Method for purifying a multispecific antibody

Abstract

A multivalent binding protein comprising four polypeptide chains is provided, wherein a first heavy chain polypeptide and a first light chain polypeptide associate to form one or more antigen-binding domains, and a second heavy chain polypeptide and a second light chain polypeptide associate and bind to the one or more antigen-binding domains. Also provided is a method for purifying such a multivalent binding protein.

Description

【Technical Field】 【0001】 Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent 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 hereby incorporated by reference in their entirety. 【0002】 Reference to Electronic Sequence Listing The contents of the electronic sequence listing (183952034140SEQLIST.xml; size: 21,251 bytes; and creation date: May 30, 2023) are hereby incorporated by reference in their entirety. 【0003】 The present disclosure relates to multivalent binding proteins with reduced binding to Protein L and / or KappaSelect chromatography materials, methods for generating such multivalent binding proteins, and methods for purifying such multivalent binding proteins from compositions containing multivalent binding proteins and impurities (e.g., misfolded polypeptides). 【Background Art】 【0004】 The development of multivalent binding proteins (e.g., multivalent and / or multispecific antibodies and antibody constructs) as therapeutic agents for human diseases has great clinical potential. However, since the antibody heavy chain has evolved to bind to the antibody light chain in a relatively disordered manner, producing multivalent binding proteins in IgG format has been a major challenge. As a result of such disordered pairing, for example, generating multivalent binding proteins that contain two or more antibody heavy chains (or heavy chain constructs) and / or two or more antibody light chains (or light chain constructs) can lead to the formation of unwanted species that include heavy chain homodimers and / or scrambled heavy chain / light chain pairs. Separating correctly assembled multivalent binding proteins from such unwanted mispaired species by chromatography can be difficult because of their similar structures and molecular weights. Furthermore, if the in vitro association reaction and / or purification method is complex, the application of many multivalent binding protein platforms, particularly their use in the high-throughput screening required for many therapeutic pipelines, is limited. 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0005】 There is a need in the art for an improved protein purification process that removes by-products of mispaired heavy / light chains and increases the yield of multivalent binding proteins. 【Means for Solving the Problems】 【0006】 In some embodiments, there is provided a multivalent binding protein comprising four polypeptide chains that form two antigen-binding domains; the four polypeptide chains are a first heavy chain polypeptide having a structure represented by the following formula: VH1-CH1 [I], a first light chain polypeptide chain having a structure represented by the following formula: VL1-CL1[II], a second heavy chain polypeptide having a structure represented by the following formula: VH2-CH1 [III], and a second light chain polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV] comprising; wherein VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain, VL2 is a κ1, κ3, or κ4 subtype light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, 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; CL1 contains one or more amino acid substitutions, thereby reducing binding to KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing binding to protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions, VH1 and VL1 associate to form a first antigen binding domain, and VH2 and VL2 associate to form a second antigen binding domain. 【0007】 In some embodiments, a multivalent binding protein is provided that comprises four polypeptide chains that form two antigen binding domains; the four polypeptide chains are 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 following formula: VH2-CH1-CH2-CH3 [III], and a second light chain polypeptide comprising a structure represented by the following formula: VL2-CL2[IV] comprising; In the formula, 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, CH2, and CH3 are immunoglobulin heavy chain constant domains; CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions, VH1 and VL1 associate to form the first antigen-binding domain, and VH2 and VL2 associate to form the second antigen-binding domain. 【0008】 In some embodiments according to (or applicable to) any embodiment of this specification, the binding of CL1 with KappaSelect containing one or more substitutions is reduced by about 90% compared to CL1 without any amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, the binding of VL2 with protein L chromatography material containing one or more substitutions is reduced by about 90% compared to VL2 without any amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 containing one or more substitutions are at positions corresponding to 109, 110, or 199, and the numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 containing one or more substitutions are T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 containing one or more substitutions are at positions corresponding to 109, 198, 199, or 202, and the numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 containing one or more substitutions are H198R substitution, Q199W substitution, or T109A-S202R substitution, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in VL2 containing one or more substitutions are framework amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in VL2 containing one or more substitutions are at positions corresponding to 12 or 18, and the numbering is according to Kabat. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in VL2 containing one or more substitutions are S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution, and the numbering is according to Kabat.In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is the CH3 domain of human IgG1 or IgG4. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, the numbering is according to the EU index, and the amino acid substitutions are S354C and T366W; the CH3 domain of the second heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, the numbering is according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, the numbering is according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V; the CH3 domain of the second heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, the numbering is according to the EU index, and the amino acid substitutions are S354C and T366W. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 of the second heavy chain polypeptide contains one or more amino acid substitutions that reduce binding to protein A. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 of the first heavy chain polypeptide contains one or more amino acid substitutions that reduce binding to protein A. In some embodiments according to (or applicable to) any embodiment of the present specification, the one or more amino acid substitutions that reduce binding to protein A are amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1, and the numbering is according to the EU index.In some embodiments according to (or applicable to) any embodiment of the present specification, the amino acid substitutions are H435R and Y436F, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of the present specification, 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 applicable to) any embodiment of the present specification, the first heavy chain polypeptide is derived from a different species than the second heavy chain polypeptide. In some embodiments according to (or applicable to) any embodiment of the present specification, 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 applicable to) any embodiment of the present specification, the first heavy chain polypeptide and the second heavy chain polypeptide each contain the CH3 domain of IgG4. In some embodiments according to (or applicable to) any embodiment of the present specification, the first heavy chain polypeptide contains the K409R amino acid substitution, and the second heavy chain polypeptide contains the F405L amino acid substitution, and the numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of the present specification, the multivalent binding protein is a bispecific antigen-binding protein. In some embodiments according to (or applicable to) any embodiment of the present specification, the first antigen-binding domain and the second antigen-binding domain bind to different antigens. 【0009】 In some embodiments according to (or applicable to) any embodiment of the present specification, the first heavy chain polypeptide chain of the multivalent binding protein has a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3-L-VL3[Ia] and includes The second heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIa] and includes In the formula, 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. 【0010】 In some embodiments according to (or applicable to) any embodiment of the present specification, the first heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3[Ib] and the second heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIb] and in the formula, VH3 is the third immunoglobulin heavy chain variable domain. 【0011】 In some embodiments according to (or applicable to) any embodiment of the present specification, VL3 contains one or more amino acid substitutions, thereby reducing the binding to protein L chromatography material as compared to VL3 that does not contain one or more amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of the present specification, VL3 is a λ subtype immunoglobulin light chain variable domain or a κ2 immunoglobulin light chain variable domain. In some embodiments according to (or applicable to) any embodiment of the present specification, the multivalent binding protein is bispecific or trispecific. In some embodiments according to (or applicable to) any embodiment of the present specification, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain bind to two or three different antigens. In some embodiments according to (or applicable to) any embodiment of the present specification, 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 applicable to) any embodiment of the present specification, 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 that includes four polypeptide chains forming three antigen-binding domains; the four polypeptide chains are A first heavy chain polypeptide having a structure represented by the following formula: VH1-L3-VH2-L4-CH1 [I], A first light chain polypeptide chain having a structure represented by the following formula: VL2-L1-VL1-L2-CL1[II], A second heavy chain polypeptide having a structure represented by the following formula: VH3-CH1 [III], And a second light chain polypeptide chain having a structure represented by the following formula: VL3-CL2[IV] including In the formula, 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; L1, L2, L3 and L4 are amino acid linkers; the polypeptide of formula I and the polypeptide of formula II form an intersected light chain-heavy chain pair; VH1 and VL1 associate to form the first antigen-binding domain, VH2 and VL2 associate to form the second antigen-binding domain, and VH3 and VL3 associate to form the third antigen-binding domain; a) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, and VL3 contains one or more amino acid substitutions, which reduce the binding to the protein L chromatography material as compared to VL3 that does not contain one or more amino acid substitutions; b) CL2 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, which reduce the binding to the protein L chromatography material as compared to VL1 and VL2 that do not contain one or more amino acid substitutions; c) CL2 contains one or more amino acid substitutions that reduce its binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions; VL1 contains one or more amino acid substitutions that reduce its binding to the Protein L chromatography material compared to VL1 that does not contain one or more amino acid substitutions; VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 contains one or more amino acid substitutions that reduce its binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions; VL2 contains one or more amino acid substitutions that reduce its binding to the Protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions; VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain. 【0013】 In some embodiments, provided is a multivalent binding protein comprising four polypeptide chains that form three antigen-binding domains; the four polypeptide chains are a first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L3-VH2-L4-CH1-CH2-CH3 [Ia], a first light chain polypeptide chain comprising a structure represented by the following formula: VL2-L1-VL1-L2-CL1[II], a second heavy chain polypeptide comprising a structure represented by the following formula: VH3-CH1-CH2-CH3 [IIIa], and a second light chain polypeptide comprising a structure represented by the following formula: VL3-CL2[IV] and In the formula, 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; L1, L2, L3 and L4 are amino acid linkers; the polypeptide of formula I and the polypeptide of formula II form an intersected light chain-heavy chain pair; VH1 and VL1 associate to form the first antigen-binding domain, VH2 and VL2 associate to form the second antigen-binding domain, and VH3 and VL3 associate to form the third antigen-binding domain; a) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, and VL3 contains one or more amino acid substitutions, which reduce the binding to the protein L chromatography material as compared to VL3 that does not contain one or more amino acid substitutions; b) CL2 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, which reduce the binding to the protein L chromatography material as compared to VL1 and VL2 that do not contain one or more amino acid substitutions; c) CL2 contains one or more amino acid substitutions, which reduce its binding to the KappaSelect chromatography material compared to CL2 without any amino acid substitutions, VL1 contains one or more amino acid substitutions, which reduce its binding to the Protein L chromatography material compared to VL1 without any amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype light chain variable immunoglobulin domain; or d) CL2 contains one or more amino acid substitutions, which reduce its binding to the KappaSelect chromatography material compared to CL2 without any amino acid substitutions, VL2 contains one or more amino acid substitutions, which reduce its binding to the Protein L chromatography material compared to VL2 without any amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype light chain variable immunoglobulin domain. 【0014】 In some embodiments according to (or applicable to) any embodiment herein, the binding protein is trispecific and can specifically bind to three different antigen targets. In some embodiments according to (or applicable to) any embodiment herein, one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments according to (or applicable to) any embodiment herein, L1, L2, L3, or L4 are each independently at least 1 amino acid in length. 【0015】 In some embodiments according to (or applicable to) any embodiment herein, the second heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH3-L5-VH4-L6-CH1 [IIIb] and the second light chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VL4-L7-VL3-L8-CL2[IVa] and In the formula, 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; the polypeptide of formula IIIa and the polypeptide of formula IVa form an intersected light chain-heavy chain pair; VH4 and VL4 associate to form the fourth antigen-binding domain; a) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, and VL3 and VL4 each contain one or more amino acid substitutions, thereby reducing the binding to the Protein L chromatography material as compared to VL3 and VL4 that do not contain one or more amino acid substitutions; b) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, VL3 contains one or more amino acid substitutions, thereby reducing the binding to the Protein L chromatography material as compared to VL3 that does not contain one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain; c) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, VL4 contains one or more amino acid substitutions, thereby reducing the binding to the Protein L chromatography material as compared to VL4 that does not contain one or more amino acid substitutions, and VL3 is a lambda subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain; d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, thereby reducing their binding to the Protein L chromatography material as compared to VL1 and VL2 that do not contain one or more amino acid substitutions; e) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; or f) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain. 【0016】 In some embodiments according to (or applicable to) any embodiment herein, the second heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH3-L5-VH4-L6-CH1-CH2-CH3 [IIIc] including 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 applicable to) any embodiment herein, the multivalent binding protein is quadrivalent and can specifically bind to four antigen targets (e.g., four different antigen targets). 【0017】 In some embodiments according to (or applicable to) any embodiment of this specification, at least one of L1, L2, L3, L4, L5, L6, L7 or L8 is independently 0 amino acids in length. In some embodiments according to (or applicable to) any embodiment of this specification, L1, L2, L3, L4, L5, L6, L7 or L8 are each independently at least 1 amino acid in length. In some embodiments according to (or applicable to) any embodiment of this specification, the binding of CL1 or CL2 containing one or more amino acid substitutions to the KappaSelect chromatography material is reduced by about 90% compared to CL1 or CL2 without any amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, the binding of VL1, VL2, VL3 and / or VL4 containing one or more substitutions to the Protein L chromatography material is reduced by about 90% compared to VL1, VL2, VL3 and / or VL4 without any amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 or CL2 are at positions corresponding to 109, 110 or 199, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 or CL2 containing one or more amino acid substitutions are T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 or CL2 containing one or more substitutions are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 or CL2 containing one or more substitutions are H198R substitution, Q199W substitution, or T109A-S202R substitution, and the amino acid numbering is according to the EU index.In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in VL1, VL2, VL3, and / or VL4 that include one or more substitutions are framework amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in VL1, VL2, VL3, and / or VL4 that include one or more substitutions are at positions corresponding to 12 or 18, numbered according to Kabat. In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in VL1, VL2, VL3, and / or VL4 that include one or more substitutions are S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution, numbered according to Kabat. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is the CH3 domain of human IgG1 or IgG4. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide includes amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, numbered according to the EU index, and the amino acid substitutions are S354C and T366W; the CH3 domain of the second heavy chain polypeptide includes amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, numbered according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide includes amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, numbered according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V; the CH3 domain of the second heavy chain polypeptide includes amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, numbered according to the EU index, and the amino acid substitutions are S354C and T366W.In some embodiments according to (or applicable to) any embodiment of this specification, the second heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A. In some embodiments according to (or applicable to) any embodiment of this specification, the CH3 of the first heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to Protein A. In some embodiments according to (or applicable to) any embodiment of this specification, the one or more amino acid substitutions that reduce binding to Protein A are amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1, and the numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, the amino acid substitutions are H435R and Y436F, and the amino acid numbering is according to the EU index. 