Pseudo-FAB-based multispecific binding proteins

Abstract

Binding proteins are provided that include a pseudo-Fab domain that includes a stabilized knockout domain and a second VH / VL that forms a first functional antigen-binding domain. Multispecific binding proteins are also provided that include at least one pseudo-Fab. Multispecific binding proteins, nucleic acids encoding 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. 18306840.2, filed December 24, 2018, and EP Application No. 19305813.8, filed June 21, 2019, the contents of which are incorporated herein by reference for all purposes. [Background technology]

[0002] The generation of asymmetry in natural antibody structure 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 Fvs on different asymmetric binding arms or Fabs, bispecific antibodies can be created that have the flexibility to simultaneously bind two different antigens or epitopes. However, despite these advantages, the wide variety of multispecific antibody technologies suffers from process and manufacturing problems due to various asymmetric mispairings of heavy and light chains. For example, many of these technologies suffer from the so-called "light chain problem," in which random pairing of two different light and heavy chains generates various combinations of chain pairings other than the desired one. In some cases, the light chain problem can be circumvented by using a common light chain that allows binding to both antigens or epitopes. However, this format requires de novo antibody generation in transgenic mice, which may not be possible for many antibodies. Furthermore, rare antibodies, such as broadly neutralizing anti-HIV antibodies from human patients, cannot be adapted to such a format. Therefore, there remains a need for alternative and creative solutions to the mismatch problem. Summary of the Invention [Problem to be solved by the invention]

[0003] The present disclosure is based on the discovery of a novel heterodimerization domain called a "stabilizing knockout domain" that is used to form a "pseudo-Fab." As disclosed herein, the pseudo-Fab can be incorporated into a wide variety of binding proteins and binding formats to confer multispecific binding properties. In certain embodiments, the pseudo-Fab portion can facilitate the preferential production, synthesis, or purification of a desired multispecific binding protein while minimizing or eliminating undesired chain mispairing commonly produced in conventional multispecific binding protein formats. In one aspect, the present disclosure provides a binding protein comprising: a first pseudo-Fab portion comprising: (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; and (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain. Including, A stabilized knockout domain provides the binding protein comprising (3) one or more inactivating mutations that abolish binding to the target antigen, and (4) one or more engineered interchain disulfide bonds.

[0004] In one aspect, the present disclosure provides a binding protein comprising: a first pseudo-Fab portion comprising: (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; and (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain. Including, The stabilized knockout domain comprises (3) one or more inactivating mutations relative to the wild-type domain that abolish its binding to the target antigen, and (4) one or more engineered interchain disulfide bonds that confer increased thermal stability (Tm) of the pseudo-Fab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudo-Fab molecule except that in the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains, providing said binding protein.

[0005] In some embodiments, the binding protein is at least a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; and a multispecific binding protein further comprising:

[0006] In one aspect, the present disclosure provides a multispecific binding protein comprising: a) a first pseudo-Fab portion comprising: (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; and (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain. b) a first Fab portion comprising (3) a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B, and (4) a first CH1 domain paired with a first CL domain. 1. A multispecific binding protein comprising: The stabilized knockout domain comprises (5) one or more inactivating mutations that abolish its binding to the target antigen, and (6) one or more engineered interchain disulfide bonds; or the stabilized knockout domain comprises (5) one or more inactivating mutations that abolish its binding to the target antigen relative to the wild-type domain, and (6) one or more engineered interchain disulfide bonds that confer increased thermal stability (Tm) of the pseudo-Fab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudo-Fab molecule except that in the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

[0007] In another aspect, the present disclosure provides a multispecific binding protein comprising: a) a first pseudo-Fab portion comprising: (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen binding site that binds to target antigen A; and (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a stabilized knockout domain, with the proviso that the first pseudo-Fab portion does not include a CH1 domain paired with a CL domain; a first pseudo-Fab portion, wherein the stabilized knockout domain comprises (3) one or more inactivating mutations that abolish its binding to the target antigen and (4) one or more engineered interchain disulfide bonds, or the stabilized knockout domain comprises (3) one or more inactivating mutations that abolish its binding to the target antigen relative to the wild-type domain and (4) one or more engineered interchain disulfide bonds that confer increased thermal stability (Tm) of the pseudo-Fab relative to a reference Fab molecule, the reference Fab molecule being identical to the pseudo-Fab molecule except that in the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains; b) (5) a first Fab portion comprising a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B, and (6) a first CH1 domain paired with a first CL domain; and c) a linker portion operably linking the first Fab portion and the first pseudo-Fab portion and

[0008] In another aspect, the present disclosure provides a multispecific binding protein comprising: a) a first pseudo-Fab portion comprising: (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; and (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain; b) a first Fab portion comprising: (3) a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; and (4) a first CH1 domain paired with a first CL domain; The stabilized knockout domain comprises (5) one or more inactivating mutations that abolish its binding to the target antigen and (6) one or more engineered interchain disulfide bonds, or the stabilized knockout domain comprises (5) one or more inactivating mutations that abolish its binding to the target antigen relative to the wild-type domain and (6) one or more engineered interchain disulfide bonds that confer increased thermal stability (Tm) of the pseudo-Fab relative to a reference Fab molecule, the reference Fab molecule comprising: a first Fab portion that is identical to the pseudo-Fab molecule except that in the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains; c) a linker portion operably linking the first Fab portion and the first pseudo-Fab portion and

[0009] In some embodiments, the linker moiety is on one or more of the heavy chains.

[0010] In some embodiments, the multispecific binding protein further comprises a third VL domain (VLc) paired with a third VH domain (VHc) to form a third functional antigen binding site that binds to target antigen C.

[0011] In some embodiments, the multispecific binding protein independently comprises one or two pseudo-Fab portions and one or two Fab portions.

[0012] In some embodiments, the linker moiety is a peptide linker. In some embodiments, the peptide linker has the formula (Gly4Ser) n (n is 1 to 10) is a Gly-Ser linker.

[0013] In some embodiments, the heterodimerization domain comprises a full-length IgG antibody. In some embodiments, the heterodimerization domain comprises the Fc domain of a full-length IgG antibody or a functional fragment thereof.

[0014] In some embodiments, the binding protein is selected from the following group: (a) VHa-CH1-L1-VHb-L2-VHX and VLa-CL and VLb-L3-VLX; (b) VHa-L2-VHX-L1-VHb-CH1 and VLa-L3-VLX and VLb-CL; (c) VHa-CH1-L1-VHa-CH1 and VHb-L2-VHX-L3-VHb-L4-VHX, as well as two chains VLb-L5-VLX Chains (a) and (b) may occur once or twice and the two chains VLa-CL comprising separate protein chains selected from one of: wherein L1, L2, L3, L4 and L5 are independently the same or different linkers.

[0015] The present disclosure provides a multispecific antibody, comprising: a) (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first antigen-binding site that binds to target antigen A; (2) a first stabilized knockout VL domain (VLX) paired with a first stabilized knockout VH domain (VHX) to form a first disulfide-stabilized knockout (dsKO) domain; (3) First heterodimerization domain (HD1) a first pseudo-Fab portion comprising a first pseudo-Fab portion that is identical to the pseudo-Fab molecule except that in the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains; b) (1) a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (2) a first CH1 domain paired with a first CL domain; and (3) Second heterodimerization domain (HD2) a first Fab portion comprising and wherein the multispecific antibody comprises:

[0016] In some embodiments, the first heterodimerization domain (HD1) is operably linked to the C-terminus of the VHX domain of the pseudo-Fab portion.

[0017] In some embodiments, the second heterodimerization domain (HD2) is operably linked to the C-terminus of the first CH1 domain of the first Fab portion.

[0018] In some embodiments, the first and second heterodimerization domains comprise a first and second Fc domain.

[0019] In some embodiments, the Fc domain comprises the general structure hinge-CH2 domain-CH3 domain.

[0020] In some embodiments, the Fc domain comprises one or more knobs-in-holes (KIH) mutations.

[0021] In some embodiments, one of the Fc domains comprises a first CH3 domain comprising one or both of an S354C and a T366W mutation, and the other Fc domain comprises a second CH3 domain comprising one or both of an Y349C, T366S, L368A and a Y407V mutation.

[0022] In some embodiments, the Fc domain comprises a H435R and / or a Y436F mutation.

[0023] In some embodiments, the first pseudo-Fab portion has the formula: (Ia)N-VHa-L1-VHX-C and a first polypeptide chain having a structure represented by the formula: (IIa)N-VLa-L2-VLX-C A second polypeptide chain having a structure represented by Including, In the formula, L1 and L2 are linkers, which may be present or absent independently, and N and C represent the N-terminus and C-terminus, respectively.

[0024] In some embodiments, the first pseudo-Fab portion has the formula: (Ib)N-VHX-L1-VHa-C and a first polypeptide chain having a structure represented by the formula: (IIb)N-VLX-L2-VLa-C A second polypeptide chain having a structure represented by Including, In the formula, L1 and L2 are linkers, which may be present or absent independently, and N and C represent the N-terminus and C-terminus, respectively.

[0025] In some embodiments, the first pseudo-Fab portion has the formula: (Ic)N-VLa-L1-VHX-C and a first polypeptide chain having a structure represented by the formula: (IIc)N-VHa-L2-VLX-C A second polypeptide chain having a structure represented by Including, In the formula, L1 and L2 are linkers, which may be present or absent independently, and N and C represent the N-terminus and C-terminus, respectively.

[0026] In some embodiments, the first pseudo-Fab portion has the formula: (Id)N-VHX-L1-VLa-C a first polypeptide chain having a structure represented by the formula: (IId)N-VLX-L2-VHa-C A second polypeptide chain having a structure represented by Including, In the formula, L1 and L2 are linkers, which may be present or absent independently, and N and C represent the N-terminus and C-terminus, respectively.

[0027] In some embodiments, at least two of target antigen A, target antigen B, and target antigen C are different target antigens.

[0028] In some embodiments, at least one of the target antigens is a ligand for a cell surface receptor, and at least one of the target antigens is a cell surface receptor.

[0029] In some embodiments, the antigen binding sites are derived from different antibodies.

[0030] In some embodiments, target antigen A, target antigen B, and target antigen C are the same target antigen.

[0031] In some embodiments, the antigen binding sites bind to different epitopes on the same target antigen.

[0032] In some embodiments, the antigen binding sites bind to the same epitope on the same target antigen.

[0033] In some embodiments, the antigen binding sites are derived from the same antibody.

[0034] In some embodiments, the melting temperature (T m ) is at least 4°C higher than the reference Fab molecule.

[0035] In some embodiments, the engineered interchain disulfide bond is VH44C-VL100C.

[0036] In some embodiments, the engineered interchain disulfide bond is VH105C-VL43C.

[0037] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in the VHX domain of the pseudo-Fab portion.

[0038] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRH3 of the VHX domain.

[0039] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRH2 of the VHX domain.

[0040] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRH1 of the VHX domain.

[0041] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in the VLX domain of the pseudo-Fab portion.

[0042] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRL3 of the VLX domain.

[0043] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRL2 of the VLX domain.

[0044] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRL1 of the VLX domain.

[0045] In some embodiments, the VHX domain of the pseudo-Fab portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79.

[0046] In some embodiments, the VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 is selected from the group consisting of:

[0047] In some embodiments, the binding protein further comprises one or more additional binding domains operably linked to the N-terminus or C-terminus of the binding protein.

[0048] In some embodiments, one or more additional binding domains are operably linked to the N-terminus of the first or second pseudo-Fab portion.

[0049] In some embodiments, one or more additional binding domains are operably linked to the N-terminus of the first or second Fab portion.

[0050] In another aspect, the present disclosure provides a multispecific binding protein comprising four polypeptide chains that form at least two antigen binding sites, (a) the first polypeptide has the formula: VLa-L1-VLX[I] The structure includes: (b) the second polypeptide has the formula: VHa-L2-VHX-FC1[II] The structure includes: (c) the third polypeptide has the formula: VLb-CL[III] The structure includes: (d) the fourth polypeptide has the formula: VHb-CH1-FC2[IV] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1 and L2 are independently the same or different amino acid linkers. (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The dsKO domain provides said multispecific binding protein comprising (i) one or more inactivating mutations that abolish its binding to a target antigen, and (ii) one or more engineered interchain disulfide bonds.

[0051] In another aspect, the present disclosure provides an antigen binding protein comprising six polypeptide chains forming four antigen binding sites, (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHa-L2-VHX-L3-VHb-CH1-FC1[V] The structure includes: (d) the sixth polypeptide has the formula: VHa-L2-VHX-L3-VHb-CH1-FC2[VI] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2 and L3 are amino acid linkers (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; A dsKO domain provides said antigen-binding protein comprising (i) one or more inactivating mutations that abolish its binding to a target antigen, and (ii) one or more engineered interchain disulfide bonds.

[0052] In another aspect, the present disclosure provides an antigen binding protein comprising six polypeptide chains forming four antigen binding sites, (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHb-CH1-L3-VHa-L2-VHX-FC1[V] The structure includes: (d) the sixth polypeptide has the formula: VHb-CH1-L3-VHa-L2-VHX-FC2[VI] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2 and L3 are amino acid linkers, (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; A dsKO domain provides said antigen-binding protein comprising (i) one or more inactivating mutations that abolish its binding to a target antigen, and (ii) one or more engineered interchain disulfide bonds.

[0053] In another aspect, the present disclosure provides an antigen binding protein comprising six polypeptide chains forming four antigen binding sites, (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHa-L2-VHX-L3-VHa-L4-VHX-FC1[V] The structure includes: (d) the sixth polypeptide has the formula: VHb-CH1-L5-VHb-CH1-FC2[VI] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, L4 and L5 are amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; A dsKO domain provides said antigen-binding protein comprising (i) one or more inactivating mutations that abolish its binding to a target antigen, and (ii) one or more engineered interchain disulfide bonds.

[0054] In another aspect, the present disclosure provides an antigen binding protein comprising four polypeptide chains forming three antigen binding sites, (a) The polypeptide has the formula: VLa-L1-VLX[I] The structure includes: (b) the second polypeptide has the formula: VHa-L2-VHX-FC1[II] The structure includes: (c) the third polypeptide has the formula: VLb-L3-VLc-L4-CL[III] The structure includes: (d) the fourth polypeptide has the formula: VHc-L5-VHb-L6-CH1-FC2[IV] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VLc is the third immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VHc is the third immunoglobulin heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; VLX is the first stabilized knockout light chain variable domain; VHX is the first stabilized knockout heavy chain variable domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, L4, L5 and L6 are amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a third VL domain (VLc) pairs with a third VH domain (VHc) to form a first functional antigen-binding site that binds to target antigen C; (4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV); (5) a stabilized knockout VL domain (VLX) paired with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; A dsKO domain provides said antigen-binding protein comprising (i) one or more inactivating mutations that abolish its binding to a target antigen, and (ii) one or more engineered interchain disulfide bonds.

[0055] In another aspect, the present disclosure provides an antigen binding protein comprising four polypeptide chains forming three antigen binding sites, (a) The polypeptide has the formula: VLa-L1-VLX[I] The structure includes: (b) the second polypeptide has the formula: VHa-L2-VHX-FC1[II] The structure includes: (c) the third polypeptide has the formula: VLb-L3-VLc-L4-CL[III] The structure includes: (d) the fourth polypeptide has the formula: VHc-L5-VHb-L6-CH1-FC2[IV] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VLc is the third immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VHc is the third immunoglobulin heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; VLX is the first stabilized knockout light chain variable domain; VHX is the first stabilized knockout heavy chain variable domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, L4, L5 and L6 are amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a third VL domain (VLc) pairs with a third VH domain (VHc) to form a first functional antigen-binding site that binds to target antigen C; (4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV); (5) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; A dsKO domain provides said antigen-binding protein comprising (i) one or more inactivating mutations that abolish the binding of a reference Fab molecule to its target antigen, and (ii) one or more engineered interchain disulfide bonds.

[0056] In another aspect, some embodiments relate to all of the binding proteins described herein, wherein the dsKO domain comprises (i) one or more inactivating mutations that abolish its binding to the target antigen relative to the wild-type domain, and (ii) one or more engineered interchain disulfide bonds that confer increased thermal stability (Tm) of the pseudo-Fab relative to a reference Fab molecule, which is identical to the pseudo-Fab molecule except that the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains in the pseudo-Fab molecule. In these embodiments, binding to the target antigen is measured by methods known in the art, such as, but not limited to, surface plasmon resonance, and thermal stability is measured by methods known in the art, such as, but not limited to, differential scanning calorimetry.

[0057] In some embodiments, the FC1 and FC2 domains comprise one or more knobs-in-holes (KIH) mutations, which mutations promote Fc domain heterodimerization of the polypeptide.

[0058] In some embodiments, one of FC1 or FC2 comprises a first CH3 domain comprising one or both of an S354C and a T366W mutation, and the other of FC1 or FC2 comprises a second CH3 domain comprising one or both of an Y349C, T366S, L368A and a Y407V mutation, wherein the mutations promote Fc domain heterodimerization.

[0059] In some embodiments, the FC1 or FC2 domain comprises an H435R and / or a Y436F mutation.

[0060] In some embodiments, at least two of target antigen A, target antigen B, and target antigen C are different target antigens.

[0061] In some embodiments, at least one of the target antigens is a ligand for a cell surface receptor, and at least one of the target antigens is a cell surface receptor.

