pairs with CL1, CH1-2 pairs with CL2, The polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair. One or more cysteine ​​residues are incorporated into one or more of the VH1 / VL1 pairs, VH2 / VL2 pairs, and VH3 / VL3 pairs to form one or more disulfide bonds. One or more of the VL1 and VH1 pairs, VL2 and VH2 pairs, and VL3 and VH3 pairs include an anti-charged mutation to promote pairing, wherein the anti-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H. Mutant residues in the VH region have the opposite charge to those in the VL region. One or both of the CL1 and CH1-1 pairs and the CL2 and CH1-2 pairs contain mutations to promote pairing. If both the CL1 and CH1-1 pair and the CL2 and CH1-2 pair contain mutations that promote pairing, the mutations in CH1-1 and CL1 are different from the mutations in CH1-2 and CL2. Here, (1) CH1-1 contains the T192E mutation, CL1 contains the N137K and S114A mutations, and / or CH1-2 contains the T192E mutation, CL2 contains the N137K and S114A mutations; (2) CH1-1 contains L143Q and S188V mutations, CL1 contains V133T and S176V mutations, and / or CH1-2 contains L143Q and S188V mutations, CL2 contains V133T and S176V mutations; (3) CH1-1 contains T192E, L143Q and S188V mutations, CL1 contains N137K, S114A, V133T and S176V mutations, and / or CH1-2 contains T192E, L143Q and S188V mutations, CL2 contains N137K, S114A, V133T and S176V mutations; (4) CH1-1 contains the K221E mutation, CL1 contains the E123K mutation, and / or CH1-2 contains the K221E mutation, CL2 contains the E123K mutation; (5) CH1-1 contains T192E and K221E mutations, CL1 contains N137K, S114A and E123K mutations, and / or CH1-2 contains T192E and K221E mutations, CL2 contains N137K, S114A and E123K mutations; (6) CH1-1 contains K221E and K228D mutations, CL1 contains D122K and E123K mutations, and / or CH1-2 contains K221E and K228D mutations, CL2 contains D122K and E123K mutations; and / or, (7) CH1-1 contains L143E, L143D, L143K, L143R or L143H mutations, CL1 contains S176E, S176D, S176K, S176R or S176H mutations, where the L143 mutation has the opposite charge to the S176 mutation, and / or CH1-2 contains L143E, L143D, L143K, L143R or L143H mutations, CL2 contains S176E, S176D, S176K, S176R or S176H mutations, where the L143 mutation has the opposite charge to the S176 mutation, and, Mutations are numbered by Kabat. The multispecific antigen-binding protein.

7. a) (1) A first VL region (VL1) paired with a first VH region (VH1) for forming a first antigen-binding site; (2) A first steady heavy chain region 1 (CH1-1) operably connected to VH1 and a first steady light chain region (CL1) operably connected to VL1 A first light chain (LC1) / heavy chain (HC1) pair including, b) (3) A second VL region (VL2) paired with a second VH region (VH2) for forming a second antigen-binding site; and (4) A second steady heavy chain region 1 (CH1-2) operably connected to VH2 and a second steady light chain region (CL2) operably connected to VL2 The second light chain (LC2) / heavy chain (HC2) pair including A multispecific antigen-binding protein containing, The C-terminus of CH1-1 is operably connected to the N-terminus of VH2. One or more cysteine ​​residues are incorporated into one or both of the VH1 / VL1 pair and the VH2 / VL2 pair to form one or more disulfide bonds. One or both of the VL1 and VH1 pairs and the VL2 and VH2 pairs include an anti-charged mutation to promote pairing, wherein the anti-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H. Mutant residues in the VH region have the opposite charge to mutant residues in the VL region. One or both of the CL1 and CH1-1 pairs, and the CL2 and CH1-2 pairs, contain mutations to promote pairing. If both the CL1 and CH1-1 pair and the CL2 and CH1-2 pair contain mutations that promote pairing, the mutations in CH1-1 and CL1 are different from the mutations in CH1-2 and CL2. Here, (1) CH1-1 contains the T192E mutation, CL1 contains the N137K and S114A mutations, and / or CH1-2 contains the T192E mutation, CL2 contains the N137K and S114A mutations; (2) CH1-1 contains L143Q and S188V mutations, CL1 contains V133T and S176V mutations, and / or CH1-2 contains L143Q and S188V mutations, CL2 contains V133T and S176V mutations; (3) CH1-1 contains T192E, L143Q and S188V mutations, CL1 contains N137K, S114A, V133T and S176V mutations, and / or CH1-2 contains T192E, L143Q and S188V mutations, CL2 contains N137K, S114A, V133T and S176V mutations; (4) CH1-1 contains the K221E mutation, CL1 contains the E123K mutation, and / or CH1-2 contains the K221E mutation, CL2 contains the E123K mutation; (5) CH1-1 contains T192E and K221E mutations, CL1 contains N137K, S114A and E123K mutations, and / or CH1-2 contains T192E and K221E mutations, CL2 contains N137K, S114A and E123K mutations; (6) CH1-1 contains K221E and K228D mutations, CL1 contains D122K and E123K mutations, and / or CH1-2 contains K221E and K228D mutations, CL2 contains D122K and E123K mutations; and / or, (7) CH1-1 contains L143E, L143D, L143K, L143R or L143H mutations, CL1 contains S176E, S176D, S176K, S176R or S176H mutations, where the L143 mutation has the opposite charge to the S176 mutation, and / or CH1-2 contains L143E, L143D, L143K, L143R or L143H mutations, CL2 contains S176E, S176D, S176K, S176R or S176H mutations, where the L143 mutation has the opposite charge to the S176 mutation, and, Mutations are numbered by Kabat. The multispecific antigen-binding protein.