【0018】 In some embodiments, provided is a multivalent binding protein comprising four polypeptide chains that form two antigen-binding domains; the four polypeptide chains comprise a first heavy chain polypeptide having a structure represented by the following formula: VH1-CH1 [I], a first light chain polypeptide chain having a structure represented by the following formula: VL1-CL1[II], a second heavy chain polypeptide having a structure represented by the following formula: VH2-CL2[III], and a second light chain polypeptide chain having a structure represented by the following formula: VL2-CH1 [IV] wherein: 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 an immunoglobulin heavy chain constant domain, a) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions, and VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material compared to VL1 that does not contain one or more amino acid substitutions; or b) CL1 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, and VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions; 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 is provided that comprises four polypeptide chains that form two antigen-binding domains; the four polypeptide chains are a first heavy-chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], a first light-chain polypeptide chain comprising a structure represented by the following formula: VL1-CL1[II], a second heavy-chain polypeptide comprising a structure represented by the following formula: VH2-CL2-CH2-CH3 [III], and a second light-chain polypeptide chain comprising a structure represented by the following formula: VL2-CH1 [IV] and; In the formula, 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; a) CL2 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions, and VL1 contains one or more amino acid substitutions, thereby reducing the binding to the Protein L chromatography material compared to VL1 that does not contain one or more amino acid substitutions; or b) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, and VL2 contains one or more amino acid substitutions, thereby reducing the binding to the Protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions; 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 is provided that comprises four polypeptide chains that form two antigen-binding domains; the four polypeptide chains are a first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1 [I], a first light chain polypeptide chain comprising a structure represented by the following formula: VL1-CL1[II], a second heavy chain polypeptide comprising a structure represented by the following formula: VL2-CH1 [III], and a second light chain polypeptide chain comprising a structure represented by the following formula: VH2-CL2[IV] comprising; 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; a) CL2 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material as compared to CL2 that does not comprise one or more amino acid substitutions, and VL1 comprises one or more amino acid substitutions that reduce binding to a protein L chromatography material as compared to VL1 that does not comprise one or more amino acid substitutions, or b) CL1 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material as compared to CL1 that does not comprise one or more amino acid substitutions, and VL2 comprises one or more amino acid substitutions that reduce binding to a protein L chromatography material as compared to VL2 that does not comprise one or more amino acid substitutions; 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, provided is a multivalent binding protein comprising four polypeptide chains that form two antigen-binding domains; the four polypeptide chains are a first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], a first light chain polypeptide chain comprising a structure represented by the following formula: VL1-CL1[II], a second heavy chain polypeptide comprising a structure represented by the following formula: VH2-CH1-CH2-CH3 [III], and a second light chain polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV] comprising; 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; a) CL2 comprises one or more amino acid substitutions, which reduce the binding to KappaSelect chromatography material as compared to CL2 that does not comprise one or more amino acid substitutions, and VL1 comprises one or more amino acid substitutions, which reduce the binding to Protein L chromatography material as compared to VL1 that does not comprise one or more amino acid substitutions, or b) CL1 comprises one or more amino acid substitutions, which reduce the binding to KappaSelect chromatography material as compared to CL1 that does not comprise one or more amino acid substitutions, and VL2 comprises one or more amino acid substitutions, which reduce the binding to Protein L chromatography material as compared to VL2 that does not comprise one or more amino acid substitutions; 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 applicable to) any embodiment of this specification, the binding of CL1 or CL2 KappaSelect chromatography material containing one or more amino acid substitutions is reduced by about 90% compared to CL1 or CL2 without one or more amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, the binding of VL1 or VL2 protein L chromatography material containing one or more amino acid substitutions is reduced by about 90% compared to VL1 or VL2 without one or more amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 or CL2 containing one or more amino acid substitutions are at positions corresponding to 109, 110, or 199, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 or CL2 containing one or more amino acid substitutions are T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 or CL2 containing one or more substitutions are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 or CL2 containing one or more substitutions are H198R substitution, Q199W substitution, or T109A-S202R substitution, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in VL1 or VL2 containing one or more amino acid substitutions are framework amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in VL1 or VL2 containing one or more amino acid substitutions are at positions corresponding to 12 or 18, numbered according to Kabat.In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in VL1 or VL2 that include one or more amino acid substitutions are S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution, and the numbering is according to Kabat. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is the CH3 domain of human IgG1 or IgG4. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide includes amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, and the numbering is according to the EU index, and the amino acid substitutions are S354C and T366W; the CH3 domain of the second heavy chain polypeptide includes amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, and the numbering is according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide includes amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, and the numbering is according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V; the CH3 domain of the second heavy chain polypeptide includes amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, and the numbering is according to the EU index, and the amino acid substitutions are S354C and T366W. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 of the second heavy chain polypeptide includes one or more amino acid substitutions that reduce binding to protein A chromatography material. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 of the first heavy chain polypeptide includes one or more amino acid substitutions that reduce binding to protein A chromatography material.In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions that reduce binding to protein A chromatography material are amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, the amino acid substitutions are H435R and Y436F, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, the multivalent binding protein is a bispecific antigen-binding protein. In some embodiments according to (or applicable to) any embodiment of this specification, the first antigen-binding domain and the second antigen-binding domain bind to different antigens. 【0023】 In some embodiments, there is provided a multivalent binding protein comprising four polypeptide chains that form four antigen-binding domains; the four polypeptide chains are A first heavy-chain polypeptide having a structure represented by the following formula: VH1-CH11-L1-VH2-CH12[I], A first light-chain polypeptide having a structure represented by the following formula: VL1-CL1-L2-VL2-CL2[II], A second heavy-chain polypeptide having a structure represented by the following formula: VH3-CH13-L3-VH4-CH14[III], A second light-chain polypeptide having a structure represented by the following formula: VL3-CL3-L4-VL4-CL4[IV] including In the formula, 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; L1, L2, L3 and L4 are amino acid linkers; a) CL1 and CL2 each contain one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 and CL2 that do not contain one or more amino acid substitutions, and VL3 and VL4 each contain one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL3 and VL4 that do not contain one or more amino acid substitutions; b) CL1 and CL2 each contain one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 and CL2 that do not contain one or more amino acid substitutions, VL3 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL3 that does not contain one or more amino acid substitutions, and VL4 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain; c) CL1 and CL2 each contain one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 and CL2 that do not contain one or more amino acid substitutions. VL4 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL4 that does not contain one or more amino acid substitutions. VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; d) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions. CL2 is a lambda subtype immunoglobulin light chain constant domain. VL3 and VL4 each contain one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL1 and VL2 that do not contain one or more amino acid substitutions; e) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions. CL2 is a lambda subtype immunoglobulin light chain constant domain. VL3 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL3 that does not contain one or more amino acid substitutions. VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; f) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions. CL2 is a lambda subtype immunoglobulin light chain constant domain. VL4 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL4 that does not contain one or more amino acid substitutions. VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; g) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. CL1 is a lambda subtype immunoglobulin light chain constant domain. VL3 and VL4 each contain one or more amino acid substitutions, thereby reducing their binding to the Protein L chromatography material as compared to VL3 and VL4 that do not contain one or more amino acid substitutions; h) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. CL1 is a lambda subtype immunoglobulin light chain constant domain. VL3 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL3 that does not contain one or more amino acid substitutions. VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; i) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. CL1 is a lambda subtype immunoglobulin light chain constant domain. VL4 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL4 that does not contain one or more amino acid substitutions. VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; 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 applicable to) any embodiment of the present specification, one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments according to (or applicable to) any embodiment of the present specification, one of L1, L2, L3, or L4 is independently at least 1 amino acid in length. 【0025】 In some embodiments, a multivalent binding protein is provided that includes four polypeptide chains that form four antigen-binding domains; the four polypeptide chains are A first heavy chain polypeptide having a structure represented by the following formula: VH1-L1-VH2-L2-CH11[I], A first light chain polypeptide having a structure represented by the following formula: VL1-L3-VL2-L4-CL1[II], A second heavy chain polypeptide having a structure represented by the following formula: VH3-L5-VH4-L6-CH12[III], A second light chain polypeptide having a structure represented by the following formula: VL3-L7-VL4-L8-CL2[IV] and 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; L1, L2, L3, L4, L5, L6, L7, and L8 are amino acid linkers; a) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions. VL3 and VL4 each contain one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material as compared to VL3 and VL4 that do not contain one or more amino acid substitutions; b) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions. VL3 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material as compared to VL3 that does not contain one or more amino acid substitutions. VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; c) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions. VL4 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material as compared to VL4 that does not contain one or more amino acid substitutions. VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; 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 applicable to) any embodiment of this specification, at least one of L1, L2, L3, L4, L5, L6, L7, or L8 is independently 0 amino acids in length. In some embodiments according to (or applicable to) any embodiment of this specification, at least one of L1, L2, L3, L4, L5, L6, L7, or L8 is independently at least 1 amino acid in length. In some embodiments according to (or applicable to) any embodiment of this specification, the binding of CL1 and / or CL2 containing one or more amino acid substitutions to KappaSelect chromatography material is reduced by about 90% compared to CL1 and / or CL2 without any amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, the binding of VL3 and / or VL4 containing one or more amino acid substitutions to protein L chromatography material is reduced by about 90% compared to VL3 and / or VL4 without any amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 and / or CL2 containing one or more amino acid substitutions are at positions corresponding to 109, 110, or 199, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 and / or CL2 containing one or more amino acid substitutions are T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 and / or CL2 containing one or more substitutions are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in CL1 and / or CL2 containing one or more substitutions are H198R substitution, Q199W substitution, or T109A-S202R substitution, and the amino acid numbering is according to the EU index.In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in VL3 and / or VL4 that include one or more amino acid substitutions are framework amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in VL3 and / or VL4 that include one or more amino acid substitutions are at positions corresponding to 12 or 18, numbered according to Kabat. In some embodiments according to (or applicable to) any embodiment of this specification, one or more amino acid substitutions in VL3 and / or VL4 that include one or more amino acid substitutions are S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution, numbered according to Kabat. 【0027】 In some embodiments according to (or applicable to) any embodiment of this specification, the first heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH1-CH11-L1-VH2-CH12-CH2-CH3 [Ia] and includes The second heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH3-CH13-L3-VH4-CH14-CH2-CH3 [IIIa] and includes. 【0028】 In some embodiments according to (or applicable to) any embodiment of this specification, the first heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH1-L1-VH2-L2-CH11-CH2-CH3 [Ia] and includes The second heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH3-L5-VH4-L6-CH12-CH2-CH3 [IIIa] and includes. 【0029】 In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is the CH3 domain of human IgG1 or IgG4. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, numbered according to the EU index, and the amino acid substitutions are S354C and T366W; the CH3 domain of the second heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, numbered according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 domain of the first heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, numbered according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V; the CH3 domain of the second heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, numbered according to the EU index, and the amino acid substitutions are S354C and T366W. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 of the second heavy chain polypeptide contains one or more amino acid substitutions that reduce binding to protein A chromatography material. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 of the first heavy chain polypeptide contains one or more amino acid substitutions that reduce binding to protein A chromatography material. In some embodiments according to (or applicable to) any embodiment of the present specification, the one or more amino acid substitutions that reduce binding to protein A chromatography material are amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1, numbered according to the EU index.In some embodiments according to (or applicable to) any embodiment of the present specification, the amino acid substitutions are H435R and Y436F, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of the present specification, the binding protein is tetravalent and can specifically bind to four different antigen targets. 【0030】 In some embodiments, a multivalent binding protein is provided that includes four polypeptide chains forming an antigen-binding domain; the four polypeptide chains are A first heavy-chain polypeptide comprising a structure represented by the following formula: VH1-CH11[I], A first light-chain polypeptide chain comprising a structure represented by the following formula: VL1-CL1[II], A second heavy-chain polypeptide comprising a structure represented by the following formula: Fusion polypeptide-L1-CH12[III], And a second light-chain polypeptide chain comprising a structure represented by the following formula: Fusion polypeptide-L2-CL2[IV] Comprising Wherein 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 the second immunoglobulin heavy-chain constant domain; L1 and L2 are amino acid linkers; CL2 contains one or more amino acid substitutions, whereby the binding to KappaSelect chromatography material is reduced compared to CL2 that does not contain one or more amino acid substitutions, and VL1 contains one or more amino acid substitutions, whereby the binding to protein L chromatography material is reduced compared to VL1 that does not contain one or more amino acid substitutions; VL1 and VH1 form an antigen-binding domain. 【0031】 In some embodiments according to (or applicable to) any embodiment of this specification, L1 or L2 is independently 0 amino acids in length. In some embodiments according to (or applicable to) any embodiment of this specification, L1 or L2 is independently at least 1 amino acid in length. 【0032】 In some embodiments, provided is a multivalent binding protein comprising four polypeptide chains that form two antigen-binding domains; the four polypeptide chains are a first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH11-L1-VH2-CH12[I], a first light chain polypeptide chain comprising a structure represented by the following formula: VL1-CL1-L2-VL2-CL2[II], a second heavy chain polypeptide comprising 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 polypeptide-L4-CL3[IV] comprising, 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; a) CL3 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL3 that does not contain one or more amino acid substitutions. VL1 and VL2 each contain one or more amino acid substitutions, thereby reducing their binding to the Protein L chromatography material as compared to VL1 and VL2 that do not contain one or more amino acid substitutions. b) CL3 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL3 that does not contain one or more amino acid substitutions. VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions. VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin domain; or c) CL3 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL3 that does not contain one or more amino acid substitutions. VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions. VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin domain; 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 applicable to) any embodiment of the present specification, one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments according to (or applicable to) any embodiment of the present specification, L1, L2, L3, or L4 is independently at least 1 amino acid in length. In some embodiments according to (or applicable to) any embodiment of the present specification, the binding of CL2 or CL3 KappaSelect chromatography material containing one or more amino acid substitutions is reduced by about 90% compared to CL2 or CL3 without any amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of the present specification, the binding of VL1 and / or VL2 protein L chromatography material containing one or more amino acid substitutions is reduced by about 90% compared to VL1 and / or VL2 without any amino acid substitutions. In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in CL2 or CL3 containing one or more amino acid substitutions are at positions corresponding to 109, 110, or 199, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in CL2 or CL3 containing one or more amino acid substitutions are T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution. In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in CL2 or CL3 containing one or more substitutions are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index. In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in CL2 or CL3 containing one or more substitutions are H198R substitution, Q199W substitution, or T109A-S202R substitution, and the amino acid numbering is according to the EU index. In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in VL1 and / or VL2 containing one or more amino acid substitutions are framework amino acid substitutions.In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in VL1 and / or VL2 that include one or more amino acid substitutions are at positions corresponding to 12 or 18, numbered according to Kabat. In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions in VL1 and / or VL2 are S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution, numbered according to Kabat. In some embodiments according to (or applicable to) any embodiment of the present specification, the first heavy chain polypeptide includes a first CH2 immunoglobulin heavy chain constant domain and a first CH3 immunoglobulin heavy chain constant domain, and the second heavy chain polypeptide includes a second CH2 immunoglobulin heavy chain constant domain and a second CH3 immunoglobulin heavy chain constant domain. In some embodiments according to (or applicable to) any embodiment of the present specification, the first CH3 domain and / or CH3 domain is the CH3 domain of human IgG1 or IgG4. In some embodiments according to (or applicable to) any embodiment of the present specification, the first CH3 domain includes amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, numbered according to the EU index, and the amino acid substitutions are S354C and T366W; the second CH3 domain includes amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, numbered according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 of the second heavy chain polypeptide includes one or more amino acid substitutions that reduce binding to protein A. In some embodiments according to (or applicable to) any embodiment of the present specification, the CH3 of the first heavy chain polypeptide includes one or more amino acid substitutions that reduce binding to protein A.In some embodiments according to (or applicable to) any embodiment of the present specification, one or more amino acid substitutions that reduce binding to Protein A are amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1, with numbering according to the EU index. In some embodiments according to (or applicable to) any embodiment of the present specification, the amino acid substitutions are H435R and Y436F, with amino acid numbering according to the EU index. In some embodiments according to (or applicable to) any embodiment of the present specification, the multivalent binding protein is a multispecific antibody or an antigen-binding fragment thereof. 