[0062] In some embodiments, the antigen binding sites are derived from different antibodies.

[0063] In some embodiments, target antigen A, target antigen B, and target antigen C are the same target antigen.

[0064] In some embodiments, the antigen binding sites bind to different epitopes on the same target antigen.

[0065] In some embodiments, the antigen binding sites bind to the same epitope on the same target antigen.

[0066] In some embodiments, the antigen binding sites are derived from the same antibody.

[0067] In some embodiments, the melting temperature (T m ) is at least 4°C higher than the reference Fab molecule.

[0068] In some embodiments, the engineered interchain disulfide bond is VH44C-VL100C.

[0069] In some embodiments, the engineered interchain disulfide bond is VH105C-VL43C.

[0070] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in the VHX domain of the pseudo-Fab portion.

[0071] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRH3 of the VHX domain.

[0072] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRH2 of the VHX domain.

[0073] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRH1 of the VHX domain.

[0074] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in the VLX domain of the pseudo-Fab portion.

[0075] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRL3 of the VLX domain.

[0076] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRL2 of the VLX domain.

[0077] In some embodiments, at least one of the one or more inactivating mutations that abolish binding to the target antigen is present in CDRL1 of the VLX domain.

[0078] In some embodiments, the VHX domain of the pseudo-Fab portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79.

[0079] In some embodiments, the VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 is selected from the group consisting of:

[0080] In another aspect, the disclosure provides the use of a stabilized knockout domain to reduce heavy chain-light chain mispairing in a multispecific binding protein, wherein the stabilized knockout domain comprises (3) a VHX and VLX domain that comprises one or more inactivating mutations that abolish its binding to a target antigen, and (4) one or more engineered interchain disulfide bonds that confer increased thermal stability (Tm) of the pseudo-Fab relative to a reference Fab molecule that is identical to the pseudo-Fab molecule except that in the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with the VHX and VLX domains.

[0081] In some embodiments, the engineered interchain disulfide bond is VH44C-VL100C.

[0082] In some embodiments, the engineered interchain disulfide bond is VH105C-VL43C.

[0083] In some embodiments, the VHX domain of the pseudo-Fab portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79.

[0084] In some embodiments, the VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; VHX comprising the amino acid sequence of SEQ ID NO: 79 is selected from the group consisting of:

[0085] In some embodiments, the pseudo-Fab lacks the CH1 and CL domains.

[0086] In some embodiments, an isolated nucleic acid molecule is provided that comprises a nucleotide sequence encoding one or more of the binding proteins. In some embodiments, a kit of isolated nucleic acid molecules is provided that comprises one or more nucleotide sequences encoding one or more of the binding proteins.

[0087] In some embodiments, an expression vector comprising the nucleic acid molecule is provided. In some embodiments, an expression vector kit comprising the nucleic acid molecule is provided.

[0088] In some embodiments, an isolated host cell is provided comprising the nucleic acid molecule or expression vector. In some embodiments, an isolated host cell is provided comprising a kit of nucleic acid molecules or a kit of expression vectors.

[0089] In some embodiments, a method of producing a binding protein is provided, the method comprising culturing a host cell under conditions such that the binding protein is expressed; and purifying the binding protein from the host cell.

[0090] In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the multispecific binding protein. In some embodiments, a multispecific binding protein is provided for use as a medicament.

[0091] In some embodiments, methods are provided for treating a disorder in which antigenic activity is detrimental, comprising administering to a subject in need thereof an effective amount of a multispecific binding protein.

[0092] The above summary of the invention is non-limiting, and other features and advantages of the disclosed compositions and methods will be apparent from the following detailed description of the invention and the claims.

[0093] A brief description of arrays SEQ ID NO: 1: Variable light chain sequence of trastuzumab.

[0094] SEQ ID NO: 2: Variable heavy chain sequence of trastuzumab.

[0095] SEQ ID NO: 3: Variable heavy chain sequence of trastuzumab ko variant 1.

[0096] SEQ ID NO: 4: Variable heavy chain sequence of trastuzumab ko mutant 2.

[0097] SEQ ID NO: 5: Variable heavy chain sequence of trastuzumab ko mutant 3.

[0098] SEQ ID NO: 6: Anti-IL13--VL-G4S-anti-Her2-(Trastuzumab-Q100C)-VL.

[0099] SEQ ID NO: 7: Anti-IL13--VH-G4S-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0100] SEQ ID NO: 8: Anti-IL13-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0101] SEQ ID NO: 9: Anti-IL13-VH-(G4S)2-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0102] SEQ ID NO: 10: anti-IL13-VL3-IGK.

[0103] SEQ ID NO: 11: Anti-IL13-VH2-IGHG1.

[0104] SEQ ID NO: 12: Anti-TNFalpha-VL-G4S-anti-Her2-(trastuzumab-Q100C)-VL.

[0105] SEQ ID NO: 13: Anti-TNFalpha-VH-G4S-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0106] SEQ ID NO: 14: Anti-TNFalpha-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0107] SEQ ID NO: 15: Anti-TNFalpha-VH-(G4S)2-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0108] SEQ ID NO: 16: anti-TNFα-VL-huIGKC.

[0109] SEQ ID NO: 17: anti-TNFα-VH-huIgG1.

[0110] SEQ ID NO: 18: Anti-IL6R-VL-G4S-anti-Her2-(trastuzumab-Q100C)-VL.

[0111] SEQ ID NO: 19: Anti-IL6R-VH-G4S-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0112] SEQ ID NO: 20: Anti-IL6R-VL-(G4S)2-Anti-Her2-(Trastuzumab-Q100C)-VL

[0113] SEQ ID NO: 21: Anti-IL6R-VH-(G4S)2-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0114] SEQ ID NO: 22: Anti-IL6R-VL-huIGKC.

[0115] SEQ ID NO: 23: Anti-IL6R-VH-huIgG1.

[0116] SEQ ID NO: 24: Anti-CTLA4-VL-G4S-anti-Her2-(trastuzumab-Q100C)-VL.

[0117] SEQ ID NO: 25: Anti-CTLA4-VH-G4S-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0118] SEQ ID NO: 26: Anti-CTLA4-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0119] SEQ ID NO: 27: Anti-CTLA4-VH-(G4S)2-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0120] SEQ ID NO: 28: Anti-CTLA4-VL-huIGKC.

[0121] SEQ ID NO: 29: Anti-CTLA4-VH-huIgG1.

[0122] SEQ ID NO: 30: Anti-PD1-VL-G4S-anti-Her2-(trastuzumab-Q100C)-VL.

[0123] SEQ ID NO: 31: Anti-PD1-VH-G4S-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0124] SEQ ID NO: 32: Anti-PD1-VL-(G4S)2-Anti-Her2-(Trastuzumab-Q100C)-VL

[0125] SEQ ID NO: 33: Anti-PD1-VH-(G4S)2-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0126] SEQ ID NO: 34: anti-huPD-1-VL-huIGKC.

[0127] SEQ ID NO: 35: anti-huPD-1-VH-huIgG1.

[0128] SEQ ID NO: 36: Anti-IL4-VL-G4S-anti-Her2-(trastuzumab-Q100C)-VL.

[0129] SEQ ID NO: 37: Anti-IL4-VH-G4S-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0130] SEQ ID NO: 38: Anti-IL4-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0131] SEQ ID NO: 39: Anti-IL4-VH-(G4S)2-anti-Her2-(trastuzumab-G44C)-VH-Fc-huIgG1.

[0132] SEQ ID NO: 40: Anti-IL4-VL1-IGKC.

[0133] SEQ ID NO: 41: Anti-IL4-VH1-IgG1.

[0134] SEQ ID NO: 42: Anti-PD1-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0135] SEQ ID NO: 43: Anti-PD1-VH-(G4S)2-anti-Her2-(trastuzumab-G44C-Var2)-VH-Fc-huIgG1.

[0136] SEQ ID NO: 44: Anti-CTLA4-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0137] SEQ ID NO: 45: Anti-CTLA4-VH-(G4S)2-anti-Her2-(trastuzumab-G44C-Var2)-VH-Fc-huIgG1.

[0138] SEQ ID NO: 46: Anti-IL4-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0139] SEQ ID NO: 47: Anti-IL4-VH-(G4S)2-anti-Her2-(trastuzumab-G44C-Var2)-VH-Fc-huIgG1.

[0140] SEQ ID NO: 48: Anti-IL13-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0141] SEQ ID NO: 49: Anti-IL13-VH-(G4S)2-Anti-Her2-(Trastuzumab-G44C-Var2)-VH-DKTHT-His6.

[0142] SEQ ID NO: 50: Anti-IL13-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0143] SEQ ID NO: 51: Anti-IL13-VH-(G4S)2-anti-Her2-(Trastuzumab-VH_Var1-G44C)-VH-Fc-huIgG1.

[0144] SEQ ID NO: 52: Anti-IL4-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0145] SEQ ID NO: 53: Anti-IL4-VH-(G4S)2-Anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-huIgG1 (knob).

[0146] SEQ ID NO: 54: anti-PD1-huIGKC.

[0147] SEQ ID NO: 55: Anti-PD1-VH-huIgG1(Whole-RF).

[0148] SEQ ID NO: 56: Anti-IL13-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0149] SEQ ID NO: 57: Anti-IL13-VH-(G4S)2-Anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-huIgG1 (knob).

[0150] SEQ ID NO: 58: anti-PD1-VL-huIGKC.

[0151] SEQ ID NO: 59: Anti-PD1-VH-huIgG1(Whole-RF).

[0152] SEQ ID NO: 60: Anti-PD1-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0153] SEQ ID NO: 61: Anti-PD1-VH-(G4S)2-anti-Her2-(trastuzumab-G44C-Var2)-VH-Fc-huIgG1 (knob).

[0154] SEQ ID NO: 62: Anti-IL13-VL huIGKC.

[0155] SEQ ID NO: 63: Anti-IL13-VH-huIgG1(Whole-RF).

[0156] SEQ ID NO: 64: Anti-CTLA4-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0157] SEQ ID NO: 65: Anti-CTLA4-VH-(G4S)2-Anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-huIgG1 (knob).

[0158] SEQ ID NO: 66: anti-PD1-VL-huIGKC.

[0159] SEQ ID NO: 67: Anti-PD1-VH-huIgG1(Whole-RF).

[0160] SEQ ID NO: 68: Anti-IL4-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0161] SEQ ID NO: 69: Anti-IL4-VH-(G4S)2-Anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-huIgG1 (knob).

[0162] SEQ ID NO: 70: Anti-IL13-VL huIGKC.

[0163] SEQ ID NO: 71: Anti-IL13-VH-huIgG1(Whole-RF).

[0164] SEQ ID NO: 72: Anti-PD1-VL-(G4S)2-anti-Her2-(trastuzumab-Q100C)-VL.

[0165] SEQ ID NO: 73: Anti-PD1-VH-(G4S)2-anti-Her2-(trastuzumab-G44C-Var2)-VH-Fc-huIgG1 (knob).

[0166] SEQ ID NO: 74: anti-PD1-VL-huIGKC.

[0167] SEQ ID NO: 75: Anti-PD1-VH-huIgG1(Whole-RF).

[0168] SEQ ID NO: 76: Variable light chain sequence of ds ko trastuzumab.

[0169] SEQ ID NO: 77: Variable heavy chain sequence of ds ko trastuzumab variant 1.

[0170] SEQ ID NO: 78: Variable heavy chain sequence of ds ko trastuzumab variant 2.

[0171] SEQ ID NO: 79: Variable heavy chain sequence of ds ko trastuzumab variant 3.

[0172] SEQ ID NO: 80: Anti-TCR alpha / beta x anti-CD123 wild type

[0173] SEQ ID NO: 81: anti-TCR alpha / beta x anti-CD123-dsTrasKO2

[0174] SEQ ID NO: 82: anti-TCRα / β-dsTrasKO2×anti-CD123

[0175] SEQ ID NO: 83: anti-CD3ε x anti-CD123 wild type

[0176] SEQ ID NO: 84: anti-CD3ε x anti-CD123-dsTrasKO2

[0177] SEQ ID NO: 85: anti-CD3ε-dsTrasKO2×anti-CD123

[0178] SEQ ID NO: 86: anti-CD3ε x anti-CD123 wild type

[0179] SEQ ID NO: 87: anti-CD3ε x anti-CD123-dsTrasKO2

[0180] SEQ ID NO: 88: anti-CD3ε-dsTrasKO2×anti-CD123

[0181] SEQ ID NO: 89: Anti-TCR alpha / beta x anti-TNP negative control - wild type

[0182] SEQ ID NO: 90: Anti-TCR alpha / beta x anti-TNP-dsTrasKO2 negative control

[0183] SEQ ID NO: 91: anti-TCRα / β-dsTrasKO2 x anti-TNP negative control

[0184] SEQ ID NO: 92: Anti-TNP x anti-CD123 negative control - wild type

[0185] SEQ ID NO: 93: Anti-TNP x anti-CD123-dsTrasKO2 negative control

[0186] SEQ ID NO: 94: anti-TNP-dsTrasKO2 x anti-CD123 negative control

[0187] SEQ ID NO: 95: Anti-CD3ε x anti-TNP negative control - wild type

[0188] SEQ ID NO: 96: anti-CD3ε x anti-TNP-dsTrasKO2 negative control

[0189] SEQ ID NO: 97: anti-CD3ε-dsTrasKO2 x anti-TNP negative control

[0190] SEQ ID NO: 98: Anti-CD3ε x anti-TNP negative control - wild type

[0191] SEQ ID NO: 99: anti-CD3ε x anti-TNP-dsTrasKO2 negative control

[0192] SEQ ID NO: 100: anti-CD3ε-dsTrasKO2×x anti-TNP negative control

[0193] The above and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file will contain at least one drawing executed in color. Copies of this patent or patent application publication and the color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0194] [Figure 1] 1A-1B are schematic diagrams of dimeric bispecific tandem molecules containing pseudo-Fab fragments in which the CH1 / CL pair has been replaced with a disulfide-stabilized knockout domain (dsKO). A tandem-(Fv-Fab x Fv-pseudo-Fab) molecule is shown in FIG. 1A, and a tandem-(Fv-pseudo-Fab x Fv-Fab) molecule is shown in FIG. 1B. [Figure 2]Figure 1 shows a schematic representation of an exemplary dimeric bispecific IgG molecule ((Fv-pseudo-Fab) x (Fv-Fab)-Fc). A disulfide-stabilized knockout domain (dsKO) replaces the CH / CL domain of one Fab arm of the IgG molecule. A peptide linker (e.g., G4S or (G4S)2) connects the first antigen-binding site (Fv) to the dsKO domain to form the pseudo-Fab portion that binds to antigen target A. An Fc heterodimerization domain with a knob-into-hole (KIH) or RF mutation links the pseudo-Fab portion to the second Fab binding arm that binds to antigen target B. [Figure 3] 3A-3C are schematic diagrams showing the monomer fraction of an antibody construct containing a trastuzumab WT VH / VL substitution in CH1 / CL (FIG. 3A) compared to an antibody construct containing a disulfide-stabilized trastuzumab knockout ("dsTrastKO") VH / VL substitution in CH1 / CL (FIG. 3B), as well as the thermal stability of both constructs (FIG. 3C). [Figure 4] FIG. 1 is a schematic representation of the PDB structure 1N8Z of trastuzumab bound to HER2. The locations of four inactivating mutations that abolish binding to HER2 (R50, R59, Y33, and Y103) are indicated. [Figure 5] 5A-5B are graphs depicting the results of binding experiments showing that dsTrastuKO mutants 1-3 no longer bind to HER2. [Figure 6] 6A-6B show pseudo-IgG and pseudo-Fab constructs used as a type of control in the experiments. [Figure 7]Figures 7A-7D show representative bispecific formats and purification results according to certain exemplary embodiments. Figure 7A shows the arrangement of individual domains within the IgG scaffold. Fv1 (anti-IL4) is fused to the VL / VH of dsTrasKO2 via a (G4S)2 linker. Fv2 (anti-IL13) retains the wild-type configuration. Figure 7B shows the reduced (one light chain and one heavy chain) and oxidized forms of the antibody using 4-12% Bis / Tris MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Product purity using analytical size exclusion chromatography is shown in Figure 7C. Molecular integrity was verified by intact mass analysis using an Agilent 6540 ultra-high definition (UHD) Q-TOF with a Jet Stream dual ESI interface and an Agilent 1290 / 1260 Infinity LC system (Figure 7D). [Figure 8] FIG. 1 shows analytical hydrophobic interaction chromatography (HIC) results, demonstrating that the dsTrasKO2-bispecific construct was correctly paired and contained no unexpected species. [Figure 9] 6A is a schematic representation of the crystal structure of the pseudo-Fab IL13-dsTrasKO2 construct with the ultrastructure of Figure 6A. The structure was solved at 3.75 Å. The structure shows the superposition of the IL13-VH / VL domains of the TrasKO2-IL13 pseudo-Fab (light grey) and the Fab anti-IL13 (dark grey). [Figure 10] 10A-10B are schematic diagrams of dimeric bispecific tandem molecules containing pseudo-Fab fragments in which the CH1 / CL pair has been replaced with a disulfide-stabilized knockout domain (dsKO). A tandem-(Fv-Fab x Fv-pseudo-Fab)-IgG molecule is shown in Figure 10A, and a tandem-(Fv-pseudo-Fab x Fv-Fab)-IgG molecule is shown in Figure 10B. [Figure 11]11A-11B are schematic diagrams of dimeric bispecific tandem molecules containing pseudo-Fab fragments in which the CH1 / CL pair has been replaced with a disulfide-stabilized knockout domain (dsKO). The ((((Fv-pseudo-Fab)[HC]-(Fv-pseudo-Fab))×((Fv-Fab)[HC]-(Fv-Fab)))-Fc molecule is shown in FIG. 11A with an Fc heterodimerization domain with knobs-into-holes (KIH) and RF mutations, and in FIG. 11B with only the RF mutation. [Figure 12] Figures 12A-12D show a representative bispecific tandem IgG design in which each Fv2 domain (with binding specificity for a second target antigen B, i.e., Ox40) of a conventional IgG antibody (pogalizumab) is appended with a first pseudo-Fab containing an Fv1 domain (with binding specificity for a first target antigen A, i.e., GITR) and a dsTrasKO domain. Figure 12A shows the arrangement of the individual domains within the IgG scaffold. Fv1 (anti-GITR) is fused to the VL / VH of dsTrasKO2 via a (G4S)2 linker. Fv2 (anti-Ox40) retains its wild-type configuration. Fv1-dsTrasKO2 is fused to Fv2-Ck / CHI via a (G4S)2 linker. Figure 12B shows the reduced (two light chains and one heavy chain) and oxidized forms of the antibody using 4-12% Bis / Tris MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Product purity using analytical size exclusion chromatography is shown in Figure 12C. The integrity of the molecule was verified by intact mass analysis using an Agilent 6540 ultra-high definition (UHD) Q-TOF with a Jet Stream Dual ESI interface and an Agilent 1290 / 1260 Infinity LC system (Figure 12D). [Figure 13]Figures 13A-13D show a representative trispecific CODV IgG design, in which a first pseudo-Fab containing an Fv3 domain (with binding specificity for the first target antigen A, i.e., CD137) and a dsTrasKO domain is paired with a CODV arm (with binding specificity for the second target antigen B, i.e., Ox40 (Fv1) and a third target antigen C, i.e., PD1 (Fv2)). Figure 13A shows the arrangement of individual domains within the CODV IgG scaffold. Fv1 (anti-Ox40) and Fv2 (anti-PD1) on the CODV arm are fused to the wild-type lambda and CH1 domains. Fv3 (anti-CD137) on the Fab arm is fused to the VL / VH of dsTrasKO2 via a (G4S)2 linker. Figure 13B shows the reduced (two light chains and two heavy chains) and oxidized forms of CODV antibodies using 4-12% Bis / Tris MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Product purity using analytical size exclusion chromatography is shown in Figure 13C. The integrity of the molecule was verified by intact mass analysis using an Agilent 6540 ultra-high definition (UHD) Q-TOF with a Jet Stream Dual ESI interface and an Agilent 1290 / 1260 Infinity LC system (Figure 13D). [Figure 14] FIG. 1 shows a schematic of the two-step purification process used to ensure correct light chain pairing with dsTrasKO2 knockout bispecific molecules. [Figure 15]Figures 15A-15B show human pan-T cell cytotoxicity assays against THP-1 target cells co-incubated with the bispecific antibody anti-CD3ε x anti-CD123. Figure 15A corresponds to bispecific antibodies with ID numbers 33, 34, and 35, with negative controls 45 and 46. Figure 15B corresponds to bispecific antibodies with ID numbers 36, 37, and 38, with negative controls 47 and 48. T effector cells and CFSE-labeled THP-1 target cells were seeded at an effector-to-target ratio of 10:1 and co-incubated with serial dilutions of each bispecific molecule (10 nM to 0 nM) for 20 hours at 37°C. 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 double positive). Data show dead target cells [%] versus bispecific molecule concentration [pM] as an average of two representative healthy donors. DETAILED DESCRIPTION OF THE INVENTION

