Purification method of multispecific antibody

Abstract

Multivalent binding proteins comprising four polypeptide chains are provided wherein a first heavy chain polypeptide and a first light chain polypeptide are associated to form one or more antigen binding domains and a second heavy chain polypeptide and a second light chain polypeptide are associated to bind the one or more antigen binding domains. Methods of purifying such multivalent binding proteins are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 350,255, filed on June 8, 2022; and European Patent Application No. 22315206.7, filed on September 8, 2022, the contents of each of which are incorporated herein by reference in their entirety. Reference Electronic Sequence Listing

[0002] The contents of the electronic sequence listing (183952034140SEQLIST.xml; size: 21,251 bytes; and creation date: May 30, 2023) are incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to multivalent binding proteins with reduced binding to Protein L and / or KappaSelect chromatography materials, methods of producing such multivalent binding proteins, and methods of purifying such multivalent binding proteins from compositions comprising the multivalent binding proteins and impurities (e.g., mismatched polypeptides). Background Art

[0004] Multivalent binding proteins (e.g., multivalent and / or multispecific antibodies and antibody constructs) have huge clinical potential as the development of therapeutic agents for human diseases. However, the production of multivalent binding proteins in the form of IgG is always challenging because antibody heavy chains have evolved to bind antibody light chains in a relatively mixed manner. Due to this type of messy pairing, the generation of multivalent binding proteins comprising, for example, two or more antibody heavy chains (or heavy chain constructs) and / or two or more antibody light chains (or light chain constructs) may lead to the formation of undesirable species comprising heavy chain homodimers and / or mixed heavy chain / light chain pairs. Due to the similarity in structure and molecular mass, it may be difficult to separate the multivalent binding proteins that are correctly assembled from this undesirable mismatched species by chromatography. In addition, complicated in vitro assembly reactions and / or purification methods limit the applicability of many multivalent binding protein platforms, particularly their purposes in the necessary high-throughput screening of many therapeutic drug pipelines.

[0005] There is a need in the art for improved protein purification methods that remove mispaired heavy chain / light chain byproducts and increase multivalent binding protein yields. Summary of the invention

[0006] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain, wherein VL2 is a κ1, κ3, or κ4 subtype light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, wherein CL1 is a Cκ subtype light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH1 is an immunoglobulin heavy chain constant domain; wherein CL1 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, wherein VL2 comprises one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VH1 associates with VL1 to form a first antigen binding domain, and VH2 associates with VL2 to form a second antigen binding domain.

[0007] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1-CH2-CH3 [III], and a second light chain polypeptide comprising a structure represented by the following formula: VL2-CL2[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH1, CH2 and CH3 are immunoglobulin heavy chain constant domains; wherein CL1 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, wherein VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VH1 associates with VL1 to form a first antigen binding domain, and VH2 associates with VL2 to form a second antigen binding domain.

[0008] In some embodiments according to (or as applicable to) any embodiment of the present invention, the binding of CL1 comprising one or more substitutions to KappaSelect is reduced by about 90% compared to CL1 without the one or more amino acid substitutions. In some embodiments according to (or as applicable to) any embodiment of the present invention, the binding of VL2 comprising one or more substitutions to protein L chromatography material is reduced by about 90% compared to VL2 without the one or more amino acid substitutions. In some embodiments according to (or as applicable to) any embodiment of the present invention, the one or more amino acid substitutions in CL1 comprising one or more substitutions are at positions corresponding to 109, 110 or 199, wherein the numbering is according to the EU index. In some embodiments according to (or as applicable to) any embodiment of the present invention, the one or more amino acid substitutions in CL1 comprising one or more substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL1 comprising one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL1 comprising one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL2 comprising one or more substitutions are substitutions of framework amino acids. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL2 comprising one or more substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in the VL2 comprising one or more substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein the numbering is according to Kabat. In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain.In some embodiments according to (or as applied to) any of the embodiments herein, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are S354C and T366W. In some embodiments according to (or as applied to) any embodiment herein, the CH3 of the second heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A. In some embodiments according to (or as applied to) any embodiment herein, the CH3 of the first heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A. In some embodiments according to (or as applicable to) any embodiment of this paper, one or more amino acid replacements that reduce the combination with protein A are amino acid replacements at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments according to (or as applicable to) any embodiment of this paper, these amino acid replacements are H435R and Y436F, wherein amino acid numbering is according to the EU index. In some embodiments according to (or as applicable to) any embodiment of this paper, the CH1, CH2 and CH3 domains of the first heavy chain polypeptide are different from the CH1, CH2 and CH3 domains of the second heavy chain polypeptide. In some embodiments according to (or as applicable to) any embodiment of this paper, the first heavy chain polypeptide is derived from a species different from the second heavy chain polypeptide. In some embodiments according to (or as applicable to) any embodiment of this paper, the first heavy chain polypeptide and the first light chain polypeptide are derived from mouse heavy chain immunoglobulin and mouse light chain immunoglobulin, and the second heavy chain polypeptide and the second light chain polypeptide are derived from rat heavy chain immunoglobulin and rat light chain immunoglobulin. In some embodiments according to (or as applied to) any of the embodiments herein, the first heavy chain polypeptide and the second heavy chain polypeptide each comprise an IgG4 CH3 domain.In some embodiments according to (or as applied to) any embodiment herein, the first heavy chain polypeptide comprises a K409R amino acid substitution and the second heavy chain polypeptide comprises a F405L amino acid substitution, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the multivalent binding protein is a bispecific antigen binding protein. In some embodiments according to (or as applied to) any embodiment herein, the first antigen binding domain and the second antigen binding domain bind different antigens.

[0009] In some embodiments according to (or as applied to) any of the embodiments herein, the first heavy polypeptide chain of the multivalent binding protein comprises a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3-L-VL3 [Ia], and The second heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIa], Wherein: VL3 is the third immunoglobulin light chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; L is an amino acid linker; wherein VH3 and VL3 associate to form a third antigen binding domain.

[0010] In some embodiments according to (or as applied to) any of the embodiments herein, the first heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3[Ib], and The second heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIb], Wherein: VH3 is the third immunoglobulin heavy chain variable domain.

[0011] In some embodiments according to (or as applicable to) any embodiment of this invention, VL3 comprises one or more amino acid substitutions that reduce binding to protein L chromatographic material compared to VL3 without one or more amino acid substitutions. In some embodiments according to (or as applicable to) any embodiment of this invention, VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 immunoglobulin light chain variable domain. In some embodiments according to (or as applicable to) any embodiment of this invention, the multivalent binding protein is bispecific or trispecific. In some embodiments according to (or as applicable to) any embodiment of this invention, the first antigen binding domain, the second antigen binding domain binds and the third antigen binding domain binds two or three different antigens. In some embodiments according to (or as applicable to) any embodiment of this invention, the first antigen binding domain binds to the first antigen, the second antigen binding domain binds to the second antigen, and the third antigen binding domain binds to the third antigen. In some embodiments according to (or as applicable to) any embodiment of this invention, the first antigen binding domain and the second antigen binding domain bind to the first antigen, and the third antigen binding domain binds to the second antigen.

[0012] In some embodiments, a multivalent binding protein is provided comprising four polypeptide chains forming three antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L3-VH2-L4-CH1 [I], A first light polypeptide chain comprising a structure represented by the formula: VL2-L1-VL1-L2-CL1 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL3-CL2 [IV] wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair; wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain.

[0013] In some embodiments, a multivalent binding protein is provided comprising four polypeptide chains forming three antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L3-VH2-L4-CH1-CH2-CH3 [Ia], A first light polypeptide chain comprising a structure represented by the formula: VL2-L1-VL1-L2-CL1 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH1-CH2-CH3 [IIIa], and a second light chain polypeptide comprising a structure represented by the following formula: VL3-CL2 [IV] wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair; wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype light chain variable immunoglobulin domain; or d) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype light chain variable immunoglobulin domain.

[0014] In some embodiments according to (or as applied to) any embodiment herein, the binding protein is trispecific and is capable of specifically binding to three different antigen targets. In some embodiments according to (or as applied to) any embodiment herein, at least one of L1, L2, L3 or L4 is each independently 0 amino acids in length. In some embodiments according to (or as applied to) any embodiment herein, L1, L2, L3 or L4 is each independently at least one amino acid in length.

[0015] In some embodiments according to (or as applied to) any of the embodiments herein, the second heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH1 [IIIb], And the second light chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VL4-L7-VL3-L8-CL2 [IVa] wherein: VL4 is a fourth immunoglobulin light chain variable domain; VH4 is a fourth immunoglobulin heavy chain variable domain; L5, L6, L7 and L8 are amino acid linkers; wherein the polypeptide of formula IIIa and the polypeptide of formula IVa form a crossed light chain-heavy chain pair; wherein VH4 and VL4 associate to form a fourth antigen binding domain; and wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and wherein VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; c) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; e) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or f) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain.

[0016] In some embodiments according to (or as applied to) any of the embodiments herein, the second heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH1-CH2-CH3 [IIIc], wherein: CH2 is an immunoglobulin CH2 heavy chain constant domain and CH3 is an immunoglobulin CH3 heavy chain constant domain. In some embodiments according to (or as applied to) any embodiment herein, the multivalent binding protein is tetraspecific and is capable of specifically binding to four antigen targets (e.g., four different target antigens).

[0017] In some embodiments according to (or as applicable to) any embodiment of the present invention, at least one of L1, L2, L3, L4, L5, L6, L7 or L8 is each independently 0 amino acids in length. In some embodiments according to (or as applicable to) any embodiment of the present invention, L1, L2, L3, L4, L5, L6, L7 or L8 is each independently at least one amino acid in length. In some embodiments according to (or as applicable to) any embodiment of the present invention, the binding of CL1 or CL2 comprising one or more amino acid substitutions to KappaSelect chromatography material is reduced by about 90% compared to CL1 or CL2 without the one or more amino acid substitutions. In some embodiments according to (or as applicable to) any embodiment of the present invention, the binding of VL1, VL2, VL3 and / or VL4 comprising one or more substitutions to protein L chromatography material is reduced by about 90% compared to VL1, VL2, VL3 and / or VL4 without the one or more amino acid substitutions. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in the CL1 or CL2 are at positions corresponding to 109, 110 or 199, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in the CL1 or CL2 comprising the one or more amino acid substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in the CL1 or CL2 comprising the one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in the CL1 or CL2 comprising the one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments according to (or as applicable to) any embodiment herein, one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4 comprising one or more substitutions are substitutions of framework amino acids. In some embodiments according to (or as applicable to) any embodiment herein, one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4 comprising one or more substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat.In some embodiments according to (or as applied to) any embodiment herein, one or more amino acid substitutions in VL1, VL2, VL3, and / or VL4 comprising one or more substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat. In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. In some embodiments according to (or as applied to) any of the embodiments herein, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are S354C and T366W. In some embodiments according to (or as applied to) any embodiment herein, the second heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A. In some embodiments according to (or as applied to) any embodiment herein, the CH3 of the first heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions that reduce binding to Protein A are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the amino acid substitutions are H435R and Y436F, wherein the amino acid numbering is according to the EU index.

[0018] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CL2[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CH1[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH1 is an immunoglobulin heavy chain constant domain, wherein a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

[0019] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CL2-CH2-CH3 [III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CH1[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH1 is an immunoglobulin heavy chain constant domain; wherein a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

[0020] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VL2-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VH2-CL2[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH1, CH2 and CH3 are immunoglobulin heavy chain constant domains; in a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

[0021] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1-CH2-CH3 [III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH1, CH2 and CH3 are immunoglobulin heavy chain constant domains; in a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

[0022] In some embodiments according to (or as applicable to) any embodiment of this invention, the binding of CL1 or CL2 comprising one or more amino acid replacements to KappaSelect chromatographic materials is reduced by about 90% compared to CL1 or CL2 without the one or more amino acid replacements. In some embodiments according to (or as applicable to) any embodiment of this invention, the binding of VL1 or VL2 comprising one or more amino acid replacements to protein L chromatographic materials is reduced by about 90% compared to VL1 or VL2 without the one or more amino acid replacements. In some embodiments according to (or as applicable to) any embodiment of this invention, the one or more amino acid replacements in CL1 or CL2 comprising one or more amino acid replacements are at positions corresponding to 109, 110 or 199, wherein the numbering is according to the EU index. In some embodiments according to (or as applicable to) any embodiment of this invention, the one or more amino acid replacements in CL1 or CL2 comprising one or more amino acid replacements are T109A replacements, V110D replacements, Q199K replacements, T109A-V110D replacements, or T109A-V110D-Q199K replacements. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL1 or CL2 comprising one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL1 or CL2 comprising one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL1 or VL2 comprising one or more amino acid substitutions are substitutions of framework amino acids. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL1 or VL2 comprising one or more amino acid substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL1 or VL2 comprising one or more amino acid substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat. In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain.In some embodiments according to (or as applied to) any of the embodiments herein, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are S354C and T366W. In some embodiments according to (or as applied to) any embodiment herein, the CH3 of the second heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A chromatographic materials. In some embodiments according to (or as applied to) any embodiment herein, the CH3 of the first heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A chromatographic materials. In some embodiments according to (or as applicable to) any embodiment of this paper, one or more amino acid replacements that reduce the combination with protein A chromatographic material are amino acid replacements at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments according to (or as applicable to) any embodiment of this paper, these amino acid replacements are H435R and Y436F, wherein amino acid numbering is according to the EU index. In some embodiments according to (or as applicable to) any embodiment of this paper, the multivalent binding protein is a bispecific antigen-binding protein. In some embodiments according to (or as applicable to) any embodiment of this paper, the first antigen-binding domain and the second antigen-binding domain bind different antigens.

[0023] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming four antigen binding domains is provided; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH11-L1-VH 2- CH12 [I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-CL1-L 2- V L 2- CL2 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH13-L3-VH 4- CH14 [III], A second light chain polypeptide comprising a structure represented by the following formula: VL3-CL3-L 4- V L 4- CL4 [IV] wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; VL4 is a fourth immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; CL3 is a third immunoglobulin light chain constant domain; CL4 is a fourth immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; VH4 is a fourth immunoglobulin heavy chain variable domain; CH11 is a first immunoglobulin heavy chain constant domain; CH12 is a second immunoglobulin heavy chain constant domain; CH13 is a third immunoglobulin heavy chain constant domain; CH14 is a fourth immunoglobulin heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein: a) CL1 and CL2 each comprise the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 and CL2 each comprise one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, VL3 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; c) CL1 and CL2 each comprise the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; e) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, VL3 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; f) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, VL4 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; g) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; h) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, VL3 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; i) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, VL4 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; And wherein VL1 and VH1 form a first antigen binding domain, VL2 and VH2 form a second antigen binding domain, VL3 and VH3 form a third antigen binding domain, and VL4 and VH4 form a fourth antigen binding domain.

[0024] In some embodiments according to (or as applied to) any embodiment herein, at least one of L1, L2, L3 or L4 is each independently 0 amino acids in length. In some embodiments according to (or as applied to) any embodiment herein, at least one of L1, L2, L3 or L4 is each independently at least one amino acid in length.

[0025] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming four antigen binding domains is provided; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L1-VH 2- L2-CH11 [I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-L3-VL 2- L4-CL1 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-L5-VH 4- L6-CH12 [III], A second light chain polypeptide comprising a structure represented by the following formula: VL3-L 7- V L 4- L8-CL2 [IV] wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; VL4 is a fourth immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; VH4 is a fourth immunoglobulin heavy chain variable domain; CH11 is a first immunoglobulin heavy chain constant domain; CH12 is a second immunoglobulin heavy chain constant domain; and L1, L2, L 3、 L4, L5, L6, L7 and L8 are amino acid linkers; wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; c) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; And wherein VL1 and VH1 form a first antigen binding domain, VL2 and VH2 form a second antigen binding domain, VL3 and VH3 form a third antigen binding domain, and VL4 and VH4 form a fourth antigen binding domain.

[0026] In some embodiments according to (or as applicable to) any embodiment of the present invention, at least one of L1, L2, L3, L4, L5, L6, L7 or L8 is each independently 0 amino acids in length. In some embodiments according to (or as applicable to) any embodiment of the present invention, at least one of L1, L2, L3, L4, L5, L6, L7 or L8 is each independently 1 amino acid in length. In some embodiments according to (or as applicable to) any embodiment of the present invention, the binding of CL1 and / or CL2 comprising one or more amino acid replacements to KappaSelect chromatography material is reduced by about 90% compared to CL1 and / or CL2 without the one or more amino acid replacements. In some embodiments according to (or as applicable to) any embodiment of the present invention, the binding of VL3 and / or VL4 comprising one or more amino acid replacements to protein L chromatography material is reduced by about 90% compared to VL3 and / or VL4 without the one or more amino acid replacements. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL1 and / or CL2 comprising one or more amino acid substitutions are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL1 and / or CL2 comprising one or more amino acid substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL1 and / or CL2 comprising one or more substitutions are at positions corresponding to 109, 198, 199, or 202, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL1 and / or CL2 comprising one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL3 and / or VL4 comprising one or more amino acid substitutions are substitutions of framework amino acids. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL3 and / or VL4 comprising one or more amino acid substitutions are at positions corresponding to 12 or 18, wherein the numbering is according to Kabat.In some embodiments according to (or as applied to) any of the embodiments herein, the one or more amino acid substitutions in VL3 and / or VL4 comprising one or more amino acid substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, where numbering is according to Kabat.

[0027] In some embodiments according to (or as applied to) any of the embodiments herein, the first heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH1-CH11-L1-VH 2- CH12-CH2-CH3 [Ia], The second heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH3-CH13-L3-VH 4- CH14-CH2-CH3 [IIIa].

[0028] In some embodiments according to (or as applied to) any of the embodiments herein, the first heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH1-L1-VH 2- L2-CH11-CH2-CH3 [Ia], The second heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH12-CH2-CH3 [IIIa].

[0029] In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or as applied to) any embodiment herein, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are S354C and T366W. In some embodiments according to (or as applied to) any embodiment herein, the CH3 of the second heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A chromatographic materials. In some embodiments according to (or as applied to) any embodiment herein, the CH3 of the first heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A chromatographic materials. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions that reduce binding to the protein A chromatographic material are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, these amino acid substitutions are H435R and Y436F, wherein amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the binding protein is tetraspecific and is capable of specifically binding to four different antigen targets.

[0030] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming an antigen binding domain is provided; wherein the four polypeptide chains comprise: The first heavy chain polypeptide comprises a structure represented by the following formula: VH1-CH11[I], The first light polypeptide chain comprises a structure represented by the following formula: VL1-CL1[II], The second heavy chain polypeptide comprises a structure represented by the following formula: Fusion polypeptide-L1-CH12 [III], and a second light polypeptide chain comprising a structure represented by the following formula: Fusion peptide-L2-CL2 [IV] wherein: VL1 is a first immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; CH11 is a first immunoglobulin heavy chain constant domain; CH12 is a second immunoglobulin heavy chain constant domain; and L1 and L2 are amino acid linkers; wherein CL2 comprises the one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions; and wherein VL1 and VH1 form an antigen binding domain.

[0031] In some embodiments according to (or as applied to) any embodiment herein, L1 or L2 are independently 0 amino acids in length. In some embodiments according to (or as applied to) any embodiment herein, L1 or L2 are independently at least 1 amino acid in length.

[0032] In some embodiments, a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: The first heavy chain polypeptide comprises a structure represented by the following formula: VH1-CH11-L1-VH2-CH12 [I], The first light polypeptide chain comprises a structure represented by the following formula: VL1-CL1-L2-VL2-CL2 [II], The second heavy chain polypeptide comprises a structure represented by the following formula: Fusion polypeptide-L3-CH13 [III], And a second light chain polypeptide comprising a structure represented by the following formula: Fusion peptide-L4-CL3 [IV] wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; CL3 is a third immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH11 is a first immunoglobulin heavy chain constant domain; CH12 is a second immunoglobulin heavy chain constant domain; CH13 is a third immunoglobulin heavy chain constant domain, and L1, L2, L3 and L4 are amino acid linkers; wherein: a) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions, b) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin domain; or c) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin domain; and Wherein VL1 and VH1 form a first antigen binding domain and VL2 and VH2 form a second antigen binding domain.