【0034】 In some embodiments, one or more polynucleotides encoding the multivalent binding proteins described herein are provided. In some embodiments, vectors comprising one or more polynucleotides described herein are provided. In some embodiments, host cells comprising one or more polynucleotides or vectors described herein are provided. In some embodiments, a method of producing a multivalent binding protein is provided, the method comprising culturing a host cell described herein such that a binding protein is produced (e.g., under conditions in which the multivalent binding protein is expressed by the host cell). In some embodiments, the method further comprises recovering the binding protein from the host cell. 【0035】 In some embodiments, pharmaceutical compositions comprising a multivalent binding protein described herein and a pharmaceutically acceptable carrier are provided. 【0036】 In some embodiments, provided herein is a method for purifying a multivalent binding protein (e.g., separating the multivalent binding protein provided herein from one or more impurities), the method comprising: a) subjecting a composition comprising the multivalent binding protein (e.g., an impurity such as the multivalent binding protein and a misfolded or misassociated polypeptide) to protein L chromatography in binding and elution modes to produce a protein L eluate; and b) subjecting the protein L eluate to KappaSelect chromatography in binding and elution modes to produce a KappaSelect eluate, the KappaSelect eluate comprising the multivalent binding protein and essentially free of misfolded (or misassociated) 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 misfolded or misassociated polypeptides). In some embodiments, less than 15%, less than 10%, or less than 5% of the polypeptides in the KappaSelect eluate are misfolded (or misassociated) polypeptides. 【0037】 In some embodiments, provided is 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 mispaired or misassociated polypeptides), the method comprising: a) subjecting a composition comprising the multivalent binding protein and the mispaired (or misassociated) polypeptide to KappaSelect chromatography in binding and elution chromatography to generate a KappaSelect eluate; and b) subjecting the KappaSelect eluate to protein L chromatography in binding and elution mode to generate a protein L eluate, the protein L eluate comprising the multivalent binding protein and essentially free of mispaired (or misassociated) 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 mispaired or misassociated polypeptides). In some embodiments, less than 15%, less than 10%, or less than 5% of the polypeptides in the protein L eluate are mispaired or misassociated polypeptides. 【0038】 In some embodiments, provided is a method of purifying a multivalent binding protein described herein (e.g., separating the multivalent binding protein provided herein from one or more impurities), the method comprising: a) subjecting a composition comprising the multivalent binding protein (e.g., impurities such as a multivalent antigen-binding protein and misfolded or misassociated polypeptides) to protein A chromatography in binding and elution modes to produce a protein A eluate; b) subjecting the protein A eluate to protein L chromatography in binding and elution modes to produce a protein L eluate; and c) subjecting the protein L eluate to KappaSelect chromatography in binding and elution modes to produce a KappaSelect eluate, the KappaSelect eluate comprising the multivalent binding protein and essentially free of misfolded (or misassociated) 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 misfolded or misassociated polypeptides). In some embodiments, less than 15%, less than 10%, or less than 5% of the polypeptides in the KappaSelect eluate are misfolded (or misassociated) polypeptides. 【0039】 In some embodiments, provided is a method of purifying a multivalent binding protein described herein (e.g., separating a multivalent binding protein provided herein from one or more impurities), the method comprising: a) subjecting a composition comprising the multivalent binding protein (e.g., a composition comprising the multivalent binding protein and misfolded or misassociated polypeptides) to Protein A chromatography in binding and elution modes to produce a Protein A eluate; b) subjecting the Protein A eluate to KappaSelect chromatography in binding and elution modes to produce a KappaSelect eluate; and c) subjecting the Protein KappaSelect eluate to Protein L chromatography in binding and elution modes to produce a Protein L eluate, the L eluate comprising the multivalent binding protein and essentially free of misfolded (or misassociated) 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 misfolded or misassociated polypeptides). In some embodiments, less than 15%, less than 10%, or less than 5% of the polypeptides in the Protein L eluate are misfolded (or misassociated) polypeptides. 【0040】 In some embodiments according to (or applicable to) any embodiment of this specification, a composition comprising a multivalent binding protein is derived from a host cell engineered to express a multispecific binding protein. In some embodiments according to (or applicable to) any embodiment of this specification, a composition comprising a multivalent binding protein is a host cell culture supernatant. In some embodiments according to (or applicable to) any embodiment of this specification, a composition comprising a multivalent binding protein further comprises mispaired polypeptides. In some embodiments according to (or applicable to) any embodiment of this specification, a composition comprising a multivalent binding protein is filtered prior to chromatography. In some embodiments according to (or applicable to this specification) any embodiment of this specification, the method further comprises a polishing step after KappaSelect or Protein L chromatography. In some embodiments according to (or applicable to) any embodiment of this specification, the polishing step is size exclusion chromatography. In some embodiments according to (or applicable to) any embodiment of this specification, Protein A chromatography is MabSelect™, MabSelect SuRe™, MabSelect SuRe™ LX, MabSelect PrismA, ProSep®-vA, ProSep® Ultra Plus, Protein A Sepharose® Fast Flow, or Toyopearl® AF-rProtein A chromatography. In some embodiments according to (or applicable to) any embodiment of this specification, Protein L chromatography is Pierce™ Protein L chromatography cartridge, Capto™ L chromatography, HiTrap® Protein L chromatography, TOYOPEARL® AF-rProtein L-650F chromatography, or KanCap™ L chromatography.In some embodiments according to (or applicable to) any embodiment of this specification, KappaSelect chromatography is HiTrap™ KappaSelect or CaptureSelect™ Kappa XL chromatography. In some embodiments according to (or applicable to) any embodiment of this specification, a composition comprising a multivalent binding protein is combined with a pharmaceutically acceptable carrier. 【0041】 It will be understood that one, some, or all features of various embodiments described herein may be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those skilled in the art. These and other embodiments of the invention are further described in the following modes for carrying out the invention. 【Brief Description of the Drawings】 【0042】 【Figure 1A】 Schematic diagram of an exemplary bivalent binding protein. 【Figure 1B】 Schematic diagram of an exemplary Triomab® bivalent binding protein. 【Figure 1C】 Schematic diagram of an exemplary Duomab® bivalent binding protein. 【Figure 1D】 Schematic diagram of an exemplary Ab-Nb trivalent binding protein comprising an scFv linked to a "knob" heavy chain. 【Figure 1E】 Schematic diagram of an exemplary Ab-Nb trivalent binding protein comprising a VHH linked to a "knob" heavy chain. 【Figure 1F】 Schematic diagram of an exemplary CODV multivalent binding protein comprising a CODV arm on the left and a Fab arm on the right. 【Figure 1G】 Schematic diagram of an exemplary CODV multivalent binding protein comprising two CODV arms. 【Figure 1H】 Schematic diagram of an exemplary CrossMab bivalent binding protein in which the CL domain and the CH1 domain of one arm of the binding protein are exchanged. 【Figure 1I】Schematic diagram of an exemplary cross - Mab bivalent binding protein in which the VL domain and the VH domain of one arm of the binding protein are exchanged. 【Figure 1J】 Schematic diagram of an exemplary tandem Fab multivalent binding protein in which each variable domain contains VL, CL, VH, and CH1 domains. 【Figure 1K】 Schematic diagram of an exemplary tandem Fab multivalent binding protein in which the N - terminal variable domain contains VL and VH domains and the C - terminal variable domain contains VL, CL, VH, and CH1 domains. 【Figure 1L】 Schematic diagram of an exemplary multivalent binding protein containing a Fab arm and a fusion protein arm. The Fab arm is composed of a single Fab. 【Figure 1M】 Schematic diagram of an exemplary multivalent binding protein containing a Fab arm and a fusion protein arm. The Fab arm is composed of tandem Fabs. 【Figure 2】 Schematic diagram of a representative three - step process for purifying a CODV multivalent binding protein. The desired product is shown within the dotted frame. CODV includes knob substitution and hole substitution. The knob arm of CODV contains a KappaSelect KO mutation (e.g., a mutation that reduces binding to the KappaSelect chromatography medium in the VL domain). The hole arm of CODV contains a Protein L KO mutation (e.g., a mutation that reduces binding to Protein L in the CL domain) and an RF mutation in the CH3 domain (e.g., H435R and 436F mutations, with amino acid numbering according to the EU index) (see, for example, Edelman et al., 1969, Proc Natl Acad Sci USA 63;78 - 85). Representative mis - paired products are shown next to the desired product in the upper left. The three steps in this process include Protein A chromatography (e.g., MabSelectSure® (MSS)), followed by Protein L chromatography, followed by KappaSelect (KS) chromatography. Removal of mis - paired proteins is shown. 【Figure 3】Alignment of the amino acid sequences of different VL kappas and lambda subtypes is shown. Citation from Graille et al., Structure, Vol. 9, 679 - 687, August, 2001. 【Figure 4】 Results of screening for potential ProL KO variants are shown. The upper panel shows the variants evaluated. The lower panel shows the results based on the protein yield after MabSelect® Sure chromatography. 【Figure 5】 Biolayer interferometry (BLI) binding evaluation of adalimumab ProL KO variant to ProL ligand is shown. 【Figure 6】 BLI binding evaluation of adalimumab ProL KO variant to TNFα is shown. 【Figure 7】 Evaluation of the binding of adalimumab ProL KO variant to protein - L resin compared to the wild - type is shown. 【Figure 8】 Results of screening for potential KS KO variants are shown. The upper panel shows the variants evaluated. The lower panel shows the results based on the protein yield after MabSelect® Sure chromatography. 【Figure 9】 BLI binding evaluation of adalimumab KS KO variant to KS ligand is shown. 【Figure 10】 BLI binding evaluation of adalimumab KS KO variant to TNFα is shown. 【Figure 11】 Evaluation of the binding of adalimumab KS KO variant to KS resin compared to the wild - type is shown. 【Figure 12】 Schematic of two - step chromatography of an antibody containing trispecific CODV having an LC of Vk2 Fab arm with proL KO mutation and an LC of Vk1 CODV arm with pro - L binding ability. The first step is MSS chromatography. Then, the MSS eluate containing not only the desired triAb but also the mis - paired species of 2×FabLC and 2×CODV LC is further purified in two steps on protein - L resin. 【Figure 13】 Shown is an SDS-PAGE gel of a sample from the two-step purification of the triple-specific CODV, stained with Coomassie. 【Figure 14】 Shown are size-exclusion chromatography (aSEC) data for the analysis of wild-type and ProL KO mutant versions of adalimumab after MSS purification. The percentages of the expected main peaks are shown. 【Figure 15】 Shown is the sample analysis of WT adalimumab and the S12P-R18P mutant taken at the end of the accelerated stability test (2 weeks at 40 °C). The upper panel shows the DS-PAGE gels of Coomassie-stained non-reduced and reduced samples. The lower panel shows the aSEC data for the respective corresponding samples. The percentages of the expected main peaks are shown. 【Figure 16】 Shown are differential scanning fluorimetry (nano-DSF) data and the derived Tm for WT adalimumab and the S12P-R18P mutant. 【Figure 17】 Shown are size-exclusion chromatography (aSEC) data for the analysis of wild-type and KS KO mutant versions of adalimumab after MSS purification. The percentages of the expected main peaks are shown. 【Figure 18】 Shown is the sample analysis of WT adalimumab and three CL mutants taken at the end of the accelerated stability test (2 weeks at 40 °C). The upper panel shows the SDS-PAGE gels of Coomassie-stained non-reduced and reduced samples. The lower panel shows the aSEC data for the respective corresponding samples. The percentage of the expected main peak. 【Figure 19】 Shown is the DSC analysis of F(ab)’2 derived from WT adalimumab and three KS KO mutants. 【Figure 20】 Shown are nano-DSF data and the derived Tm for WT adalimumab and three KS KO mutants. 【Figure 21】 Shown are SDS-PAGE gels of Coomassie-stained non-reduced and reduced samples after a three-step purification process. On the left, the identification of selected high-molecular-weight bands is shown. 【Figure 22】SDS-PAGE gels of Coomassie-stained non-reduced and reduced samples after the purification process of the 3-step process are shown. Identification of selected high molecular weight bands is shown on the left. 【Figure 23A】 Chromatograms of MSS purification of bispecific antibodies containing KS KO and ProL KO mutations as part of the 3-step purification process are shown. 【Figure 23B】 Chromatograms of the MSS step, KS step and ProL step are shown in Figure 23B. 【Figure 24】 SDS-PAGE gel of a sample from the purification of trispecific CODV stained with Coomassie is shown. 【Figure 25A】 Chromatogram of an adalimumab variant containing a light chain substituted with His198Arg with amino acid numbering according to the EU index is shown. 【Figure 25B】 Chromatogram of an adalimumab variant containing a light chain substituted with Gln199Trp with amino acid numbering according to the EU index is shown. 【Mode for Carrying Out the Invention】 【0043】 Overview The present invention provides a multivalent binding protein comprising four polypeptide chains, wherein a first heavy chain polypeptide and a first light chain polypeptide associate to form one or more antigen-binding domains, and a second heavy chain polypeptide and a second light chain polypeptide associate and bind to the one or more antigen-binding domains. In some embodiments, the multivalent binding protein binds to two or more antigens. To reduce the presence of proteins containing mispaired polypeptide chains formed during the production of the multivalent binding protein, amino acid substitutions are introduced into one light chain to essentially prevent binding to Kappa Select chromatography material, and amino acid substitutions are introduced into other light chain polypeptides to essentially prevent binding to Protein L chromatography material. Further, to further reduce mispaired polypeptides in the multivalent binding protein preparation, the Fc portion of the multivalent binding protein may include a knob-into-hole mutation and an RF mutation (e.g., H435R and 436F mutations, with amino acid numbering according to the EU index). 【0044】 The present invention also provides two-step and three-step chromatography methods for purifying the multivalent binding protein of the present invention, which utilize Kappa Select chromatography and Protein L chromatography, with or without Protein A chromatography. Proteins containing mispaired polypeptides are removed from the multivalent binding protein preparation based on their lack of Kappa Select binding and / or Protein L binding. 【0045】 General Definitions As used in accordance with the present disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated. Unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. 【0046】 As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer that is at least 10 nucleotides in length. In certain embodiments, the nucleotides that make up the polynucleotide can be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, and phosphoramidate. The term "polynucleotide" specifically includes DNA in both single-stranded and double-stranded forms. 【0047】 An "isolated polynucleotide" is a polynucleotide that is of genomic, cDNA, or synthetic origin, or a combination of some of these, and (1) is not associated with all or a portion of the polynucleotide as it is found in nature, (2) is ligated to a polynucleotide that is not ligated in nature, or (3) does not occur in nature as part of a larger sequence. 【0048】 An "isolated polypeptide" is one that (1) does not contain at least some of the other polypeptides 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 different species, (4) is separated from at least about 50% of polynucleotides, lipids, carbohydrates, or other substances that naturally associate, (5) does not associate (by covalent or non-covalent interactions) with a part of a polypeptide with which the "isolated polypeptide" naturally associates, (6) is operably associated (by covalent or non-covalent interactions) with a polypeptide that does not naturally associate, or (7) is not naturally occurring. Such isolated polypeptides can 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 found in its natural environment that interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise). 