[0195] I. Definition In order that this disclosure may be more readily understood, selected terms are defined below.

[0196] As 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, such as α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids, may also be suitable components of the polypeptide chains of the binding proteins of the invention. 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 the polypeptide notation used herein, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxyl-terminal direction, following standard usage and convention. Naturally occurring residues can be divided into classes based on common side chain properties (see Table 1).

[0197] [Table 1]

[0198] Conservative amino acid substitutions can involve exchanging one member of these classes with another member of the same class.Conservative amino acid substitutions can involve non-naturally occurring amino acid residues, and are typically incorporated by chemical peptide synthesis rather than by synthesis in living systems.These include peptidomimetics and other forms of reversion or inversion of amino acid residues.Non-conservative substitutions can involve exchanging one member of these classes with a member of another class.

[0199] As used herein, the term "mutation" or "mutant form" refers to a change in amino acid sequence due to deletion, insertion, and / or substitution of one or more amino acids. Mutations are introduced into a predetermined sequence, for example, the amino acid sequence of a VL1 and / or VH1 pair that specifically recognizes epitope 1. The term "non-mutated" refers to any amino acid sequence that exhibits functional properties, for example, any sequence that still exhibits binding properties. This is illustrated as follows: VH1 / VL1 is mutated so that VH1 / VL1 does not specifically bind to the epitope. The non-mutated form of this VH1 / VL1 still specifically binds to epitope 1. Thus, all VH / VL domains of antibodies that bind to any epitope are suitable to be mutated to serve as scaffold proteins of the present invention.

[0200] As used herein, the term "variant" refers to an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence from which it is derived, e.g., SEQ ID NO: 1 or SEQ ID NO: 2. The determination of percent identity between two sequences is accomplished using the mathematical algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Alternatively, variants can also be defined as having up to 20, 15, 10, 5, 4, 3, 2, or 1 amino acid substitutions, particularly conservative amino acid substitutions. Conservative substitutions are well known in the art (see, e.g., Creighton (1984) Proteins. W.H. Freeman and Company). A summary of the physical and chemical properties of amino acids is provided in Table 1 above. In certain embodiments, conservative substitutions are made with amino acids that share at least one property according to Table 1 in common (i.e., in columns 1 and / or 2). The term "variant" also includes fragments. Fragments have N-terminal and / or C-terminal deletions totaling up to 20, 15, 10, 5, 4, 3, 2, or 1 amino acid(s). Additionally or alternatively, the variants are modified by N-terminal and / or C-terminal amino acid additions, for example, up to a total of 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 amino acids.

[0201] As used herein, the term "binding protein" or "binding polypeptide" refers to a polypeptide (e.g., an antibody or fragment thereof) that comprises at least one binding site that is involved in selective binding to 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 comprises multiple (e.g., two, three, four, or more) binding sites. In certain embodiments, the binding protein is not a therapeutic enzyme.

[0202] As used herein, the term "Her2" or "HER2" refers to human epidermal growth factor receptor 2, a member of the epidermal growth factor receptor family.

[0203] As used herein, the term "binding protein" refers to a non-naturally occurring or recombinant or engineered molecule capable of specifically binding to at least one antigen. In certain embodiments, the binding protein comprises at least one VH / VL pair that specifically binds to the antigen.

[0204] Producing bispecific binding proteins by coexpression of two light chains and two heavy chains in a single host cell can be very challenging due to low yields of the desired bispecific binding protein and the difficulty of removing closely related mismatched binding protein contaminants (Suresh et al., Proc. Natl. Acad. Sci. USA 83:7989-7993, 1986). This is because heavy chains form homodimers as well as the desired heterodimers, referred to herein as the "heavy chain pairing problem." Furthermore, because light chains can mispair with non-cognate heavy chains, referred to herein as the "light chain pairing problem," coexpression of two antibodies can result in up to nine undesired species in addition to the desired bispecific binding protein.

[0205] As used herein, "heterodimerization domain" refers to a subunit of a bi- or multispecific binding protein that facilitates, directs, or forces the correct assembly of light chains and their cognate heavy chains to yield the desired protein, while preventing mispairing of the respective light or heavy chains.

[0206] As used herein, the term "heterodimerized Fc" or "functional fragment of heterodimerized Fc" refers to a mutant form of a constant domain, e.g., a CH2-CH3 or CH2-CH3-CH4 mutant form, which is mutant with respect to a naturally occurring Fc portion in that it no longer forms homodimers but forms heterodimers with the corresponding mutant Fc portion. Thus, the term refers to one portion of the two chains that form the heterodimer. Several such portions are known in the art and include, for example, knobs-in-holes (KIH) mutants or EV-RWT mutants.

[0207] Ridgeway and coworkers created a CH3 interface that favors heterodimer assembly by replacing small side chains on one CH3 interface with larger ones to create a knob, and replacing large side chains on the other CH3 domain with smaller ones to create a hole. Testing of such mutants demonstrated selective heterodimerization. This original knob-into-hole mutation was further expanded to identify further suitable combinations through phage display, which was used to generate bispecific IgG antibodies to test additional substitutions that allow disulfide bond formation. Knob-into-hole mutants are further described in U.S. Patent Nos. 5,732,168 and 8,216,805, which are incorporated herein by reference. Thus, in one embodiment, the CH3 domain of one Fc domain or heterodimerization domain comprises the mutations Y349C, T366S, L368A and Y407V, and the CH3 domain of another Fc domain or heterodimerization domain comprises the mutations S354C and T366W (amino acid positions are indicated by reference to the IgG1 sequence).

[0208] As used herein, the term "homodimerization domain" refers to a domain that mediates the homodimerization of two similar domains, e.g., two heavy chains. Heavy chain pairing is mediated by the last domain of the constant region, i.e., CH3 in IgG molecules, and results in a high-affinity homodimer complex (K of approximately 10 pM). D ) form a homodimer. Additional interactions exist in the hinge region, which is involved in the covalent bond between the two heavy chains formed after heavy chain assembly. As shown for the CH3 of human γ1, the CH3 homodimer interaction involves approximately 16 residues at the CH3 interface, with a patch of six residues (T366, L368, F405, Y407, and K409) formed in the center of the interface that strongly contributes to stability. Homodimerization domains include, but are not limited to, Fc regions and their effector-modified variants or fragments thereof, as well as CH2 domains or fragments thereof, CH3 domains or fragments thereof, CH4 domains or fragments thereof, etc.

[0209] Naturally occurring antibodies typically comprise tetramers. Such tetramers typically consist of two identical pairs of polypeptide chains, each pair having one full-length "light chain" (typically having a molecular weight of about 25 kDa) and one full-length "heavy chain" (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain," as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100-110 or more amino acids responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in naturally occurring antibodies, a full-length heavy chain IgG immunoglobulin polypeptide comprises a variable domain (VH) and three constant domains (CH1, CH2 and CH3), with the VH domain being at the amino-terminus of the polypeptide and the CH3 domain being at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide comprises a variable domain (VL) and a constant domain (CL), with the VL domain being at the amino-terminus of the polypeptide and the CL domain being at the carboxyl-terminus.

[0210] 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 the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses, including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, and heavy chains also contain a "D" region of about 10 or more amino acids. See, e.g., Basic Immunology (Paul, W., ed., Raven Press, 2nd ed., 1989), incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, allowing binding to a specific epitope. From the amino terminus to the carboxyl terminus, both the light chain and the heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0211] As used herein, the term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently in different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health, Bethesda, MD (1987) and (1991)) not only provides unambiguous residue numbers applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs are referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Affol. Biol. 196:901-17; Chothia et al., 1989, Nature 342:877-83) found that, despite great diversity at the amino acid sequence level, certain subportions within the Kabat CDRs adopt nearly identical peptide backbone structures. These subportions are designated L1, L2, and L3, or H1, H2, and H3, with "L" and "H" indicating the light and heavy chain regions, respectively. These regions are designated by the Kabat CDRs. The boundaries of CDRs that overlap with the Kabat CDRs are referred to as Chothia CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs are described by Padlan, 1995, FASEB J. 9:133-39; MacCallum, 1996, J. Mol. Biol. 262(5):732-45; Lefranc, 2003, Dev. Comp. Immunol. 27:55-77. Still other CDR boundary definitions may not strictly adhere to one of the systems herein, but may nonetheless overlap with the Kabat CDRs, although they may be shortened or extended 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.The methods used herein can utilize CDRs defined according to any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia. Identification of predicted CDRs using amino acid sequences is well known in the art, for example, in Martin, A.C., "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Vol. 2, Kontermann R., Dikel S. (eds.), Springer-Verlag, Berlin, pp. 33-51 (2010). The amino acid sequences of the heavy and / or light chain variable domains can also be examined to identify CDR sequences by other conventional methods, for example, by comparing them with known amino acid sequences of other heavy and light chain variable regions to determine regions of sequence hypervariability. Numbered sequences can be aligned visually or by using an alignment program, such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models are routinely used to accurately delineate the framework and CDR regions and thus refine the sequence-based assignments.

[0212] In some embodiments, the definition of the CDR / FR in an immunoglobulin light or heavy chain may be determined based on the IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0213] The term "Fc," as used herein, refers to a molecule containing the sequence of a non-antigen-binding fragment resulting from antibody digestion or produced by other means, and may include the hinge region, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is typically human and can be any immunoglobulin, although IgG1 and IgG2 are used in exemplary embodiments. Fc molecules are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from one to four, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of an Fc is a disulfide-linked dimer resulting from papain digestion of IgG. The term "native Fc" as used herein generally refers to the monomeric, dimeric and multimeric forms.

[0214] An F(ab) fragment typically contains one light chain and one heavy chain VH and CH1 domain, and the VH-CH1 heavy chain portion of the F(ab) fragment cannot form disulfide bonds with another heavy chain polypeptide. As used herein, an F(ab) fragment can also contain one light chain containing two variable domains separated by an amino acid linker, and one heavy chain containing two variable domains separated by an amino acid linker and a CH1 domain.

[0215] An F(ab') fragment typically contains one light chain and a portion of one heavy chain containing more of the constant region (between the CH1 and CH2 domains), allowing interchain disulfide bonds to form between the two heavy chains to form an F(ab')2 molecule.

[0216] As used herein, the term "Tm" refers to the melting temperature of a binding protein, antigen-binding protein, or antibody, and is an important parameter for the thermal stability of an antigen-binding protein. Tm generally refers to the thermal stability of an Fv fragment, i.e., the variable region heavy chain and light chain (VH / VL). Tm can be measured by differential scanning calorimetry (DSC).

[0217] One embodiment of the present disclosure provides binding proteins having biological and immunological specificity for one to three target antigens. Another embodiment of the present disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another embodiment of the present disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Yet another embodiment of the present disclosure provides host cells that express such binding proteins (i.e., comprise nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).

[0218] The term "antigen" or "target antigen" or "antigen target," as used herein, refers to a molecule or portion of a molecule (e.g., an epitope) that can be bound by a binding protein and that can further be used in an animal to generate antibodies that can bind to an epitope of that antigen. A target antigen can have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein can compete with intact antibodies that recognize the target antigen.

[0219] As used herein, the terms "epitope" or "target epitope" or "epitope target" refer to any determinant, e.g., a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. For example, but in no way limited to, target epitope A can be a first epitope on an antigen, and target epitope B can be a second epitope on an antigen. Alternatively, target epitope B can be a second epitope on a second antigen. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody, or an antigen-binding fragment of an antibody, or a binding protein. In certain embodiments, a binding protein is said to specifically bind 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 the equilibrium dissociation constant is less than 10 M, -9 M or dissociation constant is 10 -10 If the binding affinity is less than M, it is said to specifically bind to the antigen.

[0220] As used herein, the term "linker" refers to 0 to 100 consecutive amino acid residues. Linkers may be present or absent, and may be the same or different. The linkers may all have the same amino acid sequence, or may all have different amino acid sequences.

[0221] In some embodiments, the term "linker" refers to 1 to 15 consecutive amino acid residues. Typically, a linker provides flexibility and spatial separation between two amino acids or two polypeptide domains. Linkers can be inserted between the VH, VL, CH, and / or CL domains to provide sufficient flexibility and mobility for the light and heavy chain domains, depending on the format of the molecule, for example, to fold into a dual-variable region immunoglobulin. Linkers are typically inserted at the amino acid sequence level at the transition between variable domains, between a variable domain and a knockout domain, or between a variable domain and a constant domain, respectively. Transitions between domains can be identified because the dimensions of immunoglobulin domains are well understood. The precise location of domain transitions can be determined by placing secondary structural elements, such as peptide stretches that do not form 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 create tandem Fab antibodies. In certain embodiments, the linker is inserted between the N-terminus of the VH domain of a first Fab and the C-terminus of the CH1 domain of a second Fab.

[0222] The identity and sequence of amino acid residues in the linker can vary depending on the type of secondary structure element(s) required to be achieved in 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 are useful when an extended linker is desired. Any amino acid residue can be considered as a linker in combination with other amino acid residues to construct a larger peptide linker, if required depending on the desired properties.

[0223] In some embodiments, the linker comprises 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; SEQ ID NO: x); a peptide having five glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: x); a peptide having six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: x); a peptide having seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: x); and a peptide having eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: x).

[0224] In some embodiments, the linker comprises a small amino acid such as Gly, Ala, or Ser.

[0225] In some embodiments, the linker comprises Gly and Ser, or GS, GGS, GGGS, or GGGGS. In some embodiments, the linker comprises (Gly-Gly-Gly-Gly-Ser)2 (i.e., (GGGGS)2). In some embodiments, the linker comprises (Gly-Gly-Gly-Gly-Ser)3 (i.e., (GGGGS)3).

[0226] In some embodiments, the linker comprises Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), the peptide Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x) and the peptide Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x).