[0033] In some embodiments according to (or as applied to) any embodiment of this invention, at least one of L1, L2, L3 or L4 is independently 0 amino acids in length. In some embodiments according to (or as applied to) any embodiment of this invention, L1, L2, L3 or L4 is independently at least one amino acid in length. In some embodiments according to (or as applied to) any embodiment of this invention, the binding of CL2 or CL3 comprising one or more amino acid replacements to KappaSelect chromatographic materials is reduced by about 90% compared with CL2 or CL3 without the one or more amino acid replacements. In some embodiments according to (or as applied to) any embodiment of this invention, the binding of VL1 and / or VL2 comprising one or more amino acid replacements to protein L chromatographic materials is reduced by about 90% compared with VL1 and / or VL2 without the one or more amino acid replacements. In some embodiments according to (or as applied to) any embodiment of this invention, one or more amino acid replacements in CL2 or CL3 comprising one or more amino acid replacements are at positions corresponding to 109, 110 or 199, wherein the numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL2 or CL3 comprising one or more amino acid substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL2 or CL3 comprising one or more substitutions are at positions corresponding to 109, 198, 199, or 202, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in CL2 or CL3 comprising one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL1 and / or VL2 comprising one or more amino acid substitutions are substitutions of framework amino acids. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL1 and / or VL2 comprising one or more amino acid substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions in VL1 and / or VL2 are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.In some embodiments according to (or as applied to) any embodiment herein, the first heavy chain polypeptide comprises a first CH2 immunoglobulin heavy chain constant domain and a first CH3 immunoglobulin heavy chain constant domain, and the second heavy chain polypeptide comprises a second CH2 immunoglobulin heavy chain constant domain and a second CH3 immunoglobulin heavy chain constant domain. In some embodiments according to (or as applied to) any embodiment herein, the first CH3 domain and / or CH3 domain is a human IgG1 or IgG4 CH3 domain. In some embodiments according to (or as applicable to) any embodiment herein, the first CH3 domain comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are S354C and T366W; wherein the second CH3 domain comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or as applicable to) any embodiment herein, the CH3 of the second heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A. In some embodiments according to (or as applicable to) any embodiment herein, the CH3 of the first heavy chain polypeptide comprises one or more amino acid replacements that reduce binding to protein A. In some embodiments according to (or as applied to) any embodiment herein, the one or more amino acid substitutions that reduce binding to protein A are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the amino acid substitutions are H435R and Y436F, wherein amino acid numbering is according to the EU index. In some embodiments according to (or as applied to) any embodiment herein, the multivalent binding protein is a multispecific antibody or antigen-binding fragment thereof.

[0034] In some embodiments, one or more polynucleotides encoding multivalent binding proteins as described herein are provided. In some embodiments, a vector comprising one or more polynucleotides as described herein is provided. In some embodiments, a host cell comprising one or more polynucleotides as described herein or one or more vectors is provided. In some embodiments, a method for producing multivalent binding proteins is provided, the method comprising cultivating a host cell as described herein so that binding proteins are produced (e.g., under conditions where the multivalent binding proteins are expressed by the host cell). In some embodiments, the method further comprises recovering the binding proteins from the host cell.

[0035] In some embodiments, provided are pharmaceutical compositions comprising a multivalent binding protein described herein and a pharmaceutically acceptable carrier.

[0036] In some embodiments, a method for purifying a multivalent binding protein as provided herein (e.g., separating a multivalent binding protein as provided herein from one or more impurities) is provided, the method comprising: a) subjecting a composition comprising a multivalent binding protein (e.g., a multivalent binding protein and impurities, such as mismatched or misassembled polypeptides) to protein L chromatography in a bind and elute mode to generate a protein L eluate, and b) subjecting the protein L eluate to KappaSelect chromatography in a bind and elute mode to generate a KappaSelect eluate, wherein the KappaSelect eluate comprises the multivalent binding protein and is substantially free of mismatched (or misassembled) polypeptides. In some embodiments, the multivalent binding protein in the KappaSelect eluate is at least 85% pure, at least 90% pure, or at least 95% pure (e.g., free of mismatched or misassembled polypeptides). In some embodiments, less than 15%, less than 10%, or less than 5% of the polypeptides in the KappaSelect eluate are mismatched (or misassembled) polypeptides.

[0037] In some embodiments, a method of purifying a multivalent binding protein described herein (e.g., separating a multivalent binding protein provided herein from one or more impurities such as mismatched or misassembled polypeptides) is provided, the method comprising a) subjecting a composition comprising a multivalent binding protein and mismatched (or misassembled) polypeptides to KappaSelect chromatography in a bind and elute mode to generate a KappaSelect eluate, and b) subjecting the KappaSelect eluate to a protein L chromatography in a bind and elute mode to generate a protein L eluate, wherein the protein L eluate comprises the multivalent binding protein and is substantially free of mismatched (or misassembled) polypeptides. In some embodiments, the multivalent binding protein in the protein L eluate is at least 85% pure, at least 90% pure, or at least 95% pure (e.g., free of mismatched or misassembled polypeptides). In some embodiments, less than 15%, less than 10%, or less than 5% of the polypeptides in the protein L eluate are mismatched or misassembled polypeptides.

[0038] In some embodiments, a method of purifying a multivalent binding protein described herein (e.g., separating a multivalent binding protein provided herein from one or more impurities) is provided, the method comprising: a) subjecting a composition comprising a multivalent binding protein (e.g., a multivalent antigen binding protein and impurities, such as mispaired or misassembled polypeptides) to protein A chromatography in a bind and elute mode to produce a protein A eluate, b) subjecting the protein A eluate to protein L chromatography in a bind and elute mode to produce a protein L eluate, and c) subjecting the protein L eluate to KappaSelect chromatography in a bind and elute mode to produce a KappaSelect eluate, wherein the KappaSelect eluate comprises the multivalent binding protein and is substantially free of mispaired (or misassembled) polypeptides. In some embodiments, the multivalent binding protein in the KappaSelect eluate is at least 85% pure, at least 90% pure, or at least 95% pure (e.g., free of mispaired or misassembled polypeptides). In some embodiments, less than 15%, less than 10%, or less than 5% of the polypeptides in the KappaSelect eluate are mismatched (or misassembled) polypeptides.

[0039] In some embodiments, a method of purifying a multivalent binding protein described herein (e.g., separating a multivalent binding protein provided herein from one or more impurities) is provided, the method comprising: a) subjecting a composition comprising a multivalent binding protein (e.g., a composition comprising a multivalent binding protein and a mismatched or misassembled polypeptide) to protein A chromatography in a bind and elute mode to produce a protein A eluate, b) subjecting the protein A eluate to KappaSelect chromatography in a bind and elute mode to produce a KappaSelect eluate, and c) subjecting the protein KappaSelect eluate to protein L chromatography in a bind and elute mode to produce a protein L eluate, wherein the L eluate comprises the multivalent binding protein and is substantially free of mismatched (or misassembled) polypeptides. In some embodiments, the multivalent binding protein in the protein L eluate is at least 85% pure, at least 90% pure, or at least 95% pure (e.g., free of mismatched or misassembled polypeptides). In some embodiments, less than 15%, less than 10%, or less than 5% of the polypeptides in the protein L eluate are mismatched (or misassembled) polypeptides.

[0040] In some embodiments according to (or as applicable to) any embodiment herein, the composition comprising the multivalent binding protein is derived from a host cell engineered to express a multispecific binding protein. In some embodiments according to (or as applicable to) any embodiment herein, the composition comprising the multivalent binding protein is a host cell culture supernatant. In some embodiments according to (or as applicable to) any embodiment herein, the composition comprising the multivalent binding protein further comprises a mismatched polypeptide. In some embodiments according to (or as applicable to) any embodiment herein, the composition comprising the multivalent binding protein is filtered prior to chromatography. In some embodiments according to (or as applicable to) any embodiment herein, the method further comprises a refining step after the KappaSelect or protein L chromatography. In some embodiments according to (or as applicable to) any embodiment herein, the refining step is size exclusion chromatography. In some embodiments according to (or as applicable to) any embodiment herein, the protein A chromatography is MabSelect TM 、MabSelect SuRe TM 、MabSelect SuRe TM LX, MabSelectPrismA, Ultra Plus, Protein A Fast Flow, or AF-rProtein A chromatogram. In some embodiments according to (or as applied to) any embodiment herein, the Protein L chromatogram is Pierce TM Protein L columns, Capto TM L Chromatography, Protein L chromatography, AF-rProtein L-650F chromatogram, or KanCap TM L chromatography. In some embodiments according to (or as applied to) any embodiment herein, the KappaSelect chromatography is HiTrap TM KappaSelect or CaptureSelect TM Kappa XL chromatography. In some embodiments according to (or as applied to) any of the embodiments herein, the composition comprising the multivalent binding protein is combined with a pharmaceutically acceptable carrier.

[0041] It should be understood that one, some or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A is a schematic diagram of an exemplary bivalent binding protein.

[0043] Figure 1B is exemplary Schematic representation of a bivalent binding protein.

[0044] Figure 1C is exemplary Schematic representation of a bivalent binding protein.

[0045] Figure 1D is a schematic diagram of an exemplary Ab-Nb trivalent binding protein in which the scFv is linked to the "knob" heavy chain.

[0046] Figure 1E is a schematic diagram of an exemplary Ab-Nb trivalent binding protein in which the VHH is linked to the "knob" heavy chain.

[0047] Figure 1F is a schematic diagram of an exemplary CODV multivalent binding protein comprising a CODV arm on the left and a Fab arm on the right.

[0048] Figure 1G is a schematic diagram of an exemplary CODV multivalent binding protein comprising two CODV arms.

[0049] Figure 1H is a schematic diagram of an exemplary cross-monoclonal antibody (cross-mab) bivalent binding protein in which the CL and CH1 domains of one arm of the binding protein have been exchanged.

[0050] Fig. 1I is a schematic diagram of an exemplary cross-monoclonal antibody (cross-mab) bivalent binding protein in which the VL and VH domains of one arm of the binding protein have been exchanged.

[0051] Figure 1J is a schematic diagram of an exemplary tandem Fab multivalent binding protein, wherein each variable domain comprises a VL, CL, VH, and CH1 domain.

[0052] Figure 1K is a schematic diagram of an exemplary tandem Fab multivalent binding protein, wherein the N-terminal variable domain comprises VL and VH domains and the C-terminal variable domain comprises VL, CL, VH, and CH1 domains.

[0053] Figure 1L is a schematic diagram of an exemplary multivalent binding protein comprising a Fab arm and a fusion protein arm. The Fab arm comprises a single Fab.

[0054] Figure 1Mis a schematic diagram of an exemplary multivalent binding protein comprising a Fab arm and a fusion protein arm. The Fab arm comprises a tandem Fab.

[0055] Figure 2 It is a schematic diagram of a representative three-step process of purifying CODV multivalent binding proteins. The desired product is shown in the dotted frame. The CODV includes a pestle and mortar displacement. The pestle arm of the CODV includes a KappaSelect KO mutation (e.g., a mutation that reduces the binding of the VL domain to the KappaSelect chromatographic medium). The mortar arm of the CODV includes an RF mutation (e.g., H435R and 436F mutations, wherein the amino acid numbering is according to the EU index) of the protein L KO mutation (e.g., a mutation that reduces the binding of the CL domain to the protein L) and the CH3 domain (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States] 63:78-85). Representative mispairing products are shown in the upper left corner, next to the desired product. The three steps in the process include a protein A chromatogram (e.g., (MSS)), followed by Protein L chromatography, and then KappaSelect (KS) chromatography. Removal of mismatched proteins is shown.

[0056] Figure 3 An alignment of the amino acid sequences of the different VLκ and λ subtypes is shown. Adapted from Graille et al., Structure, Vol. 9, pp. 679–687, August 2001.

[0057] Figure 4 The results of screening for potential ProL KO mutations are shown. The top panel shows the mutations evaluated. The bottom panel shows the Sure results for protein yield after chromatography.

[0058] Figure 5 Shown is the biolayer interferometry (BLI) binding assessment of adalimumab ProL KO variants to the ProL ligand.

[0059] Figure 6 Shown is the BLI binding assessment of adalimumab ProL KO variants to TNFα.

[0060] Figure 7 Evaluation of adalimumab ProL KO variant binding to Protein L resin compared to wild type is shown.

[0061] Figure 8 Results of screening for potential KS KO mutations are shown. The top panel shows the mutations evaluated. The bottom panel shows the Results of protein yield after Sure chromatography

[0062] Fig. 9 BLI binding assessment of adalimumab KS KO variants to KS ligands is shown.

[0063] Fig.10 Shown is the BLI binding assessment of adalimumab KS KO variants to TNFα.

[0064] Fig.11 Evaluation of adalimumab KS KO variant binding to KS resin compared to wild type is shown.

[0065] Fig.12 A schematic two-step chromatogram of a trispecific CODV-containing antibody is shown with a Vk2 Fab arm LC with a proL KO mutation and a Vk1 CODV arm LC capable of binding pro-L. The first step is an MSS chromatogram. The MSS eluted material containing the target triAb and 2xFabLC and 2xCODV LC mispairing species is then further purified by a protein L resin in a second step.

[0066] Fig.13 Shown is a Coomassie-stained SDS-PAGE gel of samples from the 2-step purification of the tri-specific CODV.

[0067] Fig.14 Analytical size exclusion chromatography (aSEC) data of wild-type and ProL KO mutant adalimumab after MSS purification are shown. The percentage of the expected main peak is shown.

[0068] Fig.15 Analysis of WT adalimumab and the S12P-R18P mutant samples collected at the end of an accelerated stability test (40°C for 2 weeks) is shown. The top panel shows Coomassie-stained SDS-PAGE gels of non-reducing and reducing samples. The bottom panel shows aSEC data for each sample. The percentage of the expected main peak is shown.

[0069] Fig.16 Differential scanning fluorimetry (nano-DSF) data are shown along with the derived Tm of WT adalimumab and the S12P-R18P mutant.

[0070] Fig.17 Analytical size exclusion chromatography (aSEC) data of wild-type and KS KO mutant adalimumab after MSS purification are shown. The percentage of the expected main peak is shown.

[0071] Fig.18Analysis of WT adalimumab and three CL mutant samples collected at the end of accelerated stability testing (40°C, 2 weeks) is shown. The top panel shows Coomassie-stained SDS-PAGE gels of non-reducing and reducing samples. The bottom panel shows aSEC data for each sample shown. Percentage of expected main peak.

[0072] Fig.19 DSC analysis of F(ab)'2 derived from WT adalimumab and three KS KO mutants is shown.

[0073] Fig. 20 Shown are nano-DSF data and derived Tm for WT adalimumab and the three KS KO mutants.

[0074] Fig.21 Coomassie-stained SDS-PAGE gels of non-reducing and reducing samples after the three-step purification process are shown. The identities of selected high molecular weight bands are shown on the left.

[0075] Fig. 22 Coomassie-stained SDS-PAGE gels of non-reducing and reducing samples after the three-step purification process are shown. The identities of selected high molecular weight bands are shown on the left.

[0076] Fig.23A A chromatogram of MSS purification of a bispecific antibody with KS KO and ProL KO mutations as part of a three-step purification process is shown. The chromatograms of the MSS step, the KS step, and the ProL step are shown in Fig. 23B middle.

[0077] Fig.24 Shown is a Coomassie-stained SDS-PAGE gel of samples from the tri-specific CODV purification.

[0078] Fig.25A A chromatogram of an adalimumab variant of the light chain comprising a His198Arg substitution is provided, wherein the amino acid numbering is according to the EU index.

[0079] Fig.25B A chromatogram of an adalimumab variant of the light chain comprising a Gln199Trp substitution is provided, wherein the amino acid numbering is according to the EU index. DETAILED DESCRIPTION Overview

[0080] The present invention provides a multivalent binding protein comprising four polypeptide chains, wherein the first heavy chain polypeptide and the first light chain polypeptide are associated to form one or more antigen binding domains, and the second heavy chain polypeptide and the second light chain polypeptide are associated to bind to one or more antigen binding domains. In some embodiments, the multivalent binding protein binds more than one antigen. In order to reduce the presence of proteins with mismatched polypeptide chains formed during the production of the multivalent binding protein, amino acid substitutions are introduced into one light chain to substantially prevent it from binding to the Kappa Select chromatographic material, and amino acid substitutions are introduced into another light chain to substantially prevent it from binding to the protein L chromatographic material. Knob-into-hole and RF mutations (e.g., H435R and 436F mutations, wherein the amino acid numbering is according to the EU index) can also be included in the Fc portion of the multivalent binding protein to further reduce the mismatched polypeptides in the multivalent binding protein preparation.

[0081] The present invention also provides two-step and three-step chromatography methods for purifying the multivalent binding proteins of the present invention using Kappa Select and Protein L chromatography with or without Protein A chromatography. Proteins containing mismatched polypeptides are removed from the preparation of the multivalent binding protein based on their lack of Kappa Select binding and / or Protein L binding. General Definition

[0082] As used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise stated. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0083] 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. In certain embodiments, the nucleotides constituting the polynucleotides can be ribonucleotides or deoxyribonucleotides or modified forms of these two nucleotides. Such modifications include base modifications (such as bromouridine), ribose modifications (such as arabinoside and 2', 3'-dideoxyribose), and internucleotide connections modified, such as thiophosphates, dithiophosphates, selenophosphates, diselenphosphates, phenylaminothiophosphates, phenylaminophosphorothioates and phosphoramidates. The term "polynucleotide" particularly includes single-stranded and double-stranded forms of DNA.

[0084] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which: (1) is not associated with all or part of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0085] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it is normally found, (2) is substantially free of other polypeptides from the same source (e.g., from the same species), (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other substances with which it is associated in nature, (5) is not associated (by covalent or non-covalent interactions) with a portion of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or non-covalent interactions) with a polypeptide with which it is not associated in nature, or (7) does not exist in nature. Such an isolated polypeptide may be encoded by genomic DNA, cDNA, mRNA or other synthetically derived RNA, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants present in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0086] Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two pairs of identical polypeptide chains, each pair having a full-length "light" chain (typically having a molecular weight of about 25 kDa) and a 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 sequences to confer specificity to a target antigen. The amino terminal portion of each light chain and heavy chain typically comprises a variable domain of about 100 to 110 or more amino acids, which are typically responsible for antigen recognition. The carboxyl terminal portion of each chain typically defines a constant domain responsible for effector function. Therefore, in naturally occurring antibodies, a full-length heavy chain immunoglobulin polypeptide comprises a variable domain (V H ) and three constant domains (C H1 , C H2 and C H3 ), where V H The domain is at the amino terminus of the polypeptide and the C H3 The variable domain is at the carboxyl terminus, and the full-length light chain immunoglobulin polypeptide includes a variable domain (V L ) and a constant domain (C L ), where V L The domain is at the amino terminus of the polypeptide and the C L The domain is at the carboxyl terminus.