【0049】 Naturally occurring antibodies typically consist of tetramers. Each such tetramer typically consists of a pair of two identical polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50 - 70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity for a target antigen. The amino-terminal portion of each light chain and each heavy chain typically contains a variable domain of about 100 - 110 or more amino acids typically involved in antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain involved in effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide contains a variable domain (V H ) and three constant domains (C H1 , C H2 , and C H3 ), with V HThere is a domain and a C at the carboxyl terminus H3 There is a domain and the full-length light chain immunoglobulin polypeptide has a variable domain (V L ) and a constant domain (C L ), and there is a V domain at the amino terminus of the polypeptide and a C L domain at the carboxyl terminus L domain 【0050】 Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and 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 subclasses including, but not limited to, IgM1 and IgM2. Similarly, IgA is subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within the full-length light and heavy chains, typically, the variable and constant domains are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 or more amino acids. See, for example, Fundamental Immunology (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated herein by reference in its entirety for all purposes. The variable regions of each light chain / heavy chain pair typically form the antigen-binding site. The variable domains of naturally occurring antibodies typically have relatively conserved framework regions (FRs) of the same overall structure, linked by three hypervariable regions, also called complementarity-determining regions or CDRs. Typically, the FRs are positioned between the CDRs from the two chains of each pair, which may thereby enable binding to a specific epitope. The variable domains of both the light and heavy chains typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus to the carboxyl terminus 【0051】 The term "CDR set" refers to a group of three CDRs present within a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are variously defined according to different methods. The method represented by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) provides not only a clear residue numbering system applicable to any variable region of an antibody, but also the exact residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia et al. (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 342;877-83) discovered that, despite being highly diverse at the amino acid sequence level, certain sub-portions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These sub-portions are designated as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" mean the light chain region and the heavy chain region, respectively. These regions may be referred to as Chothia CDRs and have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs overlapping with the Kabat CDRs are described in Padlan, 1995, FASEB J. 9;133-39; MacCallum, 1996, J. Mol. Biol. 262(5);732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27;55-77. Although other definitions of CDR boundaries need not strictly follow one of the methods herein, these may be shortened or extended in consideration of predictions or experimental findings that, while overlapping with the Kabat CDRs, certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these methods, but in certain embodiments, CDRs defined by Kabat or Chothia are used.Identifying CDRs predicted using amino acid sequences is well known in the art, such as in Martin, A.C. “Protein sequence and structure analysis of antibody variable domains,” In Antibody Engineering, Vol. 2. Kontermann R., Duebel S., eds. Springer-Verlag, Berlin, p. 33-51 (2010). Also, the amino acid sequences of the heavy and / or light chain variable domains may be examined to identify the CDR sequences by comparing them to other known amino acid sequences of other heavy and light chain variable regions, and the hypervariable regions of the sequences may be determined. The numbered sequences may be aligned visually or by using an alignment program such as the CLUSTAL program suite described in Thompson, 1994, Nucleic Acids Res. 22;4673-80. Molecular models are customarily used to accurately delineate the framework and CDR regions, thereby correcting sequence-based assignments. 【0052】 As used herein, the term "Fc" refers to a molecule containing the sequence of a non-antigen-binding fragment, which may be produced by digesting an antibody or produced by other means, and may contain a hinge region, regardless of whether it is in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably of human origin and can be any immunoglobulin, but IgG1 and IgG2 are preferred. Fc molecules are composed of monomeric polypeptides and can be linked by covalent bonds (i.e., disulfide bonds) and non-covalent bonds to form dimeric or multimeric forms. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of Fc is a disulfide-linked dimer obtained by papain digestion of IgG. As used herein, the term "native Fc" collectively refers to monomeric, dimeric, and multimeric forms. 【0053】 F(ab) fragments typically contain one light chain and the V H and C H1 domains of one heavy chain, and the V of the F(ab) fragment H -C H1 heavy chain portion cannot form a disulfide bond with another heavy chain polypeptide. As used herein, an F(ab) fragment may also contain one light chain containing two variable domains separated by an amino acid linker, and one heavy chain containing two variable domains separated by an amino acid linker and the C H1 domain. 【0054】 F(ab’) fragments typically contain one light chain and a portion of one heavy chain that contains much of the constant region (between the C H1 domain and the C H2 domain) such that it can form an interchain disulfide bond between the two heavy chains to form an F(ab’)2 molecule. 【0055】 As used herein, the term "multivalent binding protein" refers to a non-naturally occurring (or recombinant or engineered) molecule that includes two or more binding domains, wherein the two or more binding domains bind to two or more target antigens. In some examples, the binding protein includes four polypeptide chains, typically a first heavy chain polypeptide and a first light chain polypeptide that associate to form at least one antigen-binding domain, and a second heavy chain polypeptide and a second light chain polypeptide that associate to form at least one antigen-binding domain. 【0056】 A "recombinant" molecule is a molecule that has been prepared, expressed, produced, or isolated by recombinant means. 【0057】 One embodiment of the present disclosure provides a multivalent binding protein having biological and immunological specificity for two or more antigens. Another embodiment of the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a multivalent binding protein. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a multivalent binding protein. Yet another embodiment of the present disclosure provides a host cell that expresses such a multivalent binding protein (i.e., that comprises a nucleic acid molecule or vector encoding a polypeptide chain that forms such a binding protein). 【0058】 As used herein, the term "exchangeability" refers to the interchangeability of variable domains within the context of the binding protein format while maintaining folding and final binding affinity. "Full exchangeability" means that within the polypeptide chain of Formula I or the polypeptide chain of Formula II, while maintaining the full functionality of the binding protein as demonstrated by maintaining binding affinity, the V H1 domain and the V H2 domain in both orders, and thus the order of the V L1 domain and the V L2 domain can be exchanged (i.e., reversed). Further, V H and VL It should be noted that such notations only refer to the positions of the domains of a specific protein chain in the final form. For example, V H1 and V H2 are derived from the V L1 and V L2 domains of the parental antibody and can be arranged at the positions of V H1 and V H2 of the binding protein. Similarly, V L1 and V L2 are derived from the V H1 and V H2 domains of the parental antibody and can be arranged at the positions of V H1 and V H2 of the binding protein. Therefore, the notations V H and V L refer to the current positions and not the original positions in the parental antibody. Therefore, the V H domain and the V L domain are "exchangeable". 【0059】 As used herein, the terms "antigen" or "target antigen" or "antigen target" refer to a molecule or a part of a molecule to which a binding protein can bind and which can be used in an animal to produce an antibody that can further bind to an epitope of the antigen. The target antigen can have one or more epitopes. For each target antigen recognized by the binding protein, the binding protein can compete with an intact antibody that recognizes the target antigen. 【0060】 The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target. 【0061】 The term "monovalent binding protein" refers to a binding protein having one antigen-binding site. 【0062】 The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. 【0063】 The term "bivalent binding protein" refers to a binding protein having two binding sites. 【0064】 The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. 【0065】 The term "trivalent binding protein" refers to a binding protein having 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. 【0066】 The term "tetraspecific binding protein" refers to a binding protein that specifically binds to four different antigen targets. 【0067】 The term "tetravalent binding protein" refers to a binding protein having four binding sites. In certain embodiments, the tetravalent binding protein can bind to one antigen target. In other embodiments, the tetravalent binding protein can bind to two antigen targets. In other embodiments, the tetravalent binding protein can bind to three antigen targets. In other embodiments, the tetravalent binding protein can bind to four antigen targets. 【0068】 An "isolated" binding protein is one that has been identified, separated, and / or recovered from the components of its natural environment. The contaminating components of its natural environment are materials that interfere with the diagnostic or therapeutic use of the binding protein and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein is purified to the extent that (1) the antibody exceeds 95% by weight, most preferably exceeds 99% by weight, as measured by the Lowry method, (2) at least 15 residues of the N-terminal or internal amino acid sequence are sufficiently obtained by use of a spinning cup sequenator, or (3) is homogeneous by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. A binding protein that is present in situ within a recombinant cell is included within the isolated binding protein because at least one component of its natural environment is absent. 【0069】 As used herein, the terms "substantially pure" or "substantially purified" refer to a compound or species that is the predominant species present (i.e., more abundant on a molar basis than any other individual 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 the species comprises at least about 50% (on a molar basis) of all polymeric species present. In other embodiments, a substantially pure composition comprises greater than about 80%, 85%, 90%, 95%, or 99% of all polymeric species present in the composition. In still other embodiments, the species is purified until it is essentially homogeneous (no contaminating species can be detected in the composition by conventional detection methods) and the composition consists essentially of a single polymeric species. In yet further embodiments, the species is present in a form of improved purity such that it is more pure than it is in its natural environment and / or than when it was first synthesized and / or amplified under laboratory conditions. 【0070】 As used herein, a "neutralizing" binding protein refers to a molecule that can inhibit or substantially reduce the effector function of the target antigen to which it binds. As used herein, "substantially reduce" means reducing the effector function of the target antigen 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%. 【0071】 The term "epitope" includes any determinant, preferably a polypeptide determinant, that can specifically bind to an immunoglobulin or a T cell receptor. In certain embodiments, the epitope determinant includes chemically active surface groups 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 the region of an antigen to which an antibody or binding protein binds. In certain embodiments, a binding protein is said to specifically bind to an antigen when it preferentially recognizes its 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, most preferably when the dissociation constant is ≦10 -10 M, the binding protein is said to specifically bind to the antigen. 【0072】 The dissociation constant (K D) can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures in real time the binding interaction between a ligand (target antigen on the 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 the biosensor matrix) and presenting the ligand (target antigen). As used herein, the term "K D " refers to the dissociation constant of the interaction between a specific binding protein and a target antigen. 【0073】 As used herein, the term "specifically binds" means that a binding protein or an antigen-binding fragment thereof binds to an antigen containing an epitope with a Kd of at least about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 M, or greater, and / or the ability to bind to the epitope with an affinity at least 2-fold greater than the affinity for a non-specific antigen. 【0074】 As used herein, the term "linker" refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold and cross over to form an immunoglobulin of the dual variable domain. The linker is inserted at the sequence level at the transition between the variable domains or between the variable domain and the constant domain, respectively. Since the approximate size of the immunoglobulin domain is well understood, the transition between domains can be identified. The exact position of the domain transition can be determined by identifying a peptide stretch that does not form secondary structure elements such as β-sheets or α-helices, as demonstrated by experimental data or as can be estimated by techniques for modeling or predicting secondary structure. For example, with respect to the exemplary multivalent binding protein shown in Figure 1F, the linker is located in the light chain between the C-terminus of L1(V L2 and the N-terminus of the V L1 domain); and between the C-terminus of L2(V L1 and the N-terminus of the C L domain). The heavy chain linker is known as L3, located between the C-terminus of V H1 and the N-terminus of the V H2 domain; and L4, located between the C-terminus of V H2 and the N-terminus of the C H1 domain. 【0075】 As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to introduce coding information into a host cell. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, into which additional DNA segments can be inserted into the viral genome. Certain vectors are capable of self-replicating in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell when introduced into the host cell and can thereby replicate with the host genome. Furthermore, certain vectors are capable of inducing the expression of a gene operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vectors, the terms "plasmid" and "vector" may be used interchangeably herein. However, the present disclosure is intended to include other forms of expression vectors such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions. 【0076】 As used herein, the term "recombinant host cell" (or "host cell") refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to a particular target cell, but also to the progeny of such a cell. Because of mutations or environmental influences, specific modifications may occur in later generations, and such progeny may not be identical to the parental cells in fact, but nevertheless, such cells are included within the scope of the term "host cell" as used herein. A variety of host cell expression systems can be used to express the binding protein, including bacteria, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To recombinantly express the binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors having a DNA fragment encoding the polypeptide chain of the binding protein so as to express the polypeptide chain in the host cell, and preferably, the binding protein can be recovered from the medium in which the host cell is cultured by secreting it into the medium. 【0077】 As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, and a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed when it has been genetically modified from its natural state. After transformation, this transformed DNA may be recombined with the cell's DNA by physically integrating it into the cell's chromosome, or it may be transiently maintained as an episomal element without replication, or it may replicate independently as a plasmid. When the DNA is replicated during cell division, the cell is considered to be stably transformed. As used herein, the term "transfection" refers to the uptake of exogenous or foreign DNA by a cell, and a cell is "transfected" when the foreign DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art. Using such techniques, one or more exogenous DNA molecules can be introduced into a suitable host cell. 【0078】 As used herein, the term "naturally occurring" as applied to an object refers to the fact that the object can be found 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 natural source and has not been intentionally modified by humans is naturally occurring. Similarly, as used herein, "not naturally occurring" refers to an object that is not found in nature or has been structurally modified or synthesized by humans. 【0079】 As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the 20 conventional amino acids (e.g., d - amino acids); unnatural amino acids and analogs such as α-,α - disubstituted amino acids, N - alkyl amino acids, and lactic acid, and other non - conventional amino acids can also be suitable components of the polypeptide chains of conjugated proteins. Examples of non - conventional amino acids include 4 - hydroxyproline, γ - carboxyglutamate, 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 notation used herein, in accordance with standard usage and convention, the left - hand direction is the amino - terminal direction and the right - hand direction is the carboxyl - terminal direction. 【0080】 Naturally occurring residues can be subdivided into the following classifications based on common side - chain characteristics: (1) Hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) Aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues affecting chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyr. 【0081】 Conservative amino acid substitutions can involve the exchange of one member of one of these classifications with another member of the same classification. Non-conservative substitutions can involve the exchange of one member of one of these classifications with a member of another classification. 【0082】 One of ordinary skill in the art can determine suitable variants of the polypeptide chain of the multivalent binding protein using well-known techniques. For example, one of ordinary skill in the art can identify suitable regions of the polypeptide chain that can be altered without impairing activity by targeting regions that do not appear to be important for activity. Alternatively, one of ordinary skill in the art can identify residues and portions of molecules that are conserved among similar polypeptides. In addition, even in regions that may be important for biological activity or structure, conservative amino acid substitutions can be made without impairing biological activity or causing a detrimental effect on the polypeptide structure. 【0083】 As used herein, the terms "pharmaceutical composition" or "therapeutic composition" refer to a compound or composition that can induce a desired therapeutic effect when appropriately administered to a patient. 【0084】 As used herein, the terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refer to one or more formulation materials suitable for achieving or enhancing the delivery of the binding protein. 【0085】 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 the binding protein sufficient to inhibit one or more of the clinically defined pathological processes associated with the condition being treated over a period of time. The effective amount can vary depending on the particular binding protein used, as well as on various factors and conditions related to the patient being treated and the severity of the disorder. For example, when administering a binding protein in vivo, factors such as the patient's age, weight, and health status, as well as the dose-response curve and toxicity data obtained from preclinical animal studies, are considered among those factors. Determining the effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of one of ordinary skill in the art. 【0086】 According to one embodiment of the present disclosure, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein. 【0087】 All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety as if each individual reference were specifically and individually indicated and incorporated by reference. 【0088】 A multivalent binding protein with reduced binding to KappaSelect and / or Protein L chromatography materials Exemplary bivalent binding proteins In some embodiments, there is provided a multivalent binding protein (e.g., a bispecific antibody) comprising four polypeptide chains that form two antigen-binding domains; the four polypeptide chains comprise a first heavy chain polypeptide having a structure represented by the following formula: VH1-CH1 [I], a first light chain polypeptide chain having a structure represented by the following formula: VL1-CL1[II], a second heavy chain polypeptide having a structure represented by the following formula: VH2-CH1 [III], and a second light chain polypeptide chain comprising a structure represented by the following formula: VL2-CL2[IV] comprising; wherein VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain, VL2 is a κ1, κ3, or κ4 subtype light chain variable domain, CL1 is a first immunoglobulin light chain constant domain, 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; CL1 contains one or more amino acid substitutions, thereby reducing binding to KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing binding to protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions, VH1 and VL1 associate to form a first antigen-binding domain, and VH2 and VL2 associate to form a second antigen-binding domain. 