[0227] 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; Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), Thr-Val-Ala-Ala-Pro (SEQ ID NO: x), Gln-Pro-Lys-Ala-Ala, (SEQ ID NO: x), Gln-Arg-Ile-Glu-Gly (SEQ ID NO: x); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: x), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x), His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: x), and Asp-Lys-Thr-His-Thr (SEQ ID NO: x).

[0228] In some embodiments, the two tandem Fabs are linked via a (Gly-Gly-Gly-Gly-Ser) linker. In some embodiments, the linker between the stabilized knockout domain and the VH / VL pair is a (Gly-Gly-Gly-Gly-Ser) linker.

[0229] In some embodiments of a CODV-Fab portion in which L1 and L2 are on the light chain and L3 and L4 are on the heavy chain, L1 is 3-12 amino acid residues in length, L2 is 3-14 amino acid residues in length, L3 is 1-8 amino acid residues in length, and L4 is 1-3 amino acid residues in length. In some embodiments, L1 is 5-10 amino acid residues in length, L2 is 5-8 amino acid residues in length, L3 is 1-5 amino acid residues in length, and L4 is 1-2 amino acid residues in length. In some embodiments, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length. In some embodiments, L1 is 10 amino acid residues in length, L2 is 10 amino acid residues in length, L3 is 0 amino acid residue in length, and L4 is 0 amino acid residue in length. 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 (SEQ ID NO: x). In some embodiments, the linker comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x), L2 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: x), L3 comprises the sequence Ser, and L4 comprises the sequence Arg-Thr. In some embodiments, L3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x).In some embodiments, L1 comprises the sequence Ser, L2 comprises the sequence Arg-Thr, L3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Pro (SEQ ID NO: x), and L4 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: x).

[0230] In some embodiments, L1, L2, L3, and L4 are each independently (Gly-Gly-Gly-Gly-Ser) n(n is an integer from 0 to 5; SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-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-Gly-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x), L2 comprises the sequence Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), L3 comprises the sequence Ser, and L4 comprises the sequence Arg-Thr. In some embodiments, L1 comprises the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), L2 comprises the sequence Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x), L2 comprises the sequence Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), L2 is 0 amino acids in length, L3 comprises the sequence Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), and L4 is 0 amino acids in length. In some embodiments, L1 and L2 are 0 amino acids in length, and L3 and L4 are each (Gly-Gly-Gly-Gly-Ser) n(SEQ ID NO: x) (n is an integer between 0 and 5; SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-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-Gly-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x). In some embodiments, L3 and L4 are 0 amino acids in length, and L1 and L2 each comprise a sequence independently selected from (Gly-Gly-Gly-Gly-Ser) n (wherein n is an integer from 0 to 5; SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-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-Gly-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x).

[0231] In some embodiments, the linker(s) comprise a sequence derived from a naturally occurring sequence at the junction between an antibody variable domain and an antibody constant domain (e.g., as described in WO 2012 / 135345). For example, in some embodiments, the linker comprises a sequence found at the transition between an endogenous VH and CH1 domain, or between an endogenous VL and CL domain (e.g., kappa or lambda). In some embodiments, the linker comprises a sequence found at the transition between an endogenous human VH and CH1 domain, or between an endogenous human VL and CL domain (e.g., human kappa or lambda).

[0232] The examples listed above are not intended to limit the scope of the disclosure in any way, and linkers comprising randomly selected amino acids selected 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 description of linker sequences, see, for example, WO2012135345, WO2017 / 180913, which are incorporated by reference.

[0233] As used herein, the term "valency" refers to the number of binding sites of a binding protein, epitope, antigen-binding protein, or antibody. For example, the term "monovalent binding protein" refers to a binding protein with one antigen-binding site. The term "bivalent binding protein" refers to a binding protein with two binding sites. The term "trivalent binding protein" refers to a binding protein with three binding sites. The term "tetravalent binding protein" refers to a binding protein with 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., is monospecific. In other embodiments, a trivalent binding protein can bind to two different antigen targets, i.e., is bispecific. In other embodiments, a trivalent binding protein can bind to three different antigen targets, i.e., is trispecific. In certain embodiments, a tetravalent binding protein can bind to one antigen target, i.e., is monospecific. In other embodiments, the tetravalent binding protein is capable of binding to two different antigen targets, i.e., bispecific. In other embodiments, the tetravalent binding protein is capable of binding to three different antigen targets, i.e., trispecific. In other embodiments, the tetravalent binding protein is capable of binding to four different antigen targets, i.e., tetraspecific.

[0234] 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 "monospecific 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 "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. The term "tetraspecific binding protein" refers to a binding protein that specifically binds to four different antigen targets, etc.

[0235] As used herein, the term "selective recognition site" refers to a modification in a binding protein that allows it to be selectively recognized by an affinity reagent that binds to the selective recognition site. Examples of selective recognition sites include the binding site for protein A in the Fc portion of immunoglobulins.

[0236] As used herein, the term "affinity reagent" refers to a reagent containing a ligand immobilized on a matrix that specifically binds to surface groupings of molecules, such as amino acids or sugar side chains, and typically has specific three-dimensional structural characteristics as well as specific charge characteristics. Affinity reagents are tools for affinity chromatography, where specific interactions between the ligand and the product enable purification. "Protein L" is an example of an affinity reagent and refers to recombinant Protein L immobilized on a matrix and forming a ligand with affinity for a subset of the variable domain of the immunoglobulin kappa light chain. Such a matrix can be a resin. Another example of an affinity reagent is "KappaSelect," which refers to a recombinant 13 kDa camelid-derived single-chain antibody immobilized on a matrix and forming a ligand with 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 42 kDa surface protein originally found on the cell wall of the bacterium Staphylococcus aureus. Crystallographic refinements have shown that the primary binding site of 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, including IgG F(ab')2 fragments derived from the human VH3 gene family. Protein A can bind with high affinity to the Fc portion of certain immunoglobulins.

[0237] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis uses a BIAcore system (Pharmacia Biosensor; Piscataway, NJ) to measure real-time binding interactions between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term "KD" as used herein refers to the dissociation constant of the interaction between a specific binding protein and a target antigen.

[0238] As used herein, the term "specifically binds" means that a binding protein or antigen-binding fragment thereof binds to at least about 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 It refers to the ability to bind to an antigen containing the epitope with a Kd of M or greater, and / or the ability to bind to the epitope with an affinity at least two-fold greater than its affinity for a nonspecific antigen. The binding affinity of an antigen to a binding protein or antibody can be determined by surface plasmon resonance (SPR) using a BIAcore instrument.

[0239] As used herein, the term "reference Fab molecule" refers to a molecule that is identical to a pseudo-Fab molecule, except that in the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains. A "reference Fab molecule" refers to a molecule whose variable domains are identical to those of the pseudo-Fab and which has CH1 and CL domains. In the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

[0240] As used herein, the term "nucleic acid" refers to a polymeric or oligomeric macromolecule or large biomolecule essential to all known forms of life. Nucleic acids, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made of monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymer chains wound 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 nitrogenous nucleobase, a monosaccharide called deoxyribose or ribose, and a phosphate group. Naturally occurring nucleobases include guanine (G), adenine (A), thymine (T), uracil (U), or cytosine (C). Nucleotides are connected to each other in the chain by covalent bonds 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.

[0241] 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 the nucleotides comprising polynucleotides can be ribonucleotides or deoxyribonucleotides, or modified forms of any type of nucleotide.Such modifications include base modifications such as bromolysine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate and phosphoramidate.The term "polynucleotide" particularly includes single-stranded and double-stranded forms of DNA.

[0242] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and which (1) is not associated with all or part of a polynucleotide with which the isolated polynucleotide is found in nature; (2) is associated with a polynucleotide with which it is not naturally linked; or (3) is not found in nature as part of a larger sequence.

[0243] An "isolated polypeptide" is one that (1) is free from at least some other polypeptides with which it is normally found; (2) is substantially free from other polypeptides from the same source, e.g., the same species; (3) is expressed by cells from a different species; (4) is separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) is not associated (covalently or noncovalently) with portions of polypeptides with which it is naturally associated; (6) is operably linked (covalently or noncovalently) to polypeptides with which it is not naturally associated; or (7) is not naturally occurring. Such isolated polypeptides are encoded by genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, 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, preventative, research, or otherwise).

[0244] As used herein, the term "expression vector," also referred to as an expression construct, typically refers to a plasmid or virus designed for protein expression in cells. The term "vector" refers to a protein or polynucleotide, or a mixture thereof, that can or will introduce a protein and / or nucleic acid contained in the 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, e.g., foreign or heterologous DNA, into a suitable host cell. A vector may contain a "replicon" polynucleotide sequence that facilitates autonomous replication of the vector in the host cell. Foreign DNA is defined as DNA not naturally found in the host cell, e.g., heterologous DNA that replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene. Once inside a host cell, the vector can replicate independently of or simultaneously with the host chromosomal DNA, generating several copies of the vector and its inserted DNA. Furthermore, vectors can also contain the necessary elements to allow the inserted DNA to be transcribed into mRNA molecules or otherwise replicate the inserted DNA into multiple copies of RNA. Vectors can also include "expression control sequences" that regulate the expression of a gene of interest. Typically, expression control sequences are polypeptides or polynucleotides, such as, but not limited to, promoters, enhancers, silencers, insulators, or repressors. In vectors containing more than one polynucleotide encoding one or more gene products of interest, expression is controlled jointly or separately by one or more expression control sequences. More specifically, each polynucleotide contained on a vector is controlled by a separate expression control sequence, or all polynucleotides contained on a vector are controlled by a single expression control sequence. Polynucleotides contained on a single vector controlled by a single expression control sequence can form an open reading frame.Some expression vectors further contain sequence elements flanking the inserted DNA that increase the half-life of the expressed mRNA and / or allow translation of the mRNA into protein molecules, thus allowing rapid synthesis of many molecules of mRNA and polypeptide encoded by the inserted DNA.

[0245] 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 the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but such cells are included within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express binding proteins, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To recombinantly express a binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein, such that the polypeptide chains of the binding protein are expressed in the host cell and, in an exemplary embodiment, secreted into the medium in which the host cell is cultured, from which the binding protein can be recovered.

[0246] As used herein, the term "pharmaceutical composition" refers to a compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient.

[0247] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier," as used herein, refers to one or more formulation materials suitable for achieving or enhancing delivery of a binding protein.

[0248] The terms "effective amount" and "therapeutically effective amount," when used in reference to a pharmaceutical composition containing one or more binding proteins (e.g., antibodies or antigen-binding fragments thereof), refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of binding protein (e.g., antibodies or antigen-binding fragments thereof) sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated for a period of time. The effective amount can vary depending on the specific antibody-like binding protein being used and also depends 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 or therapeutically effective amount of a given pharmaceutical composition is within the capabilities of one of ordinary skill in the art.

[0249] As used herein, the term "method of producing a binding protein" refers to recombinant methods of protein expression using techniques well known in the art.

[0250] II. Pseudo FAB part In certain embodiments, the binding molecules described herein comprise at least one pseudo-Fab portion. As used herein, a "pseudo-Fab" portion is similar to the Fab portion of a conventional antibody in that it comprises a functional antigen-binding portion formed by pairing a variable light (VL) domain with a variable heavy (VH) domain. However, while the VL and VH domains of a conventional Fab are directly fused or linked to a constant light (CL) domain and a constant heavy 1 (CH1) domain, respectively, the pseudo-Fab portion lacks the CH1 and CL domains. Instead, the VL and VH domains of the pseudo-Fab are operably linked to a second pair of stabilized knockout VL and VH domains (referred to herein as VLX and VHX) that form an inactive or non-functional binding portion (referred to herein as a "stabilized knockout" portion or domain) that cannot specifically bind to a target antigen (e.g., any target antigen). In certain embodiments, the pseudo-Fab portion cannot bind to the target antigen of the corresponding Fab portion from which it is derived. The pseudo-Fab portion lacks the CH and CL domains.

[0251] Although unable to selectively bind to a target antigen, the VLX and VHX domains of the pseudo-Fab nevertheless preferentially associate with each other to form stable chain pairing. Thus, by appending the pseudo-Fab to one or more additional binding domains with different specificities, the inherent stability of the VLX / VHX chain pairing of the pseudo-Fab can drive heterodimerization of the chains of a desired multispecific binding molecule.

[0252] Thus, the pseudo-Fab of the present disclosure has the formula: (I) VHa-L1-VHX; or (II) VHb-L1-VHX and a first polypeptide chain having a structure represented by the formula: (III) VLa-L2-VLX; or (IV) VLb-L2-VLX A second polypeptide chain having a structure represented by comprising or consisting of During the ceremony, VHX associates with VLX to form the knockout domain, VH associates with VL to form a first functional antigen-binding domain; L1 and L2 are linkers and are either present or absent.

[0253] In certain embodiments, the first polypeptide chain of the pseudo-Fab has the structure VH-L1-VHX and the second polypeptide chain of the pseudo-Fab has the structure VL-L2-VLX.

[0254] In other embodiments, the first polypeptide of the pseudo-Fab has the structure VHX-L1-VH and the second polypeptide chain has the structure VLX-L2-VL.

[0255] In some embodiments, the binding protein is selected from the following group: (a) VHa-CH1-L1-VHb-L2-VHX and VLa-CL and VLb-L3-VLX; (b) VHa-L2-VHX-L1-VHb-CH1 and VLa-L3-VLX and VLb-CL; (c) VHa-CH1-L1-VHa-CH1 and VHb-L2-VHX-L3-VHb-L4-VHX and two chains VLb-L5-VLX and two chains VLa-CL and Strands (a) and (b) can occur once or twice, and L1, L2, L3, L4 and L5 are independently the same or different linkers.

[0256] (a) Knockout domain The "knockout" domain of a pseudo-Fab can be generated by any means that results in the abrogation or reduction of the binding affinity or specificity of a normally functional antigen-binding site. In certain embodiments, the knockout domain of a pseudo-Fab is rendered inactive or non-functional by one or more mutations in one or both of the VLX and VHX domains of the knockout domain. In one embodiment, the knockout modification is an amino acid substitution. In another embodiment, the knockout modification is an amino acid insertion or deletion. In another embodiment, the knockout modification is a combination of one or more amino acid substitutions, insertions, and deletions. In yet other embodiments, the knockout domain is rendered non-functional by, for example, covalent modification with a moiety that interferes with the ability of the variable domain to bind to the target antigen.

[0257] In certain embodiments, knockout modifications abolish binding by creating repulsion or disruption of a stabilized antigen-binding protein complex. In certain embodiments, knockout modifications can involve substituting residues that normally contact the target antigen with amino acids that create charge-charge repulsion with the target antigen. Additionally or alternatively, mutations can be introduced that destabilize the π-π interacting complex.

[0258] In certain embodiments, the knockout modification is a substitution of a charged amino acid with an uncharged amino acid. In other embodiments, the knockout modification is a substitution of an uncharged amino acid with a charged amino acid. In other embodiments, the knockout modification is a substitution of a polar amino acid with a nonpolar amino acid. In other embodiments, the knockout modification is a substitution of a charged amino acid with a polar and uncharged amino acid, and a substitution of a polar uncharged amino acid with a nonpolar hydrophobic amino acid.

[0259] In certain embodiments, knockout modifications are introduced at amino acid positions that form binding interactions with antigens. For example, knockout modifications can be located on the protein surface where antigen-antibody interactions normally occur. In certain exemplary embodiments, modifications can be introduced into the complementarity-determining regions (CDRs) of one or both VLX or VHX domains.

[0260] In certain embodiments, the knockout modification is a substitution of a residue in CDRH1 of the VHX domain. In other embodiments, the knockout modification is a substitution of a residue in CDRH2 of the VHX domain. In other embodiments, the knockout modification is a substitution of a residue in CDRH3 of the VHX domain.

[0261] In certain embodiments, the knockout modification is a substitution of a residue in CDRL1 of the VLX domain. In other embodiments, the knockout modification is a substitution of a residue in CDRL2 of the VLX domain. In other embodiments, the knockout modification is a substitution of a residue in CDRL3 of the VLX domain.

[0262] In certain exemplary embodiments, an arginine in the CDR of a VHX or VLX domain is mutated to glutamic acid, hi other embodiments, one or more tyrosines in the CDR of a VHX or VLX domain are mutated to alanine.

[0263] In certain embodiments, the modifications result in a knockout domain that completely lacks the target antigen binding activity of the functional, non-knockout (i.e., wild-type) counterpart from which it is derived. Alternatively, the binding functionality of the knockout domain can be substantially reduced compared to its functional, non-knockout counterpart, but can nevertheless retain some level of detectable binding.

[0264] The scaffold for generating knockout domains can be obtained, for example, from the Protein Data Bank (PDB). The PDB is a crystallographic database of three-dimensional structural data of large biomolecules, such as proteins and nucleic acids. Knockout domains can be generated by specifically mutating amino acids predicted to be involved in antigen binding by computer-based modeling of the antigen-binding domain and its cognate target antigen. Subsequent binding studies can be performed using methods known in the art, such as surface plasmon resonance, to determine with high accuracy whether the binding function is lost and the interaction between the binding domain and its target is disrupted.