8. An antigen-binding domain containing at least one VH / VL pair; and Steady-state light chain CL region paired with steady-state heavy chain CH1 region An antigen-binding protein containing, The antigen-binding domain selectively binds to the target antigen, and the CH1 region and CL region are L143E, L143D, L143K, L143R or L143 in the CH1 region H mutations, and S176E, S176D, S176K, S176R, or S176H mutations in the CL region. Includes, Mutant residues in the CH1 region have the opposite charge to mutant residues in the CL region. At least one VH / VL pair includes an opposite-charged mutation to facilitate pairing, the opposite-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, wherein the mutant residue in the VH region has the opposite charge to the mutant residue in the VL region, and Mutations are numbered by Kabat. The aforementioned antigen-binding protein.

9. (1) T192E(CH1) mutation, and N137K and S114A(CL) mutations, (2) L143Q and S188V(CH1) mutations, and V133T and S176V(CL) mutations, (3) T192E, L143Q and S188V(CH1) mutations, and N137K, S114A, V133T and S176V(CL) mutations, (4) K221E(CH1) mutation and E123K(CL) mutation, (5) K228D(CH1) mutation and D122K(CL) mutation, and (6) K221E and K228D (CH1) mutations, and D122K and E123K (CL) mutations Further comprising CH1 / CL mutations to promote pairing selected from one or more of the following groups, If two CH1 / CL pairs contain mutations that facilitate pairing for two different VH / VL pairs, then the two CH1 / CL pairs do not contain the same mutation, and, Mutations are numbered by Kabat. The antigen-binding protein according to claim 8.

10. An antigen-binding domain containing at least one VH / VL pair; and Steady-state light chain CL region paired with steady-state heavy chain CH1 region An antigen-binding protein containing, The antigen-binding domain selectively binds to the target antigen, and the CH1 region and CL region are as follows: a) L143E, L143D, L143K, L143R or L143H mutations in the CH1 region, and S176E, S176D, S176K, S176R or S176H mutations in the CL region; and b) K221E and K228D mutations in the CH1 region, and D122K and E123K mutations in the CL region Including one or both of the following: Mutant residues in the CH1 region have the opposite charge to mutant residues in the CL region. At least one VH / VL pair includes an opposite-charged mutation to facilitate pairing, the opposite-charged mutation includes (1) a mutant residue in the VH region at Kabat position 39 selected from E, D, K, R, or H, and (2) a mutant residue in the VL region at Kabat position 38 selected from E, D, K, R, or H, wherein the mutant residue in the VH region has the opposite charge to the mutant residue in the VL region, and Mutations are numbered by Kabat. The aforementioned antigen-binding protein.

11. One or more VH / to form one or more disulfide bonds A multispecific antigen-binding protein according to any one of claims 1 to 7, further comprising one or more cysteine ​​residues incorporated into a VL pair, or an antigen-binding protein according to any one of claims 8 to 10.

12. The multispecific antigen-binding protein or antigen-binding protein according to claim 11, wherein one or both VH regions contain one or both of the 44C and 105C mutations, and one or both VL regions contain one or both of the 100C and 43C mutations.

13. A kit comprising one or more isolated nucleic acid molecules, each comprising one or more nucleotide sequences encoding a multispecific antigen-binding protein according to any one of claims 1 to 7, 11, and 12, or an antigen-binding protein according to any one of claims 8 to 10.

14. A kit comprising one or more expression vectors containing one or more nucleic acid molecules as described in claim 13.

15. An isolated host cell comprising one or more nucleic acid molecules as described in claim 13, or one or more expression vectors as described in claim 14.

16. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a multispecific antigen-binding protein according to any one of claims 1 to 7, 11, and 12, or an antigen-binding protein according to any one of claims 8 to 10.

17. A pharmaceutical agent for treating a disorder in which antigen activity is harmful, comprising an effective amount of a multispecific antigen-binding protein according to any one of claims 1 to 7, 11, and 12 or an antigen-binding protein according to any one of claims 8 to 10.

18. A multispecific antigen-binding protein or antigen-binding protein according to any one of claims 1 to 12, 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.

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