【0089】 In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that comprises four polypeptide chains that form two antigen-binding domains; the four polypeptide chains comprise a first heavy chain polypeptide having a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], a first light chain polypeptide chain having a structure represented by the following formula: VL1-CL1[II], a second heavy chain polypeptide having a structure represented by the following formula: VH2-CH1-CH2-CH3 [III], and a second light chain polypeptide chain having a structure represented by the following formula: VL2-CL2[IV] comprising; In the formula, 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, CH2, and CH3 are immunoglobulin heavy chain constant domains; CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions, VH1 and VL1 associate to form the first antigen-binding domain, and VH2 and VL2 associate to form the second antigen-binding domain. An example of this embodiment is shown in FIG. 1A. 【0090】 In some embodiments, the binding of the CL1 of a multivalent protein (e.g., a bispecific antibody) to a KappaSelect chromatography material is reduced by about 90% compared to the binding of the CL1 of a multivalent protein (e.g., a bispecific antibody) that does not contain one or more amino acid substitutions. In some embodiments, the binding of the VL2 of a multivalent protein to a protein L chromatography material is reduced by about 90% compared to the binding of the VL2 of a multivalent protein that does not contain one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in the CL1 of a multivalent protein are at positions corresponding to 109, 110, or 199, and the amino acid numbering is according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63;78-85). In some embodiments, one or more amino acid substitutions in the CL1 are a T109A substitution, a V110D substitution, a Q199K substitution, a T109A-V110D substitution, or a T109A-V110D-Q199K substitution, and the amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in the VL2 of a multivalent protein are framework amino acid substitutions. In some embodiments, one or more amino acid substitutions in the VL2 of a multivalent protein are at positions corresponding to 12 or 18, and 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 a multivalent protein are an S12P substitution, an R18P substitution, an R18Q substitution, an S12P-R18P substitution, or an S12P-R18Q substitution, and the amino acid numbering is according to Kabat. 【0091】 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 the CH3 domain of human IgG1 or IgG4. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, with amino acid numbering according to the EU index. See, for example, Spiess et al. (2013) JBC, 288(37); P26583-26593. In some embodiments, the amino acid substitutions are S354C and T366W of human IgG1 (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W (or corresponding thereto), with amino acid numbering according to the EU index. 【0092】 In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (e.g., 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 (e.g., further comprises) one or more amino acid substitutions that reduce binding to Protein A. In some embodiments, one or more amino acid substitutions in the CH3 domain of the first or second heavy chain polypeptide that reduce binding to Protein A chromatography material are amino acid substitutions at positions corresponding to (e.g., relative to) positions 435 and 436 of human IgG1, and the numbering is according to the EU index. In some embodiments, the amino acid substitutions are H435R and Y436F (or corresponding thereto), and the amino acid numbering is according to the EU index. 【0093】 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 different species than the second heavy chain polypeptide. Additionally or alternatively, in some embodiments, the first light chain polypeptide is derived from a different species than the second light chain polypeptide. An example of this embodiment is shown in FIG. 1B. In some embodiments, the first heavy chain polypeptide and the first light chain polypeptide are each derived from mouse heavy chain immunoglobulin and mouse light chain immunoglobulin, and the second heavy chain polypeptide and the second light chain polypeptide are each derived from rat heavy chain immunoglobulin and rat light chain immunoglobulin. In some embodiments, the first heavy chain polypeptide and the first light chain polypeptide are each derived from rat heavy chain immunoglobulin and rat light chain immunoglobulin, and the second heavy chain polypeptide and the second light chain polypeptide are each derived from mouse heavy chain immunoglobulin and mouse light chain immunoglobulin. 【0094】 In some embodiments, the first heavy chain polypeptide and the second heavy chain polypeptide of the multivalent binding protein each comprise the CH3 domain of IgG4. An example of this embodiment is shown in FIG. 1C. In some embodiments, the first heavy chain polypeptide comprises the K409R amino acid substitution and the second heavy chain polypeptide comprises the F405L amino acid substitution, numbering according to the EU index (see FIG. 1C). In some embodiments, the first heavy chain polypeptide comprises the F405L amino acid substitution and the second heavy chain polypeptide comprises the K409R amino acid substitution, numbering 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 an antigen-binding fragment thereof. 【0095】 Exemplary trivalent binding protein In some embodiments, the first heavy chain polypeptide chain of the multivalent binding protein has a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3-L-VL3[Ia] comprising the first light chain polypeptide chain has a structure represented by the following formula: VL1-CL1[II] comprising the second heavy chain polypeptide has a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIa] comprising the second light chain polypeptide chain has a structure represented by the following formula: VL2-CL2[IV] comprising; In the formula, 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; 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; CH1, CH2, and CH3 are immunoglobulin heavy chain constant domains; CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL1 without one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material compared to VL2 without one or more amino acid substitutions, VH1 and VL1 associate to form the first antigen-binding domain, VH2 and VL2 associate to form the second antigen-binding domain, VH3 and VL3 associate to form the third antigen-binding domain, and VH3 and VL3 are linked via the amino acid linker L. In some embodiments, VL3 does not bind to the protein L chromatography material. In some embodiments, VL3 is a lambda subtype immunoglobulin light chain variable domain or a κ2 immunoglobulin light chain variable domain. In some embodiments, L has a length of 0 amino acids. In some embodiments, a linker with a length of 0 amino acids indicates that there is no linker in the binding protein. In some embodiments, L has a length of at least 1 amino acid. An example of this embodiment is shown in FIG. 1D. In some embodiments, the first heavy chain polypeptide chain of the multivalent binding protein has a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3[Ib] comprises The second heavy chain polypeptide has a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIb] comprises. 【0096】 An example of this embodiment is shown in FIG. 1E. In 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 to two or three different antigens. In some embodiments, 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, 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. In some embodiments, the multivalent binding protein is a multispecific antibody or an antigen-binding fragment thereof. 【0097】 In some embodiments, the binding of the CL1 of a multivalent protein (e.g., a trispecific antibody) to a KappaSelect chromatography material is reduced by about 90% compared to the binding of the CL1 of a multivalent protein that does not contain one or more amino acid substitutions (e.g., a bispecific antibody). In some embodiments, the binding of the VL2 of a multivalent protein to a protein L chromatography material is reduced by about 90% compared to the binding of the VL2 of a multivalent protein that does not contain one or more amino acid substitutions. In some embodiments, the binding of the VL3 of a multivalent protein to a protein L chromatography material is reduced by about 90% compared to the binding of the VL3 of a multivalent protein that does not contain one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in the CL1 of a multivalent protein are at positions corresponding to 109, 110, or 199, with numbering according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63;78-85). In some embodiments, one or more amino acid substitutions in the CL1 are a T109A substitution, a V110D substitution, a Q199K substitution, a T109A-V110D substitution, or a T109A-V110D-Q199K substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in the CL1 of a multivalent protein are at positions corresponding to 109, 198, 199, or 202, with numbering according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63;78-85). In some embodiments, one or more amino acid substitutions in the CL1 are an H198R substitution, a Q199W substitution, or a T109A-S202R substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in the VL2 of a multivalent protein are framework amino acid substitutions.In some embodiments, one or more amino acid substitutions in VL2 of the multivalent protein are at positions corresponding to 12 or 18, numbered 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 VL2 of the multivalent protein are an S12P substitution, an R18P substitution, an R18Q substitution, an S12P-R18P substitution, or an S12P-R18Q substitution, numbered according to Kabat. In some embodiments, one or more amino acid substitutions in VL3 of the multivalent protein are framework amino acid substitutions. In some embodiments, one or more amino acid substitutions in VL3 of the multivalent protein are at positions corresponding to 12 or 18, numbered 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 VL3 of the multivalent protein are an S12P substitution, an R18P substitution, an R18Q substitution, an S12P-R18P substitution, or an S12P-R18Q substitution, numbered according to Kabat. 【0098】 In some embodiments, the binding of a multivalent protein (e.g., a trispecific antibody) to a KappaSelect chromatography material of CL2 is reduced by about 90% compared to the binding of a multivalent protein (e.g., a bispecific antibody) without one or more amino acid substitutions to CL2. In some embodiments, the binding of VL1 of a multivalent protein to a protein L chromatography material is reduced by about 90% compared to the binding of VL1 of a multivalent protein without one or more amino acid substitutions. In some embodiments, the binding of VL3 of a multivalent protein to a protein L chromatography material is reduced by about 90% compared to the binding of VL3 of a multivalent protein without one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in CL2 of a multivalent protein are at positions corresponding to 109, 110, or 199, numbered according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63;78-85). In some embodiments, one or more amino acid substitutions in CL2 are a T109A substitution, a V110D substitution, a Q199K substitution, a T109A-V110D substitution, or a T109A-V110D-Q199K substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in CL2 of a multivalent protein are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63;78-85). In some embodiments, one or more amino acid substitutions in CL2 are an H198R substitution, a Q199W substitution, or a T109A-S202R substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in VL1 of a multivalent protein are framework amino acid substitutions.In some embodiments, one or more amino acid substitutions in VL1 of the multivalent binding protein are at positions corresponding to 12 or 18, numbered 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 VL1 of the multivalent binding protein are an S12P substitution, an R18P substitution, an R18Q substitution, an S12P-R18P substitution, or an S12P-R18Q substitution, numbered according to Kabat. In some embodiments, one or more amino acid substitutions in VL3 of the multivalent binding protein are framework amino acid substitutions. In some embodiments, one or more amino acid substitutions in VL3 of the multivalent binding protein are at positions corresponding to 12 or 18, numbered 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 VL3 of the multivalent binding protein are an S12P substitution, an R18P substitution, an R18Q substitution, an S12P-R18P substitution, or an S12P-R18Q substitution, numbered according to Kabat. 【0099】 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 the CH3 domain of human IgG1 or IgG4. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, with amino acid numbering according to the EU index. See, for example, Spiess et al. (2013) JBC, 288(37); P26583-26593. In some embodiments, the amino acid substitutions are S354C and T366W of human IgG1 (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W (or corresponding thereto), with amino acid numbering according to the EU index. 【0100】 In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (e.g., 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 (e.g., further comprises) one or more amino acid substitutions that reduce binding to Protein A. In some embodiments, one or more amino acid substitutions in the CH3 domain of the first or second heavy chain polypeptide that reduce binding to Protein A chromatography material are amino acid substitutions at positions corresponding to (e.g., relative to) positions 435 and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are H435R and Y436F (or corresponding thereto), with amino acid numbering according to the EU index. 【0101】 Exemplary CODV multivalent binding proteins In some embodiments, there is provided a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains that form three antigen-binding domains; the four polypeptide chains comprise a first heavy chain polypeptide having a structure represented by the following formula: VH1-L3-VH2-L4-CH1 [I], a first light chain polypeptide chain having a structure represented by the following formula: VL2-L1-VL1-L2-CL1[II], a second heavy chain polypeptide having a structure represented by the following formula: VH3-CH1 [III], and a second light chain polypeptide chain having a structure represented by the following formula: VL3-CL2[IV] and In the formula, 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; L1, L2, L3, and L4 are amino acid linkers; the polypeptide of formula I and the polypeptide of formula II form an intersected light chain-heavy chain pair; VH1 and VL1 associate to form the first antigen-binding domain, VH2 and VL2 associate to form the second antigen-binding domain, and VH3 and VL3 associate to form the third antigen-binding domain; a) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, and VL3 contains one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material compared to VL3 that does not contain one or more amino acid substitutions; b) CL2 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material compared to VL1 and VL2 that do not contain one or more amino acid substitutions; c) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions. VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material compared to VL1 that does not contain one or more amino acid substitutions. VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions. VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions. VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain. In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that comprises four polypeptide chains forming three antigen-binding domains; the four polypeptide chains comprise a first heavy chain polypeptide having a structure represented by the following formula: VH1-L3-VH2-L4-CH1-CH2-CH3 [Ia], a first light chain polypeptide chain having a structure represented by the following formula: VL2-L1-VL1-L2-CL1[II], a second heavy chain polypeptide having a structure represented by the following formula: VH3-CH1-CH2-CH3 [IIIa], and a second light chain polypeptide chain having a structure represented by the following formula: VL3-CL2[IV] and Wherein, 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; L1, L2, L3 and L4 are amino acid linkers; the polypeptide of formula I and the polypeptide of formula II form an intersected light chain-heavy chain pair; VH1 and VL1 associate to form the first antigen-binding domain, VH2 and VL2 associate to form the second antigen-binding domain, and VH3 and VL3 associate to form the third antigen-binding domain; a) CL1 contains one or more amino acid substitutions, whereby the binding to the KappaSelect chromatography material is reduced as compared to CL1 that does not contain one or more amino acid substitutions, and VL3 contains one or more amino acid substitutions, whereby the binding to the Protein L chromatography material is reduced as compared to VL2 that does not contain one or more amino acid substitutions; b) CL2 contains one or more amino acid substitutions, whereby the binding to the KappaSelect chromatography material is reduced as compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, whereby the binding to the Protein L chromatography material is reduced as compared to VL1 and VL2 that do not contain one or more amino acid substitutions; c) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions. VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material compared to VL1 that does not contain one or more amino acid substitutions. VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; or d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions. VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions. VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain. An example of this embodiment is shown in Figure 1F. In some embodiments, the binding protein is trispecific and can specifically bind to three different antigen targets. In some embodiments, one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, a 0 amino acid long linker indicates the absence of a linker in the binding protein. In some embodiments, L1, L2, L3, or L4 are each independently at least 1 amino acid in length. 【0102】 In some embodiments, the second heavy chain polypeptide of the binding protein (e.g., a multivalent binding protein) has a structure represented by the following formula: VH3-L5-VH4-L6-CH1 [IIIb] and includes The second light chain polypeptide chain of the binding protein (e.g., a multivalent binding protein) has a structure represented by the following formula: VL4-L7-VL3-L8-CL2 [IVa] and includes In the formula, 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, the polypeptide of formula IIIa and the polypeptide of formula IVa form an intersected light chain-heavy chain pair, and VH4 and VL4 associate to form the fourth antigen-binding domain; a) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, and VL3 and VL4 each contain one or more amino acid substitutions, thereby reducing the binding to the Protein L chromatography material compared to VL3 and VL4 that do not contain one or more amino acid substitutions; b) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, VL3 contains one or more amino acid substitutions, thereby reducing the binding to the Protein L chromatography material compared to VL3 that does not contain one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a κ2 subtype light chain variable immunoglobulin domain; c) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, VL4 contains one or more amino acid substitutions, thereby reducing the binding to the Protein L chromatography material compared to VL4 that does not contain one or more amino acid substitutions, and VL3 is a lambda subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain; d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, thereby reducing their binding to the Protein L chromatography material as compared to VL1 and VL2 that do not contain one or more amino acid substitutions; e) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; or f) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 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) has a structure represented by the following formula: VH3-L5-VH4-L6-CH1-CH2-CH3 [IIIc] comprising In the formula, CH2 is the immunoglobulin CH2 heavy chain constant domain, and CH3 is the immunoglobulin CH3 heavy chain constant domain. An example of this embodiment is shown in FIG. 1G. In some embodiments, the binding protein is quadrivalent and can specifically bind to four different antigen targets. In some embodiments, 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 no linker is present in the binding protein. In some embodiments, L1, L2, L3, L4, L5, L6, L7, or L8 are each independently at least 1 amino acid in length. 【0103】 In one embodiment, the binding protein of the present disclosure is a trispecific and / or trivalent binding protein comprising four polypeptide chains that form 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 has a structure represented by the following formula: VL2-L1-VL1-L2-CL1[I] comprising The second polypeptide chain has a structure represented by the following formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II] comprising The third polypeptide chain has a structure represented by the following formula: VH3-CH1-hinge-CH2-CH3 [III] comprising The fourth polypeptide chain has a structure represented by the following formula: VL3-CL2[IV] comprising wherein 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 hinge region of the immunoglobulin that links the CH1 domain and the CH2 domain; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form an intersected light chain-heavy chain pair, VH1 and VL1 associate to form a first antigen-binding domain, VH2 and VL2 associate to form a second antigen-binding domain, and VH3 and VL3 associate to form a third antigen-binding domain; a) CL1 contains one or more amino acid substitutions, which reduce the binding to KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, and VL3 contains one or more amino acid substitutions, which reduce the binding to protein L chromatography material compared to VL3 that does not contain one or more amino acid substitutions; b) CL2 contains one or more amino acid substitutions, which reduce the binding to KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, which reduce the binding to protein L chromatography material compared to VL1 and VL2 that do not contain one or more amino acid substitutions; c) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions. VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions. VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain. 