[0265] (b) Stabilizing knockout domain In certain embodiments, the knockout domain is a stabilized knockout domain that has increased thermal stability compared to its wild-type counterpart. For example, in certain exemplary embodiments, the melting temperature (T m ) is within at least 0.25°C, at least 0.5°C, at least 0.75°C, at least 1°C, at least 2°C, at least 3°C, at least 4°C, at least 5°C, or at least 10°C compared to the wild-type counterpart from which it is derived. In other exemplary embodiments, the T m was increased relative to its wild-type counterpart. For example, T m may be increased by at least 0.25°C, at least 0.5°C, at least 0.75°C, at least 1°C, at least 2°C, at least 3°C, at least 4°C, at least 5°C, or at least 10°C compared to the wild-type counterpart from which it is derived. Thermal stability can be measured by differential scanning fluorimetry (DSF) or other bioanalytical methods routinely used by those of skill in the art.

[0266] In certain embodiments, the pseudo-Fab contains an engineered intrachain disulfide bond between the VHX or VLX domains of the knockout domain of the pseudo-Fab, which confers increased stability. Typically, this modification involves substituting at least one amino acid in the VHX domain with a cysteine ​​(Cys) residue and at least one amino acid in the VLX domain with a Cys residue. The Cys residue can be introduced into the VHX and VLX domains at a position that allows disulfide bond formation after dimerization. In certain embodiments, mutations can be made at the VH and VL interfaces to improve stability between the VH / VL interfaces. A specific series of amino acid mutations in the VH and VL domains can improve stability through the introduction of non-natural cysteine ​​residues that form disulfide bridges.

[0267] A first set of disulfide-stabilizing mutations can be made to amino acid residues in the VH and VL domains. In an exemplary embodiment, the disulfide bond is formed by Cys at position 44 in the VHX domain and Cys at position 100 in the VLX domain. This 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. 14:1239-1245, 1996, incorporated herein by reference for all purposes.

[0268] A second set of disulfide-stabilizing mutations can be made to the amino acid residues of the VH and VL domains. In an exemplary embodiment, the disulfide bond is formed by Cys at position 105 of the VHX domain and Cys at position 43 of the VLX domain. This second set of disulfide-stabilizing mutations is alternatively referred to as the "VH105C / VL43C" mutation set. The other disulfide-stabilizing mutation is described in more detail in U.S. Patent No. 9,527,927, which is incorporated herein by reference for all purposes.

[0269] In certain embodiments, the VLX domain of the pseudo-Fab comprises a variant of SEQ ID NO: 1 having at least one knockout modification. For example, the VLX domain can comprise an amino acid sequence that, excluding the knockout modification, is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0270] In certain embodiments, the VHX domain of the pseudo-Fab comprises a variant of SEQ ID NO: 2 with at least one knockout modification. For example, the VHX domain can comprise an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, excluding the knockout modification.

[0271] In one embodiment, the VLX domain of the pseudo-Fab comprises the amino acid sequence of SEQ ID NO:76 and the VHX domain of the pseudo-Fab comprises an amino acid sequence selected from the group of SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79.

[0272] II. Multispecific pseudo-Fab-containing binding polypeptides In another aspect, a multispecific binding protein is provided that comprises a pseudo-Fab portion as described herein. The highly modular nature of the pseudo-Fab portion allows for the formation of a wide variety of multispecific structures.

[0273] In certain embodiments, the multispecific binding proteins of the disclosure comprise additional binding specificities added to the N-terminus and / or C-terminus of one or both chains of the pseudo-Fab portion to form a multivalent pseudo-Fab-containing binding protein.

[0274] In some embodiments, the multispecific binding protein comprises: a) (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first antigen-binding site that binds to target antigen A; (2) a first stabilized knockout VL domain (VLX) paired with a first stabilized knockout VH domain (VHX) to form a first disulfide-stabilized knockout (dsKO) domain; (3) First heterodimerization domain (HD1) a first pseudo-Fab portion comprising The first dsKO domain comprises a first pseudo-Fab portion containing (i) one or more inactivating mutations that abolish its binding to the target antigen and (ii) one or more engineered interchain disulfide bonds; b) (1) a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (2) a first CH1 domain paired with a first CL domain; and (3) Second heterodimerization domain (HD2) a first Fab portion comprising Includes.

[0275] In some embodiments, the first and second heterodimerization domains of the binding protein comprise first and second Fc domains, hi some embodiments, the Fc domains comprise the general structure hinge-CH2 domain-CH3 domain.

[0276] (a) Fc-heterodimerization domain In certain embodiments, the multispecific binding protein of the present disclosure may further comprise an Fc-heterodimerization C1 or C2 domain. In some embodiments, the Fc dimerization domain is selected from the group consisting of a heterodimerization Fc or a fragment thereof, particularly a knobs-in-holes (KIH) mutant of the Fc portion and its effector-modified mutants, or a heterodimerization Fc or a fragment thereof, particularly an EV-RWT mutant of the Fc and its effector-modified mutants. In some embodiments, the Fc comprises one or more amino acid mutations. One possibility is to remove the selective recognition site for the first affinity reagent and introduce the selective recognition site for the second affinity reagent, for example, by a mutation selected from the group consisting of H435R and Y436F. Alternatively, only the selective recognition site for the second affinity reagent can be introduced.

[0277] b) Homodimerization domain In certain embodiments, the multispecific binding proteins of the present disclosure can further comprise a homodimerization domain. In some embodiments, the homodimerization domain is selected from the group consisting of an Fc region and its effector-modified variants; one or more CH2 domains, e.g., one or more CH2 domains of IgG, IgE, or IgM; one or more CH3 domains, e.g., one or more CH3 domains of IgG, IgA, or IgD; and one or more CH4 domains, e.g., one or more CH4 domains of IgE or IgM.

[0278] III. Multimeric pseudo-FAB-containing binding proteins (a) Symmetric tetravalent construct In another embodiment, the binding polypeptide comprises a pseudo-Fab-containing binding protein comprising additional polypeptide chains that associate with the polypeptide chains of the pseudo-Fab to form additional binding domains. These pseudo-Fab-containing binding polypeptides are further fused to an Fc heterodimerization domain to form one half of a traditional Y-shaped antibody.

[0279] In some embodiments, the antigen binding protein comprises six polypeptide chains that form four antigen binding sites; (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHa-L2-VHX-L3-VHb-CH1-FC1[V] The structure includes: (d) the sixth polypeptide has the formula: VHa-L2-VHX-L3-VHb-CH1-FC2[VI] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2 and L3 are amino acid linkers, (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) paired with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The dsKO domain contains (i) one or more inactivating mutations that abolish its binding to the target antigen and (ii) one or more engineered interchain disulfide bonds.

[0280] These molecules are also called "tandem-(Fv-pseudo-Fab x Fv-Fab)" (see Figure 10B).

[0281] In some embodiments, the antigen binding protein comprises six polypeptide chains that form four antigen binding sites; (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHb-CH1-L3-VHa-L2-VHX-FC1[V] The structure includes: (d) the sixth polypeptide has the formula: VHb-CH1-L3-VHa-L2-VHX-FC2[VI] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2 and L3 are amino acid linkers, (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The dsKO domain contains (i) one or more inactivating mutations that abolish its binding to the target antigen and (ii) one or more engineered interchain disulfide bonds.

[0282] These molecules are also called "tandem-(Fv-Fab x Fv-pseudo-Fab-IgG)" (see Figure 10A).

[0283] (b) Asymmetric tetraspecific molecules Dimerization of binding polypeptides, where only one contains a pseudo-Fab, results in asymmetric constructs with additional specificity. These pseudo-Fab-containing binding polypeptides are further fused to an Fc heterodimerization domain to form half of a traditional full-length Y-shaped IgG antibody.

[0284] In some embodiments, the multispecific binding protein comprises four polypeptide chains that form at least two antigen binding sites; (a) the first polypeptide has the formula: VLa-L1-VLX[I] The structure includes: (b) the second polypeptide has the formula: VHa-L2-VHX-FC1[II] The structure includes: (c) the third polypeptide has the formula: VLb-CL[III] The structure includes: (d) the fourth polypeptide has the formula: VHb-CH1-FC2[IV] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1 and L2 are independently the same or different amino acid linkers. (1) pairing a first VL domain (VLa) with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The dsKO domain contains (i) one or more inactivating mutations that abolish its binding to the target antigen and (ii) one or more engineered interchain disulfide bonds.

[0285] These molecules are also called dimeric bispecific IgG molecules (Fv-pseudo-Fab) x (Fv-Fab)-Fc (see Figures 2 and 7).

[0286] In another embodiment, the binding polypeptide comprises a pseudo-Fab-containing binding protein, which comprises an additional polypeptide chain that associates with a polypeptide chain of the pseudo-Fab to form an additional binding domain. These pseudo-Fab-containing binding polypeptides are further fused to an Fc heterodimerization domain to form one half of a conventional Y-shaped antibody. Dimerization of such molecules results in constructs with additional specificity.

[0287] In some embodiments, the antigen binding protein comprises six polypeptide chains that form four antigen binding sites; (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHa-L2-VHX-L3-VHa-L4-VHX-FC1[V] The structure includes: (d) the sixth polypeptide has the formula: VHb-CH1-L5-VHb-CH1-FC2[VI] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, L4 and L5 are amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The dsKO domain contains (i) one or more inactivating mutations that abolish its binding to the target antigen and (ii) one or more engineered interchain disulfide bonds.

[0288] The C-terminal heterodimerization domain can be a conventional Fc-knob-hole heterodimerization domain, a conventional Fc-RF heterodimerization domain, or a combination thereof. These molecules are also referred to as "Fv-pseudo-Fab([HC]-(Fv-pseudo-Fab)) x (((Fv-Fab)[HC]-(Fv-Fab)))-Fc" (see Figure 11).

[0289] In some embodiments, the antigen binding protein comprises four polypeptide chains that form three antigen binding sites: (a) the first polypeptide has the formula: VLa-L1-VLX[I] The structure includes: (b) the second polypeptide has the formula: VHa-L2-VHX-FC1[II] The structure includes: (c) the third polypeptide has the formula: VLb-L3-VLc-L4-CL[III] The structure includes: (d) the fourth polypeptide has the formula: VHc-L5-VHb-L6-CH1-FC2[IV] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VLc is the third immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VHc is the third immunoglobulin heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, L4, L5 and L6 are amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a third VL domain (VLc) pairs with a third VH domain (VHc) to form a third functional antigen-binding site that binds to target antigen C; (4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV); (5) The stabilized knockout VL domain (VLX) pairs with the stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO2) domain; The dsKO domain contains (i) one or more inactivating mutations that abolish its binding to the target antigen and (ii) one or more engineered interchain disulfide bonds.

[0290] In some embodiments, the antigen binding protein comprises four polypeptide chains that form three antigen binding sites: (a) the first polypeptide has the formula: VLa-L1-VLX[I] The structure includes: (b) the second polypeptide has the formula: VHa-L2-VHX-FC1[II] The structure includes: (c) the third polypeptide has the formula: VLb-L3-VLc-L4-CL[III] The structure includes: (d) the fourth polypeptide has the formula: VHc-L5-VHb-L6-CH1-FC2[IV] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VLc is the third immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VHc is the third immunoglobulin heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; FC1 and FC2 are Fc domains containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, L4, L5 and L6 are amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a third VL domain (VLc) pairs with a third VH domain (VHc) to form a third functional antigen-binding site that binds to target antigen C; (4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV); (5) The stabilized knockout VL domain (VLX) pairs with the stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO2) domain; The dsKO domain contains (i) one or more inactivating mutations that abolish the binding of the reference Fab molecule to its target antigen, and (ii) one or more engineered interchain disulfide bonds.

[0291] These molecules are also called "(CODV-Fab) x (pseudo-Fab)-Fc" (see Figure 13).

[0292] In particular embodiments of the first and second aspects of the invention, the binding proteins are selected from the group consisting of SEQ ID NOs: 6 and 7; SEQ ID NOs: 8 and 9; SEQ ID NOs: 10 and 11; SEQ ID NOs: 12 and 13; SEQ ID NOs: 14 and 15; SEQ ID NOs: 16 and 17; SEQ ID NOs: 18 and 19; SEQ ID NOs: 20 and 21; SEQ ID NOs: 22 and 23; SEQ ID NOs: 24 and 25; SEQ ID NOs: 26 and 27; SEQ ID NOs: 28 and 29; SEQ ID NOs: 30 and 31; SEQ ID NOs: 32 and 33; SEQ ID NOs: 34 and 35; SEQ ID NOs: 36 and 37; SEQ ID NOs: 38 and 39; SEQ ID NOs: 40 and SEQ ID NOs: 41; SEQ ID NOs: 42 and 43; SEQ ID NOs: 44 and 45; SEQ ID NOs: 46 and 47 and SEQ ID NOs: 48 and 49; SEQ ID NOs: 50 and 51; SEQ ID NOs: 52 and 53 and SEQ ID NOs: 54 and 55; SEQ ID NOs: 56 and 57 and SEQ ID NOs: 58 and 59; SEQ ID NOs: 60 and 61 and SEQ ID NOs: 62 and 63; SEQ ID NOs: 64 and 65 and SEQ ID NOs: 66 and 67; SEQ ID NOs: 68 and 69 and SEQ ID NOs: 70 and 71; SEQ ID NOs: 72 and 73; and SEQ ID NOs: 74 and 75.

[0293] In some embodiments, the first and second CL are independently selected from the group consisting of constant region light chain kappa (CLκ) and constant region light chain lambda (CLλ).

[0294] In some embodiments, HD1 and HD2 each comprise an Fc region and effector-modified variants thereof; a heterodimerized Fc portion, particularly a knobs-in-holes (KIH) variant of the Fc portion and its effector-modified variants; one or more CH2 domains, e.g., those of IgG, IgE, or IgM; one or more CH3 domains, e.g., those of IgG, IgA, or IgD; or one or more CH4 domains, e.g., those of IgE or IgM.

[0295] In some embodiments, one of the Fc domains comprises a first CH3 domain comprising one or both of an S354C and a T366W mutation, and the other Fc domain comprises a second CH3 domain comprising one or both of an Y349C, T366S, L368A and a Y407V mutation.

[0296] In one embodiment, the Fc region of HD1 or HD2 comprises one or more amino acid mutations that result in the elimination of a selective recognition site for a second affinity reagent, e.g., an amino acid mutation selected from the group consisting of H435R or Y436F, or one or more amino acid mutations that result in the introduction of a selective recognition site for a third affinity reagent.

[0297] [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 17

[0298]

Table 18

Table 19

[0299] (e) Nucleic acid In a fifth aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a pseudo-Fab comprising one or both of the binding proteins and a nucleotide sequence encoding the multispecific binding protein of any of the first, second or third aspects of the invention.

[0300] One aspect of the invention relates to a polynucleotide encoding the binding protein of any one of the first to third aspects of the invention.

[0301] Standard recombinant DNA methodologies are used to construct polynucleotides encoding polypeptides that form binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with and in the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein is that which is well known and commonly used in the art. Similarly, conventional techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0302] Another aspect of the present disclosure relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments of the fifth aspect of the invention, the isolated nucleic acid molecule comprises a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleic acid encoding any of the binding proteins described herein.

[0303] Certain aspects of the present disclosure relate to kits of polynucleotides. In some embodiments, one or more polynucleotides are vectors (e.g., expression vectors). The kits may find use, inter alia, in producing one or more of the binding proteins described herein, such as the heterodimerization domains, bivalent, trivalent, tetravalent, or multivalent binding proteins of the present disclosure.

[0304] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct the transcription of the nucleic acid sequence encoding the binding protein. A promoter can refer to a nucleic acid control sequence that directs the transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to the coding sequence of a binding protein when the promoter affects the transcription or expression of the coding sequence. Exemplary promoters may include, but are not limited to, promoters obtained from the genomes of viruses (such as polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40)), heterologous eukaryotic promoters (such as actin promoters, immunoglobulin promoters, heat shock promoters), the CAG promoter (Niwa et al., Gene 108(2):193-9, 1991), the phosphoglycerate kinase (PGK) promoter, a tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), the lac system, the trp system, the tac system, the trc system, the major operator and promoter regions of phage lambda, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast alpha mating factor. A polynucleotide encoding a binding protein of the present disclosure can be under the control of a constitutive promoter, an inducible promoter, or other suitable promoter described herein, or other suitable promoters readily recognized by one of skill in the art.

[0305] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. An expression vector may include one or more regulatory sequences operably linked to the polynucleotide to be expressed. The term "regulatory sequence," as used herein, includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers include, but are not limited to, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.) and eukaryotic viruses (e.g., the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.). Examples of suitable vectors include, for example, plasmid, cosmid, episome, transposon, and virus vector (for example, adenovirus, vaccinia virus, Sindbis virus, measles virus, herpes virus, lentivirus, retrovirus, adeno-associated virus vector, etc.).Expression vector can be used to transfect host cells, for example, bacterial cells, yeast cells, insect cells and mammalian cells.Biologically functional virus and plasmid DNA vectors that can express and replicate in hosts are known in the art and can be used to transfect any cell of interest.

[0306] In a sixth aspect, the present invention relates to an expression vector comprising the nucleic acid molecule of the fifth aspect of the invention.

[0307] Another aspect of the present disclosure relates to a vector system, such as one or more vectors encoding the first, second, third, and fourth polypeptide chains of any of the binding proteins described herein. In some embodiments of the sixth aspect of the invention, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. One or more vectors of the vector system can be any of the vectors described herein. In some embodiments, one or more vectors are expression vectors.