[0087] Human light chains are usually classified as kappa and lambda light chains, and human heavy chains are usually classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has multiple subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into multiple subclasses, including but not limited to IgA1 and IgA2. Within the full-length light chain and heavy chain, the variable domain and the constant domain are usually connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, for example, Fundamental Immunology [Basic Immunology] (Paul, W. Editor, Raven Press [Raven Press], 2nd Edition, 1989), which is incorporated herein by reference in its entirety for all purposes. The variable region of each light / heavy chain pair usually forms an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same overall structure of relatively conservative framework regions (FRs) joined by three hypervariable regions (also referred to as complementary determining regions or CDRs). Each pair of CDRs of the two chains is typically arranged by the framework regions, which can enable binding to a specific epitope. From the amino terminus to the carboxyl terminus, both the light chain variable domain and the heavy chain variable domain typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0088] The term "CDR set" refers to a group of three CDRs that appear in a single variable region that is capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest t [protein sequences with immunological significance] (National Institute of Health [National Institutes of Health], Bethesda, Md. (Bethesda, Maryland)) (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 342:877-83) found that, despite great diversity at the amino acid sequence level, certain sub-portions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These sub-portions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light chain region and the heavy chain region, respectively. These regions may be referred to as Chothia CDRs, whose boundaries overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs have been described in the following literature: Padlan, 1995, FASEB J. [Journal of the Federation of American Societies for Experimental Biology] 9: 133-39; MacCallum, 1996, J. Mol. Biol. [Journal of Molecular Biology] 262 (5): 732-45; and Lefranc, 2003, Dev. Comp. Immunol. [Development and Comparative Immunology] 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the systems described herein, but will still overlap with Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, will not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, but certain embodiments use CDRs defined according to Kabat or Chothia.The use of amino acid sequences to identify predicted CDRs is well known in the art, as described in Martin, AC "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R., Dübel S., eds. Springer-Verlag, Berlin, pp. 33–51 (2010). The amino acid sequences of the heavy and / or light chain variable domains may also be examined to identify the sequences of the CDRs by other conventional methods (e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to identify regions of sequence hypervariability). The numbered sequences may be aligned by eye, or by using an alignment program such as one of the CLUSTAL program suite, as described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular modeling is often used to correctly delineate the framework and CDR regions, thereby correcting sequence-based assignments.

[0089] The term "Fc" as used herein refers to a molecule (whether in monomeric or multimeric form) comprising a sequence of a non-antigen binding fragment produced by antibody digestion or produced by other means, and may contain a hinge region. The original immunoglobulin source of native Fc is preferably human-derived, and may be any immunoglobulin, although IgG1 and IgG2 are preferred. Fc molecules are composed of monomeric polypeptides, which can be connected into dimer or multimer forms by covalent (i.e., disulfide bonds) and non-covalent associations. According to category (e.g., IgG, IgA and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1 and IgGA2), the number of intermolecular disulfide bonds between monomer subunits of native Fc molecules is in the range of 1 to 4. An example of Fc is a disulfide-bonded dimer produced by papain digestion of IgG. The term "native Fc" as used herein is a general term for monomeric, dimer and multimer forms.

[0090] The F(ab) fragment usually consists of a light chain and a heavy chain V H and C H1 domain, in which the V H -C H1 The heavy chain portion cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment may also include a light chain containing two variable domains separated by an amino acid linker, and a light chain containing two variable domains separated by an amino acid linker and a CH1 domain of a heavy chain.

[0091] The F(ab') fragment usually consists of a light chain and a portion of a heavy chain containing the more constant region (in C H1 With C H2 domains), allowing for the formation of an interchain disulfide bond between the two heavy chains to form a F(ab')2 molecule.

[0092] The term "multivalent binding protein" as used herein refers to a non-naturally occurring (or recombinant or engineered) molecule comprising more than one binding domain, wherein the more than one binding domain binds more than one target antigen. In some examples, the binding protein comprises four polypeptide chains, typically a first heavy chain polypeptide and a first light chain polypeptide associated to form at least one antigen binding domain and a second heavy chain polypeptide and a second light chain polypeptide associated to form at least one antigen binding domain.

[0093] A "recombinant" molecule is one that is prepared, expressed, created or isolated by recombinant means.

[0094] Embodiments of the present disclosure provide multivalent binding proteins with biological and immunological specificity for more than one antigen. Another embodiment of the present disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains forming such multivalent binding proteins. Another embodiment of the present disclosure provides expression vectors comprising nucleic acid molecules, which comprise nucleotide sequences encoding polypeptide chains forming such multivalent binding proteins. Another embodiment of the present disclosure provides host cells expressing such multivalent binding proteins (i.e., nucleic acid molecules or vectors encoding polypeptide chains forming such binding proteins).

[0095] As used herein, the term "interchangeability" refers to the interchangeability of variable domains within the binding protein format while retaining folding and ultimate binding affinity. "Fully interchangeable" refers to the ability to exchange V domains in a polypeptide chain of Formula I or a polypeptide chain of Formula II while maintaining full functionality of the binding protein (as evidenced by retention of binding affinity). H1 and V H2 The order of the two domains is thus swapped. L1 and V L2 The ability to change the order of the domains (i.e., to reverse the order). In addition, it should be noted that the name V H and V L Refers only to the location of the domain on a particular protein chain in the final format. For example, V H1 and V H2 Can be derived from V in the parent antibody L1 and V L2 domain, and is placed in the V H1 and VH2 Position. Similarly, V L1 and V L2 Can be derived from V in the parent antibody H1 and V H2 domain, and is placed in the V H1 and V H2 position. Therefore, the V H and V L The names refer to the current position in the parent antibody rather than the original position. H and V L The domains are "exchangeable".

[0096] As used herein, the term "antigen" or "target antigen" or "antigenic target" refers to a molecule or a portion of a molecule that can be bound by a binding protein and that can additionally be used in an animal to produce an antibody that can bind to an epitope of the 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 an intact antibody that recognizes the target antigen.

[0097] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0098] The term "monovalent binding protein" refers to a binding protein with one antigen binding site.

[0099] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigenic targets.

[0100] The term "bivalent binding protein" refers to a binding protein having two binding sites.

[0101] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigenic targets.

[0102] The term "trivalent binding protein" refers to a binding protein with three binding sites. In certain embodiments, the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.

[0103] The term "tetraspecific binding protein" refers to a binding protein that specifically binds to four different antigenic targets.

[0104] The term "tetravalent binding protein" refers to a binding protein with four binding sites. In specific embodiments, a tetravalent binding protein can bind to one antigen target. In other embodiments, a tetravalent binding protein can bind to two antigen targets. In other embodiments, a tetravalent binding protein can bind to three antigen targets. In other embodiments, a tetravalent binding protein can bind to four antigen targets.

[0105] An "isolated" binding protein refers to a binding protein that has been identified, separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the binding protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated binding protein includes a binding protein in situ within a recombinant cell, since at least one component of the binding protein's natural environment will not be present.

[0106] As used herein, the term "substantially pure" or "substantially purified" refers to the major compound or species present (i.e., on a molar basis, it is more than any other single species in the composition). In some embodiments, such terms are relative and do not necessarily mean absolute purity. In some embodiments, a substantially purified fraction is a composition in which these species account for at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will contain more than about 80%, 85%, 90%, 95% or 99% of all macromolecular species present in the composition. In other embodiments, the species is purified to basic homogeneity (contaminant species in the composition cannot be detected by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. In still further embodiments, the purity of the species is improved so that it exists in a purer form than when it was initially synthesized and / or amplified in its natural environment and / or under laboratory conditions.

[0107] As used herein, a "neutralizing" binding protein refers to a molecule that is capable of blocking or significantly reducing the effector function of a target antigen to which it binds. As used herein, "significantly reducing" means that the effector function of the target antigen is reduced by at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, still more preferably at least about 85%, and most preferably at least about 90%.

[0108] The term "epitope" includes any determinant (preferably a polypeptide determinant) that is capable of specific binding to an immunoglobulin or T cell receptor. In certain embodiments, epitope determinants include chemically active surface clusters of molecules (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups), and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind to an antigen when it preferentially recognizes a target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, when the equilibrium dissociation constant is ≤10 -8 M, more preferably when the equilibrium dissociation constant is ≤10 -9 M, and most preferably when the dissociation constant is ≤10 -10 When M, the binding protein is considered to specifically bind the antigen.

[0109] The dissociation constant (K) of the binding protein D ) can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures the real-time binding interaction between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). As used herein, the term " D ” refers to the dissociation constant of the interaction between a specific binding protein and the target antigen.

[0110] As used herein, the term "specific binding" refers to binding of a protein or antigen-binding fragment thereof to an antigen binding site of at least about 1 x 10 - 6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 The ability to bind to an antigen containing the epitope with a Kd of M or higher and / or the ability to bind to the epitope with an affinity at least two times greater than the affinity of the binding protein or antigen-binding fragment thereof for a non-specific antigen.

[0111] As used herein, the term "linker" refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the light chain and heavy chain domains to fold into a crossover on the dual variable region immunoglobulin. At the sequence level, linkers are inserted into the transition region between variable domains or between variable domains and constant domains, respectively. Since the approximate size of immunoglobulin domains is well understood, the transition region between domains can be identified. The exact position of the domain transition can be determined by locating peptide stretches that do not form secondary structural elements (such as beta sheets or alpha helices) (as shown by experimental data or as can be assumed by modeling or secondary structure prediction techniques). For example, with respect to Figure 1F For the exemplary multivalent binding protein shown in FIG. 1 , the linker is referred to as L1 and is located between the V L2 The C-terminus of L1 domain; and L2, which is located between the V L1 The C-terminus and C L The heavy chain linker is called L3 and is located between the N-termini of the V H1 The C-terminus and V H2 domain; and L4, which is located between the N-termini of the V H2 The C-terminus and C H1 between the N-termini of the domain.

[0112] As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid or virus) used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule that can transport another nucleic acid molecule connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule into which another DNA segment can be inserted. Another type of vector is a viral vector, in which another DNA segment can be inserted into the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome. In addition, some vectors can direct the expression of genes that are operably connected to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, useful expression vectors in recombinant DNA technology are usually in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably herein because plasmids are the most commonly used vector forms. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), that serve equivalent functions.

[0113] As used herein, the phrase "recombinant host cell" (or "host cell") refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cells or host cells refer not only to specific subject cells, but also to the offspring of such cells. Since certain modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from parental cells, but such cells are still included in the scope of the term "host cell" used herein. A 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. In order to recombinantly express binding proteins, host cells are transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding binding protein polypeptide chains, so that the polypeptide chains are expressed in host cells and preferably secreted into the culture medium in which the host cells are cultured, from which the binding proteins can be recovered.

[0114] As used herein, the term "transformation" refers to the change of the genetic characteristics of a cell, and when a cell is modified to contain new DNA, the cell has been transformed. For example, a cell is transformed from its native state through genetic modification. After transformation, the transforming DNA can be physically integrated into the cell chromosome and recombined with the DNA of the cell, or can be temporarily maintained as a free element that is not replicated, or can be replicated independently as a plasmid. When the DNA is replicated as the cell divides, the cell is considered to have been stably transformed. As used herein, the term "transfection" refers to the cell taking in foreign or exogenous DNA, and when exogenous DNA has been introduced into the cell membrane, the cell has been "transfected". Many transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.

[0115] The term "naturally occurring" as used herein and applied to an object refers to the fact that the object can exist in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by humans is naturally occurring. Similarly, as used herein, "non-naturally occurring" refers to an object that is not found in nature or an object that has been artificially modified or synthesized.

[0116] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids); unnatural amino acids and analogs (such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids) can also be suitable components of binding protein polypeptide chains. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide symbols used herein, according to standard usage and convention, the left-hand direction is the amino terminal direction, and the right-hand direction is the carboxyl terminal direction.

[0117] Naturally occurring residues can be classified according to common side-chain properties: (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) Polar hydrophilicity: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues that affect chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobicity: Phe, Trp, Tyr.

[0118] Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class. Non-conservative substitutions may involve exchanging a member of one of these classes for a member from another class.

[0119] The skilled person will be able to determine suitable variants of the polypeptide chain of the multivalent binding protein using well-known techniques. For example, one skilled in the art can identify suitable regions of the polypeptide chain that can be changed without destroying the activity by targeting regions that are considered unimportant to the activity. Alternatively, one skilled in the art can identify conserved residues and molecular parts in similar polypeptides. In addition, even regions that may be important for biological activity or structure may be subjected to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0120] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0121] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing the delivery of a binding protein.

[0122] When used in connection with a pharmaceutical composition comprising one or more binding proteins, the terms "effective amount" and "therapeutically effective amount" refer to an amount or dosage sufficient to produce the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated for a period of time. The effective amount may vary depending on the specific binding protein used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo, factors such as the patient's age, weight, and health, as well as dose response curves and toxicity data obtained in preclinical animal work, will be among those considered. The determination of the effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of those skilled in the art.

[0123] Embodiments of the present disclosure provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0124] All references cited herein (including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers) are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Multivalent binding proteins with reduced binding to KappaSelect and / or Protein L chromatography materials Exemplary Bivalent Binding Proteins

[0125] In some embodiments, a multivalent binding protein (e.g., a bispecific antibody) comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: a first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain, wherein VL2 is a κ1, κ3, or κ4 subtype light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, wherein CL1 is a Cκ subtype light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH1 is an immunoglobulin heavy chain constant domain; wherein CL1 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, wherein VL2 comprises one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VH1 associates with VL1 to form a first antigen binding domain, and VH2 associates with VL2 to form a second antigen binding domain.

[0126] In some embodiments, a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains forming two antigen binding domains is provided; wherein the four polypeptide chains comprise: a first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1-CH2-CH3 [III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CH1, CH2 and CH3 are immunoglobulin heavy chain constant domains; wherein CL1 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, wherein VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VH1 associates with VL1 to form a first antigen binding domain, and VH2 associates with VL2 to form a second antigen binding domain. An example of this embodiment is Figure 1A Shown in.

[0127] In some embodiments, the binding of CL1 of the multivalent binding protein (e.g., bispecific antibody) to the KappaSelect chromatographic material is reduced by about 90% compared to the binding of CL1 of the multivalent binding protein (e.g., bispecific antibody) without the one or more amino acid substitutions. In some embodiments, the binding of VL2 of the multivalent binding protein to the Protein L chromatographic material is reduced by about 90% compared to the binding of VL2 of the multivalent binding protein without the one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions in CL1 of the multivalent binding protein are at positions corresponding to 109, 110, or 199, wherein the amino acid numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 63:78-85). In some embodiments, the one or more amino acid substitutions in CL1 are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in the VL2 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, one or more amino acid substitutions in the VL2 of the multivalent binding protein are at positions corresponding to 12 or 18, wherein the amino acid numbering is according to Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). In some embodiments, one or more amino acid substitutions in the VL2 of the multivalent binding protein are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein the amino acid numbering is according to Kabat.

[0128] In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 354 and position 366 of human IgG1, wherein the amino acid numbering is according to the EU index. See, e.g., Spiess et al. (2013) JBC [Journal of Biological Chemistry], 288 (37): P26583-26593. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W of human IgG1, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 349, position 366, position 368, position 407, position 435 and position 436, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 354 and position 366, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W, wherein the amino acid numbering is according to the EU index.

[0129] In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the one or more amino acid substitutions that reduce binding to protein A chromatographic material in the CH3 domain of the first or second heavy chain polypeptide are amino acid substitutions at positions corresponding to (e.g., such as relative to) position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments, these amino acid substitutions are (or correspond to) H435R and Y436F, wherein the amino acid numbering is according to the EU index.

[0130] In some embodiments, the CH1, CH2, and CH3 domains of the first heavy chain polypeptide of the multivalent binding protein are different from the CH1, CH2, and CH3 domains of the second heavy chain polypeptide. In some embodiments, the first heavy chain polypeptide is derived from a species different from the second heavy chain polypeptide. Additionally or alternatively, in some embodiments, the first light chain polypeptide is derived from a species different from the second light chain polypeptide. Examples of this embodiment are Figure 1B In some embodiments, the first heavy chain polypeptide and the first light chain polypeptide are derived from mouse heavy chain immunoglobulin and mouse light chain immunoglobulin, respectively, and the second heavy chain polypeptide and the second light chain polypeptide are derived from rat heavy chain immunoglobulin and rat light chain immunoglobulin, respectively. In some embodiments, the first heavy chain polypeptide and the first light chain polypeptide are derived from rat heavy chain immunoglobulin and rat light chain immunoglobulin, respectively, and the second heavy chain polypeptide and the second light chain polypeptide are derived from mouse heavy chain immunoglobulin and mouse light chain immunoglobulin, respectively.

[0131] In some embodiments, the first heavy chain polypeptide and the second heavy chain polypeptide of the multivalent binding protein each comprise an IgG4 CH3 domain. Figure 1C In some embodiments, the first heavy chain polypeptide comprises a K409R amino acid substitution and the second heavy chain polypeptide comprises a F405L amino acid substitution, wherein numbering is according to the EU index. (See Figure 1C . ) In some embodiments, the first heavy chain polypeptide comprises a F405L amino acid substitution and the second heavy chain polypeptide comprises a K409R amino acid substitution, wherein numbering is according to the EU index. In some embodiments, the multivalent binding protein is a bispecific antigen binding protein. In some embodiments, the first antigen binding domain and the second antigen binding domain bind to different antigens. In some embodiments, the first antigen binding domain and the second antigen binding domain bind to different epitopes on the same antigen. In some embodiments, the multivalent binding protein is a multispecific antibody or antigen binding fragment thereof. Exemplary trivalent binding proteins

[0132] In some embodiments, the first heavy polypeptide chain of the multivalent binding protein comprises a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3-L-VL3 [Ia], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1-CH2-CH3 [IIIa], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CH1, CH2 and CH3 are immunoglobulin heavy chain constant domains; wherein CL1 comprises and does not contain one or more The invention relates to a polypeptide comprising a polypeptide comprising at least one amino acid substitution that reduces binding to a KappaSelect chromatographic material compared to a CL1 without at least one amino acid substitution, wherein VL2 comprises at least one amino acid substitution that reduces binding to a protein L chromatographic material compared to a VL2 without at least one amino acid substitution, and wherein VH1 is associated with VL1 to form a first antigen binding domain, and VH2 is associated with VL2 to form a second antigen binding domain, and VH3 is associated with VL3 to form a third antigen binding domain, and wherein VH3 is connected to VL3 via an amino acid linker L. In some embodiments, VL3 does not bind to a protein L chromatographic material. In some embodiments, VL3 is a λ subtype immunoglobulin light chain variable domain or a κ2 immunoglobulin light chain variable domain. In some embodiments, the length of L is 0 amino acids. In some embodiments, a linker having a length of 0 amino acids indicates that the linker is not present in the binding protein. In some embodiments, the length of L is at least one amino acid. Examples of this embodiment are Figure 1D In some embodiments, the first heavy chain polypeptide chain of the multivalent binding protein comprises a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3 [Ib], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1-CH2-CH3 [IIIb].

[0133] An example of this embodiment is Figure 1EIn some embodiments, the multivalent binding protein is bispecific. In some embodiments, the multivalent binding protein is trispecific. In some embodiments, the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain bind two or three different antigens. In some embodiments, the first antigen binding domain binds to a first antigen, the second antigen binding domain binds to a second antigen, and the third antigen binding domain binds to a third antigen. In some embodiments, the first antigen binding domain and the second antigen binding domain bind to a first antigen and the third antigen binding domain binds to a second antigen. In some embodiments, the multivalent binding protein is a multispecific antibody or antigen binding fragment thereof.

[0134] In some embodiments, the binding of CL1 of a multivalent binding protein (e.g., a trispecific antibody) to a KappaSelect chromatographic material is reduced by about 90% compared to the binding of CL1 of a multivalent binding protein (e.g., a bispecific antibody) without one or more amino acid substitutions. In some embodiments, the binding of VL2 of a multivalent binding protein to a protein L chromatographic material is reduced by about 90% compared to the binding of VL2 of a multivalent binding protein without one or more amino acid substitutions. In some embodiments, the binding of VL3 of a multivalent binding protein to a protein L chromatographic material is reduced by about 90% compared to the binding of VL3 of a multivalent binding protein without one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions in CL1 of a multivalent binding protein are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 63:78-85). In some embodiments, the one or more amino acid substitutions in CL1 are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL1 of the multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, wherein the numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States] 63:78-85). In some embodiments, the one or more amino acid substitutions in CL1 are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in VL2 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, the one or more amino acid substitutions in the VL2 of the multivalent binding protein are at positions corresponding to 12 or 18, where numbering is according to Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991). In some embodiments, the one or more amino acid substitutions in the VL2 of the multivalent binding protein are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, where numbering is according to Kabat.In some embodiments, one or more amino acid substitutions in the VL3 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, one or more amino acid substitutions in the VL3 of the multivalent binding protein are at positions corresponding to 12 or 18, wherein numbering is according to Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). In some embodiments, one or more amino acid substitutions in the VL3 of the multivalent binding protein are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.