【0104】 In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-orientation that forms two individual 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, a third polypeptide and a 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. 【0105】 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 of the antigen-binding sites bind to the same antigen target. In these embodiments, the binding protein comprises two identical binding domains or different binding domains and / or specifically binds to different antigens or different epitopes on the same antigen target. In some embodiments, three of the antigen-binding sites bind to the same antigen target. In these embodiments, the binding protein comprises three identical binding domains or different binding domains and / or specifically binds to different antigens or different epitopes on the same antigen target. 【0106】 In some embodiments, the binding of CL1 of the multivalent protein to the KappaSelect chromatography material is reduced by about 90% compared to the binding of CL1 of the multivalent protein that does not contain one or more amino acid substitutions. In some embodiments, the binding of VL4 (if present) and / or VL3 of the multivalent protein 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 protein that does not contain one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in CL1 of the multivalent protein are at positions corresponding to 109, 110, or 199, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 are the T109A substitution, the V110D substitution, the Q199K substitution, the T109A-V110D substitution, or the T109A-V110D-Q199K substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 of the multivalent protein are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index (see, for example, Edelman et al., 1969, Proc Natl Acad Sci USA 63;78-85). In some embodiments, one or more amino acid substitutions in CL1 are the H198R substitution, the Q199W substitution, or the T109A-S202R substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in VL4 (if present) and / or VL3 are framework amino acid substitutions. In some embodiments, one or more amino acid substitutions in VL4 (if present) and / or VL3 of the multivalent protein are at positions corresponding to 12 or 18, numbered according to Kabat. In some embodiments, one or more amino acid substitutions in VL4 (if present) and / or VL3 are the S12P substitution, the R18P substitution, the R18Q substitution, the S12P-R18P substitution, or the S12P-R18Q substitution, with amino acid numbering according to Kabat. 【0107】 In some embodiments, the binding of CL2 of the multivalent protein to the KappaSelect chromatography material is reduced by about 90% compared to the binding of CL2 of the multivalent protein that does not contain one or more amino acid substitutions. In some embodiments, the binding of VL1 and / or VL2 of the multivalent protein to the protein L chromatography material is reduced by about 90% compared to the binding of VL1 and / or VL2 of the multivalent protein that does not contain one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in CL2 of the multivalent protein are at positions corresponding to 109, 110, or 199, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in CL2 are the T109A substitution, the V110D substitution, the Q199K substitution, the T109A-V110D substitution, or the T109A-V110D-Q199K substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 of the multivalent protein are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index (see, e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63;78-85). In some embodiments, one or more amino acid substitutions in CL1 are the H198R substitution, the Q199W substitution, or the T109A-S202R substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 are framework amino acid substitutions. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 of the multivalent protein are at positions corresponding to 12 or 18, numbered according to Kabat. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 are the S12P substitution, the R18P substitution, the R18Q substitution, the S12P-R18P substitution, or the S12P-R18Q substitution, with amino acid numbering according to Kabat. 【0108】 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 the CH3 domain of human IgG1 or IgG4. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, with amino acid numbering according to the EU index. See, for example, Spiess et al. (2013) JBC, 288(37); P26583-26593. In some embodiments, the amino acid substitutions are S354C and T366W of human IgG1 (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W (or corresponding thereto), with amino acid numbering according to the EU index. 【0109】 In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (e.g., 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 (e.g., further comprises) one or more amino acid substitutions that reduce binding to Protein A. In some embodiments, one or more amino acid substitutions in the CH3 domain of the first or second heavy chain polypeptide that reduce binding to Protein A chromatography material are amino acid substitutions at positions corresponding to (e.g., relative to) positions 435 and 436 of human IgG1, numbered according to the EU index. In some embodiments, the amino acid substitutions are H435R and Y436F (or corresponding thereto), and the amino acid numbering is according to the EU index. 【0110】 In some embodiments, the multivalent binding protein is a multispecific antibody or an antigen-binding fragment thereof. 【0111】 Exemplary crossmab bivalent binding protein In some embodiments, the binding protein is a multivalent binding protein comprising four polypeptide chains that form two antigen-binding domains; the four polypeptide chains comprise a first heavy chain polypeptide having a structure represented by the following formula: VH1-CH1 [I], a first light chain polypeptide chain having a structure represented by the following formula: VL1-CL1[II], a second heavy chain polypeptide having a structure represented by the following formula: VH2-CL2[III], and a second light chain polypeptide chain having a structure represented by the following formula: VL2-CH1 [IV] and; In the formula, 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, and CH1 is the immunoglobulin heavy chain constant domain. a) CL2 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, and VL1 contains one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions, or b) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, and VL2 contains one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions; VH1 and VL1 associate to form the first antigen-binding domain, and VH2 and VL2 associate to form the second antigen-binding domain. 【0112】 In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that includes four polypeptide chains that form two antigen-binding domains; the four polypeptide chains A first heavy chain polypeptide having a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light chain polypeptide chain having a structure represented by the following formula: VL1-CL1[II], A second heavy chain polypeptide having a structure represented by the following formula: VH2-CL2-CH2-CH3 [III], And a second light chain polypeptide chain having a structure represented by the following formula: VL2-CH1 [IV] comprising; 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; CH3 is an immunoglobulin CH3 heavy chain constant domain; a) CL2 contains one or more amino acid substitutions, thereby reducing the binding to KappaSelect chromatography material as compared to CL2 without one or more amino acid substitutions, VL1 contains one or more amino acid substitutions, thereby reducing the binding to protein L chromatography material as compared to VL1 without one or more amino acid substitutions, or b) CL1 contains one or more amino acid substitutions, thereby reducing the binding to KappaSelect chromatography material as compared to CL1 without one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing the binding to protein L chromatography material as compared to VL2 without one or more amino acid substitutions; 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 shown in FIG. 1H. 【0113】 In some embodiments, the binding protein is a multivalent binding protein comprising four polypeptide chains that form two antigen-binding domains; the four polypeptide chains are a first heavy chain polypeptide having a structure represented by the following formula: VH1-CH1 [I], a first light chain polypeptide chain having a structure represented by the following formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the following formula: VL2-CH1 [III], and a second light chain polypeptide chain comprising a structure represented by the following formula: VH2-CL2[IV] comprising; 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; a) CL2 contains one or more amino acid substitutions, thereby reducing binding to KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions, and VL1 contains one or more amino acid substitutions, thereby reducing binding to Protein L chromatography material compared to VL1 that does not contain one or more amino acid substitutions, or b) CL1 contains one or more amino acid substitutions, thereby reducing binding to KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, and VL2 contains one or more amino acid substitutions, thereby reducing binding to Protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions; VH1 and VL1 associate to form a first antigen-binding domain, and VH2 and VL2 associate to form a second antigen-binding domain. 【0114】 In some embodiments, a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains that form two antigen-binding domains is provided; the four polypeptide chains are a first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light chain polypeptide chain comprising a structure represented by the following formula: VL1-CL1[II], A second heavy chain polypeptide comprising a structure represented by the following formula: VL2-CH1-CH2-CH3 [III], And a second light chain polypeptide chain comprising a structure represented by the following formula: VH2-CL2[IV] Comprising; Wherein 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, and 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, and CH1, CH2, and CH3 are immunoglobulin heavy chain constant domains; a) CL2 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions, and VL1 contains one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material compared to VL1 that does not contain one or more amino acid substitutions, or b) CL1 contains one or more amino acid substitutions, thereby reducing the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, and VL2 contains one or more amino acid substitutions, thereby reducing the binding to the protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions; 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 shown in FIG. 1I. 【0115】 In some embodiments, the binding of CL1 or CL2 of a multivalent binding protein to a KappaSelect chromatography material is reduced by about 90% compared to the binding of CL1 or CL2 of a multivalent binding protein that does not contain one or more amino acid substitutions. In some embodiments, the binding of VL1 or VL2 of a multivalent binding protein to a protein L chromatography material is reduced by about 90% compared to the binding of VL1 or VL2 of a multivalent binding protein that does not contain one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in CL1 or CL2 of a multivalent binding protein are at positions corresponding to 109, 110, or 199, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 or CL2 are the T109A substitution, the V110D substitution, the Q199K substitution, the T109A-V110D substitution, or the T109A-V110D-Q199K substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 or CL2 of a multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 or CL2 are the H198R substitution, the Q199W substitution, or the T109A-S202R substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in VL3 or VL4 of a multivalent binding protein are framework amino acid substitutions. In some embodiments, one or more amino acid substitutions in VL3 or VL4 are at positions corresponding to 12 or 18, numbered according to Kabat. In some embodiments, one or more amino acid substitutions in VL3 or VL4 of a multivalent binding protein are the S12P substitution, the R18P substitution, the R18Q substitution, the S12P-R18P substitution, or the S12P-R18Q substitution, with numbering according to Kabat. 【0116】 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 the CH3 domain of human IgG1 or IgG4. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, and the amino acid numbering is according to the EU index. See, for example, Spiess et al. (2013) JBC, 288(37); P26583 - 26593. In some embodiments, the amino acid substitutions are S354C and T366W of human IgG1 (or corresponding thereto), and the amino acid numbering is according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, and the numbering is according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), and 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 substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, and the numbering is according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), and the amino acid numbering is according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, and the numbering is according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W (or corresponding thereto), and the amino acid numbering is according to the EU index. 【0117】 In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (e.g., 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 (e.g., further comprises) one or more amino acid substitutions that reduce binding to protein A. In some embodiments, one or more amino acid substitutions in the CH3 domain of the first or second heavy chain polypeptide that reduce binding to protein A chromatography material are amino acid substitutions at positions corresponding to (e.g., relative to) positions 435 and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are H435R and Y436F (or corresponding thereto), with amino acid numbering according to the EU index. 【0118】 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 of the multivalent binding protein domain bind to different antigens. In some embodiments, the multivalent binding protein is a multispecific antibody or an antigen-binding fragment thereof. 【0119】 Exemplary tandem Fab binding protein In some embodiments, there is provided a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains that form four antigen-binding domains, the four polypeptide chains being A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-CH11-L1-VH2-CH12[I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-CL1-L2-VL2-CL2[II], A second heavy chain polypeptide comprising a structure represented by the following formula: VH3-CH13-L3-VH4-CH14[III], A second light chain polypeptide comprising a structure represented by the following formula: VL3-CL3-L4-VL4-CL4[IV] comprising 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; a) CL1 and CL2 each contain one or more amino acid substitutions, which reduce the binding to KappaSelect chromatography material compared to CL1 and CL2 that do not contain one or more amino acid substitutions, and VL3 and VL4 each contain one or more amino acid substitutions, which reduce the binding to protein L chromatography material compared to VL3 and VL4 that do not contain one or more amino acid substitutions; b) CL1 and CL2 each contain one or more amino acid substitutions, which reduce the binding to KappaSelect chromatography material compared to CL1 and CL2 that do not contain one or more amino acid substitutions, VL3 contains one or more amino acid substitutions, which reduce the binding to protein L chromatography material compared to VL3 that does not contain one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a κ2 subtype light chain variable immunoglobulin domain; c) CL1 and CL2 each contain one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 and CL2 that do not contain one or more amino acid substitutions. VL4 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL4 that does not contain one or more amino acid substitutions. VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; d) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions. CL2 is a lambda subtype immunoglobulin light chain constant domain. VL3 and VL4 each contain one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL3 and VL4 that do not contain one or more amino acid substitutions; e) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions. CL2 is a lambda subtype immunoglobulin light chain constant domain. VL3 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL3 that does not contain one or more amino acid substitutions. VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; f) CL1 contains one or more amino acid substitutions, which reduce the binding to the KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions. CL2 is a lambda subtype immunoglobulin light chain constant domain. VL4 contains one or more amino acid substitutions, which reduce the binding to the Protein L chromatography material compared to VL4 that does not contain one or more amino acid substitutions. VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; g) CL2 contains one or more amino acid substitutions, which reduce its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. CL1 is a lambda subtype immunoglobulin light chain constant domain. VL3 and VL4 each contain one or more amino acid substitutions, which reduce their binding to the Protein L chromatography material as compared to VL3 and VL4 that do not contain one or more amino acid substitutions; h) CL2 contains one or more amino acid substitutions, which reduce its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. CL1 is a lambda subtype immunoglobulin light chain constant domain. VL3 contains one or more amino acid substitutions, which reduce its binding to the Protein L chromatography material as compared to VL3 that does not contain one or more amino acid substitutions. VL4 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; i) CL2 contains one or more amino acid substitutions, which reduce its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. CL1 is a lambda subtype immunoglobulin light chain constant domain. VL4 contains one or more amino acid substitutions, which reduce its binding to the Protein L chromatography material as compared to VL4 that does not contain one or more amino acid substitutions. VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; VL1 and VH1 form the first antigen-binding domain, VL2 and VH2 form the second antigen-binding domain, VL3 and VH3 form the third antigen-binding domain, and VL4 and VH4 form the fourth antigen-binding domain. In some embodiments, 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 the absence of a linker in the binding protein. In some embodiments, L2 and / or L4 are absent. In some embodiments, L1, L2, L3, or L4 are each independently at least 1 amino acid in length. 【0120】 In some embodiments, there is provided a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains that form four antigen-binding domains; the four polypeptide chains are A first heavy chain polypeptide comprising a structure represented by the following formula: VH1-L1-VH2-L2-CH11[I], A first light chain polypeptide comprising a structure represented by the following formula: VL1-L3-VL2-L4-CL1[II], A second heavy chain polypeptide comprising a structure represented by the following formula: VH3-L5-VH4-L6-CH12[III], A second light chain polypeptide comprising a structure represented by the following formula: VL3-L7-VL4-L8-CL2[IV] and In the formula, 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 CH1 heavy chain constant domain, CH12 is the second immunoglobulin CH1 heavy chain constant domain, and L1, L2, L3, L4, L5, L6, L7, and L8 are amino acid linkers; a) CL1 contains one or more amino acid substitutions, thereby reducing binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, and VL3 and VL4 each contain one or more amino acid substitutions, thereby reducing binding to the Protein L chromatography material as compared to VL3 and VL4 that do not contain one or more amino acid substitutions; b) CL1 contains one or more amino acid substitutions, thereby reducing binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions, VL3 contains one or more amino acid substitutions, thereby reducing binding to the Protein L chromatography material as compared to VL3 that does not contain one or more amino acid substitutions, and VL4 is a lambda subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain; c) CL1 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL1 that does not contain one or more amino acid substitutions. VL4 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL4 that does not contain one or more amino acid substitutions. VL3 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. VL1 and VL2 each contain one or more amino acid substitutions, thereby reducing their binding to the Protein L chromatography material as compared to VL1 and VL2 that do not contain one or more amino acid substitutions; e) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions. VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; f) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions. VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions. VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; VL1 and VH1 form the first antigen-binding domain, VL2 and VH2 form the second antigen-binding domain, VL3 and VH3 form the third antigen-binding domain, and VL4 and VH4 form the fourth antigen-binding domain. In some embodiments, one of L1, L2, L3, L4, L5, L6, L7, or L8 is independently 0 amino acids in length. In some embodiments, a 0 amino acid linker indicates the absence of a linker in the binding protein. In some embodiments, at least one of L3, L4, L7, or L8 is absent. In some embodiments, L1, L2, L3, L4, L5, L6, L7, or L8 are each independently at least 1 amino acid in length. In some embodiments, the binding of the CL1 or CL2 of the multivalent binding protein to the KappaSelect chromatography material is reduced by about 90% compared to the binding of the CL1 or CL2 of the multispecific binding protein that does not contain one or more amino acid substitutions. In some embodiments, the binding of the VL3 and / or VL4 of the multivalent binding protein to the protein L chromatography material is reduced by about 90% compared to the binding of the VL3 and / or VL4 of the multivalent binding protein that does not contain one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in the CL1 or CL2 of the multivalent binding protein are at positions corresponding to 109, 110 or 199, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 or CL2 are T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution, and the amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in the CL1 of the multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in CL1 are H198R substitution, Q199W substitution, or T109A-S202R substitution, and the amino acid numbering is according to the EU index. In some embodiments, one or more amino acid substitutions in the VL3 and / or VL4 of the multivalent binding protein are framework amino acid substitutions.