[0308] (f) Isolated host cells In a seventh aspect, the present invention relates to an isolated host cell comprising a nucleic acid molecule of the fifth aspect of the invention or an expression vector of the sixth aspect of the invention.

[0309] Another aspect of the present disclosure relates to an isolated host cell comprising one or more isolated polynucleotides, polynucleotide kits, vectors, and / or vector systems described herein. In some embodiments of the seventh aspect of the invention, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is an E. coli DH5α cell. In some embodiments, the host cell is a yeast cell (e.g., a S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture), Expi293™ cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL2), canine kidney cells (e.g., MDCK, ATCC CCL34), buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL1442), human lung cells (e.g., W138, ATCC CCL75), human liver cells (e.g., Hep G2, HB8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC5 cells, FS4 cells, human hepatoma lines (e.g., Hep G2), myeloma cells (e.g., NS0 and Sp2 / 0 cells), and the like.

[0310] IV. Preparation method 1. Expression method Another aspect of the present disclosure relates to a method of producing any of the binding proteins described herein. In some embodiments, the method comprises: a) culturing a host cell (e.g., any of the host cells described herein) containing an isolated nucleic acid, vector, and / or vector system (e.g., any of the isolated nucleic acids, vectors, and / or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell Methods for culturing host cells under conditions for protein expression are well known to those skilled in the art. Methods for isolating proteins from cultured host cells are well known to those skilled in the art, such as by affinity chromatography (e.g., two-step affinity chromatography including Protein A affinity chromatography followed by size exclusion chromatography).

[0311] 2. Purification method In a fourth aspect, the present invention provides a method for producing a composition comprising: (i) applying a solution containing the multispecific binding protein to a first, second, or third affinity reagent that specifically binds to a recognition site of the multispecific binding protein for the first, second, or third affinity reagent; (ii) recovering either the multispecific binding protein that does not bind to the first, second, or third affinity reagent, or the multispecific binding protein that binds to the first or third affinity reagent. wherein each of the first, second and third affinity reagents binds to a different recognition site on the multispecific binding protein.

[0312] In some embodiments, the method comprises the further step of applying the solution comprising the multispecific binding protein recovered in step (ii) to a first, second, or third affinity reagent, wherein the affinity reagent is different from the affinity reagent used in step (i), recovering either the multispecific binding protein that does not bind to the first, second, or third affinity reagent, or the multispecific binding protein that binds to the first or third affinity reagent.

[0313] In some embodiments, the first affinity reagent binds to Ckappa; the second affinity reagent is Protein A; and / or the third affinity reagent is Protein G.

[0314] In some embodiments, the binding proteins of the disclosure are purified by Protein A affinity chromatography, kappa light chain affinity chromatography (e.g., using Kappa Select resin according to the manufacturer's instructions; GE Healthcare), and optionally lambda light chain affinity chromatography (e.g., using Lambda Fab Select resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, the binding proteins of the disclosure are purified by Protein A affinity chromatography, lambda light chain affinity chromatography (e.g., using Lambda Fab Select resin according to the manufacturer's instructions; GE Healthcare), and optionally kappa light chain affinity chromatography (e.g., using Kappa Select resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, the binding proteins comprise two Fc regions, each comprising a CH3 domain, wherein only one of the CH3 domains comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being H435R and Y436F. In some embodiments, binding proteins of the disclosure are purified sequentially by Protein A affinity chromatography, followed by kappa light chain affinity chromatography (e.g., using Kappa Select resin according to the manufacturer's instructions; GE Healthcare), and then optionally by lambda light chain affinity chromatography (e.g., using Lambda Fab Select resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, binding proteins of the disclosure are purified sequentially by Protein A affinity chromatography, followed by lambda light chain affinity chromatography (e.g., using Lambda Fab Select resin according to the manufacturer's instructions; GE Healthcare), and then optionally by kappa light chain affinity chromatography (e.g., using Kappa Select resin according to the manufacturer's instructions; GE Healthcare).For example, in some embodiments, the binding protein is contacted with Protein A, eluted from Protein A under conditions suitable to isolate the binding protein from binding proteins containing either zero or two CH3 domains containing the amino acid substitutions H435R and Y436F, contacted with a kappa light chain affinity medium (e.g., as used in Kappa Select resin; GE Healthcare), and eluted from the kappa light chain affinity medium under conditions suitable to isolate the binding protein from binding proteins containing only lambda CL domains (e.g., according to the manufacturer's instructions).

[0315] Suitable conditions for Protein A elution include, but are not limited to, a stepwise elution gradient from pH 4.5 to 2.8 and are known in the art. In some embodiments, Protein A or Protein A variants useful for protein purification are employed. In some embodiments, Protein A is bound to a matrix or resin, e.g., as part of a chromatography medium. In some embodiments, after elution from the kappa light chain affinity medium, the binding protein is contacted with a lambda light chain affinity medium (e.g., Lambda Fab Select Resin; such as used by GE Healthcare) and eluted from the lambda light chain affinity medium under conditions suitable for isolating the binding protein from binding proteins containing only the kappa CL domain (e.g., according to the manufacturer's instructions). In some embodiments, the binding proteins of the present disclosure are detected using HIC chromatography. In some embodiments, the binding protein comprises a first polypeptide chain comprising a lambda CL domain; a CH3 domain of a second polypeptide chain comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W; a CH3 domain of a third polypeptide chain comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and a fourth polypeptide chain comprising a kappa CL domain. In some embodiments, the binding protein is produced by a host cell. In some embodiments, the binding protein is purified from cell culture medium or a host cell extract. In some embodiments, the binding protein is secreted by the host cell (e.g., before contacting with Protein A) or produced and extracted from the host cell. In some embodiments, the binding protein is present in cell culture medium or a host cell extract when contacted with Protein A. In some embodiments, the binding protein is purified away from other binding proteins, polypeptides, and / or other cellular components.

[0316] In some embodiments, stabilized knockout domains are used to facilitate the preferential synthesis or purification of desired multispecific binding proteins, wherein the stabilized knockout domain comprises (1) one or more inactivating mutations that abolish binding to the target antigen, and (2) one or more engineered interchain disulfide bonds that confer enhanced thermal stability (Tm) relative to a reference Fab molecule, which is identical to the pseudo-Fab molecule except that in the pseudo-Fab molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

[0317] V. Formulations / Pharmaceutical Compositions In an eighth aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a protein according to any one of the first to third aspects of the present invention.

[0318] Therapeutic or pharmaceutical compositions comprising the binding proteins are within the scope of this disclosure. Some embodiments of the eighth aspect of the invention include pharmaceutical compositions comprising a therapeutically effective amount of any one of the binding proteins or binding protein-drug conjugates described herein in admixture with a pharmaceutically or physiologically acceptable formulation selected to suit the mode of administration.

[0319] Acceptable formulation materials are typically nontoxic to recipients at the dosages and concentrations employed.

[0320] In some embodiments, pharmaceutical compositions may include formulation materials to modify, maintain, or prevent, for example, the pH, osmolality, 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., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (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, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives ( For example, 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 tyloxapal), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, e.g., sodium chloride or potassium chloride, or mannitol sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed., AR Gennaro, ed., Mack Publishing Company, 1990) and subsequent editions).

[0321] In some embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

[0322] In some embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, suitable vehicles or carriers for injection can be water, saline, or artificial cerebrospinal fluid, optionally supplemented with other substances commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the present disclosure, binding protein compositions can be prepared for storage by blending selected compositions having the desired degree of purity with any formulation in the form of a lyophilized cake or aqueous solution. Additionally, binding proteins can be formulated as lyophilized preparations using appropriate excipients, such as sucrose.

[0323] In some embodiments, the pharmaceutical compositions of the present disclosure can be selected for parenteral or subcutaneous delivery. Alternatively, the compositions can be selected for inhalation or delivery through the digestive tract, for example, orally. The preparation of such pharmaceutically acceptable compositions is within the skill of those in the art.

[0324] In some embodiments, formulation components are present in concentrations that are acceptable to the site of administration. For example, buffering agents are used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0325] When intended for parenteral administration, the therapeutic composition for use can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable vehicle.A particularly suitable vehicle for parenteral injection is sterile distilled water, and the binding protein is formulated as a sterile, isotonic solution that is appropriately preserved.Yet another preparation can include formulating the desired molecule with drugs, such as injectable microspheres, bioerodible particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, and then providing controlled or sustained release of the product delivered via depot injection.Hyaluronic acid can also be used, which can have the effect of promoting duration in circulation.Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0326] 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 nebulized.

[0327] It is also contemplated that certain formulations may be administered orally. In one embodiment of the present disclosure, multispecific binding proteins administered in this manner can be formulated with or without carriers commonly used in the compounding of solid dosage forms, such as tablets and capsules. For example, capsules can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional substances can be included to facilitate absorption of the binding protein. Diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0328] Another pharmaceutical composition can involve an effective amount of the multispecific binding protein in a mixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another suitable vehicle, a solution can be prepared in unit dosage form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0329] Further pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art and include formulations involving the binding protein in sustained- or controlled-delivery formulations. Techniques for formulating various other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and 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 shaped articles, such as films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactides, copolymers of 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 and may be prepared by any of several methods known in the art.

[0330] In some embodiments, pharmaceutical compositions should be used for in vivo administration and typically must be sterile.This can be achieved by filtering through a sterile filtration membrane.If the composition is lyophilized, sterilization can be carried out using this method either before or after lyophilization and reconstitution.Compositions for parenteral administration can be stored in lyophilized form or in solution.In addition, parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0331] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[0332] The present disclosure also encompasses kits for producing single-dosage units. The kits can each include both a first container with a dried multispecific binding protein and a second container with an aqueous formulation. Also included within the scope of the present disclosure are kits that include single- and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0333] The therapeutically effective amount of a pharmaceutical composition of a binding protein depends, for example, on the therapeutic context and purpose. Therefore, those skilled in the art will understand that the appropriate dosage level for treatment will vary depending in part on the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the patient's size (weight, body surface, or organ size) and condition (age and general health). Therefore, clinicians can titrate the dosage and modify the route of administration to achieve optimal therapeutic effect.

[0334] The dosing frequency depends on the pharmacokinetic parameters of the binding protein in the formulation used. Typically, the clinician administers the composition until a dosage that achieves the desired effect is reached. Thus, the composition can be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule), over time, or as continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of the tasks routinely performed by those skilled in the art. The appropriate dosage can be confirmed by using appropriate dose-response data.

[0335] The route of administration of the pharmaceutical composition follows known methods, for example, oral; intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional injection; by sustained release system; or by implantation device. If desired, the composition can be administered by bolus injection or continuously by infusion or by implantation device.

[0336] In some embodiments, the compositions can also be administered locally via implantation of a membrane, sponge, or other suitable material into which the desired molecule has been absorbed or encapsulated. When an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, sustained-release bolus, or continuous administration.

[0337] VI. Treatment / Usage In a ninth aspect, the invention relates to a method of treating a disorder in which antigenic activity is detrimental, comprising administering to a subject in need thereof an effective amount of the binding protein of any one of the first to third aspects of the invention. In some embodiments, there is provided a multispecific binding protein for use as a medicament.

[0338] The binding proteins 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 quantitation of one or more target antigens. The binding proteins bind to one or more target antigens with an affinity appropriate for the assay method being used.

[0339] For diagnostic applications, in some embodiments, the binding protein can be labeled with a detectable moiety. The detectable moiety can be any that is capable of producing, directly or indirectly, a detectable signal. For example, the detectable moiety can be a radioisotope, e.g., 3 H, 14 C. 32 P, 35 S, 125 I,99 Tc, 111 In or 67 Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0340] The binding protein is also useful for in vivo imaging. The binding protein labeled with a detectable moiety can be administered to an animal, for example, into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. The binding protein can be labeled with any moiety that can be detected in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0341] For clinical or research use, in some embodiments, the binding protein is conjugated to a cytotoxic agent.A variety of antibodies conjugated to a cytotoxic agent (i.e., antibody-drug conjugate) have been used to target cytotoxic payloads to specific tumor cells.Cytotoxic agents and the linkers that conjugate drugs to antibodies are known in the art; for example, see Parslow, AC et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des.Devel.Ther. 11:2265-2276.

[0342] The present disclosure also refers to kits containing binding proteins and other reagents useful for detecting target antigen levels in biological samples. Such reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit includes a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and / or host cells described herein. In some embodiments, the kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container can hold a composition by itself or in combination with another composition effective for treating, preventing, and / or diagnosing a condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used for preventing, diagnosing, and / or treating the selected condition. Alternatively, or additionally, the article of manufacture or kit can further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0343] In some embodiments, the binding proteins of the present disclosure are administered to a patient in need thereof to treat or prevent cancer. In some embodiments, the present disclosure relates to a method for preventing and / or treating a proliferative disease or disorder (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 pharmaceutical compositions related thereto described herein. In some embodiments, the patient is a human.

[0344] In some embodiments, at least one binding protein is administered in combination with one or more anti-cancer therapies (e.g., any anti-cancer chemotherapeutic agent or therapy known in the art). In some embodiments, at least one binding protein is administered prior to one or more anti-cancer therapies. In some embodiments, at least one binding protein is administered simultaneously with one or more anti-cancer therapies. In some embodiments, at least one binding protein is administered after one or more antiretroviral therapies. [Example]

[0345] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is 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 one of ordinary skill in the art. In certain embodiments, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," edited by Leuenberger, H.G.W., 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 commonly understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or exogenous definitions. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise stated. The use of the term "including," as well as other forms such as "includes" and "included," is not limiting. As used herein, unless otherwise stated, the singular forms "a," "an," and "the" include plural references. Thus, for example, a reference to a "protein" includes a plurality of protein molecules.

[0346] Furthermore, unless otherwise specified, the experiments described herein use conventional molecular and cell biological and immunological techniques within the scope of those skilled in the art.Such techniques are well known to skilled workers and are fully described in the literature.For example, see Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including supplements, Molecular Cloning: A Laboratory Manual (4th edition); MR Green, J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0347] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0348] 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 manner and in any number to create additional embodiments. The various described examples and preferred / specific embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context dictates otherwise, all permutations and combinations of elements described herein should be considered to be disclosed by the specification of this application.

[0349] In general, the nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclatures used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as the experimental procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0350] Example 1: Identification of suitable replacement scaffolds The binding proteins disclosed herein comprise the replacement of one CH1 / CL pair with a knockout VH / VL domain in various antibody formats (see Figures 1, 2, 7, and 10-13). To identify a suitable replacement scaffold for replacing the CH1 / CL dimer, the introduction of a disulfide bridge into certain VH / VL pairs was investigated. Furthermore, the expression and fusion to other VH / VL pairs were investigated.

[0351] Trastuzumab was determined to be a suitable replacement scaffold to replace the CH1 / CL dimer to solve the problem of correct light chain pairing.

[0352] Example 2: Use of trastuzumab variable domains to replace CH1 / CL The cysteine ​​residues at positions VH44 and VL100 were determined to be compatible with generating a disulfide bond between the VH and VL of trastuzumab (referred to herein as ds-trastuzumab), thereby stabilizing the heterodimer. To determine its thermal stability, the ds-trastuzumab domain was incubated at 40°C for 14 days at a concentration of 1 mg / mL in D-PBS buffer (GIBCO). Control samples at the same concentration were kept at -80°C and 4°C. After completion of the stress test, the samples were analyzed for their aggregation content by analytical size exclusion chromatography (SEC). Analytical SEC was performed using a BioSECcurity instrument (PSS Polymer) at 25°C using a TSKgel SuperSW3000 column (4.6 mm x 300 mm) and a TSKgel SuperSW HPLC guard column (Tosoh Bioscience). Analytes were run at a flow rate of 0.25 ml / min using 250 mM NaCl, 100 mM Na phosphate, pH 6.7, with detection at 280 and 260 nm. Static light scattering was detected at 436 nm. Five 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 protein standards ranging from 6.5 to 670 kDa.

[0353] The ds-trastuzumab scaffold (with VH44 / VL100 cysteine ​​modifications) was found to increase thermal stability by 4° C. (see FIGS. 3A-C).

[0354] Example 3: Investigation of VH / VL connections in pseudo-IgG design Pseudo-IgG1 constructs were generated that contained a disulfide-stabilized trastuzumab backbone as a replacement scaffold for CH1 / CL. The ds-trastuzumab scaffold was fused to the VH / VL pair of interest with a G4S or (G4S)2 linker (see Figure 6).

[0355] Determination of melting point (T m ) Melting point (T m) was measured using differential scanning fluorimetry (DSF). Samples were diluted in D-PBS buffer (Invitrogen) to a final concentration of 0.2 μg / μl containing a 4x 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 duplicate using a MyiQ2 real-time PCR instrument (BIORAD). The negative first derivative of the melting curve (-d(RFU) / dT) was generated using iQ5 software v2.1 (BIORAD). Data were then exported to Excel for T determination and data graphic display.

[0356] Six antibody sequences were expressed in fusion with ds-trastuzumab, and the % monomer following Protein A purification and melting temperature of each fusion protein, as well as the pseudo-IgG antibody without the ds-trastuzumab domain, are listed below in Table 4. Expression levels were slightly lower compared to WT-IgG, and the monomer content was slightly lower compared to WT-IgG.