[0135] In some embodiments, the binding of CL2 of a multivalent binding protein (e.g., a trispecific antibody) to a KappaSelect chromatographic material is reduced by about 90% compared to the binding of CL2 of a multivalent binding protein (e.g., a bispecific antibody) without one or more amino acid substitutions. In some embodiments, the binding of VL1 of a multivalent binding protein to a protein L chromatographic material is reduced by about 90% compared to the binding of VL1 of a multivalent binding protein without one or more amino acid substitutions. In some embodiments, the binding of VL3 of a multivalent binding protein to a protein L chromatographic material is reduced by about 90% compared to the binding of VL3 of a multivalent binding protein without one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions in CL2 of a multivalent binding protein are at positions corresponding to 109, 110, or 199, where numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 63:78-85). In some embodiments, the one or more amino acid substitutions in CL2 are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL2 of the multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, wherein the numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States] 63:78-85). In some embodiments, the one or more amino acid substitutions in CL2 are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in VL1 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, the one or more amino acid substitutions in the VL1 of the multivalent binding protein are at positions corresponding to 12 or 18, where numbering is according to Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991). In some embodiments, the one or more amino acid substitutions in the VL1 of the multivalent binding protein are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, where numbering is according to Kabat.In some embodiments, one or more amino acid substitutions in the VL3 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, one or more amino acid substitutions in the VL3 of the multivalent binding protein are at positions corresponding to 12 or 18, wherein numbering is according to Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). In some embodiments, one or more amino acid substitutions in the VL3 of the multivalent binding protein are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.

[0136] In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 354 and position 366 of human IgG1, wherein the amino acid numbering is according to the EU index. See, e.g., Spiess et al. (2013) JBC [Journal of Biological Chemistry], 288 (37): P26583-26593. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W of human IgG1, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 349, position 366, position 368, position 407, position 435 and position 436, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 354 and position 366, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W, wherein the amino acid numbering is according to the EU index.

[0137] In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the one or more amino acid substitutions that reduce binding to protein A chromatographic material in the CH3 domain of the first or second heavy chain polypeptide are amino acid substitutions at positions corresponding to (e.g., such as relative to) position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments, these amino acid substitutions are (or correspond to) H435R and Y436F, wherein the amino acid numbering is according to the EU index. Exemplary CODV multivalent binding proteins

[0138] In some embodiments, a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains forming three antigen binding domains is provided; wherein the four polypeptide chains comprise: a first heavy chain polypeptide comprising a structure represented by the formula: VH1-L3-VH2-L4-CH1 [I], A first light polypeptide chain comprising a structure represented by the formula: VL2-L1-VL1-L2-CL1 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL3-CL2 [IV] wherein VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair; wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatography material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatography material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain. In some embodiments, a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains forming three antigen binding domains is provided; wherein the four polypeptide chains comprise a first heavy chain polypeptide, the first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L3-VH2-L4-CH1-CH2-CH3 [Ia], A first light polypeptide chain comprising a structure represented by the formula: VL2-L1-VL1-L2-CL1 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH1-CH2-CH3 [IIIa], and a second light polypeptide chain comprising a structure represented by the following formula: VL3-CL2 [IV] wherein VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL1 is a first immunoglobulin light chain constant domain; CL2 is a second immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair; wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain. Examples of this embodiment are Figure 1F In some embodiments, the binding protein is trispecific and is capable of specifically binding to three different antigen targets. In some embodiments, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, a linker of 0 amino acids in length indicates that the linker is not present in the binding protein. In some embodiments, L1, L2, L3, or L4 is each independently at least one amino acid in length.

[0139] In some embodiments, the second heavy chain polypeptide of the binding protein (eg, a multivalent binding protein) comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH1 [IIIb], And the second light polypeptide chain of the binding protein (eg, a multivalent binding protein) comprises a structure represented by the following formula: VL4-L7-VL3-L8-CL2 [IVa] wherein VL4 is a fourth immunoglobulin light chain variable domain, VH4 is a fourth immunoglobulin heavy chain variable domain, L5, L6, L7 and L8 are amino acid linkers, wherein the polypeptide of Formula IIIa and the polypeptide of Formula IVa form a crossover light chain-heavy chain pair, wherein VH4 associates with VL4 to form a fourth antigen binding domain; and wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and wherein VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype light chain variable immunoglobulin domain; c) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; e) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or f) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatography material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatography material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain. In some embodiments, the second heavy chain polypeptide chain of the binding protein (e.g., a multivalent binding protein) comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH1-CH2-CH3 [IIIc], wherein CH2 is an immunoglobulin CH2 heavy chain constant domain and CH3 is an immunoglobulin CH3 heavy chain constant domain. An example of this embodiment is Figure 1G In some embodiments, the binding protein is tetraspecific and is capable of specifically binding to four different antigen targets. In some embodiments, at least one of L1, L2, L3, L4, L5, L6, L7 or L8 is independently 0 amino acids in length. In some embodiments, a linker of 0 amino acids in length indicates that the linker is not present in the binding protein. In some embodiments, L1, L2, L3, L4, L5, L6, L7 or L8 is each independently at least one amino acid in length.

[0140] In one embodiment, the binding protein of the present disclosure is a trispecific and / or trivalent binding protein comprising four polypeptide chains forming three antigen binding sites that specifically bind to one or more (e.g., three) different antigen targets or target proteins, wherein the first polypeptide chain comprises a structure represented by the following formula: VL2-L1-VL1-L2-CL1 [I] And the second polypeptide chain comprises a structure represented by the following formula: VH1-L3-VH2-L4-CH1-Hinge-CH2-CH3 [II] And the third polypeptide chain comprises a structure represented by the following formula: VH3-CH1-hinge-CH2-CH3 [III] And the fourth polypeptide chain comprises a structure represented by the following formula: VL3-CL2 [IV] in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; CH2 is the immunoglobulin CH2 heavy chain constant domain; CH3 is the immunoglobulin CH3 heavy chain constant domain; The hinge is the immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers; and wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair, wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain.

[0141] In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross orientation that forms two different antigen binding sites. In some embodiments, VH1 and VL1 form a binding pair and form a first antigen binding site. In some embodiments, VH2 and VL2 form a binding pair and form a second antigen binding site. In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen binding site. In some embodiments, VH3 (e.g., VH3 of the third polypeptide) and VL3 (e.g., VL3 of the fourth polypeptide) form a binding pair and form a third antigen binding site.

[0142] In some embodiments, the binding protein of the present disclosure comprises three antigen binding sites that specifically bind to one, two or three antigen targets or target proteins (e.g., one antigen target, two different antigen targets, or three different antigen targets). In some embodiments, the binding protein binds to three antigen targets. In some embodiments, the binding protein binds to three different antigen targets. In some embodiments, two antigen binding sites bind to the same antigen target. In those embodiments, the binding protein includes the same binding domain twice, or includes different binding domains, and / or specifically binds to different antigens or epitopes on the same antigen target. In some embodiments, three antigen binding sites bind to the same antigen target. In those embodiments, the binding protein includes the same binding domain three times, or includes different binding domains, and / or specifically binds to different antigens or epitopes on the same antigen target.

[0143] In some embodiments, the binding of CL1 of the multivalent binding protein to the KappaSelect chromatography material is reduced by about 90% compared to the binding of CL1 of the multivalent binding protein without one or more amino acid substitutions. In some embodiments, the binding of VL4 (if present) and / or VL3 to the protein L chromatography material is reduced by about 90% compared to the binding of VL4 (if present) and / or VL3 of the multivalent binding protein without one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions in CL1 of the multivalent binding protein are at positions corresponding to 109, 110, or 199, wherein the numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL1 are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL1 of the multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, wherein numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 63:78-85). In some embodiments, the one or more amino acid substitutions in CL1 are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in VL4 (if present) and / or VL3 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, the one or more amino acid substitutions in VL4 (if present) and / or VL3 of the multivalent binding protein are at positions corresponding to 12 or 18, wherein numbering is according to Kabat. In some embodiments, one or more amino acid substitutions in VL4 (if present) and / or VL3 are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein the amino acid numbering is according to Kabat.

[0144] In some embodiments, the binding of CL2 of the multivalent binding protein to the KappaSelect chromatography material is reduced by about 90% compared to the binding of CL2 of the multivalent binding protein without one or more amino acid substitutions. In some embodiments, the binding of VL1 and / or VL2 to the protein L chromatography material is reduced by about 90% compared to the binding of VL1 and / or VL2 of the multivalent binding protein without one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions in CL2 of the multivalent binding protein are at positions corresponding to 109, 110, or 199, wherein the numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL2 are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 of the multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, wherein numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 63:78-85). In some embodiments, one or more amino acid substitutions in CL1 are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 are substitutions of framework amino acids. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 of the multivalent binding protein are at positions corresponding to 12 or 18, wherein numbering is according to Kabat. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein amino acid numbering is according to Kabat.

[0145] In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 354 and position 366 of human IgG1, wherein the amino acid numbering is according to the EU index. See, e.g., Spiess et al. (2013) JBC [Journal of Biological Chemistry], 288 (37): P26583-26593. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W of human IgG1, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 349, position 366, position 368, position 407, position 435 and position 436, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 354 and position 366, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W, wherein the amino acid numbering is according to the EU index.

[0146] In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the one or more amino acid substitutions that reduce binding to protein A chromatographic material in the CH3 domain of the first or second heavy chain polypeptide are amino acid substitutions at positions corresponding to (e.g., such as relative to) position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments, these amino acid substitutions are (or correspond to) H435R and Y436F, wherein the amino acid numbering is according to the EU index.

[0147] In some embodiments, the multivalent binding protein is a multispecific antibody or antigen-binding fragment thereof. Exemplary Cross-Monoclonal Antibody Bivalent Binding Proteins

[0148] In some embodiments, the binding protein is a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein the four polypeptide chains comprise a first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CL2[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CH1[IV]; wherein VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, CH1 is an immunoglobulin heavy chain constant domain, and wherein a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

[0149] In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that comprises four polypeptide chains forming two antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CL2-CH2-CH3 [III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CH1[IV]; wherein VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain. CH1 is an immunoglobulin CH1 heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; and CH3 is an immunoglobulin CH3 heavy chain constant domain; wherein a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain. An example of this embodiment is Figure 1H Shown in.

[0150] In some embodiments, the binding protein is a multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VL2-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VH2-CL2[IV]; wherein VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, and CH1 is an immunoglobulin CH1 heavy chain constant domain; wherein a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

[0151] In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that comprises four polypeptide chains forming two antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VL2-CH1-CH2-CH3 [III], and a second light polypeptide chain comprising a structure represented by the following formula: VH2-CL2[IV]; wherein VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain; VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, CH1, CH2 and CH3 are immunoglobulin heavy chain constant domains; wherein a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain. An example of this embodiment is Fig. 1I Shown in.

[0152] In some embodiments, the binding of CL1 or CL2 of the multivalent binding protein to the KappaSelect chromatography material is reduced by about 90% compared to the binding of CL1 or CL2 of the multivalent binding protein without one or more amino acid substitutions. In some embodiments, the binding of VL1 or VL2 of the multivalent binding protein to the protein L chromatography material is reduced by about 90% compared to the binding of VL1 or VL2 of the multivalent binding protein without one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions in CL1 or CL2 of the multivalent binding protein are at positions corresponding to 109, 110 or 199, wherein the numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL1 or CL2 of the multivalent binding protein are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL1 or CL2 of the multivalent binding protein are at positions corresponding to 109, 198, 199 or 202, wherein the numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 or CL2 are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in VL3 or VL4 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, one or more amino acid substitutions in VL3 or VL4 are located at positions corresponding to 12 or 18, wherein the numbering is according to Kabat. In some embodiments, one or more amino acid substitutions in VL3 or VL4 of the multivalent binding protein are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein the numbering is according to Kabat.

[0153] In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 354 and position 366 of human IgG1, wherein the amino acid numbering is according to the EU index. See, e.g., Spiess et al. (2013) JBC [Journal of Biological Chemistry], 288 (37): P26583-26593. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W of human IgG1, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 349, position 366, position 368, position 407, position 435 and position 436, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 354 and position 366, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W, wherein the amino acid numbering is according to the EU index.

[0154] In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the one or more amino acid substitutions that reduce binding to protein A chromatographic material in the CH3 domain of the first or second heavy chain polypeptide are amino acid substitutions at positions corresponding to (e.g., such as relative to) position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments, these amino acid substitutions are (or correspond to) H435R and Y436F, wherein the amino acid numbering is according to the EU index.

[0155] In some embodiments, the multivalent binding protein is a bispecific antigen binding protein. In some embodiments, the first antigen binding domain and the second antigen binding domain of the multivalent binding protein domain bind different antigens. In some embodiments, the multivalent binding protein is a multispecific antibody or antigen binding fragment thereof. Exemplary Tandem Fab Binding Proteins

[0156] In some embodiments, provided are binding proteins (e.g., multivalent binding proteins) comprising four polypeptide chains forming four antigen binding domains, wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH11-L1-VH 2- CH12 [I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-CL1-L 2- V L 2- CL2 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH13-L3-VH 4- CH14 [III], A second light chain polypeptide comprising a structure represented by the following formula: VL3-CL3-L 4- V L 4- CL4 [IV] wherein VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, VL3 is a third immunoglobulin light chain variable domain, VL4 is a fourth immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, CL3 is a third immunoglobulin light chain constant domain, CL4 is a fourth immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, VH3 is a third immunoglobulin heavy chain variable domain, VH4 is a fourth immunoglobulin heavy chain variable domain, CH11 is a first immunoglobulin heavy chain constant domain, CH12 is a second immunoglobulin heavy chain constant domain, CH13 is a third immunoglobulin heavy chain constant domain, CH14 is a fourth immunoglobulin heavy chain constant domain, and L1, L2, L3 and L4 are amino acid linkers; wherein: a) CL1 and CL2 each comprise the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 and CL2 each comprise one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, VL3 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype light chain variable immunoglobulin domain; c) CL1 and CL2 each comprise the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; e) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, VL3 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; f) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, VL4 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; g) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; h) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, VL3 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; i) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, VL4 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; And wherein VL1 and VH1 form a first antigen binding domain, VL2 and VH2 form a second antigen binding domain, VL3 and VH3 form a third antigen binding domain, and VL4 and VH4 form a fourth antigen binding domain. In some embodiments, at least one of L1, L2, L3 or L4 is independently 0 amino acids in length. In some embodiments, a linker with a length of 0 amino acids indicates that the linker is not present in the binding protein. In some embodiments, L2 and / or L4 do not exist. In some embodiments, L1, L2, L3 or L4 are each independently at least one amino acid in length.

[0157] In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that comprises four polypeptide chains forming four antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L1-VH 2- L2-CH11 [I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-L3-VL 2- L4-CL1 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-L5-VH 4- L6-CH12 [III], A second light chain polypeptide comprising a structure represented by the following formula: VL3-L 7- V L 4- L8-CL2 [IV] wherein VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, VL3 is a third immunoglobulin light chain variable domain, VL4 is a fourth immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, VH3 is a third immunoglobulin heavy chain variable domain, VH4 is a fourth immunoglobulin heavy chain variable domain, CH11 is a first immunoglobulin CH1 heavy chain constant domain, CH12 is a second immunoglobulin CH1 heavy chain constant domain, and L1, L2, L 3、 L4, L5, L6, L7 and L8 are amino acid linkers; wherein: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; c) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; e) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; f) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; And wherein VL1 forms a first antigen binding domain with VH1, VL2 forms a second antigen binding domain with VH2, VL3 forms a third antigen binding domain with VH3, and VL4 forms a fourth antigen binding domain with VH4. In some embodiments, at least one of L1, L2, L3, L4, L5, L6, L7 or L8 is independently 0 amino acids in length. In some embodiments, a linker having a length of 0 amino acids indicates that the linker is not present in the binding protein. In some embodiments, wherein at least one of L3, L4, L7 or L8 is not present. In some embodiments, L1, L2, L3, L4, L5, L6, L7 or L8 is each independently at least one amino acid in length. In some embodiments, the binding of CL1 or CL2 of the multivalent binding protein to the KappaSelect chromatographic material is reduced by about 90% compared to the binding of CL1 or CL2 of the multispecific binding protein without one or more amino acid substitutions. In some embodiments, the binding of VL3 and / or VL4 of the multivalent binding protein to the protein L chromatographic material is reduced by about 90% compared to the binding of VL3 and / or VL4 of the multivalent binding protein without one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in CL1 or CL2 of the multivalent binding protein are at positions corresponding to 109, 110, or 199, wherein the numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 or CL2 of the multivalent binding protein are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 of the multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, wherein the numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in VL3 and / or VL4 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, one or more amino acid substitutions in VL3 and / or VL4 of the multivalent binding protein are at positions corresponding to 12 or 18, wherein the numbering is according to Kabat. In some embodiments, one or more amino acid substitutions in VL3 or VL4 are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein the numbering is according to Kabat.

[0158] In some embodiments, the first heavy chain polypeptide of the multivalent binding protein comprises a structure represented by the following formula: VH1-CH11-L1-VH 2-CH12-CH2-CH3 [Ia], And the second heavy chain polypeptide comprises a structure represented by the following formula: VH3-CH13-L3-VH 4- CH14-CH2-CH3 [IIIa]. An example of this embodiment is Figure 1J Shown in.

[0159] In some embodiments, the first heavy chain polypeptide of the multivalent binding peptide comprises a structure represented by the following formula: VH1-L1-VH 2- L2-CH11-CH2-CH3 [Ia], And the second heavy chain polypeptide comprises a structure represented by the following formula: VH3-L5-VH 4- L 6- CH12-CH2-CH3 [IIIa]. An example of this embodiment is Figure 1K Shown in.

[0160] In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 354 and position 366 of human IgG1, wherein the amino acid numbering is according to the EU index. See, e.g., Spiess et al. (2013) JBC [Journal of Biological Chemistry], 288 (37): P26583-26593. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W of human IgG1, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., such as relative to) position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 349, position 366, position 368, position 407, position 435 and position 436, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) Y349C, T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to (e.g., such as relative to) human IgG1, position 354 and position 366, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W, wherein the amino acid numbering is according to the EU index.

[0161] In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the CH3 domain of the first heavy chain polypeptide of the multivalent binding protein comprises (such as further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the one or more amino acid substitutions that reduce binding to protein A chromatographic material in the CH3 domain of the first or second heavy chain polypeptide are amino acid substitutions at positions corresponding to (e.g., such as relative to) position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments, these amino acid substitutions are (or correspond to) H435R and Y436F, wherein the amino acid numbering is according to the EU index.

[0162] In some embodiments, the binding protein is tetraspecific and is capable of specifically binding to four different antigenic targets.