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, numbered according to Kabat. In some embodiments, one or more amino acid substitutions in VL3 or VL4 are S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution, numbered according to Kabat. 【0121】 In some embodiments, the first heavy chain polypeptide of the multivalent binding protein has a structure represented by the following formula: VH1-CH11-L1-VH2-CH12-CH2-CH3 [Ia] and the second heavy chain polypeptide has a structure represented by the following formula: VH3-CH13-L3-VH4-CH14-CH2-CH3 [IIIa] An example of this embodiment is shown in FIG. 1J. 【0122】 In some embodiments, the first heavy chain polypeptide of the multivalent binding peptide has a structure represented by the following formula: VH1-L1-VH2-L2-CH11-CH2-CH3 [Ia] and the second heavy chain polypeptide has a structure represented by the following formula: VH3-L5-VH4-L6-CH12-CH2-CH3 [IIIa] An example of this embodiment is shown in FIG. 1K. 【0123】 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 the CH3 domain of human IgG1 or IgG4. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, with amino acid numbering according to the EU index. See, for example, Spiess et al. (2013) JBC, 288(37); P26583-26593. In some embodiments, the amino acid substitutions are S354C and T366W of human IgG1 (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. In some embodiments, the CH3 domain of the first heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W (or corresponding thereto), with amino acid numbering according to the EU index. 【0124】 In some embodiments, the CH3 domain of the second heavy chain polypeptide of the multivalent binding protein comprises (e.g., 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 (e.g., further comprises) one or more amino acid substitutions that reduce binding to Protein A. In some embodiments, one or more amino acid substitutions in the CH3 domain of the first or second heavy chain polypeptide that reduce binding to Protein A chromatography material are amino acid substitutions at positions corresponding to (e.g., relative to) positions 435 and 436 of human IgG1, and the numbering is according to the EU index. In some embodiments, the amino acid substitutions are H435R and Y436F (or corresponding thereto), and the amino acid numbering is according to the EU index. 【0125】 In some embodiments, the binding protein is tetravalent and can specifically bind to four different antigen targets. 【0126】 In some embodiments, the multivalent binding protein is a multispecific antibody or an antigen-binding fragment thereof. 【0127】 Exemplary multivalent binding proteins comprising fusion proteins In some embodiments, there is provided a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains that form an antigen-binding domain and a target-binding domain; the four polypeptide chains are A first polypeptide chain (e.g., a first heavy chain polypeptide) comprising a structure represented by the following formula: VH1-CH11[I], A second polypeptide chain (e.g., a first light chain polypeptide) comprising a structure represented by the following formula: VL1-CL1[II], A third polypeptide (e.g., 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 polypeptide-L2-CL2[IV] or L2-CL2[IVa] comprising Wherein, 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 CH1 immunoglobulin heavy chain constant domain, CH12 is the second CH1 immunoglobulin heavy chain constant domain; L1 and L2 are amino acid linkers; CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material compared to CL2 that does not contain one or more amino acid substitutions, and VL1 contains one or more amino acid substitutions, thereby reducing its binding to the protein L chromatography material compared to VL1 that does not contain one or more amino acid substitutions; VL1 and VH1 form an antigen-binding domain. In some embodiments, the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide containing [III] and a fourth polypeptide containing formula [IV]. In some embodiments, the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide containing [IIIa] and a fourth polypeptide containing formula [IV]. In some embodiments, the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide containing [III] and a fourth polypeptide containing formula [IVa]. In some embodiments, the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide containing [III] and a fourth polypeptide containing formula [IV], and each fusion polypeptide binds to the target antigen. In some embodiments, the target antigens are the same. In some embodiments, the target antigens are different. In some embodiments, the binding protein (e.g., a multivalent binding protein) comprises a third polypeptide containing [III] and a fourth polypeptide containing formula [IV], and the fusion polypeptides dimerize and bind to a single target. An example of the embodiment described above is shown in Figure 1L. In some embodiments, L1 and / or L2 are independently 0 amino acids in length. In some embodiments, a linker of 0 amino acids in length indicates the absence of a linker in the binding protein.In some embodiments, L1 and / or L2 are each independently at least 1 amino acid in length. 【0128】 In some embodiments, the binding of the CL2 of the multivalent binding protein to the KappaSelect chromatography material is reduced by about 90% compared to the binding of the CL2 of the multivalent binding protein that does not contain one or more amino acid substitutions. In some embodiments, the binding of the VL1 of the multivalent binding protein to the protein L chromatography material is reduced by about 90% compared to the binding of the VL1 that does not contain one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in the CL2 of the multivalent binding protein are at positions corresponding to 109, 110, or 199, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in the CL2 are the T109A substitution, the V110D substitution, the Q199K substitution, the T109A-V110D substitution, or the T109A-V110D-Q199K substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in the CL2 of the multivalent binding protein are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in the CL2 are the H198R substitution, the Q199W substitution, or the T109A-S202R substitution, with amino acid numbering according to the EU index. In some embodiments, one or more amino acid substitutions in the VL1 of the multivalent binding protein are framework amino acid substitutions. In some embodiments, one or more amino acid substitutions in the VL1 are at positions corresponding to 12 or 18, numbered according to Kabat. In some embodiments, one or more amino acid substitutions in the VL1 are the S12P substitution, the R18P substitution, the R18Q substitution, the S12P-R18P substitution, or the S12P-R18Q substitution, with numbering according to Kabat. 【0129】 In some embodiments, there is provided a binding protein (e.g., a multivalent binding protein) comprising four polypeptide chains that form two antigen-binding domains and a target-binding domain; the four polypeptide chains are 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], A second polypeptide chain (e.g., a first light chain polypeptide) comprising a structure represented by the following formula: VL1-CL1-L2-VL2-CL2[II], A third polypeptide chain (e.g., 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] Comprising 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; a) CL3 contains one or more amino acid substitutions, thereby reducing binding to a KappaSelect chromatography material as compared to CL3 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, thereby reducing binding to a protein L chromatography material as compared to VL1 and VL2 that do not contain one or more amino acid substitutions, b) CL3 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL3 that does not contain one or more amino acid substitutions. VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions. VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or c) CL3 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL3 that does not contain one or more amino acid substitutions. VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions. VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; VL1 and VH1 form a first antigen-binding domain, and VL2 and VH2 form a second antigen-binding domain. 【0130】 In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that includes four polypeptide chains that form two antigen-binding domains and a target-binding domain; the four polypeptide chains are a first polypeptide chain (e.g., a first heavy chain polypeptide) that includes a structure represented by the following formula: VH1-CH11-L1-VH2-CH12[I], a second polypeptide chain (e.g., a first light chain polypeptide) that includes a structure represented by the following formula: VL1-CL1-L2-VL2-CL2[II], a third polypeptide chain (e.g., a second heavy chain polypeptide) that includes a structure represented by the following formula: CH13[IIIa], and a fourth polypeptide chain (e.g., a second light chain polypeptide) that includes a structure represented by the following formula: fusion polypeptide-L3-CL3[IVb] comprising 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; a) CL3 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material as compared to CL3 that does not comprise one or more amino acid substitutions, and VL1 and VL2 each comprise one or more amino acid substitutions that reduce binding to a Protein L chromatography material as compared to VL1 and VL2 that do not comprise one or more amino acid substitutions, b) CL3 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material as compared to CL3 that does not comprise one or more amino acid substitutions, VL1 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatography material as compared to VL1 that does not comprise one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain; or c) CL3 comprises one or more amino acid substitutions that reduce binding to a KappaSelect chromatography material as compared to CL3 that does not comprise one or more amino acid substitutions, VL2 comprises one or more amino acid substitutions that reduce binding to a Protein L chromatography material as compared to VL2 that does not comprise one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain; VL1 and VH1 form a first antigen-binding domain, and VL2 and VH2 form a second antigen-binding domain. 【0131】 In some embodiments, at least one of L1, L2, or L3 is independently 0 amino acids in length. In some embodiments, a linker of 0 amino acids in length indicates that there is no linker in the binding protein. In some embodiments, L1, L2, or L3 is independently at least 1 amino acid in length. In some embodiments, the binding of CL3 of the multivalent binding protein to the KappaSelect chromatography material is reduced by about 90% compared to the binding of CL3 of the multivalent binding protein that does not contain 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 that does not contain one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions in CL3 of the multivalent binding protein are at positions corresponding to 109, 110, or 199, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in CL3 are the T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution, numbered 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, numbered according to the EU index. In some embodiments, one or more amino acid substitutions in CL3 are the H198R substitution, Q199W substitution, or T109A-S202R substitution, and the amino acids are numbered 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 framework amino acid substitutions. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 are at positions corresponding to 12 or 18, numbered according to Kabat. In some embodiments, one or more amino acid substitutions in VL1 and / or VL2 are the S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution, numbered according to Kabat. 【0132】 In some embodiments, a binding protein (e.g., a multivalent binding protein) is provided that includes four polypeptide chains forming two antigen-binding domains and a target-binding domain; the four polypeptide chains are a first polypeptide chain (e.g., a first heavy chain polypeptide) having a structure represented by the following formula: VH1-CH11-L1-VH2-CH12[I], a second polypeptide chain (e.g., a first light chain polypeptide) having a structure represented by the following formula: VL1-CL1-L2-VL2-CL2[II], a third polypeptide chain (e.g., a second heavy chain polypeptide) having a structure represented by the following formula: fusion polypeptide-L3-CH13[III], and a fourth polypeptide chain (e.g., a second light chain polypeptide) having a structure represented by the following formula: fusion polypeptide-L4-CL3[IV] wherein, in the formula, 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; a) CL3 includes one or more amino acid substitutions, thereby reducing binding to a KappaSelect chromatography material as compared to CL3 that does not include one or more amino acid substitutions, and VL1 and VL2 each include one or more amino acid substitutions, thereby reducing binding to a protein L chromatography material as compared to VL1 and VL2 that do not include one or more amino acid substitutions, b) CL3 contains one or more amino acid substitutions, which reduce its binding to the KappaSelect chromatography material as compared to CL3 that does not contain one or more amino acid substitutions. VL1 contains one or more amino acid substitutions, which reduce its binding to the Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions. VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; or c) CL3 contains one or more amino acid substitutions, which reduce its binding to the KappaSelect chromatography material as compared to CL3 that does not contain one or more amino acid substitutions. VL2 contains one or more amino acid substitutions, which reduce its binding to the Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions. VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; 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 to 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 to a single target. An example of the embodiments described above is shown in Figure 1M. 【0133】 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 CH3 domain is the CH3 domain of human IgG1 or IgG4. In some embodiments, the first CH3 domain comprises amino acid substitutions at positions corresponding to (e.g., opposite to) positions 354 and 366 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W (or corresponding thereto), with amino acid numbering according to the EU index. Further or alternatively, in some embodiments, the second CH3 domain comprises amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, with numbering according to the EU index, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (or corresponding thereto), with amino acid numbering according to the EU index. 【0134】 In some embodiments, the second CH3 domain of the multivalent binding protein comprises (e.g., further comprises) 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 (e.g., 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 chromatography material are amino acid substitutions at positions corresponding to (e.g., relative to) positions 435 and 436 of human IgG1, with numbering according to the EU index. In some embodiments, the amino acid substitutions are H435R and Y436F (or corresponding thereto), with amino acid numbering according to the EU index. In some embodiments, the multivalent binding protein is a multispecific antibody or an antigen-binding fragment thereof. 【0135】 Linker Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide of four glycine residues (Gly-Gly-Gly-Gly (SEQ ID NO: 1)); a peptide of five glycine residues (Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 2)); a peptide of six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 3)); a peptide of seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 4)); a peptide of eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5)). As combinations of other amino acid residues, peptides such as Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 6), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 8), and Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: 6) can be used. In some embodiments, the linker is (GGGGS) n , for example, (SEQ ID NO: 6) n(wherein n is an integer from 0 to 5), for example, GGGGSGGGGS (SEQ ID NO: 9), GGGGSGGGGSGGGGS (SEQ ID NO: 10), etc. or including these. In some embodiments, the linker is S, RT, TKGPS (SEQ ID NO: 11), GQPKAAP (SEQ ID NO: 12), or GGSGSSGSGG (SEQ ID NO: 13) or including these. Other suitable linkers include a single Ser residue or a single Val residue; 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); 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 containing 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 for the binding protein. For further explanation of the linker sequence, see, for example, WO 2012 / 135345 pamphlet. 【0136】 The identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structure element required to be achieved within the linker. For example, glycine, serine, and alanine are best as linkers with maximum flexibility. Some combinations of glycine, proline, threonine, and serine are useful when a stronger and more extended linker is required. If desired, any amino acid residue can be combined with other amino acid residues to be considered as a linker and a larger peptide linker can be constructed according to the desired properties. 【0137】 In some embodiments, the linker of the multivalent binding protein disclosed herein comprises a sequence derived from a sequence that naturally occurs at the junction between the variable domain and the constant domain of an antibody (as described, for example, in the pamphlet of International Publication No. WO 2012 / 135345). For example, in some embodiments, the linker is the endogenous V H domain and C H1 domain, or between the endogenous V L domain and C L domain (e.g., kappa or lambda), including the sequences found at the transition region. In some embodiments, the linker is the endogenous human V H domain and C H1 domain, or between the endogenous human V L domain and C L domain (e.g., human kappa or lambda), including the sequences found at the transition region. 【0138】 Fc region and constant domain In some embodiments, the multivalent binding proteins of the present disclosure include one or more Fc variants. In some examples, as used herein, the term "Fc variant" refers to a molecule or sequence that has been modified from native Fc but still contains the binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Thus, the term "Fc variant" can include molecules or sequences that have been humanized from non-human native Fc. In some examples, native Fc contains regions that can be removed to provide structural features or biological activities that are not required for the antibody-like binding proteins of the invention. Thus, the term "Fc variant" refers to a molecule or sequence that lacks one or more native Fc sites or residues, or has one or more Fc sites or residues modified, that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cell cytotoxicity (ADCC). 【0139】 To improve the yield of the binding protein, the C H3 domains can be modified by, for example, the "knob-into-hole" technology described in detail in some examples in WO 96 / 027011 pamphlet, Ridgway et al., 1996, Protein Eng. 9; 617-21; and Merchant et al., 1998, Nat. Biotechnol. 