[0357] [Table 40]

[0358] Example 4: Generation of ds-Tras knockout (dsTrasKO) mutants The binding activity of trastuzumab was knocked out by introducing mutations at four key amino acid residues in the paratopic region of the trastuzumab antibody to affect the antibody's binding ability to the receptor tyrosine-protein kinase erbB-2 (HER2). Trastuzumab was modeled based on the PDB structure 1N8Z, available in the public PDB database. Four positions on the heavy chain were identified that potentially disrupt the interaction of ds-trastuzumab with its antigen. These positions include both arginines R59 and R50, which connect the glutamic acid and aspartic acid residues on HER2. Charge reversal by mutating the arginine residues to glutamic acid residues should result in repulsion of HER2. Without intending to be bound by scientific theory, two other positions, tyrosines Y33 and Y105, appeared to stabilize the complex through π-π interactions with phenylalanine. Without intending to be bound by scientific theory, mutation of alanine should disrupt this interaction. The crystallographic data from the 1N8Z file was analyzed in the BIOVIA Discovery Studio suite, with particular attention paid to the existing interface region between the antibody (trastuzumab fab) and its target (HER2 protein domain) (see Figure 4).

[0359] Analysis of the 1N8Z cocrystal interfacial region During antibody and target interface area analysis, all non-covalent interactions were qualified using the BIOVIA Discovery Studio Non-bonded Interaction Monitor. An extract of the resulting table is shown in Table 5. For each interaction, Table 5 shows the distance between the interacting atoms, the nature of the interaction type, and the characteristics of the interacting atoms.

[0360] [Table 41]

[0361] Based on a table of non-covalent interactions occurring between trastuzumab fab and HER2 protein, certain interactions were selected to be eliminated in order to disrupt the binding interaction.

[0362] Recommended combinations of mutations that disrupt binding interactions The following four amino acid residue mutations were selected based on non-bonded interaction data to maintain the original trastuzumab sequence:

[0363] For trastuzumab heavy chain arginine 50 residue: This arginine residue of the antibody makes salt-bridge interactions with HER2 residues glutamic acid 558 and aspartic acid 560. To create a repulsion between HER2 residues glutamic acid 558 and aspartic acid 560, arginine 50 was mutated to a glutamic acid residue.

[0364] For trastuzumab heavy chain arginine 59 residue: This arginine residue of the antibody makes salt-bridge interactions with HER2 residues glutamic acid 558 and aspartic acid 560. To create a repulsion between HER2 residues glutamic acid 558 and aspartic acid 560, arginine 59 was mutated to a glutamic acid residue.

[0365] For tyrosine heavy chain arginine 33 residue: This tyrosine 33 residue of the antibody makes π interactions with HER2 residue phenylalanine 573. To disrupt these π interactions between the antibody and its target, tyrosine 33 was mutated to an alanine residue.

[0366] For tyrosine heavy chain arginine 105 residue: This tyrosine 105 residue of the antibody makes π interactions with the HER2 residue phenylalanine 573. To disrupt these π interactions between the antibody and its target, tyrosine 105 was mutated to an alanine residue.

[0367] The specific combinations of amino acid residues recommended for mutation are shown in Table 6. These combinations result in two variants of the trastuzumab antibody.

[0368] [Table 42]

[0369] Mutant 1 Fab contains all four point mutations. Mutant 2 Fab, which changes two positively charged residues back to negatively charged residues, has a repulsive region for HER2 protein in the original trastuzumab HER2 binding region. Mutant 3 Fab, which disrupts the π interaction with HER2 phenylalanine 573, induces destabilization of the trastuzumab-HER2 complex. All three dsTrasKO mutants were found to abolish binding to HER2 (see Figure 5A-B). Mutant 2 (dsTrasKO2) was identified as the best producer.

[0370] Example 5: Evaluation of dsTrasKO2 as a replacement scaffold for CH1 / CL The dsTrasKO mutant was evaluated in pseudo-IgG (monospecific) format (see Figure 6A) and pseudo-Fab (see Figure 6B). The pseudo-IgG and pseudo-Fab formats tested had similar expression and purification characteristics to the parent antibody (see Table 7).

[0371] [Table 43]

[0372] Example 6: Evaluation of bispecific antibodies with native structure using dsTrasKO2 as a replacement scaffold for CH1 / CL The dsTrasKO2 domain was evaluated as a replacement scaffold in bispecific antibodies with the native IgG structure. In particular, an anti-IL4-dsTrasKO(Var2) x anti-IL13-huIgG1 bispecific design with RF mutations in the Fc was synthesized (see Figure 7).

[0373] Expression of i.dsTrasKO2 bispecific molecules Expression plasmids encoding both heavy chains (dsTrasKO2-knob and WT-hole) and both light chains (dsTrasKO and WT-kappa / lambda) of the corresponding constructs were propagated in Escherichia coli (DH5α). Plasmids used for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega kit.

[0374] HEK293-FS cells grown in F17 serum-free suspension culture (Invitrogen) were transfected with the indicated light chain (LC) and heavy chain (HC) plasmids using polyethyleneimine transfection reagent. After 7 days of culture at 37°C, cells were removed by centrifugation, and the supernatant was passed over a 0.22 μm filter to remove particles.

[0375] For purification, the antibody was captured on a MabSelect SuRe column (catalog number: 11-0034-93, GE Healthcare), eluted with 0.1 M citrate buffer pH 3.0, and directly desalted using a HiPrep 26 / 10 desalting column (catalog number: 17-05087-02, GE Healthcare). Potential homodimeric dsTrasKO2 monospecific molecules were selected by using an additional KappaSelect capture step (0.1 M glycine pH 2.7 elution buffer) (see Figure 14). After final protein polishing by size exclusion chromatography (SEC, performed as described in Example 2 above) using Superdex 200 26 / 60 (GE) and a final ultrafiltration concentration step, the protein was used for further characterization. See Table 8 for purification results of dsTrasKO2 bispecific constructs. See Figures 7A-7D for representative bispecific design and purification results.

[0376] [Table 44]

[0377] Evaluation of antigen binding of a bispecific antibody containing the dsTrasKO2 substitution of the CH1 / CL pair Antigen binding to the antibody constructs was assessed using surface plasmon resonance (SPR). Antigen binding to the antibody constructs was measured using HBS-EP buffer (GE Healthcare) on a BIAcore 3000 instrument (GE Healthcare). Human IL4 (IL004, Millipore) and human IL13 (IL012, Millipore), human HER2 (11292-ER, R&D Systems), human TNFα (H8916, SIGMA-Aldrich), human CTLA4 (CT4-H5229, ACRO Biosystems), human PD-1 (8986-PD, R&D Systems), and human IL6Ra (227-SR / CF, R&D Systems) were used as antigens. Anti-human Fc capture antibodies (Human Antibody Capture Kit, GE Life Sciences) were immobilized via primary amine groups (11,000 RU) on a research-grade CM5 chip (GE Life Sciences) using standard procedures. The ligand was captured at a flow rate of 10 μl / min, and the adjusted RU value was used to determine a maximum analyte binding of 30 RU. HER2 binding studies were performed by capturing the antigen with anti-human Fc antibodies. The tested antibody constructs were used as analytes at a 100 nM concentration and injected for 240 seconds at a flow rate of 30 μL / min, with a dissociation time of 300 seconds. Binding kinetics measurements were performed using the captured antibodies by injecting two-fold serial dilutions of the analyte from 3 nM to 100 nM. Dilution series of 0.1 nM to 3 nM and 0.8 nM to 25 nM were used for human IL4 and IL13, respectively. The chip surface was regenerated by injecting the regeneration buffer provided with the capture kit for 2 minutes. Sensorgrams were double-referenced with a blank chip surface and an HBS-EP buffer blank. Data analysis was performed using BIAevaluation software v4.1. The binding characteristics of the mAb, pseudo-IgG, and bispecifics are shown in Table 9 below.

[0378] [Table 45]

[0379] SPR results showed that various VH / VL fused to dsTrasKO2 retained the parent antigen-binding affinity as a bispecific antibody.

[0380] Protein integrity analysis The protein integrity and potential mispairing of the heterodimer constructs were analyzed by LC-mass spectrometry (LC-MS). Protein samples were deglycosylated using 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) for 15 hours at 37°C. LC-MS analysis was performed using a 6540 UHD accurate mass Q-TOF LC / MS instrument (Agilent). Reversed-phase (RP) chromatography was performed using a Poroshell 300SB-C8, 5 μm, 75 × 0.5 mm (Agilent) with a guard column, Poroshell 300SB-C8, 5 μm, 2.1 × 12.5 mm (Agilent), at 180 μL / min. The eluents 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 onto the column and eluted using a linear gradient from 0% to 100% B over 13 minutes. Data analysis was performed using MassHunter software B.06 (Agilent). Molecular weights were calculated based on the amino acid sequence of the protein using GPMAW software version 9.13a2 (Lighthouse data). The dsTrasKO2-antibody fusion protein was shown to be largely intact and exhibit correct heterodimeric pairing (see Table 10 below).

[0381] [Table 46]

[0382] In addition to LC-MS analysis, the bispecific samples were also analyzed by hydrophobic interaction chromatography (HIC) to detect potential mismatches or unexpected species. Analytical HIC was performed at 25 °C using an LC10 HPLC system (Shimadzu) equipped with a TSKgel Ether-5PW 10 μm, 2 × 75 mm column (Tosoh Bioscience). The analysis was performed at a flow rate of 0.1 ml / min, with a detection wavelength of 280 nm. Five μg of undiluted protein sample was applied to the column. Gradient elution was 0–30 min (0–100% B), followed by 10 min of 100% B and 15 min of re-equilibration. Buffer A consisted of 1.5 M ammonium sulfate, 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). The data showed that the dsTrasKO2-bispecific sample had a homogenous HIC profile indicating the absence of unexpected species and that the bispecific sample had the correct match (see Figure 8).

[0383] Thermal stability: Comparison of pseudo-IgG and bispecifics The thermal stability of dsTrasKO2 pseudo-IgG and bispecific samples was assessed as described above in Example 2. Melting temperatures (Tm) were determined using differential scanning fluorimetry (DSF) as described in Example 3. The dsTrasKO2-based constructs exhibited reduced melting temperatures compared to the parental monoclonal IgG, and no impairment of stability was detected in a 2-week thermal stability assay (40°C, 4°C, and -80°C) (see Table 11 below).

[0384] [Table 47]

[0385] Example 7: Crystal structure of pseudo-Fab IL-13 Crystallization of dsTrasKO2-IL13-pseudo-Fab was performed for structural studies. Crystallization experiments were set up as standard two-drop, seated drop experiments in 96-well MRC plates using a 1:1 protein:reservoir ratio and incubated at 20 °C. Both TrasKO2-CH1 / CL and anti-IL13-TrasKO2 were screened for crystallization hits against various commercially available sparse matrix screens. Screening drops for TrasKO2-CH1 / CL (15 mg / ml stock) and anti-IL13-TrasKO2 (15.6 mg / ml stock) in 20 mM HEPES pH 7.5, 0.1 M sodium chloride were prepared by mixing 100 nl of protein solution with 100 nl of reservoir solution and equilibrated in a seated drop vapor diffusion experiment against an 80 μl reservoir. Final crystals of TrasKO2-CH1 / CL suitable for data collection and structural solution were grown at 20 °C using a reservoir solution consisting of 0.1 M sodium phosphate citrate pH 4.2, 20% (w / v) polyethylene glycol 8000, and 0.2 M sodium chloride. Diffraction-quality anti-IL13-TrasKO2 crystals were grown in a reservoir solution consisting of 0.1 M CHES pH 9.5 and 20% (w / v) polyethylene glycol 8000. All crystals were cryoprotected before flash cooling in liquid nitrogen by the addition of 25% (w / v) ethylene glycol (final concentration).

[0386] Data collection and structure determination Diffraction data were collected at beamline PSII at the Swiss Light Source (SLS), Villingen, Switzerland. Diffraction data were processed using a combination of XDS (Kabsch, 2010) and AIMLESS (Evans & Mushudov, 2013) from the CCP4 program suite (Winn et al., 2011). TrasKO2-CH1 / CL was crystallized in space group I23 with cell parameters of 153.23 Å, 153.23 Å, 153.23 Å, 90.00°, 90.00°, and 90.00°. Data were extended to a resolution of 3.70 Å. Crystals of anti-IL13-TrasKO2 belonged to space group P3221 with cell parameters 145.37 145.37 52.06 90.00 90.00 120.00 and diffracted to 1.85 Å.

[0387] The structures were solved by molecular replacement using the CCP4 implementation of Phaser (McCoy et al., 2007). For TrasKO2-CH1 / CL, a modified version of the trastuzumab-VH-VL domain and the CH1 / CL domain from pdb-entry 1n8z were used as search models. Molecular replacement on anti-IL13-TrasKO2 was performed using the anti-IL13 VH / VL domain from the Sanofi internal structure of Fab anti-IL13 and a modified version of the 1n8z VH / VL domain as the topological model. Atomic models were constructed by iterative rounds of manual model building and refinement using Coot (Emsley et al., 2010) and Refine (Bricogne, 2017). The R-factors and R-free factors of the final models are 19.2 / 23.2 for TrasKO2-CH1 / CL and 21.0 / 31.2 for anti-IL13-TrasKO2 (see Figure 9 for the crystal structures).

[0388] Example 8: Evaluation of bispecific antibodies with tandem architecture using dsTrasKO2 as replacement scaffold CH1 / CL The dsTrasKO2 domain was evaluated as a replacement scaffold in bispecific tandem IgG designs. In particular, the anti-GITR-dsTrasKO2 × anti-OX40-kappa]-huIgG1 tandem IgG design was synthesized and tested (see Figures 10-12).

[0389] Expression of dsTrasKO2 bispecific tandem molecules Expression plasmids encoding the heavy and both light chains (dsTrasKO and WT-kappa / lambda) of the corresponding constructs were propagated in E. coli DH5a. Plasmids used for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega kit. HEK293-FS cells grown in F17 serum-free suspension culture (Invitrogen) were transfected with the indicated LC and HC plasmids using polyethylenimine transfection reagent. After 7 days of incubation at 37°C, cells were removed by centrifugation, and the supernatant was passed through a 0.22 μm filter to remove particles. For purification, antibodies were captured on a MabSelect SuRe column (catalog number: 11-0034-93, GE Healthcare), eluted with 0.1 M citrate buffer, pH 3.0, and directly desalted using a HiPrep 26 / 10 desalting column (catalog number: 17-05087-02, GE Healthcare). Potential homodimeric dsTrasKO2 monospecific molecules were selected by using an additional KappaSelect capture step (0.1 M glycine pH 2.7 elution buffer) (see Figure 14). After final purification of the protein by size exclusion chromatography (SEC) using Superdex 200 26 / 60 (GE) and a final ultrafiltration concentration step, the protein was used for further characterization. See Figures 12A-12D for representative bispecific design and purification results.

[0390] Example 9: Evaluation of trispecific antibodies with CODV structure using dsTrasKO2 as a replacement scaffold for CH1 / CL The dsTrasKO2 domain was evaluated as a replacement scaffold in trispecific crossover mutant domain (CODV) design. Specifically, the CODV-anti-Ox40 × anti-PD1 × anti-CD137-dsTrasKO2-hulgHuG1-LALA-KIH-RF construct was synthesized and tested (see Figure 13).

[0391] Expression of dsTrasKO2 trispecific CODV molecules Expression plasmids encoding the heavy and both light chains (dsTrasKO and WT) and the corresponding constructs encoding both heavy chains (CODV-knob and dsTrasKO2-hole) and both light chains (CODV and dsTrasKO) were propagated in E. coli DH5a. Plasmids used for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega kit. HEK293-FS cells grown in F17 serum-free suspension culture (Invitrogen) were transfected with the indicated LC and HC plasmids using polyethylenimine transfection reagent. After 7 days of incubation at 37°C, cells were removed by centrifugation, and the supernatant was passed through a 0.22 μm filter to remove particles. For purification, the antibody was captured on a MabSelect SuRe column (catalog number: 11-0034-93, GE Healthcare), eluted with 0.1 M citrate buffer, pH 3.0, and directly desalted using a HiPrep 26 / 10 desalting column (catalog number: 17-05087-02, GE Healthcare). The sample was further purified on a MonoS cation exchange column (catalog number: 17-5169-01, GE Healthcare, with a 0-1 M NaCl salt gradient in 0.01 M L-histidine, pH 6.0 buffer). After an ultrafiltration concentration step, the protein was used for further characterization. See Figures 13A-D for representative trispecific design and purification results.

[0392] Example 10: Evaluation of bispecific T cell-inducing antibodies using dsTrasKO2 as a replacement scaffold for CH1 / CL General method 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 x 300 mm) and an AdvanceBio 300 guard column (Agilent Technologies). Analysis was performed at a flow rate of 0.5 ml / min using 2x concentrated D-PBS buffer (Thermo Fisher Scientific) with detection at 280 nm. Ten 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 weights, the SEC column was calibrated with a protein calibration standard mix (Agilent Technologies).

[0393] Analytical Hydrophobic Interaction Chromatography (HIC) 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). The analysis was performed at a flow rate of 1 ml / min with detection at 280 nm. Five μg of undiluted protein sample was applied to the column. Gradient elution was performed from 15% B to 85% B for 7 min, followed by 100% B for 1 min, then 15% B for 1 min, followed by equilibration at 15% B for 3 min. Buffer A consisted of 1.5 M ammonium sulfate, 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).