[0163] In some embodiments, the multivalent binding protein is a multispecific antibody or antigen-binding fragment thereof. Exemplary multivalent binding proteins comprising fusion proteins

[0164] In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that comprises four polypeptide chains that form an antigen binding domain and a target binding domain; wherein the four polypeptide chains comprise: A first polypeptide chain (e.g., a first heavy chain polypeptide) comprising a structure represented by the following formula: VH1-CH11[I], The second polypeptide chain (e.g., the first light chain polypeptide) comprises a structure represented by the following formula: VL1-CL1[II], A third polypeptide (eg, a second heavy chain polypeptide) comprising a structure represented by the following formula: Fusion polypeptide-L1-CH12[III] or L1-CH12 [IIIa] And a fourth polypeptide (e.g., a second light chain polypeptide) comprising a structure represented by the following formula: Fusion peptide-L2-CL2 [IV] or L2-CL2 [IVa] Wherein VL1 is a first immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, CH11 is a first CH1 immunoglobulin heavy chain constant domain, and CH12 is a second CH1 immunoglobulin heavy chain constant domain; and L1 and L2 are amino acid linkers; wherein CL2 comprises one or more amino acid replacements that reduce binding to KappaSelect chromatographic material compared to CL2 without one or more amino acid replacements, and VL1 comprises one or more amino acid replacements that reduce binding to protein L chromatographic material compared to VL1 without one or more amino acid replacements; and wherein VL1 and VH1 form an antigen binding domain. In some embodiments, the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide comprising formula [III] and a fourth polypeptide comprising formula [IV]. In some embodiments, the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide comprising formula [IIIa] and a fourth polypeptide comprising formula [IV]. In some embodiments, the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide comprising formula [III] and a fourth polypeptide comprising formula [IVa]. In some embodiments, wherein the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide comprising [III] and a fourth polypeptide comprising formula [IV], each fusion polypeptide binds to a target antigen. In some embodiments, the target antigens are the same. In some embodiments, the target antigens are different. In some embodiments, wherein the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide comprising [III] and a fourth polypeptide comprising formula [IV], the fusion polypeptide dimerizes and binds to a single target. Examples of the above embodiments are described in Figure 1L In some embodiments, L1 and / or L2 are independently 0 amino acids in length. In some embodiments, a linker having a length of 0 amino acids indicates that the linker is not present in the binding protein. In some embodiments, L1 and / or L2 are independently at least one amino acid in length.

[0165] In some embodiments, the binding of CL2 of the multivalent binding protein to the KappaSelect chromatographic material is reduced by about 90% compared to the binding of CL2 of the multivalent binding protein without one or more amino acid substitutions. In some embodiments, the binding of VL1 to the protein L chromatographic material is reduced by about 90% compared to the binding of VL1 without one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions in CL2 of the multivalent binding protein are at positions corresponding to 109, 110, or 199, wherein the numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL2 of the multivalent binding protein are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL2 of the multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, wherein the numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in CL2 are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. In some embodiments, the one or more amino acid substitutions in VL1 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, the one or more amino acid substitutions in VL1 are located at positions corresponding to 12 or 18, wherein the numbering is according to Kabat. In some embodiments, the one or more amino acid substitutions in VL1 are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein the numbering is according to Kabat.

[0166] In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided comprising four polypeptide chains forming two antigen binding domains and a target binding domain; wherein the four polypeptide chains comprise: A first polypeptide chain (e.g., a first heavy chain polypeptide) comprising a structure represented by the following formula: VH1-CH11-L1-VH2-CH12 [I], The second polypeptide chain (e.g., the first light chain polypeptide) comprises a structure represented by the following formula: VL1-CL1-L2-VL2-CL2 [II], A third polypeptide chain (eg, a second heavy chain polypeptide) comprising a structure represented by the following formula: Fusion polypeptide-L3-CH13 [III], And a fourth polypeptide chain (e.g., a second light chain polypeptide) comprising a structure represented by the following formula: CL3 [IVa] wherein: VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, CL3 is a third immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, CH11 is a first immunoglobulin heavy chain constant domain, CH12 is a second immunoglobulin heavy chain constant domain, CH13 is a third immunoglobulin heavy chain constant domain, and L1, L2 and L3 are amino acid linkers; wherein: a) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions, b) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or c) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; and Wherein VL1 and VH1 form a first antigen binding domain and VL2 and VH2 form a second antigen binding domain.

[0167] In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided comprising four polypeptide chains forming two antigen binding domains and a target binding domain; wherein the four polypeptide chains comprise: A first polypeptide chain (e.g., a first heavy chain polypeptide) comprising a structure represented by the following formula: VH1-CH11-L1-VH2-CH12 [I], The second polypeptide chain (eg, the first light chain polypeptide) comprises a structure represented by the following formula: VL1-CL1-L2-VL2-CL2 [II], A third polypeptide chain (eg, a second heavy chain polypeptide) comprising a structure represented by the following formula: CH13 [IIIa], And a fourth polypeptide chain (e.g., a second light chain polypeptide) comprising a structure represented by the following formula: Fusion peptide-L3-CL3 [IVb] wherein: VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, CL3 is a third immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, CH11 is a first immunoglobulin heavy chain constant domain, CH12 is a second immunoglobulin heavy chain constant domain, CH13 is a third immunoglobulin heavy chain constant domain, and L1, L2 and L3 are amino acid linkers; wherein: a) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions, b) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or c) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; and Wherein VL1 and VH1 form a first antigen binding domain and VL2 and VH2 form a second antigen binding domain.

[0168] In some embodiments, at least one of L1, L2 or L3 is independently 0 amino acid in length. In some embodiments, a joint with a length of 0 amino acid indicates that the joint is not present in the binding protein. In some embodiments, L1, L2 or L3 is independently at least one amino acid in length. In some embodiments, the binding of CL3 of a multivalent binding protein to a KappaSelect chromatographic material is reduced by about 90% compared to the binding of CL3 of a multivalent binding protein without one or more amino acid substitutions. In some embodiments, the binding of VL1 and / or VL2 to a protein L chromatographic material is reduced by about 90% compared to the binding of VL1 or VL2 without one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in CL3 of a multivalent binding protein are in positions corresponding to 109, 110 or 199, wherein the numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in CL3 are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in CL3 of the multivalent binding protein are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in CL3 are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 of the multivalent binding protein are substitutions of framework amino acids. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 are at positions corresponding to 12 or 18, wherein numbering is according to Kabat. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.

[0169] In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided comprising four polypeptide chains forming two antigen binding domains and a target binding domain; wherein the four polypeptide chains comprise: A first polypeptide chain (e.g., a first heavy chain polypeptide) comprising a structure represented by the following formula: VH1-CH11-L1-VH2-CH12 [I], The second polypeptide chain (e.g., the first light chain polypeptide) comprises a structure represented by the following formula: VL1-CL1-L2-VL2-CL2 [II], A third polypeptide chain (eg, a second heavy chain polypeptide) comprising a structure represented by the following formula: Fusion polypeptide-L3-CH13 [III], And a fourth polypeptide chain (e.g., a second light chain polypeptide) comprising a structure represented by the following formula: Fusion peptide-L4-CL3 [IV] wherein: VL1 is a first immunoglobulin light chain variable domain, VL2 is a second immunoglobulin light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, CL2 is a second immunoglobulin light chain constant domain, CL3 is a third immunoglobulin light chain constant domain, VH1 is a first immunoglobulin heavy chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, CH11 is a first immunoglobulin heavy chain constant domain, CH12 is a second immunoglobulin heavy chain constant domain, CH13 is a third immunoglobulin heavy chain constant domain, and L1, L2, L3 and L4 are amino acid linkers; wherein: a) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions, b) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or c) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; and Wherein VL1 and VH1 form a first antigen binding domain and VL2 and VH2 form a second antigen binding domain. In some embodiments, each fusion polypeptide binds a target antigen. In some embodiments, the target antigens are the same. In some embodiments, the target antigens are different. In some embodiments, the fusion polypeptide dimerizes and binds a single target. Examples of the above embodiments are Figure 1M Shown in.

[0170] In some embodiments, the first heavy chain polypeptide of the multivalent binding protein comprises a first CH2 immunoglobulin heavy chain constant domain and a first CH3 immunoglobulin heavy chain constant domain, and the second heavy chain polypeptide of the multivalent binding protein comprises a second CH2 immunoglobulin heavy chain constant domain and a second CH3 immunoglobulin heavy chain constant domain. In some embodiments, the first CH3 domain and / or the CH3 domain are human IgG1 or IgG4 CH3 domains. In some embodiments, the first CH3 domain comprises amino acid replacements at positions corresponding to (e.g., relative to) position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) S354C and T366W, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the second CH3 domain comprises amino acid replacements at positions corresponding to (e.g., relative to) position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein these amino acid replacements are (or correspond to) Y349C, T366S, L368A, and Y407V, wherein the amino acid numbering is according to the EU index.

[0171] In some embodiments, the second CH3 domain of the multivalent binding protein comprises (such as further comprising) one or more amino acid replacements that reduce binding to protein A. In some embodiments, the CH3 of the first heavy chain polypeptide of the multivalent binding protein comprises (such as further comprising) one or more amino acid replacements that reduce binding to protein A. In some embodiments, the one or more amino acid replacements that reduce binding to protein A chromatographic material are amino acid replacements at positions corresponding to (e.g., relative to) position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments, these amino acid replacements are (or correspond to) H435R and Y436F, wherein amino acid numbering is according to the EU index. In some embodiments, the multivalent binding protein is a multispecific antibody or antigen-binding fragment thereof. Connectors

[0172] Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-Gly-Gly-Gly (SEQ ID NO: 1)); a peptide with five glycine residues (Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 2)); a peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 3)); a peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 4)); a peptide with eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5)). Other combinations of amino acid residues can be used, such as the peptide Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 6), the peptide Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7), the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 8), and the peptide Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: 6). In some embodiments, the linker is or comprises (GGGGS) n , for example (SEQ ID NO: 6) n(wherein n is an integer between 0 and 5), e.g., GGGGSGGGGS (SEQ ID NO: 9), GGGGSGGGGSGGGGS (SEQ IS NO: 10), etc. In some embodiments, the linker is or comprises S, RT, TKGPS (SEQ ID NO: 11), GQPKAAP (SEQ ID NO: 12), or GGSGSSGSGG (SEQ ID NO: 13). Other suitable linkers include a single Ser residue or a single Val residue; the dipeptides Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 13); Thr-Val-Ala-Ala-Pro (SEQ ID NO: 14); Gln-Pro-Lys-Ala-Ala (SEQ ID NO: 15); Gln-Arg-Ile-Glu-Gly (SEQ ID NO: 16); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 17); Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: 18); Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 19); Asp-Lys-Thr-His-Thr (SEQ ID NO: 20); NO: 20); Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 21); Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: 22); and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: 23). The examples listed above are not intended to limit the scope of the present 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 have been shown to be suitable in binding proteins. For additional description of linker sequences, see, for example, WO2012135345.

[0173] The identity and sequence of the amino acid residues in the joint can vary according to the type of secondary structure element required for realization in the joint. For example, glycine, serine and alanine are best suited for joints with maximum flexibility. If a more rigid and extensible joint is desired, some combinations of glycine, proline, threonine and serine are useful. Any amino acid residue may be considered as a joint in combination with other amino acid residues to build a larger peptide joint according to desired characteristics.

[0174] In some embodiments, the linker of the multivalent binding protein disclosed herein comprises 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 an endogenous V H With C H1 between domains or endogenous V L With C L In some embodiments, the linker comprises a sequence present at a transition between endogenous human V H With C H1 between domains or endogenous human V L With C L Sequences present at transitions between domains (e.g., human kappa or lambda). Fc region and constant domains

[0175] In some embodiments, the multivalent binding protein of the present disclosure comprises one or more Fc variants. In some instances, the term "Fc variant" as used herein refers to a molecule or sequence modified from a native Fc but still comprising a binding site for a salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Therefore, the term "Fc variant" may include molecules or sequences humanized from non-human native Fc. In some instances, the native Fc includes regions that can be removed because they provide structural features or biological activities that are not required by the antibody-like binding protein of the present invention. Therefore, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues or that have been modified, and the one or more Fc sites or residues affect or participate in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0176] To improve the yield of bound protein, the C H3 domain, a technique described in detail with several examples in, for example, International Publication No. WO 96 / 027011, Ridgway et al., 1996, Protein Eng. 9:617-21; and Merchant et al., 1998, Nat. Biotechnol. 16:677-81. Specifically, altering two C H3 domains to increase the interaction surface containing these two C H3Heterodimerization of the two heavy chains of the domain. H3 Each of the domains can be a "knob" and the other a "hole". The introduction of disulfide bonds further stabilizes the heterodimer (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270:26-35) and improves yield. In certain embodiments, the knob is located on the second pair of polypeptides having a single variable domain. In other embodiments, the knob is located on the first pair of polypeptides having a cross-orientation. In other embodiments, C H3 The domain does not include a knob in a hole. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., relative to) position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index. See, e.g., Spiess et al. (2013) JBC [Journal of Biological Chemistry], 288(37): P26583-26593. In some embodiments, the amino acid replacement is (or corresponds to) S354C and T366W (i.e., a knob replacement), wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises an amino acid replacement at a position corresponding to (e.g., relative to) position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index. In some embodiments, the amino acid replacement is (or corresponds to) Y349C, T366S, L368A, and Y407V (i.e., a hole replacement), wherein the amino acid numbering is according to the EU index.

[0177] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve serum half-life (see, e.g., Hinton, PR et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutation comprises a substitution at a position corresponding to (e.g., as relative to) position 428 and position 434 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid substitutions are (or correspond to) M428L and N434S, wherein the amino acid numbering is according to the EU index.

[0178] In certain embodiments, the binding protein of the present disclosure includes one or more mutations to improve purification, for example, by adjusting the affinity to chromatographic materials. For example, it is known that if one of the two Fc regions of heterodimer form contains one or more mutations that reduce or eliminate the combination with protein A, then heterodimer binding protein can be selectively purified from its homodimer form, because compared with any homodimer form, heterodimer form will have medium affinity for purification based on protein A, and can be selectively eluted from protein A, for example, by using different pH (see, for example, Smith, EJ et al. (2015) Sci.Rep. [Science Report] 5: 17943). In certain embodiments, mutations include displacements at positions corresponding to (for example, such as relative to) human IgG1 position 435 and position 436, wherein numbering is according to EU index, wherein these amino acid replacements are (or correspond to) H435R and Y436F, wherein amino acid numbering is according to EU index.

[0179] In some embodiments, the multivalent binding protein of the present disclosure comprises one or more mutations to reduce effector function, e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the mutation comprises an amino acid replacement at a position corresponding to (e.g., relative to) position 234, position 235, and / or position 239 of human IgG1, wherein the numbering is according to the EU index. In some embodiments, the amino acid replacement is (or corresponds to) L234A, L235A, and / or P329A, wherein the amino acid numbering is according to the EU index.

[0180] In some embodiments, the types of mutations described above can be combined in any order or combination. For example, the binding proteins of the present disclosure can comprise two or more "knob" and "hole" mutations, one or more mutations that improve serum half-life, one or more mutations that improve IgG4 stability, one or more mutations that improve purification, and / or one or more mutations that reduce effector function described above. Methods for purifying multivalent binding proteins Exemplary Chromatographic Materials Protein A

[0181] Staphylococcal protein A (SPA or "Protein A") was one of the first immunoglobulin binding molecules discovered and has been extensively studied over the past few decades. Due to its affinity for immunoglobulins, Protein A has been widely used as a tool for detecting and purifying antibodies, antibody constructs, and Fc-containing fusion proteins. It consists of five immunoglobulin binding domains, each of which is able to bind to proteins from many mammalian species, especially immunoglobulin G (IgG), through the heavy chain in the Fc region. Although the native form of Protein A was used as a ligand for the first generation of Protein A resins, the recombinant form (rProtein A) produced in Escherichia coli (E. coli) is the most common today. Commercially available Protein A chromatographic materials include, but are not limited to, those described in detail below. KappaSelect

[0182] KappaSelect is an affinity chromatography material that specifically binds to the constant region of a kappa light chain (LC). The KappaSelect ligand comprises a camelid univariate heavy chain (HC) Ig (VHH) domain that binds with high affinity to all human Cκ-LCs, regardless of the VL sequence. The KappaSelect ligand interacts primarily with residues in Cκ and, to a lesser extent, with residues in the Vκ-Cκ hinge region. Commercially available KappaSelect chromatography materials include, but are not limited to, those described in detail below. Protein L

[0183] Protein L is a cell wall protein of the bacterium Peptostreptococcus magnus ( et al. (1988) "Novel Bacterial-Cell Wall Protein with Affinity for Ig L-Chains." J. Immunol. 140: 1194-1197), which binds to the variable region of a kappa light chain without interfering with the antigen binding site of an antibody or antibody construct (e.g., a multivalent binding protein described herein) (Nilson et al. (1992) "Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain." J Biol Chem. 267: 2234-2239). Protein L interacts with FW1 in the kappa light chain V region, and its binding is limited to VL of the kappa 1, kappa 3, and kappa 4 subtypes. Commercially available protein L chromatography materials include, but are not limited to, those described in detail below. Exemplary Two-Step Purification Process

[0184] Provided herein is a method for purifying a multivalent binding protein described herein, the method comprising (a) subjecting a composition comprising the multivalent binding protein to protein L chromatography in a bind and elute mode to generate a protein L eluate, and (b) subjecting the protein L eluate to KappaSelect chromatography in a bind and elute mode to generate a KappaSelect eluate, wherein the KappaSelect eluate comprises the multivalent binding protein and is substantially free of mispaired polypeptides. For example, in some embodiments, the multivalent binding protein is contacted with protein L under conditions suitable for separating the binding protein from undesirable protein species comprising 0 or 2 VL domains comprising one or more amino acid substitutions that reduce binding of the VL to the protein L chromatography material (e.g., compared to a VL domain without the one or more amino acid substitutions). The Protein L eluate thus generated is then contacted with a KappaSelect chromatography material under conditions suitable for separating the binding protein from undesirable protein species comprising 0 or 2 CL domains comprising one or more amino acid substitutions that reduce binding of CL to the KappaSelect chromatography material (e.g., compared to a CL domain without the one or more amino acid substitutions). Also provided herein is a method for purifying a multivalent binding protein disclosed herein, the method comprising (a) subjecting a composition comprising a multivalent binding protein and a mismatched antibody to KappaSelect chromatography in a bind and elute mode to generate a KappaSelect eluate, and (b) subjecting the KappaSelect eluate to Protein L chromatography in a bind and elute mode to generate a Protein L eluate, wherein the Protein L eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides.

[0185] Conditions suitable for using Protein L and KappaSelect chromatography materials in binding and elution modes known in the art. In some embodiments, the Protein L and / or KappaSelect is attached to a matrix or resin, for example, as part of a chromatography material. In some embodiments, the Protein L chromatography is a Pierce TM Protein L columns, Capto TM L Chromatography, Protein L chromatography, AF-rProtein L-650F chromatogram, or KanCap TM Additionally or alternatively, in some embodiments of any of the purification methods disclosed herein, the KappaSelect chromatogram is a HiTrap TM KappaSelect or CaptureSelect TM Kappa XL chromatography.

[0186] The KappaSelect eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides. In some embodiments, the multivalent binding protein in the KappaSelect eluate is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or any one of 99% purity, including any range between these values. In some embodiments, the purity of the multivalent binding protein in the KappaSelect eluate is higher than about 99%. In some embodiments, less than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the multivalent binding protein in the KappaSelect eluate is a mismatched polypeptide. In some embodiments, less than about 1% of the multivalent binding proteins in the KappaSelect eluate are mismatched polypeptides. Exemplary Three-Step Purification Process

[0187] Provided herein is a method for purifying a multivalent binding protein described herein, the method comprising (a) subjecting a composition comprising the multivalent binding protein to protein A chromatography in a bind and elute mode to generate a protein A eluate, (b) subjecting the protein A eluate to protein L chromatography in a bind and elute mode to generate a protein L eluate, and (c) subjecting the protein L eluate to KappaSelect chromatography in a bind and elute mode to generate a KappaSelect eluate, wherein the KappaSelect eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides. For example, in some embodiments, the multivalent binding protein is contacted with protein A under conditions for separating the binding protein from undesirable protein species comprising 2 CH3 domains, each CH3 domain comprising Y349C, T366S, L368A, and Y407V substitutions (relative to human IgG1), wherein the amino acid numbering is according to the EU index, and wherein. In some embodiments, the multivalent binding protein is contacted with protein A under conditions for separating the binding protein from undesirable protein species comprising 2 CH3 domains, each CH3 domain comprising H435R and Y436F substitutions (relative to human IgG1), wherein the amino acid numbering is according to the EU index. In some embodiments, the multivalent binding protein is contacted with protein A under conditions for separating the binding protein from undesirable protein species comprising 2 CH3 domains, each CH3 domain comprising Y349C, T366S, L368A and Y407V substitutions and H435R and Y436F substitutions (relative to human IgG1), wherein the amino acid numbering is according to the EU index. In some embodiments, the second CH3 domain of the multivalent binding protein comprises (such as further comprising) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the CH3 of the first heavy chain polypeptide of the multivalent binding protein comprises (such as further comprising) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, the one or more amino acid substitutions that reduce binding to the Protein A chromatographic material are (or correspond to) amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. In some embodiments, these amino acid substitutions are (or correspond to) H435R and Y436F, wherein amino acid numbering is according to the EU index. In some embodiments, the multivalent binding protein is a multispecific antibody or antigen-binding fragment thereof.

[0188] The Protein A eluate is then contacted with Protein L under conditions suitable for separating the binding protein from undesirable protein species comprising 0 or 2 VL domains comprising one or more amino acid substitutions that reduce binding of VL to a Protein L chromatography material (e.g., compared to a VL domain without the one or more amino acid substitutions). The Protein L eluate is then contacted with a KappaSelect chromatography material under conditions suitable for separating the binding protein from undesirable protein species comprising 0 or 2 CL domains comprising one or more amino acid substitutions that reduce binding of CL to a KappaSelect chromatography material (e.g., compared to a CL domain without the one or more amino acid substitutions). Also provided is a method for purifying a multivalent binding protein described herein, the method comprising (a) subjecting a composition comprising the multivalent binding protein to protein A chromatography in a bind and elute mode to produce a protein A eluate, (b) subjecting the protein A eluate to KappaSelect chromatography in a bind and elute mode to produce a KappaSelect eluate, and (c) subjecting the protein KappaSelect eluate to protein L chromatography in a bind and elute mode to produce a protein L eluate, wherein the L eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides.

[0189] Conditions suitable for using Protein A, Protein L, and KappaSelect chromatography materials in binding and elution modes known in the art. In some embodiments, Protein A, Protein L, and / or KappaSelect ligands are attached to a matrix or resin, for example as part of the chromatography material. In some embodiments of any of the purification methods disclosed herein, Protein A chromatography is MabSelect TM 、MabSelect SuRe TM 、MabSelect SuRe TM LX, MabSelect PrismA, Ultra Plus, Protein A Fast Flow, or AF-rProtein A chromatogram. In some embodiments, the protein L chromatogram is a Pierce TM Protein L columns, Capto TM L Chromatography, Protein L chromatography, AF-rProtein L-650F chromatogram, or KanCap TM Additionally or alternatively, in some embodiments of any of the purification methods disclosed herein, the KappaSelect chromatogram is a HiTrap TMKappaSelect or CaptureSelect TM Kappa XL chromatography.

[0190] The KappaSelect eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides. In some embodiments, the multivalent binding protein in the KappaSelect eluate is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or any one of 99% purity, including any range between these values. In some embodiments, the purity of the multivalent binding protein in the KappaSelect eluate is higher than about 99%. In some embodiments, less than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the multivalent binding protein in the KappaSelect eluate is a mismatched polypeptide. In some embodiments, less than about 1% of the multivalent binding proteins in the KappaSelect eluate are mismatched polypeptides.

[0191] In some embodiments of any purification method disclosed herein, the composition comprising the multivalent binding protein is derived from a host cell engineered to produce a multispecific binding protein. In some embodiments, the composition comprising the multivalent binding protein is a host cell culture supernatant. In some embodiments, the composition comprising the multivalent binding protein further comprises a mispaired polypeptide. In some embodiments, the composition comprising the multivalent binding protein is filtered before chromatography (e.g., a first chromatography step). In some embodiments, the purification method disclosed herein further includes a refining step after KappaSelect chromatography or protein L chromatography. In some embodiments, the refining step is size exclusion chromatography.

[0192] In some embodiments of any of the purification methods disclosed herein, the composition comprising the multivalent binding protein is combined with a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers and excipients are described in more detail elsewhere herein. Methods for producing multivalent binding proteins with reduced binding to KappaSelect and / or Protein L chromatography materials Nucleic acids and vectors

[0193] Also encompassed are nucleic acid molecules encoding multivalent binding proteins as described herein. In some embodiments, nucleic acids (or a group of nucleic acids) encoding multivalent binding proteins as described herein are provided. Standard recombinant DNA methods are used to construct polynucleotides encoding polypeptides forming binding proteins, these polynucleotides are incorporated into recombinant expression vectors, and such vectors are introduced into host cells. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual] (Cold Spring Harbor Laboratory Press, 3rd Edition). Enzymatic reactions and purification techniques can be performed as generally implemented in the art or as described herein according to the manufacturer's instructions. Unless a specific definition is provided, the nomenclature and laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry as described herein are those well known and commonly used in the art. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and patient treatment.

[0194] Other aspects of the present disclosure relate to isolated nucleic acid molecules, which comprise nucleotide sequences encoding any binding protein described herein (e.g., multivalent binding protein). In certain embodiments, the isolated nucleic acid is operably connected to a heterologous promoter to instruct the transcription of the binding protein encoding nucleic acid sequence. Promoter may refer to a nucleic acid control sequence that instructs nucleic acid transcription. When the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence is operably connected to the second nucleic acid sequence. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably connected to the binding protein coding sequence. Examples of promoters may include, but are not limited to, promoters obtained from the genomes of viruses (e.g., polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), heterologous eukaryotic promoters (e.g., actin promoter, immunoglobulin promoter, heat shock promoter, etc.), CAG promoter (Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK) promoter, tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), lac system, trp system, tac system, trc system, major operator and promoter region of bacteriophage λ, promoter of 3-phosphoglycerate kinase, promoter of yeast acid phosphatase, and promoter of yeast α mating factor. The polynucleotides encoding the binding proteins of the disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or that will be readily identified by one of skill in the art.

[0195] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector may include one or more regulatory sequences operably connected to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (e.g., globulin, elastase, albumin, alpha-fetoprotein, insulin, etc.), and enhancer sequences from eukaryotic cell viruses (e.g., SV40 enhancer (bp 100-270), cytomegalovirus early promoter enhancer, polyoma enhancer on the rear side of the replication origin, adenovirus enhancer, etc.). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons and viral vectors (e.g., adenovirus, vaccinia virus, sindbis virus, measles, herpes virus, lentivirus, retrovirus, adeno-associated virus vector, etc.). Expression vectors may be used to transfect host cells, such as bacterial cells, yeast cells, insect cells and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art and can be used to transfect any cell of interest.

[0196] Other aspects of the present disclosure relate to a vector system comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of any binding protein described herein. In some embodiments, 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 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 polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the binding protein. One or more vectors of the vector system may be any vector described herein. In some embodiments, one or more vectors are expression vectors. Isolated host cells

[0197] Other aspects of the disclosure relate to isolated host cells comprising one or more isolated polynucleotides, vectors, and / or vector systems described herein. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., a Saccharomyces cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five TM 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 can include, for example, human embryonic kidney cells (e.g., 293 cells or 293 cells subcloned for growth in suspension culture), Expi293TM cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), 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 cancer cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0198] Other aspects of the present disclosure relate to methods for producing any binding protein described herein. In some embodiments, the method includes a) culturing a host cell (e.g., any host cell described herein) containing an isolated nucleic acid, a vector, and / or a vector system (e.g., any isolated nucleic acid, a vector, and / or a vector system 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 of expressing proteins are well known to those of ordinary skill in the art. Methods for isolating multivalent binding proteins described herein from cultured host cells are well known to those of ordinary skill in the art. Methods for purifying multivalent binding proteins described herein are described in detail elsewhere herein. Exemplary Uses of Multivalent Binding Proteins

[0199] The multivalent binding proteins can be used in any known assay methods for detecting and quantifying one or more target antigens, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. The binding proteins will bind to one or more target antigens with an affinity suitable for the assay method employed.

[0200] For diagnostic applications, in certain embodiments, the binding protein can be labeled with a detectable moiety. The detectable moiety can be any moiety that is capable of directly or indirectly producing a detectable signal. For example, the detectable moiety can be a radioactive isotope, such as 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.

[0201] The binding protein can also be used for in vivo imaging. The binding protein labeled with a detectable moiety can be administered to an animal, preferably in the bloodstream, and the presence and location of the labeled antibody in the host is determined. The binding protein can be labeled with any moiety detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection methods known in the art.

[0202] Binding proteins can also be used for cell activation, tumor targeting, neutralization of cytokine activity, neutralization of viral infection, combination of multiple signal transduction events to treat cancer, arthritis, and / or inflammatory disorders. For example, in some embodiments, the binding protein specifically binds to one, two or three antigen targets selected from the following: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), C CL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2,Receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin ), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, exonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF 1R, IL2Rβ, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as ligand of IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NK G2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments,One or more of the above antigenic targets are human antigenic targets.

[0203] In some embodiments, the binding protein of the present disclosure is applied to a patient in need thereof to treat or prevent cancer. For example, in some embodiments, the binding protein comprises an antigen binding site that specifically binds to a T cell surface protein and another antigen binding site that specifically binds to a tumor target protein (for example, two antigen binding sites that specifically bind to a T cell surface protein and one antigen binding site that specifically binds to a tumor target protein, or two antigen binding sites that specifically bind to a tumor target protein and one antigen binding site that specifically binds to a T cell surface protein). In certain embodiments, the binding protein comprises an antigen binding site that specifically binds to CD3, an antigen binding site that specifically binds to CD28, and an antigen binding site that specifically binds to a tumor target protein selected from CD19, CD20, CD38, Her2 and LAMP1. In some embodiments, the binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the patient is a person.

[0204] In some embodiments, the binding protein of the present disclosure is administered to a patient in need thereof to treat or prevent an inflammatory disease or disorder. In some embodiments, the binding protein comprises three antigen binding sites, each of which specifically binds to a cytokine target protein selected from IL-4, IL-13, and TNFa. In some embodiments, the binding protein is co-administered with an anti-inflammatory agent. In some embodiments, the patient is a human.

[0205] The present disclosure also relates to a kit comprising binding proteins and other reagents that can be used to detect target antigen levels in biological samples. Such reagents may include detectable labels, closed serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and / or host cell described herein. In some embodiments, the kit comprises 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 may be formed of various materials (such as glass or plastic). The container holds the composition, which can be effectively treated, prevented, and / or diagnosed alone or in combination with another composition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used to prevent, diagnose, and / or treat a selected disease. Alternatively or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes. Therapeutic compositions comprising multivalent binding proteins and their administration

[0206] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the present disclosure. Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of a binding protein or binding protein-drug conjugate admixed with a pharmaceutically or physiologically acceptable formulation selected to be appropriate for the mode of administration.

[0207] Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.

[0208] Pharmaceutical compositions may contain formulation materials used to alter, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, 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, β-cyclodextrin or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose or dextrin), proteins (e.g., serum albumin, gelatin or immunoglobulins), colorants, flavorings and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium ), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronic acid; PEG; sorbitan esters; polysorbates, such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol tyloxapal), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides—preferably sodium chloride or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical Sciences). Sciences [Remington's Pharmaceutical Sciences] (18th edition, AR Gennaro ed., Mack Publishing Company, 1990) and subsequent editions, which are incorporated herein by reference for any purpose).

[0209] The optimal pharmaceutical composition will be determined by a skilled person based on, for example, the intended route of administration, delivery form, and desired dosage. Such composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0210] The main medium or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, the medium or carrier suitable for injection can be water, physiological saline solution or artificial cerebrospinal fluid, which may supplement other materials common in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or suitable substitutes. In one embodiment of the present disclosure, the protein-binding composition can be prepared for storage by mixing the selected composition with the desired purity with the optional preparation in the form of lyophilized cake or aqueous solution. In addition, the protein-binding composition can be formulated as a lyophilized product using a suitable excipient (such as sucrose).

[0211] The pharmaceutical composition of the present disclosure can be selected for parenteral delivery or subcutaneous delivery. Alternatively, the composition can be selected for inhalation or delivery (such as oral) by the digestive tract. The preparation of such pharmaceutically acceptable compositions is within the technical scope of the art.

[0212] The formulation components are present in concentrations acceptable to the site of administration. For example, a buffer is used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

[0213] When considering parenteral administration, the therapeutic composition used can be in the form of a pyrogen-free parenteral acceptable aqueous solution, which is included in the desired protein-binding protein in a pharmaceutically acceptable vehicle. The vehicle specifically suitable for parenteral injection is sterile distilled water, in which the protein-binding protein is formulated into a sterile, isotonic solution, and is properly preserved. Another preparation can involve the preparation of the desired molecule with a controlled or sustained release reagent (such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes) providing a product, which can then be delivered via an injection stored therein. Hyaluronic acid can also be used, which can play a role in promoting the duration of circulation. Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0214] 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 another embodiment, the solution can be atomized.

[0215] It is also expected that some preparations can be administered orally. In one embodiment of the present disclosure, the associated protein used in this way may be formulated with or without those carriers commonly used in the compounding of solid dosage forms (such as tablets and capsules). For example, the capsule can be designed to release the active part of the preparation at the point of the gastrointestinal tract when the bioavailability is maximized and the pre-systemic degradation is minimized. Other medicaments may be included to promote the absorption of the associated protein. Diluents, flavoring agents, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants and adhesives may also be used.

[0216] Another pharmaceutical composition may comprise an effective amount of a binding protein in admixture with a non-toxic excipient suitable for making tablets. A solution in unit dosage form may be prepared by dissolving the tablet in sterile water or other suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or gum arabic; or lubricants such as magnesium stearate, stearic acid or talc.

[0217] The other pharmaceutical compositions disclosed herein are obvious to those skilled in the art, including formulations related to protein-bound formulations in sustained or controlled delivery formulations. The technology for preparing a variety of other sustained or controlled delivery means (such as liposome carriers, bioerodible microparticles or porous beads and deposit injection) is also known to those skilled in the art. Other examples of sustained-release formulations include semipermeable polymer matrices in the form of molded articles, such as films or microcapsules. The sustained-release matrix may include copolymers of polyester, hydrogel, polylactic acid, L-glutamic acid and γ-ethyl-L-glutamic acid, poly-(2-hydroxyethyl-methacrylate), ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also include liposomes, which may be prepared by any of several methods known in the art.

[0218] The pharmaceutical composition for in vivo administration typically must be sterile. This can be achieved by filtering with a sterile filtration membrane. When the composition is lyophilized, sterilization using the method can be performed before or after lyophilization and reconstruction. Compositions for parenteral administration can be stored in lyophilized form or solution form. In addition, parenteral compositions are usually placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be pierced by hypodermic needles.

[0219] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that requires reconstitution (e.g., lyophilized form) prior to administration.

[0220] The present disclosure also encompasses kits for producing single-dose administration units. These kits may each comprise a first container with a dried protein and a second container with an aqueous formulation. Kits comprising single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes) are also included within the scope of the present disclosure.

[0221] The effective amount of the protein-binding pharmaceutical composition used in treatment will depend on, for example, treatment context and target. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will therefore depend in part on the size (body weight, body surface area or organ size) and condition (age and general health) of the molecule being delivered, the indication of the protein-binding protein being used, the route of administration, and the patient. Therefore, the clinician can adjust the dosage and modify the route of administration to obtain the best therapeutic effect.

[0222] The frequency of administration will depend on the pharmacokinetic parameters of the binding protein in the formulation used. Typically, the clinician will administer the composition until the dosage for achieving the desired effect is reached. Therefore, the composition can be given in a single dose, over time with two or more doses (which may or may not contain the same amount of the desired molecule), or via an implant or catheter continuous infusion. Further refinement of the appropriate dosage is routinely performed by those of ordinary skill in the art, and within the scope of their routinely performed tasks. Appropriate dosage can be determined by using appropriate dose response data.

[0223] The administration route of the pharmaceutical composition is according to known methods, such as oral administration; injection by intravenous, intraperitoneal, intracerebral (intra-organ parenchyma), intracerebroventricular, intramuscular, intraocular, intra-arterial, intra-portal vein or intralesional route; by sustained release system; or by implant device. When desired, the composition can be administered by push injection or continuously by infusion or by implant device.

[0224] The composition can also be applied locally by implanting a membrane, sponge or other suitable material that has absorbed or encapsulated the desired molecule. Where an implant device is used, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be carried out via diffusion, timed release bolus, or continuous administration.

[0225] In some embodiments, the 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 binding protein as described herein. In some embodiments, the patient is a human. In some embodiments, the at least one binding protein is administered in combination with one or more anticancer therapies (e.g., any anticancer therapy known in the art). In some embodiments, at least one binding protein is administered before one or more anticancer therapies. In some embodiments, at least one binding protein is administered simultaneously with one or more anticancer therapies. In some embodiments, at least one binding protein is administered after one or more antiretroviral therapies.

[0226] In some embodiments, the disclosure relates to a method for preventing and / or treating an inflammatory disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one binding protein as described herein. In some embodiments, the patient is a human. In some embodiments, at least one binding protein is administered in combination with one or more anti-inflammatory therapies (e.g., any anti-inflammatory therapies known in the art). In some embodiments, at least one binding protein is administered before one or more anti-inflammatory therapies. In some embodiments, at least one binding protein is administered simultaneously with one or more anti-inflammatory therapies. In some embodiments, at least one binding protein is administered after one or more anti-inflammatory therapies.