[0394] nanoDSF The onset temperature of protein denaturation (T onset) and melting temperature (T m ) were measured using nanodifferential scanning fluorimetry (nanoDSF). Samples were diluted to a final concentration of 0.5 μg / μl in formulation buffer and loaded in duplicate into nanoDSF capillaries (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).

[0395] Surface Plasmon Resonance (SPR) Antigen binding to the antibody constructs was measured using surface plasmon resonance (SPR) on a BIAcore 8K instrument (GE Healthcare) using HBS-EP+ buffer (GE Healthcare). For binding kinetics and affinity determination, human CD3εδ-Fc-His and human CD123-Fc-His fusion proteins (both from internal sources) were used as antigens. Anti-His capture antibodies (His capture kit, GE Life Science) were immobilized via primary amine groups (11,000 RU) on a research-grade CM5 chip (GE Life Science) using standard procedures. The antigen was captured by the anti-His capture chip surface at a flow rate of 10 μL / min for 90 s to reach antibody binding levels between 10 and 30 RU. Antibodies were injected at 30 μL / min for 240 s in two-fold dilutions from 100 nM to 3.1 nM to determine CD123 affinity, and in two-fold dilutions from 400 nM to 3.1 nM or 100 nM to 3.1 nM to determine CD3 binding affinity. Dissociation was measured at 30 μL / min by injecting HBS-EP+ buffer for 1200 s. The chip surface was regenerated by injecting regeneration buffer (His Capture Kit, GE Life Sciences). Sensorgrams were double-referenced with a blank chip surface and an HBS-EP+ buffer blank. Data were fitted to a 1:1 Langmuir binding model to determine kinetics and affinity constants ka, kd, and KD using Biacore 8K Evaluation software v1.11.7442 (GE Healthcare).

[0396] To assess the relative binding levels (Rmax%) of antibodies to CD3 and CD123, the antibodies were captured by anti-Fc affinity capture onto a sensor chip. Human CD3εδ-FLAG-His (#CT038-H2508H, Sino Biological) and human CD123 (#301-R3 / CF, R&D Systems) proteins were used in this assay. Anti-human Fc capture antibodies (Human Antibody Capture Kit, GE Life Sciences) were immobilized via primary amine groups (11,000 RU) on a research-grade CM5 chip (GE Life Sciences) using standard procedures. Antibodies were captured at a flow rate of 10 μl / min, resulting in a maximum analyte binding of 10–30 RU, using adjusted RU values. Antigen was used as the analyte, injected at either 400 nM or 100 nM concentrations for CD3εδ-FLAG-His and at 100 nM for CD123. The antigen was 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. Sensorgrams were double-referenced with 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). R% values ​​were calculated using the maximum binding level divided by the theoretical R value. The theoretical R value was calculated from the capture level R, the measured binding stoichiometry N, and the molecular weights of the antibody Mw (Ab) and antigen Mw (Ag), with R = Rcapture × N × (Mw(Ag) / Mw(Ab)).

[0397] mass spectrometry The protein integrity and potential mispairing of the heterodimer constructs were analyzed by LC-mass spectrometry (LC-MS). Protein samples were deglycosylated using 12.5 μg of protein diluted to 0.17 mg / mL in LC-MS-grade water (Thermo Scientific) and treated with 0.5 μL of PNGaseF (glycerol-free, New England Biolabs) for 16 hours at 37°C. LC-MS analysis was performed using an Orbitrap Fusion Lumos Tribrid mass spectrometer. Reverse-phase (RP) chromatography was performed at 300 μL / min using a MabPac RP HPLC column, 4 μm analytical particle size, 2.1 × 100 mm (Thermo Scientific). The eluents were LC water, 0.1% formic acid (A) and 90% acetonitrile, 10% LC water, 0.1% formic acid (B). 2 μg of protein solution was injected onto the column and eluted using a linear gradient from 0% to 95% B over 12 min. Data analysis was performed using Expressionist software 13.0.3 (Genedata). Molecular weights were calculated based on the amino acid sequences of the proteins using GPMAW software version 10.32b1 (Lighthouse data).

[0398] Cytotoxicity assay with bispecific TrasKO2 molecules The bispecific TrasKO2 molecule was analyzed in a cytotoxicity assay using primary human T cells. Human peripheral mononuclear cells (PBMCs) 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 x g for 10 minutes. 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 a MACSpro separator (Miltenyi Biotec) and a Pan T cell isolation kit (Miltenyi Biotec, #130-096-535) according to the manufacturer's protocol. Isolated human T cells were cultured at 5 × 10 in RPMI GlutaMAX I medium (Gibco, #72400) supplemented with 10% FCS HI (Gibco, #10082-147). 6 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. Cells were washed twice in RPMI + GlutaMAX I medium and centrifuged at 400 × g for 5 minutes. THP-1 target cells were resuspended at 5 × 10 cells / mL in RPMI medium supplemented with 10% FCS HI. 5CFSE-labeled THP-1 cells and human pan-T cells were mixed at a 10:1 effector-to-target ratio and seeded in a total volume of 100 μl / well in a 96-well assay plate (Greiner BioOne, #650185). The bispecific TrasKO2 molecule was added to the cells in an 11-dilution series starting from 10 nM to 0 nM (1:6 dilution) in 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 on CFSE / 7-AAD double-positive THP-1 cells on an LSRII flow cytometer (BD), and EC50 values ​​were determined using Xlfit software.

[0399] The dsTrasKO2 domain was evaluated as a replacement scaffold in the design of bispecific T cell-inducing antibodies. Bispecific T cell-inducing agents were generated by using anti-TCRα / β or one of two different anti-CD3ε as the effector arm and anti-CD123 as the targeting arm. A negative control for both arms was generated using the TNP antibody sequence (trinitrophenol antibody). Bispecific proteins were purified using MabSelect Sure, followed by KappaSelect and SEC. Bispecific antibodies were expressed with either a TrasKO2 replacement on one of the Fab arms (both possible re-engineered Fab arms were generated) or wild-type bispecific IgG (detection of naturally occurring mismatches). Biophysical characterization of the bispecific antibodies is described in Table 12 below.

[0400] [Table 48] [Table 49]

[0401] In addition to the biophysical characterization described above, the bispecific TrasKO2 molecules were analyzed in cell-based cytotoxicity assays. As shown in Figure 15A, all three anti-CD3ε x anti-CD123 bispecific antibodies exhibited comparable and potent activity. The presence of the dsTrasKO2 domain in antibodies ID Nos. 34 and 35 reduced chain mispairing without negatively affecting activity. Negative control antibodies ID Nos. 45 and 46, containing the TNP antibody sequence, were used. As shown in Figure 15B, all three anti-CD3ε x anti-CD123 bispecific antibodies, which have alternative anti-CD3ε binding domains, also exhibited comparable and potent activity. The presence of the dsTrasKO2 domain in antibodies ID Nos. 37 and 38 reduced chain mispairing without negatively affecting activity. Antibodies ID Nos. 47 and 48, containing the TNP antibody sequence, were used as negative controls.

Claims

1. A binding protein comprising: At least one pseudo-Fab portion comprising: (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; and (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain. and the VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 The binding protein is selected from the group consisting of:

2. 2. The binding protein of claim 1, wherein the binding protein is a multispecific binding protein further comprising at least a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen binding site that binds to target antigen B.

3. 1. A multispecific binding protein comprising: a) a first pseudo-Fab portion comprising: (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; and (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain; b) a first Fab portion comprising (3) a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B, and (4) a first CH1 domain paired with a first CL domain. Including, The VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 The multispecific binding protein is selected from the group consisting of:

4. 1. A multispecific binding protein comprising: a) a first pseudo-Fab portion comprising: (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; and (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain; b) a first Fab portion comprising (3) a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B, and (4) a first CH1 domain paired with a first CL domain; and c) a linker portion operably linking the first Fab portion and the first pseudo-Fab portion; The VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 The multispecific binding protein is selected from the group consisting of:

5. The binding protein of any one of claims 1 to 4, wherein the linker moiety is a peptide linker.

6. The peptide linker has the formula (Gly 4 Ser) n 6. The binding protein of claim 5, wherein n is a Gly-Ser linker of the formula: (n is 1 to 10).

7. The binding protein of any one of claims 1 to 6, wherein the linker moiety is a heterodimerization domain.

8. 8. The binding protein of any one of claims 1 to 7, comprising independently one or two first pseudo-Fab portions and one or two first Fab portions.

9. The following groups: (a) VHa-CH1-L1-VHb-L2-VHX and VLa-CL and VLb-L3-VLX; (b) VHa-L2-VHX-L1-VHb-CH1 and VLa-L3-VLX and VLb-CL; (c) VHa-CH1-L1-VHa-CH1 and VHb-L2-VHX-L3-VHb-L4-VHX, as well as two chains VLb-L5-VLX and two chains VLa-CL and wherein the protein chains are selected from one of:

9. The binding protein of any one of claims 1 to 8, wherein chains (a) and (b) can occur once or twice, and L1, L2, L3, L4 and L5 are independently the same or different linkers.

10. The first pseudo-Fab portion has the formula: (Ia) N-VHa-L1-VHX-C and a first polypeptide chain having a structure represented by the formula: (IIa) N-VLa-L2-VLX-C A second polypeptide chain having a structure represented by Including, 10. The binding protein of claim 1, wherein L1 and L2 are linkers, which are independently present or absent, and N and C represent the N-terminus and C-terminus, respectively.

11. The first pseudo-Fab portion has the formula: (Ib)N-VHX-L1-VHa-C and a first polypeptide chain having a structure represented by the formula: (IIb)N-VLX-L2-VLa-C A second polypeptide chain having a structure represented by Including, wherein L1 and L2 are linkers, which are independently present or absent, and N and C represent the N-terminus and C-terminus, respectively.

12. The first pseudo-Fab portion has the formula: (Ic)N-VLa-L1-VHX-C and a first polypeptide chain having a structure represented by the formula: (IIc)N-VHa-L2-VLX-C A second polypeptide chain having a structure represented by Including, 12. The binding protein of claim 1, wherein L1 and L2 are linkers, which are independently present or absent, and N and C represent the N-terminus and C-terminus, respectively.

13. The first pseudo-Fab portion has the formula: (Id)N-VHX-L1-VLa-C and a first polypeptide chain having a structure represented by the formula: (IId)N-VLX-L2-VHa-C A second polypeptide chain having a structure represented by Including, wherein L1 and L2 are linkers, which are independently present or absent, and N and C represent the N-terminus and C-terminus, respectively.

14. 14. The binding protein of any one of claims 1 to 13, further comprising one or more additional binding domains operably linked to the N-terminus or C-terminus of the binding protein.

15. 15. The binding protein of any one of claims 1 to 14, wherein one or more additional binding domains are operably linked to the N-terminus of the first or second pseudo-Fab portion. quality.

16. 1. A multispecific binding protein comprising: a) (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first antigen-binding site that binds to target antigen A; (2) a first stabilized knockout VL domain (VLX) paired with a first stabilized knockout VH domain (VHX) to form a first disulfide-stabilized knockout (dsKO) domain; (3) First heterodimerization domain (HD1) a first pseudo-Fab portion comprising: where the VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 selected from the group consisting of: b) (1) a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (2) a first CH1 domain paired with a first CL domain; and (3) Second heterodimerization domain (HD2) a first Fab portion comprising 2. The multispecific binding protein comprising:

17. 17. The binding protein of claim 16, wherein the first and second heterodimerization domains comprise first and second Fc domains.

18. 18. The binding protein of claim 16 or 17, wherein the Fc domain comprises the general structure hinge-CH2 domain-CH3 domain.

19. 1. A multispecific binding protein comprising four polypeptide chains that form at least two antigen binding sites, (a) the first polypeptide has the formula: VLa-L1-VLX[I] The structure includes: (b) the second polypeptide has the formula: VHa-L2-VHX-FC1 [II] The structure includes: (c) the third polypeptide has the formula: VLb-CL [III] The structure includes: (d) the fourth polypeptide has the formula: VHb-CH1-FC2 [IV] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; and L1 and L2 are independently the same or different amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 The multispecific binding protein is selected from the group consisting of:

20. 1. An antigen-binding protein comprising six polypeptide chains that form four antigen-binding sites, (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHa-L2-VHX-L3-VHb-CH1-FC1[V] The structure includes: (d) the sixth polypeptide has the formula: VHa-L2-VHX-L3-VHb-CH1-FC2 [VI] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains comprising the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2 and L3 are amino acid linkers; (1) A first VL domain (VLa) pairs with a first VH domain (VHa) to form a target forming a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 The antigen-binding protein is selected from the group consisting of:

21. 1. An antigen-binding protein comprising six polypeptide chains that form four antigen-binding sites, (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHb-CH1-L3-VHa-L2-VHX-FC1[V] The structure includes: (d) the sixth polypeptide has the formula: VHb-CH1-L3-VHa-L2-VHX-FC2[VI] The structure includes: During the ceremony: VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains comprising the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2 and L3 are amino acid linkers; During the ceremony, (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 The antigen-binding protein is selected from the group consisting of:

22. 1. An antigen-binding protein comprising six polypeptide chains that form four antigen-binding sites, (a) the first and second polypeptides have the formula: VLa-L1-VLX[I] and [II] The structure includes: (b) the third and fourth polypeptides have the formula: VLb-CL[III] and [IV] The structure includes: (c) the fifth polypeptide has the formula: VHa-L2-VHX-L3-VHa-L4-VHX-FC1 [V] The structure includes: (d) the sixth polypeptide has the formula: VHb-CH1-L5-VHb-CH1-FC2 [VI] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin heavy chain constant domain; FC1 and FC2 are Fc domains comprising the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, L4 and L5 are amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a stabilized knockout VL domain (VLX) pairs with a stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO) domain; The VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 The antigen-binding protein is selected from the group consisting of:

23. 1. An antigen-binding protein comprising four polypeptide chains that form three antigen-binding sites, (a) the first polypeptide has the formula: VLa-L1-VLX[I] The structure includes: (b) the second polypeptide has the formula: VHa-L2-VHX-FC1 [II] The structure includes: (c) the third polypeptide has the formula: VLb-L3-VLc-L4-CL [III] The structure includes: (d) the fourth polypeptide has the formula: VHc-L5-VHb-L6-CH1-FC2 [IV] The structure includes: During the ceremony, VLa is the first immunoglobulin light chain variable domain; VLb is the second immunoglobulin light chain variable domain; VLc is the third immunoglobulin light chain variable domain; VHa is the first immunoglobulin heavy chain variable domain; VHb is the second immunoglobulin heavy chain variable domain; VHc is the third immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; VLX is a stabilized knockout light chain variable domain; VHX is a stabilized knockout heavy chain variable domain; FC1 and FC2 are Fc domains comprising the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains; L1, L2, L3, L4, L5 and L6 are amino acid linkers; (1) a first VL domain (VLa) pairs with a first VH domain (VHa) to form a first functional antigen-binding site that binds to target antigen A; (2) a second VL domain (VLb) pairs with a second VH domain (VHb) to form a second functional antigen-binding site that binds to target antigen B; (3) a third VL domain (VLc) pairs with a third VH domain (VHc) to form a third functional antigen-binding site that binds to target antigen C; (4) the polypeptide of Formula III and the polypeptide of Formula IV form a cross-linked light chain-heavy chain pair (CODV); (5) the stabilized knockout VL domain (VLX) pairs with the stabilized knockout VH domain (VHX) to form a disulfide-stabilized knockout (dsKO2) domain; The VLX / VHX pair is (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 The antigen-binding protein is selected from the group consisting of:

24. 24. The binding protein of any one of claims 17 to 23, wherein the Fc domain comprises one or more knobs-in-holes (KIH) mutations.

25. The melting temperature (T m 25. The binding protein of any one of claims 1 to 24, wherein the temperature of the binding protein is at least 4°C higher than that of a reference Fab molecule.

26. 26. The binding protein of any one of claims 1 to 25, wherein at least one of the one or more engineered interchain disulfide bonds is VH44C-VL100C.

27. 27. The binding protein of any one of claims 1 to 26, wherein target antigen A and target antigen B are different epitopes of the same antigen.

28. 1. Use of a stabilizing knockout domain to reduce heavy chain-light chain mispairing in a multispecific binding protein, wherein the stabilizing knockout domain comprises a VHK and a VLX domain, and the VLX / VHX pair is: (i) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and VHX comprising the amino acid sequence of SEQ ID NO: 77; (ii) a VLX comprising the amino acid sequence of SEQ ID NO: 76; and A VHX comprising the amino acid sequence of SEQ ID NO: 78; and (iii) a VLX comprising the amino acid sequence of SEQ ID NO: 76, and VHX comprising the amino acid sequence of SEQ ID NO: 79 and the reference Fab molecule is identical to the pseudo-Fab molecule except that in the pseudo-Fab reference molecule, the CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

29. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein of any one of claims 1 to 27.

30. 30. An expression vector comprising the nucleic acid molecule of claim 29.

31. 31. An isolated host cell comprising the nucleic acid molecule of claim 29 or the expression vector of claim 30.

32. 38. A method of producing a binding protein according to any one of claims 1 to 27, comprising culturing a host cell according to claim 31 under conditions such that the binding protein is expressed; and purifying the binding protein from the host cell.

33. 28. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the multispecific binding protein of any one of claims 1 to 27.

34. 28. A pharmaceutical composition for use in the treatment of a disorder in which antigenic activity is detrimental, said pharmaceutical composition comprising administering to a subject in need thereof an effective amount of the multispecific binding protein of any one of claims 1 to 27.

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