[0227] Without limiting the present disclosure, various embodiments of the present disclosure are described below for purposes of illustration. Exemplary Embodiments

[0228] The present invention provides exemplary embodiments listed below. 1. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; in: VL1 is the first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain, wherein VL2 is a κ1, κ3 or κ4 subtype light chain variable domain, CL1 is the first immunoglobulin light chain constant domain, wherein CL1 is a Cκ subtype light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH1 is the immunoglobulin heavy chain constant domain; wherein CL1 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material compared to CL1 without the one or more amino acid substitutions, wherein VL2 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatography material compared to VL2 without the one or more amino acid substitutions, and Wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain. 2. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1-CH2-CH3 [III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH1 is the immunoglobulin CH1 heavy chain constant domain, CH2 is an immunoglobulin CH2 heavy chain constant domain and CH3 is the immunoglobulin CH3 heavy chain constant domain; wherein CL1 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material compared to CL1 without the one or more amino acid substitutions, wherein VL2 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatography material compared to VL2 without the one or more amino acid substitutions, and Wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain. 3. The multivalent binding protein of embodiment 1 or embodiment 2, wherein the binding of CL1 comprising the one or more substitutions to KappaSelect is reduced by about 90% compared to CL1 without the one or more amino acid substitutions. 4. The multivalent binding protein of any one of embodiments 1-3, wherein the binding of the VL2 comprising the one or more substitutions to the protein L chromatography material is reduced by about 90% compared to the VL2 without the one or more amino acid substitutions. 5. The multivalent binding protein of any one of embodiments 1-4, wherein the one or more amino acid substitutions in CL1 comprising the one or more substitutions are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index. 6. The multivalent binding protein of embodiment 5, wherein the one or more amino acid substitutions in CL1 comprising the one or more substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. 7. The multivalent binding protein of any one of embodiments 1-4, wherein the one or more amino acid substitutions in CL1 comprising the one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index. 8. The multivalent binding protein of embodiment 7, wherein the one or more amino acid substitutions in CL1 comprising the one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. 9. The multivalent binding protein according to any one of embodiments 1-8, wherein the one or more amino acid substitutions in VL2 comprising the one or more substitutions are substitutions of framework amino acids. 10. The multivalent binding protein of any one of embodiments 1-9, wherein the one or more amino acid substitutions in VL2 comprising the one or more substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat. 11. The multivalent binding protein of embodiment 10, wherein the one or more amino acid substitutions in VL2 comprising the one or more substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein the numbering is according to Kabat. 12. The multivalent binding protein according to any one of embodiments 2-11, wherein the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. 13. The multivalent binding protein according to any one of embodiments 2-12, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435 and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A and Y407V. 14. The multivalent binding protein according to any one of embodiments 2-13, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W. 15. The multivalent binding protein of embodiment 13, wherein the CH3 of the second heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to protein A. 16. The multivalent binding protein of embodiment 14, wherein the CH3 of the first heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to protein A. 17. The multivalent binding protein of claim 15 or 16, wherein the one or more amino acid substitutions that reduce binding to Protein A are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index. 18. The multivalent binding protein of claim 17, wherein the amino acid substitutions are H435R and Y436F, wherein the amino acid numbering is according to the EU index. 19. The multivalent binding protein of any one of claims 2-18, wherein the CH1, CH2, and CH3 domains of the first heavy chain polypeptide are different from the CH1, CH2, and CH3 domains of the second heavy chain polypeptide. 20. The multivalent binding protein of any one of claims 2-19, wherein the first heavy chain polypeptide is derived from a different species than the second heavy chain polypeptide. 21. The multivalent binding protein of any one of claims 2-11, 19 or 20, wherein the first heavy chain polypeptide and the first light chain polypeptide are derived from a mouse heavy chain immunoglobulin and a mouse light chain immunoglobulin, and the second heavy chain polypeptide and the second light chain polypeptide are derived from a rat heavy chain immunoglobulin and a rat light chain immunoglobulin. 22. The multivalent binding protein of any one of claims 2-11, wherein the first heavy chain polypeptide and the second heavy chain polypeptide each comprise an IgG4 CH3 domain. 23. The multivalent binding protein of claim 22, wherein the first heavy chain polypeptide comprises a K409R amino acid substitution and the second heavy chain polypeptide comprises a F405L amino acid substitution, wherein numbering is according to the EU index. 24. The multivalent binding protein of any one of claims 1-23, wherein the multivalent binding protein is a bispecific antigen binding protein. 25. The multivalent binding protein of any one of claims 1-24, wherein the first antigen binding domain and the second antigen binding domain bind different antigens. 26. The multivalent binding protein according to any one of claims 2 to 18, Wherein the first heavy chain polypeptide chain comprises a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3-L-VL3 [Ia], Wherein the second heavy chain polypeptide comprises a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIa], in: VL3 is the third immunoglobulin light chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; L is an amino acid linker; VH3 and VL3 associate to form the third antigen binding domain. 27. The multivalent binding protein according to any one of claims 2 to 18, Wherein the first heavy chain polypeptide chain comprises a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3 [Ib], Wherein the second heavy chain polypeptide comprises a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIb], in: VH3 is the third immunoglobulin heavy chain variable domain. 28. The multivalent binding protein of claim 26, wherein VL3 comprises the one or more amino acid substitutions that reduce binding to the Protein L chromatography material compared to VL3 without the one or more amino acid substitutions. 29. The multivalent binding protein of claim 26, wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 immunoglobulin light chain variable domain. 30. The multivalent binding protein of any one of claims 26-29, wherein the multivalent binding protein is bispecific or trispecific. 31. The multivalent binding protein of any one of claims 26-30, wherein the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain bind two or three different antigens. 32. The multivalent binding protein of any one of claims 26-30, wherein the first antigen binding domain binds a first antigen, the second antigen binding domain binds a second antigen, and the third antigen binding domain binds a third antigen. 33. The multivalent binding protein of any one of claims 26-30, wherein the first antigen binding domain and the second antigen binding domain bind a first antigen and the third antigen binding domain binds a second antigen. 34. A multivalent binding protein comprising four polypeptide chains forming three antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L3-VH2-L4-CH1 [I], A first light polypeptide chain comprising a structure represented by the formula: VL2-L1-VL1-L2-CL1 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL3-CL2 [IV] in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair; wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and in: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain. 35. A multivalent binding protein comprising four polypeptide chains forming three antigen binding domains; wherein the four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L3-VH2-L4-CH1-CH2-CH3 [Ia], A first light polypeptide chain comprising a structure represented by the formula: VL2-L1-VL1-L2-CL1 [II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH1-CH2-CH3 [IIIa], and a second light polypeptide chain comprising a structure represented by the following formula: VL3-CL2 [IV] in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; CH2 is the immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair; wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and in: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype light chain variable immunoglobulin domain; or d) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype light chain variable immunoglobulin domain. 36. The multivalent binding protein of embodiment 34 or 35, wherein the binding protein is trispecific and is capable of specifically binding to three different antigenic targets. 37. The multivalent binding protein of any one of embodiments 34-36, wherein at least one of L1, L2, L3 and / or L4 is each independently 0 amino acids in length. 38. The multivalent binding protein of any one of embodiments 34-36, wherein L1, L2, L3 and / or L4 are each independently at least one amino acid in length. 39. The multivalent binding protein of embodiment 34 or 35, wherein: The second heavy chain polypeptide comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH1 [IIIb], and a second light polypeptide chain comprising a structure represented by the following formula: VL4-L7-VL3-L8-CL2 [IVa] in: VL4 is the fourth immunoglobulin light chain variable domain; VH4 is the fourth immunoglobulin heavy chain variable domain; L5, L6, L7, and L8 are amino acid linkers; wherein the polypeptide of formula IIIa and the polypeptide of formula IVa form a crossed light chain-heavy chain pair; wherein VH4 and VL4 associate to form a fourth antigen binding domain; and in: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and wherein VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; c) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; e) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or f) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain. 40. The multivalent binding protein of embodiment 39, wherein: The second heavy chain polypeptide comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH1-CH2-CH3 [IIIc], in: CH2 is an immunoglobulin CH2 heavy chain constant domain and CH3 is the immunoglobulin CH3 heavy chain constant domain. 41. The multivalent binding protein of embodiment 39 or 40, wherein the binding protein is tetraspecific and is capable of specifically binding to four different antigenic targets. 42. The multivalent binding protein of any one of embodiments 39-41, wherein at least one of L1, L2, L3, L4, L5, L6, L7 and / or L8 is each independently 0 amino acids in length. 43. The multivalent binding protein of any one of embodiments 39-41, wherein L1, L2, L3, L4, L5, L6, L7 and / or L8 are each independently at least one amino acid in length. 44. The multivalent binding protein of any one of embodiments 34-43, wherein binding of CL1 or CL2 comprising the one or more amino acid substitutions to the KappaSelect chromatography material is reduced by about 90% compared to CL1 or CL2 without the one or more amino acid substitutions. 45. The multivalent binding protein of any one of embodiments 34-44, wherein the binding of VL1, VL2, VL3 and / or VL4 comprising the one or more substitutions to the protein L chromatography material is reduced by about 90% compared to VL1, VL2, VL3 and / or VL4 without the one or more amino acid substitutions. 46. ​​The multivalent binding protein of any one of embodiments 34-45, wherein the one or more amino acid substitutions in the CL1 or the CL2 are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index. 47. The multivalent binding protein of embodiment 46, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more amino acid substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index. 48. The multivalent binding protein of any one of embodiments 34-45, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index. 49. The multivalent binding protein of embodiment 48, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index. 50. The multivalent binding protein of any one of embodiments 34-49, wherein the one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4 comprising the one or more substitutions are substitutions of framework amino acids. 51. The multivalent binding protein of any one of embodiments 34-50, wherein the one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4 comprising the one or more substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat. 52. The multivalent binding protein of embodiment 51, wherein the one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4 comprising the one or more substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein the numbering is according to Kabat. 53. The multivalent binding protein according to any one of embodiments 35-38 and 40-52, wherein the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain. 54. The multivalent binding protein of any one of embodiments 35-38 and 40-53, wherein the CH3 domain of the first heavy chain ...

Claims

1. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein: The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; in: VL1 is the first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain, wherein VL2 is a κ1, κ3 or κ4 subtype light chain variable domain, CL1 is the first immunoglobulin light chain constant domain, wherein CL1 is a Cκ subtype light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH1 is the immunoglobulin heavy chain constant domain; wherein CL1 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material compared to CL1 without the one or more amino acid substitutions, wherein VL2 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatography material compared to VL2 without the one or more amino acid substitutions, and Wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

2. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein: The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1-CH2-CH3[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH1 is the immunoglobulin CH1 heavy chain constant domain, CH2 is an immunoglobulin CH2 heavy chain constant domain and CH3 is the immunoglobulin CH3 heavy chain constant domain; wherein CL1 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material compared to CL1 without the one or more amino acid substitutions, wherein VL2 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatography material compared to VL2 without the one or more amino acid substitutions, and Wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

3. The multivalent binding protein of claim 1 or claim 2, wherein the binding of CL1 comprising the one or more substitutions to KappaSelect is reduced by about 90% compared to CL1 without the one or more amino acid substitutions.

4. The multivalent binding protein of any one of claims 1-3, wherein binding of the VL2 comprising the one or more substitutions to the Protein L chromatography material is reduced by about 90% compared to the VL2 without the one or more amino acid substitutions.

5. The multivalent binding protein of any one of claims 1-4, wherein the one or more amino acid substitutions in CL1 comprising the one or more substitutions are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index.

6. The multivalent binding protein of claim 5, wherein the one or more amino acid substitutions in CL1 comprising the one or more substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index.

7. The multivalent binding protein of any one of claims 1-4, wherein the one or more amino acid substitutions in CL1 comprising the one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index.

8. The multivalent binding protein of claim 7, wherein the one or more amino acid substitutions in CL1 comprising the one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index.

9. The multivalent binding protein of any one of claims 1-8, wherein the one or more amino acid substitutions in VL2 comprising the one or more substitutions are substitutions of framework amino acids.

10. The multivalent binding protein of any one of claims 1-9, wherein the one or more amino acid substitutions in VL2 comprising the one or more substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat.

11. The multivalent binding protein of claim 10, wherein the one or more amino acid substitutions in VL2 comprising the one or more substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.

12. The multivalent binding protein of any one of claims 2-11, wherein the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain.

13. The multivalent binding protein of any one of claims 2-12, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435 and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A and Y407V.

14. The multivalent binding protein of any one of claims 2-13, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W.

15. The multivalent binding protein of claim 13, wherein CH3 of the second heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A.

16. The multivalent binding protein of claim 14, wherein CH3 of the first heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A.

17. The multivalent binding protein of claim 15 or 16, wherein the one or more amino acid substitutions that reduce binding to Protein A are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index.

18. The multivalent binding protein of claim 17, wherein the amino acid substitutions are H435R and Y436F, wherein the amino acid numbering is according to the EU index.

19. The multivalent binding protein of any one of claims 2-18, wherein the CH1, CH2, and CH3 domains of the first heavy chain polypeptide are different from the CH1, CH2, and CH3 domains of the second heavy chain polypeptide.

20. The multivalent binding protein of any one of claims 2-19, wherein the first heavy chain polypeptide is derived from a different species than the second heavy chain polypeptide.

21. The multivalent binding protein of any one of claims 2-11, 19 or 20, wherein the first heavy chain polypeptide and the first light chain polypeptide are derived from a mouse heavy chain immunoglobulin and a mouse light chain immunoglobulin, and the second heavy chain polypeptide and the second light chain polypeptide are derived from a rat heavy chain immunoglobulin and a rat light chain immunoglobulin.

22. The multivalent binding protein of any one of claims 2-11, wherein the first heavy chain polypeptide and the second heavy chain polypeptide each comprise an IgG4 CH3 domain.

23. The multivalent binding protein of claim 22, wherein the first heavy chain polypeptide comprises a K409R amino acid substitution and the second heavy chain polypeptide comprises a F405L amino acid substitution, wherein numbering is according to the EU index.

24. The multivalent binding protein of any one of claims 1-23, wherein the multivalent binding protein is a bispecific antigen binding protein.

25. The multivalent binding protein of any one of claims 1-24, wherein the first antigen binding domain and the second antigen binding domain bind different antigens.

26. The multivalent binding protein according to any one of claims 2 to 18, Wherein the first heavy chain polypeptide chain comprises a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3-L-VL3[Ia], Wherein the second heavy chain polypeptide comprises a structure represented by the following formula: VH2-CH1-CH2-CH3[IIIa], in: VL3 is the third immunoglobulin light chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; L is an amino acid linker; VH3 and VL3 associate to form the third antigen binding domain.

27. The multivalent binding protein according to any one of claims 2 to 18, Wherein the first heavy chain polypeptide chain comprises a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3[Ib], Wherein the second heavy chain polypeptide comprises a structure represented by the following formula: VH2-CH1-CH2-CH3[IIIb], in: VH3 is the third immunoglobulin heavy chain variable domain.

28. The multivalent binding protein of claim 26, wherein VL3 comprises the one or more amino acid substitutions that reduce binding to the Protein L chromatography material compared to VL3 without the one or more amino acid substitutions.

29. The multivalent binding protein of claim 26, wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 immunoglobulin light chain variable domain.

30. The multivalent binding protein of any one of claims 26-29, wherein the multivalent binding protein is bispecific or trispecific.

31. The multivalent binding protein of any one of claims 26-30, wherein the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain bind two or three different antigens.

32. The multivalent binding protein of any one of claims 26-30, wherein the first antigen binding domain binds a first antigen, the second antigen binding domain binds a second antigen, and the third antigen binding domain binds a third antigen.

33. The multivalent binding protein of any one of claims 26-30, wherein the first antigen binding domain and the second antigen binding domain bind a first antigen and the third antigen binding domain binds a second antigen.

34. A multivalent binding protein comprising four polypeptide chains forming three antigen binding domains; wherein: The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L3-VH2-L4-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL2-L1-VL1-L2-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL3-CL2[IV] in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair; wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and in: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain.

35. A multivalent binding protein comprising four polypeptide chains forming three antigen binding domains; wherein: The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L3-VH2-L4-CH1-CH2-CH3[Ia], A first light polypeptide chain comprising a structure represented by the formula: VL2-L1-VL1-L2-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH3-CH1-CH2-CH3[IIIa], and a second light polypeptide chain comprising a structure represented by the following formula: VL3-CL2[IV] in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; CH2 is the immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light chain-heavy chain pair; wherein VH1 associates with VL1 to form a first antigen binding domain, VH2 associates with VL2 to form a second antigen binding domain, and VH3 associates with VL3 to form a third antigen binding domain; and in: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions; b) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; c) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype light chain variable immunoglobulin domain; or d) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype light chain variable immunoglobulin domain.

36. The multivalent binding protein of claim 34 or 35, wherein the binding protein is trispecific and is capable of specifically binding to three different antigenic targets.

37. The multivalent binding protein of any one of claims 34-36, wherein at least one of L1, L2, L3 and / or L4 is each independently 0 amino acids in length.

38. The multivalent binding protein of any one of claims 34-36, wherein L1, L2, L3 and / or L4 are each independently at least one amino acid in length.

39. The multivalent binding protein of claim 34 or 35, wherein: The second heavy chain polypeptide comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH1[IIIb], and a second light polypeptide chain comprising a structure represented by the following formula: VL4-L7-VL3-L8-CL2[IVa] in: VL4 is the fourth immunoglobulin light chain variable domain; VH4 is the fourth immunoglobulin heavy chain variable domain; L5, L6, L7, and L8 are amino acid linkers; wherein the polypeptide of formula IIIa and the polypeptide of formula IVa form a crossed light chain-heavy chain pair; wherein VH4 and VL4 associate to form a fourth antigen binding domain; and in: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and wherein VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; c) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; e) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and wherein VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or f) CL2 comprises one or more amino acid substitutions that reduce binding to KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and wherein VL1 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain.

40. The multivalent binding protein of claim 39, wherein: The second heavy chain polypeptide comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH1-CH2-CH3[IIIc], in: CH2 is an immunoglobulin CH2 heavy chain constant domain and CH3 is the immunoglobulin CH3 heavy chain constant domain.

41. The multivalent binding protein of claim 39 or 40, wherein the binding protein is tetraspecific and is capable of specifically binding to four different antigenic targets.

42. The multivalent binding protein of any one of claims 39-41, wherein at least one of L1, L2, L3, L4, L5, L6, L7 and / or L8 is each independently 0 amino acids in length.

43. The multivalent binding protein of any one of claims 39-41, wherein L1, L2, L3, L4, L5, L6, L7 and / or L8 are each independently at least one amino acid in length.

44. The multivalent binding protein of any one of claims 34-43, wherein binding of CL1 or CL2 comprising the one or more amino acid substitutions to the KappaSelect chromatography material is reduced by about 90% compared to CL1 or CL2 without the one or more amino acid substitutions.

45. The multivalent binding protein of any one of claims 34-44, wherein binding of VL1, VL2, VL3 and / or VL4 comprising the one or more substitutions to the Protein L chromatography material is reduced by about 90% compared to VL1, VL2, VL3 and / or VL4 without the one or more amino acid substitutions.

46. ​​The multivalent binding protein of any one of claims 34-45, wherein one or more amino acid substitutions in the CL1 or CL2 are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index.

47. The multivalent binding protein of claim 46, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more amino acid substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index.

48. The multivalent binding protein of any one of claims 34-45, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index.

49. The multivalent binding protein of claim 48, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index.

50. The multivalent binding protein of any one of claims 34-49, wherein one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4 comprising the one or more substitutions are substitutions of framework amino acids.

51. The multivalent binding protein of any one of claims 34-50, wherein the one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4 comprising the one or more substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat.

52. The multivalent binding protein of claim 51 , wherein the one or more amino acid substitutions in VL1 , VL2, VL3 and / or VL4 comprising the one or more substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.

53. The multivalent binding protein of any one of claims 35-38 and 40-52, wherein the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain.

54. The multivalent binding protein of any one of claims 35-38 and 40-53, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V.

55. The multivalent binding protein of any one of claims 35-38 and 40-53, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W.

56. The multivalent binding protein of claim 54, wherein the CH3 of the second heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A.

57. The multivalent binding protein of claim 55, wherein the CH3 of the first heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A.

58. The multivalent binding protein of claim 56 or 57, wherein the one or more amino acid substitutions that reduce binding to Protein A are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index.

59. The multivalent binding protein of claim 58, wherein the amino acid substitutions are H435R and Y436F, wherein the amino acid numbering is according to the EU index.

60. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein: The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CL2[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CH1[IV]; in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH1 is the immunoglobulin heavy chain constant domain in a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

61. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein: The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CL2-CH2-CH3[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CH1[IV]; in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH1 is the immunoglobulin heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; and CH3 is the immunoglobulin CH3 heavy chain constant domain in a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

62. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein: The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VL2-CH1[III], and a second light polypeptide chain comprising a structure represented by the following formula: VH2-CL2[IV]; in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH1 is the immunoglobulin heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; and CH3 is the immunoglobulin CH3 heavy chain constant domain; in a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

63. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein: The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3[I], A first light polypeptide chain comprising a structure represented by the formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: VH2-CH1-CH2-CH3[III], and a second light polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV]; in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH1 is the immunoglobulin heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; and CH3 is the immunoglobulin CH3 heavy chain constant domain; in a) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, or b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions; And wherein VH1 and VL1 associate to form a first antigen binding domain and VH2 and VL2 associate to form a second antigen binding domain.

64. The multivalent binding protein of any one of claims 60-63, wherein binding of CL1 or CL2 comprising the one or more amino acid substitutions to the KappaSelect chromatography material is reduced by about 90% compared to CL1 or CL2 without the one or more amino acid substitutions.

65. The multivalent binding protein of any one of claims 60-64, wherein binding of VL1 or VL2 comprising the one or more amino acid substitutions to the Protein L chromatography material is reduced by about 90% compared to VL1 or VL2 without the one or more amino acid substitutions.

66. The multivalent binding protein of any one of claims 60-65, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more amino acid substitutions are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index.

67. The multivalent binding protein of claim 66, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more amino acid substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions.

68. The multivalent binding protein of any one of claims 60-65, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index.

69. The multivalent binding protein of claim 68, wherein the one or more amino acid substitutions in CL1 or CL2 comprising the one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index.

70. The multivalent binding protein of any one of claims 60-69, wherein the one or more amino acid substitutions in VL1 or VL2 comprising the one or more amino acid substitutions are substitutions of framework amino acids.

71. The multivalent binding protein of any one of claims 60-70, wherein the one or more amino acid substitutions in VL1 or VL2 comprising the one or more amino acid substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat.

72. The multivalent binding protein of claim 71, wherein the one or more amino acid substitutions in VL1 or VL2 comprising the one or more amino acid substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.

73. The multivalent binding protein of any one of claims 57-66, wherein the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain.

74. The multivalent binding protein of any one of claims 61-73, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V.

75. The multivalent binding protein of any one of claims 61-74, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W.

76. The multivalent binding protein of claim 74, wherein the CH3 of the second heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A chromatography material.

77. The multivalent binding protein of claim 75, wherein the CH3 of the first heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A chromatography material.

78. The multivalent binding protein of claim 76 or 77, wherein the one or more amino acid substitutions that reduce binding to the Protein A chromatographic material are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index.

79. The multivalent binding protein of claim 78, wherein the amino acid substitutions are H435R and Y436F, wherein the amino acid numbering is according to the EU index.

80. The multivalent binding protein of any one of claims 56-73, wherein the multivalent binding protein is a bispecific antigen binding protein.

81. The multivalent binding protein of any one of claims 60-80, wherein the first antigen binding domain and the second antigen binding domain bind different antigens.

82. A multivalent binding protein comprising four polypeptide chains forming four antigen binding domains; wherein, The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH11-L1-VH 2- CH12[I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-CL1-L 2- VL 2- CL2[II], A second heavy chain polypeptide comprising a structure represented by the formula: <h2 style=";text-align:left;direction:ltr">VH3-CH13-L3-VH<h2 style=";text-align:left;direction:ltr"> 4- <h2 style=";text-align:left;direction:ltr"> CH14[III], A second light chain polypeptide comprising a structure represented by the following formula: VL3-CL3-L 4- VL 4- CL4[IV] in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VL4 is the fourth immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; CL3 is the third immunoglobulin light chain constant domain; CL4 is the fourth immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; VH4 is the fourth immunoglobulin heavy chain variable domain; CH11 is the first immunoglobulin heavy chain constant domain; CH12 is the second immunoglobulin heavy chain constant domain; CH13 is the third immunoglobulin heavy chain constant domain; CH14 is the fourth immunoglobulin heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; in: a) CL1 and CL2 each comprise the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 and CL2 each comprise one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, VL3 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; c) CL1 and CL2 each comprise the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 and CL2 without the one or more amino acid substitutions, VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions; e) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, VL3 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; f) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, CL2 is a lambda subtype immunoglobulin light chain constant domain, VL4 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; g) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; h) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, VL3 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; i) CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, CL1 is a lambda subtype immunoglobulin light chain constant domain, VL4 comprises the one or more amino acid substitutions that reduce binding to a protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; And wherein VL1 and VH1 form a first antigen binding domain, VL2 and VH2 form a second antigen binding domain, VL3 and VH3 form a third antigen binding domain, and VL4 and VH4 form a fourth antigen binding domain.

83. The multivalent binding protein of claim 82, wherein at least one of L1, L2, L3, or L4 is each independently 0 amino acids in length.

84. The binding protein of claim 82 or 83, wherein at least one of L1, L2, L3 or L4 is each independently at least one amino acid in length.

85. A multivalent binding protein comprising four polypeptide chains forming four antigen binding domains; wherein, The four polypeptide chains comprise: A first heavy chain polypeptide comprising a structure represented by the following formula: <h2 style=";text-align:left;direction:ltr">VH1-L1-VH<h2 style=";text-align:left;direction:ltr"> 2- <h2 style=";text-align:left;direction:ltr"> L2-CH11[I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-L3-VL 2- L4-CL1[II], A second heavy chain polypeptide comprising a structure represented by the formula: <h2 style=";text-align:left;direction:ltr">VH3-L5-VH<h2 style=";text-align:left;direction:ltr"> 4- <h2 style=";text-align:left;direction:ltr"> L6-CH12[III], A second light chain polypeptide comprising a structure represented by the following formula: VL3-L 7- VL 4- L8-CL2[IV] in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VL3 is the third immunoglobulin light chain variable domain; VL4 is the fourth immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; VH3 is the third immunoglobulin heavy chain variable domain; VH4 is the fourth immunoglobulin heavy chain variable domain; CH11 is the first immunoglobulin heavy chain constant domain; CH12 is the second immunoglobulin heavy chain constant domain; and L1, L2, L 3、 L4, L5, L6, L7 and L8 are amino acid linkers; in: a) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, and VL3 and VL4 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 and VL4 without the one or more amino acid substitutions; b) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, VL3 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL3 without the one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; c) CL1 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL1 without the one or more amino acid substitutions, VL4 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL4 without the one or more amino acid substitutions, and wherein VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; And wherein VL1 and VH1 form a first antigen binding domain, VL2 and VH2 form a second antigen binding domain, VL3 and VH3 form a third antigen binding domain, and VL4 and VH4 form a fourth antigen binding domain.

86. The multivalent binding protein of claim 85, wherein at least one of L1, L2, L3, L4, L5, L6, L7, or L8 is each independently 0 amino acids in length.

87. The binding protein of claim 85 or 86, wherein at least one of L1, L2, L3, L4, L5, L6, L7 or L8 is each independently at least one amino acid in length.

88. The multivalent binding protein of any one of claims 82-87, wherein binding of CL1 and / or CL2 comprising the one or more amino acid substitutions to the KappaSelect chromatography material is reduced by about 90% compared to CL1 and / or CL2 without the one or more amino acid substitutions.

89. The multivalent binding protein of any one of claims 82-88, wherein binding of the VL3 and / or VL4 comprising the one or more amino acid substitutions to the Protein L chromatography material is reduced by about 90% compared to the VL3 and / or VL4 without the one or more amino acid substitutions.

90. The multivalent binding protein of any one of claims 82-89, wherein the one or more amino acid substitutions in CL1 and / or CL2 comprising the one or more amino acid substitutions are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index.

91. The multivalent binding protein of claim 90, wherein the one or more amino acid substitutions in CL1 and / or CL2 comprising the one or more amino acid substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions, wherein the amino acid numbering is according to the EU index.

92. The multivalent binding protein of any one of claims 82-89, wherein the one or more amino acid substitutions in CL1 and / or CL2 comprising the one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index.

93. The multivalent binding protein of claim 92, wherein the one or more amino acid substitutions in CL1 and / or CL2 comprising the one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index.

94. The multivalent binding protein of any one of claims 82-93, wherein the one or more amino acid substitutions in VL3 and / or VL4 comprising the one or more amino acid substitutions are substitutions of framework amino acids.

95. The multivalent binding protein of any one of claims 82-94, wherein the one or more amino acid substitutions in VL3 and / or VL4 comprising the one or more amino acid substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat.

96. The multivalent binding protein of claim 95, wherein the one or more amino acid substitutions in VL3 and / or VL4 comprising the one or more amino acid substitutions are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.

97. The multivalent binding protein of any one of claims 82 or 86-96, wherein The first heavy chain polypeptide comprises a structure represented by the following formula: VH1-CH11-L1-VH 2- CH12-CH2-CH3[Ia], The second heavy chain polypeptide comprises a structure represented by the following formula: <h2 style=";text-align:left;direction:ltr">VH3-CH13-L3-VH<h2 style=";text-align:left;direction:ltr"> 4- <h2 style=";text-align:left;direction:ltr"> CH14-CH2-CH3[IIIa]。 98. The multivalent binding protein of any one of claims 83-96, wherein The first heavy chain polypeptide comprises a structure represented by the following formula: <h2 style=";text-align:left;direction:ltr">VH1-L1-VH<h2 style=";text-align:left;direction:ltr"> 2- <h2 style=";text-align:left;direction:ltr"> L2-CH11-CH2-CH3[Ia], The second heavy chain polypeptide comprises a structure represented by the following formula: VH3-L5-VH4-L6-CH12-CH2-CH3[IIIa].

99. The multivalent binding protein of claim 97, wherein the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is a human IgG1 or IgG4 CH3 domain.

100. The multivalent binding protein of claim 97 or 99, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V.

101. The multivalent binding protein of any one of claims 96-100, wherein the CH3 domain of the first heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V; wherein the CH3 domain of the second heavy chain polypeptide comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W.

102. The multivalent binding protein of claim 101, wherein the CH3 of the second heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A chromatography material.

103. The multivalent binding protein of claim 101, wherein the CH3 of the first heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A chromatography material.

104. The multivalent binding protein of claim 102 or 103, wherein the one or more amino acid substitutions that reduce binding to the Protein A chromatographic material are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index.

105. The multivalent binding protein of claim 104, wherein the amino acid substitutions are H435R and Y436F, wherein the amino acid numbering is according to the EU index.

106. The multivalent binding protein of any one of claims 82-105, wherein the binding protein is tetraspecific and is capable of specifically binding to four different antigenic targets.

107. A multivalent binding protein comprising four polypeptide chains forming an antigen binding domain; wherein, The four polypeptide chains comprise: The first heavy chain polypeptide comprises a structure represented by the following formula: VH1-CH11[I], The first light polypeptide chain comprises a structure represented by the following formula: VL1-CL1[II], The second heavy chain polypeptide comprises a structure represented by the following formula: Fusion polypeptide-L1-CH12[III], and a second light polypeptide chain comprising a structure represented by the following formula: Fusion peptide-L2-CL2[IV] in: VL1 is the first immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; CH11 is the first immunoglobulin heavy chain constant domain; CH12 is a second immunoglobulin heavy chain constant domain; and L1 and L2 are amino acid linkers; wherein CL2 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL2 without the one or more amino acid substitutions, and VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions; and Among them, VL1 and VH1 form the antigen binding domain.

108. The multivalent binding protein of claim 107, wherein L1 or L2 is independently 0 amino acids in length.

109. The binding protein of claim 107 or 108, wherein L1 or L2 is independently at least one amino acid in length.

110. A multivalent binding protein comprising four polypeptide chains forming two antigen binding domains; wherein: The four polypeptide chains comprise: The first heavy chain polypeptide comprises a structure represented by the following formula: VH1-CH11-L1-VH2-CH12[I], The first light polypeptide chain comprises a structure represented by the following formula: VL1-CL1-L2-VL2-CL2[II], The second heavy chain polypeptide comprises a structure represented by the following formula: Fusion polypeptide-L3-CH13[III], And a second light chain polypeptide comprising a structure represented by the following formula: Fusion peptide-L4-CL3[IV] in: VL1 is the first immunoglobulin light chain variable domain; VL2 is the second immunoglobulin light chain variable domain; CL1 is the first immunoglobulin light chain constant domain; CL2 is the second immunoglobulin light chain constant domain; CL3 is the third immunoglobulin light chain constant domain; VH1 is the first immunoglobulin heavy chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CH11 is the first immunoglobulin heavy chain constant domain; CH12 is the second immunoglobulin heavy chain constant domain; CH13 is the third immunoglobulin heavy chain constant domain, and L1, L2, L3 and L4 are amino acid linkers; in: a) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, and VL1 and VL2 each comprise the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 and VL2 without the one or more amino acid substitutions, b) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL1 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL1 without the one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin domain; or c) CL3 comprises the one or more amino acid substitutions that reduce binding to a KappaSelect chromatographic material compared to CL3 without the one or more amino acid substitutions, VL2 comprises the one or more amino acid substitutions that reduce binding to a Protein L chromatographic material compared to VL2 without the one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin domain; and Wherein VL1 and VH1 form a first antigen binding domain and VL2 and VH2 form a second antigen binding domain.

111. The multivalent binding protein of claim 110, wherein L1, L2, L 3、 or L4 are each independently 0 amino acids in length.

112. The binding protein of claim 110 or 111, wherein L1, L2, L3 or L4 are each independently at least one amino acid in length.

113. The multivalent binding protein of any one of claims 99-104, wherein binding of CL2 or CL3 comprising the one or more amino acid substitutions to the KappaSelect chromatography material is reduced by about 90% compared to CL2 or CL3 without the one or more amino acid substitutions.

114. The multivalent binding protein of any one of claims 107-113, wherein binding of VL1 and / or VL2 comprising the one or more amino acid substitutions to the Protein L chromatography material is reduced by about 90% compared to VL1 and / or VL2 without the one or more amino acid substitutions.

115. The multivalent binding protein of any one of claims 107-114, wherein the one or more amino acid substitutions in CL2 or CL3 comprising the one or more amino acid substitutions are at positions corresponding to 109, 110, or 199, wherein numbering is according to the EU index.

116. The multivalent binding protein of claim 115, wherein the one or more amino acid substitutions in CL2 or CL3 comprising the one or more amino acid substitutions are T109A substitutions, V110D substitutions, Q199K substitutions, T109A-V110D substitutions, or T109A-V110D-Q199K substitutions.

117. The multivalent binding protein of any one of claims 107-114, wherein the one or more amino acid substitutions in CL2 or CL3 comprising the one or more substitutions are at positions corresponding to 109, 198, 199 or 202, wherein numbering is according to the EU index.

118. The multivalent binding protein of claim 117, wherein the one or more amino acid substitutions in CL2 or CL3 comprising the one or more substitutions are H198R substitutions, Q199W substitutions, or T109A-S202R substitutions, wherein the amino acid numbering is according to the EU index.

119. The multivalent binding protein of any one of claims 107-118, wherein the one or more amino acid substitutions in VL1 and / or VL2 comprising the one or more amino acid substitutions are substitutions of framework amino acids.

120. The multivalent binding protein of any one of claims 107-119, wherein the one or more amino acid substitutions in VL1 and / or VL2 comprising the one or more amino acid substitutions are at positions corresponding to 12 or 18, wherein numbering is according to Kabat.

121. The multivalent binding protein of claim 120, wherein one or more amino acid substitutions in the VL1 and / or the VL2 are S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions, wherein numbering is according to Kabat.

122. The multivalent binding protein of any one of claims 107-121, wherein the first heavy chain polypeptide comprises a first CH2 immunoglobulin heavy chain constant domain and a first CH3 immunoglobulin heavy chain constant domain, and the second heavy chain polypeptide comprises a second CH2 immunoglobulin heavy chain constant domain and a second CH3 immunoglobulin heavy chain constant domain.

123. The multivalent binding protein of claim 122, wherein the first CH3 domain and / or the CH3 domain is a human IgG1 or IgG4 CH3 domain.

124. The multivalent binding protein of any one of claims 122 or 123, wherein the first CH3 domain comprises amino acid replacements at positions corresponding to position 354 and position 366 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are S354C and T366W; wherein the second CH3 domain comprises amino acid replacements at positions corresponding to position 349, position 366, position 368, position 407, position 435, and position 436 of human IgG1, wherein the numbering is according to the EU index, wherein the amino acid replacements are Y349C, T366S, L368A, and Y407V.

125. The multivalent binding protein of claim 124, wherein the CH3 of the second heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A.

126. The multivalent binding protein of claim 125, wherein the CH3 of the first heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A.

127. The multivalent binding protein of claim 126, wherein the one or more amino acid substitutions that reduce binding to Protein A are amino acid substitutions at positions corresponding to position 435 and position 436 of human IgG1, wherein numbering is according to the EU index.

128. The multivalent binding protein of claim 127, wherein the amino acid substitutions are H435R and Y436F, wherein the amino acid numbering is according to the EU index.

129. The multivalent binding protein of any one of claims 1-128, wherein the multivalent binding protein is a multispecific antibody or antigen-binding fragment thereof.

130. A polynucleotide encoding the multivalent binding protein of any one of claims 1-129.

131. A vector comprising the polynucleotide of claim 130.

132. A host cell comprising the polynucleotide of claim 130 or the vector of claim 131.

133. A method for producing a multivalent binding protein, the method comprising culturing the host cell of claim 122 such that the binding protein is produced.

134. The method of claim 133, further comprising recovering the binding protein from the host cell.

135. A pharmaceutical composition comprising the multivalent binding protein of any one of claims 1-129 and a pharmaceutically acceptable carrier.

136. A method for purifying a multivalent binding protein as described in any one of claims 1 to 129, the method comprising a) subjecting the composition comprising the multivalent binding protein to protein L chromatography in a bind and elute mode to produce a protein L eluate, and b) subjecting the Protein L eluate to KappaSelect chromatography in bind and elute mode to produce a KappaSelect eluate, The KappaSelect eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides.

137. The method of claim 136, wherein the multivalent binding protein in the KappaSelect eluate is at least 85% pure, at least 90% pure, or at least 95% pure.

138. The method of claim 136 or 137, wherein less than 15%, less than 10% or less than 5% of the polypeptides in the KappaSelect eluate are mismatched polypeptides.

139. A method for purifying a multivalent binding protein as described in any one of claims 1 to 129, the method comprising a) subjecting the composition comprising the multivalent binding protein and the mismatched antibody to KappaSelect chromatography in a bind and elute mode to produce a KappaSelect eluate, and b) subjecting the KappaSelect eluate to Protein L chromatography in bind and elute mode to produce a Protein L eluate, The protein L eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides.

140. The method of claim 139, wherein the multivalent binding protein in the Protein L eluate is at least 85% pure, at least 90% pure, or at least 95% pure.

141. The method of claim 139 or 140, wherein less than 15%, less than 10% or less than 5% of the polypeptides in the Protein L eluate are mismatched polypeptides.

142. A method for purifying a multivalent binding protein as described in any one of claims 1 to 129, the method comprising a) subjecting the composition comprising the multivalent binding protein to Protein A chromatography in a bind and elute mode to produce a Protein A eluate, b) subjecting the Protein A eluate to Protein L chromatography in bind and elute mode to produce a Protein L eluate, and c) subjecting the Protein L eluate to KappaSelect chromatography in bind and elute mode to produce a KappaSelect eluate, The KappaSelect eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides.

143. The method of claim 142, wherein the multivalent binding protein in the KappaSelect eluate is at least 85% pure, at least 90% pure, or at least 95% pure.

144. The method of claim 142 or 143, wherein less than 15%, less than 10% or less than 5% of the polypeptides in the KappaSelect eluate are mismatched polypeptides.

145. A method for purifying a multivalent binding protein as described in any one of claims 1 to 129, the method comprising a) subjecting the composition comprising the multivalent binding protein to Protein A chromatography in a bind and elute mode to produce a Protein A eluate, b) subjecting the Protein A eluate to KappaSelect chromatography in bind and elute mode to produce a KappaSelect eluate, and c) subjecting the Protein KappaSelect eluate to Protein L chromatography in bind and elute mode to produce a Protein L eluate, Wherein the L eluate comprises the multivalent binding protein and is substantially free of mismatched polypeptides.

146. The method of claim 145, wherein the multivalent binding protein in the Protein L eluate is at least 85% pure, at least 90% pure, or at least 95% pure.

147. The method of claim 145 or 146, wherein less than 15%, less than 10% or less than 5% of the polypeptides in the Protein L eluate are mismatched polypeptides.

148. The method of any one of claims 136-147, wherein the composition comprising the multivalent binding protein is derived from a host cell engineered to express the multispecific binding protein.

149. The method of any one of claims 136-148, wherein the composition comprising the multivalent binding protein is a host cell culture supernatant.

150. The method of any one of claims 136-149, wherein the composition comprising the multivalent binding protein further comprises a mismatched polypeptide.

151. The method of any one of claims 136-150, wherein the composition comprising the multivalent binding protein is filtered prior to chromatography.

152. The method of any one of claims 136-151, further comprising a polishing step after the KappaSelect or Protein L chromatography.

153. The method of claim 152, wherein the polishing step is size exclusion chromatography.

154. The method of any one of claims 146-153, wherein the Protein A chromatography is MabSelect TM 、MabSelect SuRe TM 、MabSelect SuRe TM LX, MabSelect PrismA, Ultra Plus, Protein A Fast Flow or AF-rProtein A chromatogram.

155. The method of any one of claims 136-154, wherein the Protein L chromatography is a Pierce TM Protein L columns, Capto TM L Chromatography, Protein L chromatography, AF-rProtein L-650F Chromatography or KanCap TM L Chromatography.

156. The method of any one of claims 136-155, wherein the KappaSelect chromatogram is a HiTrap TM KappaSelect or CaptureSelect TM Kappa XL chromatography.

157. The method of any one of claims 136-156, wherein the composition comprising the multivalent binding protein is combined with a pharmaceutically acceptable carrier.

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