16; 677-81. Specifically, the interaction surface of these two C H3 domains is modified such that the heterodimerization of the heavy chains containing the two C H3 domains is improved. The interaction surface of the two C H3Each of the domains can be a "knob" while the other is a "hole". Introduction of disulfide bridges further stabilizes the heterodimer (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270; 26-35) and improves the yield. In certain embodiments, the knob is present on the second pair of polypeptides having a single variable domain. In other embodiments, the knob is present on the first pair of polypeptides having a cross-orientation. In still other embodiments, C H3 The domain does not include a knob in hole. In some embodiments, the CH3 domain of the first heavy chain polypeptide contains amino acid substitutions at positions corresponding to (e.g., relative to) positions 354 and 366 of human IgG1, numbered according to the EU index. See, for example, Spiess et al. (2013) JBC, 288(37); P26583-26593. In some embodiments, the amino acid substitutions are S354C and T366W (e.g., knob substitutions) (or corresponding thereto), and the amino acid numbering is according to the EU index. Further or alternatively, in some embodiments, the CH3 domain of the second heavy chain polypeptide contains amino acid substitutions at positions corresponding to (e.g., relative to) positions 349, 366, 368, 407, 435, and 436 of human IgG1, numbered according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V (e.g., hole substitutions) (or corresponding thereto), and the amino acid numbering is according to the EU index. 【0140】 In some embodiments, the binding proteins of the present disclosure include one or more mutations to improve serum half-life (see, e.g., Hinton, P.R. et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutations include substitutions at positions corresponding to (e.g., relative to) positions 428 and 434 of human IgG1, numbering is by the EU index, the amino acid substitutions are M428L and N434S (or corresponding thereto), and the amino acid numbering is by the EU index. 【0141】 In some embodiments, the binding proteins of the present disclosure include one or more mutations to improve purification, for example, by modulating the affinity for chromatographic materials. For example, it is known that heterodimeric binding proteins can be selectively purified by separating them from their homodimers when one of the two Fc regions in the heterodimeric form contains a mutation that reduces or eliminates binding to protein A. This is because the heterodimeric form will have an intermediate affinity for protein A-based purification compared to either homodimeric form and can be selectively eluted from protein A, for example, by using different pHs (see, e.g., Smith, E.J. et al. (2015) Sci. Rep. 5:17943). In some embodiments, the mutations include substitutions at positions corresponding to (e.g., relative to) positions 435 and 436 of human IgG1, numbering is by the EU index, the amino acid substitutions are H435R and Y436F (or corresponding thereto), and the amino acid numbering is by the EU index. 【0142】 In some embodiments, the multivalent binding proteins of the present disclosure include one or more mutations to reduce effector functions, such as antibody-dependent cell phagocytosis (ADCP) mediated by Fc receptors, complement-dependent cytotoxicity (CDC), and / or antibody-dependent cell cytotoxicity (ADCC). In some embodiments, the mutations include amino acid substitutions at positions corresponding to (e.g., relative to) positions 234, 235, and / or 239 of human IgG1, numbered according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A (or corresponding thereto), and the amino acids are numbered according to the EU index. 【0143】 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 include two or more of the "knob" and "hole" mutations, one or more mutations to improve serum half-life, one or more mutations to improve the stability of IgG4, one or more mutations to improve purification, and / or one or more mutations to reduce the effector functions described above. 【0144】 Method for purifying multivalent binding protein Exemplary chromatography materials Protein A 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. Protein A has an affinity for immunoglobulins and thus has found extensive use as a tool in the detection and purification of antibodies, antibody constructs, and Fc-containing fusion proteins. It consists of five immunoglobulin-binding domains, each of which can bind to proteins from many mammalian species, particularly immunoglobulin G (IgG), via the heavy chain within the Fc region. Native protein A was used as the ligand for first-generation protein A resins, but currently, recombinant protein A (r-protein A) produced in Escherichia coli (E. coli) is the most prevalent. Commercially available protein A chromatography materials include, but are not limited to, those detailed below in this specification. 【0145】 KappaSelect KappaSelect is an affinity chromatography material that specifically binds to the constant region of the kappa light chain (LC). The ligand of KappaSelect is composed of a single variable heavy chain (HC) immunoglobulin (Ig) domain (VHH) of the camelid family that binds with high affinity to all human Cκ-LCs regardless of the VL sequence. The ligand of KappaSelect interacts mainly with the residues of Cκ and to some extent with the residues of the Vκ-Cκ hinge region. Commercially available KappaSelect chromatography materials include, but are not limited to, those detailed below in this specification. 【0146】 Protein L Protein L is a protein in the cell wall of the bacterium Peptostreptococcus magnus (Bjoerck et al. (1988) “Novel Bacterial-Cell Wall Protein with Affinity for Ig L-Chains.” J. Immunol. 140;1194-1197), and binds to the variable region of the kappa light chain without interfering with the antigen-binding site of an antibody or antibody construct (e.g., the multivalent binding proteins 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 V region of the kappa light chain, and its binding is limited to VL of the κ1, κ3, and κ4 subtypes. Commercially available Protein L chromatography materials include, but are not limited to, those detailed below in this specification. 【0147】 Exemplary two-step purification process A method for purifying a multivalent binding protein is provided herein, the method comprising: (a) subjecting a composition comprising the multivalent binding protein to protein L chromatography in binding and elution modes to produce a protein L eluate; and (b) subjecting the protein L eluate to KappaSelect chromatography in binding and elution chromatography to produce a KappaSelect eluate, the KappaSelect eluate comprising the multivalent binding protein and essentially free of mispaired polypeptides. For example, in some embodiments, the multivalent binding protein is contacted with protein L under conditions suitable to separate and isolate a binding protein comprising either 0 or 2 VL domains, each comprising one or more amino acid substitutions that reduce the binding of VL to the protein L chromatography material (e.g., as compared to a VL domain that does not contain one or more amino acid substitutions) from unwanted protein species. The protein L eluate thus produced is then contacted with a KappaSelect chromatography material under conditions suitable to separate and isolate a binding protein comprising either 0 or 2 CL domains, each comprising one or more amino acid substitutions that reduce the binding of CL to the KappaSelect chromatography material (e.g., as compared to a CL domain that does not contain one or more amino acid substitutions) from unwanted protein species. Also provided herein is a method for purifying a multivalent binding protein, the method comprising: (a) subjecting a composition comprising the multivalent binding protein and mispaired antibodies to KappaSelect chromatography in binding and elution modes to produce a KappaSelect eluate; and (b) subjecting the KappaSelect eluate to protein L chromatography in binding and elution modes to produce a protein L eluate, the protein L eluate comprising the multivalent binding protein and essentially free of mispaired polypeptides. 【0148】 Conditions suitable for using Protein L and KappaSelect chromatography materials in known binding and elution modes in the art. In some embodiments, Protein L and / or KappaSelect is bound to a substrate or resin, for example, as part of a chromatography material. In some embodiments, Protein L chromatography is Pierce™ Protein L chromatography cartridge, Capto™ L chromatography, HiTrap® Protein L chromatography, TOYOPEARL® AF-rProtein L-650F chromatography, or KanCap™ L chromatography. Further or alternatively, in some embodiments of any of the purification processes disclosed herein, KappaSelect chromatography is HiTrap™ KappaSelect or CaptureSelect™ Kappa XL chromatography. 【0149】 The KappaSelect eluate contains multivalent binding proteins and substantially no mispaired polypeptides. In some embodiments, the multivalent binding proteins in the KappaSelect eluate are pure at any one of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, including any range between these values. In some embodiments, the multivalent binding proteins in the KappaSelect eluate are more than about 99% pure. 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 polypeptides in the KappaSelect eluate are mispaired polypeptides. In some embodiments, less than about 1% of the multivalent binding proteins in the KappaSelect eluate are mispaired polypeptides. 【0150】 Exemplary three-step purification process A method for purifying a multivalent binding protein is provided herein. The method includes (a) subjecting a composition comprising the multivalent binding protein to Protein A chromatography in binding and elution modes to generate a Protein A eluate; (b) subjecting the Protein A eluate to Protein L chromatography in binding and elution modes to generate a Protein L eluate; and (c) subjecting the Protein L eluate to KappaSelect chromatography in binding and elution modes to generate a KappaSelect eluate, wherein the KappaSelect eluate comprises the multivalent binding protein and is essentially free of mispaired polypeptides. For example, in some embodiments, the multivalent binding protein is contacted with Protein A under conditions for separating and isolating a binding protein comprising two CH3 domains each comprising a Y349C, T366S, L368A, and Y407V substitution (with respect to human IgG1) (amino acid numbering is according to the EU index) from unwanted protein species. In some embodiments, the multivalent binding protein is contacted with Protein A under conditions for separating and isolating a binding protein comprising two CH3 domains each comprising an H435R and Y436F substitution (with respect to human IgG1) (amino acid numbering is according to the EU index) from unwanted protein species. In some embodiments, the multivalent binding protein is contacted with Protein A under conditions for separating and isolating a binding protein comprising two CH3 domains each comprising a Y349C, T366S, L368A, and Y407V substitution and an H435R and Y436F substitution (with respect to human IgG1) (amino acid numbering is according to the EU index) from unwanted protein species. In some embodiments, the second CH3 domain of the multivalent binding protein comprises (e.g., further comprises) 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 (e.g., further comprises) one or more amino acid substitutions that reduce binding to Protein A.In some embodiments, one or more amino acid substitutions that reduce binding to Protein A chromatography material are amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 (e.g., relative thereto), with numbering according to the EU index. In some embodiments, the amino acid substitutions are H435R and Y436F (or corresponding thereto), with amino acid numbering according to the EU index. In some embodiments, the multivalent binding protein is a multispecific antibody or an antigen-binding fragment thereof. 【0151】 (e.g., as compared to a VL domain that does not contain one or more amino acid substitutions) Under conditions suitable for separating and isolating a binding protein comprising either 0 or 2 VL domains that contain one or more amino acid substitutions that reduce the binding of the VL to Protein L chromatography material from unwanted protein species, the Protein A eluate is then contacted with Protein L. (e.g., as compared to a CL domain that does not contain one or more amino acid substitutions) Under conditions suitable for separating and isolating a binding protein comprising either 0 or 2 CL domains that contain one or more amino acid substitutions that reduce the binding of the CL to KappaSelect chromatography material from unwanted protein species, the Protein L eluate is then contacted with KappaSelect chromatography material. 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 binding and elution modes to produce a Protein A eluate; b) subjecting the Protein A eluate to KappaSelect chromatography in binding and elution modes to produce a KappaSelect eluate; and (c) subjecting the Protein KappaSelect eluate to Protein L chromatography in binding and elution modes to produce a Protein L eluate, the L eluate comprising the multivalent binding protein and essentially free of mispaired polypeptides. 【0152】 Suitable conditions for using Protein A, Protein L, and KappaSelect chromatography materials in binding and elution modes are known in the art. In some embodiments, the ligands of Protein A, Protein L, and / or KappaSelect are bound to a substrate or resin, for example, as part of the chromatography material. In some embodiments of any of the purification processes disclosed herein, Protein A chromatography is MabSelect™, MabSelect SuRe™, MabSelect SuRe™ LX, MabSelect PrismA, ProSep®-vA, ProSep® Ultra Plus, Protein A Sepharose® Fast Flow, or Toyopearl® AF-rProtein A chromatography. In some embodiments, Protein L chromatography is Pierce™ Protein L chromatography cartridge, Capto™ L chromatography, HiTrap® Protein L chromatography, TOYOPEARL® AF-rProtein L-650F chromatography, or KanCap™ L chromatography. Further or alternatively, in some embodiments of any of the purification processes disclosed herein, KappaSelect chromatography is HiTrap™ KappaSelect or CaptureSelect™ Kappa XL chromatography. 【0153】 The KappaSelect eluate contains multivalent binding proteins and substantially no mispaired polypeptides. In some embodiments, the multivalent binding proteins in the KappaSelect eluate are pure to any one of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, including any range between these values. In some embodiments, the multivalent binding proteins in the KappaSelect eluate are pure at greater 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 polypeptides in the KappaSelect eluate are mispaired polypeptides. In some embodiments, less than about 1% of the multivalent binding proteins in the KappaSelect eluate are mispaired polypeptides. 【0154】 In some embodiments of any of the purification processes disclosed herein, a composition containing a multivalent binding protein is derived from a host cell engineered to produce a multispecific binding protein. In some embodiments, the composition containing the multivalent binding protein is a host cell culture supernatant. In some embodiments, the composition containing the multivalent binding protein further contains mispaired polypeptides. In some embodiments, the composition containing the multivalent binding protein is filtered prior to chromatography (e.g., a first chromatography step). In some embodiments, the purification processes disclosed herein further include a polishing step after KappaSelect chromatography or Protein L chromatography. In some embodiments, the polishing step is size exclusion chromatography. 【0155】 In some embodiments of any of the purification processes disclosed herein, a composition containing a multivalent binding protein is combined with a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers and excipients are described in more detail elsewhere herein. 【0156】 Method for producing a multivalent binding protein with reduced binding to KappaSelect and / or Protein L chromatography materials Nucleic acids and vectors Nucleic acid molecules encoding the multivalent binding proteins described herein are also contemplated. In some embodiments, nucleic acids (or a series of nucleic acids) encoding the multivalent binding proteins described herein are provided. Standard recombinant DNA techniques are used to construct polynucleotides encoding the polypeptides that form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques can be performed according to the manufacturer's specifications, as commonly practiced in the art or as described herein. Unless specific definitions are provided, the terminology used in connection with the analytical chemistry, synthetic organic chemistry, and experimental procedures and techniques of medicinal and pharmaceutical chemistry described herein is well known and commonly used in the art. Similarly, conventional techniques may be used for chemical synthesis, chemical analysis, the preparation, formulation, delivery, and treatment of patients with pharmaceuticals. 【0157】 Other aspects of the disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins (e.g., multivalent binding proteins) described herein. In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter so as to induce transcription of the nucleic acid sequence encoding the binding protein. A promoter can refer to a nucleic acid control sequence that induces transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence of a binding protein when the promoter affects transcription or expression of the coding sequence. Examples of promoters include promoters obtained from viral genomes (e.g., polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus type 2), bovine papillomavirus, Rous 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 regions of bacteriophage lambda, promoter of 3-phosphoglycerate kinase, promoter of yeast acid phosphatase, and promoter of yeast α mating factor, but are not limited thereto. The polynucleotide encoding the binding protein of the disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein, or any other suitable promoter readily recognized by those skilled in the art. 【0158】 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 linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Examples of suitable enhancers may include enhancer sequences derived from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, insulin, etc.), and enhancer sequences derived from eukaryotic viruses (e.g., the SV40 enhancer (bp 100-270) behind the origin of replication, the cytomegalovirus immediate early promoter enhancer, the polyoma enhancer behind the origin of replication, the adenovirus enhancer, etc.), but are not limited thereto. Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus vectors, vaccinia virus vectors, sindbis virus vectors, measles vectors, herpes virus vectors, lentivirus vectors, retrovirus vectors, adeno-associated virus vectors, etc.). Host cells such as bacterial cells, yeast cells, insect cells, and mammalian cells can be transfected using the expression vector. Biologically functional viral and plasmid DNA vectors capable of expressing and replicating in a host are known in the art and can be used to transfect any cell of interest. 【0159】 Other aspects of the disclosure relate to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any of the binding proteins 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, second, third, and fourth polypeptide chains of the binding protein. One or more vectors of the vector system can be any of the vectors described herein. In some embodiments, the one or more vectors are expression vectors. 【0160】 Isolated host cell Other aspects of the disclosure relate to isolated host cells comprising one or more of the 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., an S. 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™ cells), and Spodoptera frugiperda cells (e.g., Sf21 cells or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells may include, for example, human fetal kidney cells (e.g., 293 cells or 293 cells subcloned to grow 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 CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical cancer cells (e.g., HELA, ATCC CCL 2), dog 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 hepatocarcinoma cell lines (e.g., Hep G2), and myeloma cells (e.g., NS0 cells and Sp2 / 0 cells). 【0161】 Other aspects of the disclosure relate to methods of producing any of the binding proteins described herein. In some embodiments, the method comprises: a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and / or vector system (e.g., any of the isolated nucleic acids, vectors, and / or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods of culturing host cells under conditions to express a protein are well known to those of skill in the art. Methods of isolating the multivalent binding proteins described herein from cultured host cells are well known to those of skill in the art. Methods of purifying the multivalent binding proteins described herein are detailed elsewhere in this specification. 【0162】 Exemplary uses of the multivalent binding protein The multivalent binding protein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, for detecting and quantifying one or more target antigens. The binding protein binds to one or more target antigens with an affinity suitable for the assay method used. 【0163】 For diagnostic uses, in certain embodiments, the binding protein may be labeled with a detectable moiety. The detectable moiety can be anything capable of generating a directly or indirectly detectable signal. For example, the detectable moiety can be 3 H, 14 C, 32 P, 35 S, 125 I, 99 Tc, 111 In, or 67 a radioisotope such as 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. 【0164】 Binding proteins are also useful for in vivo imaging. A binding protein labeled with a detectable moiety can be administered to an animal, preferably into its bloodstream, to assay for the presence and location of a labeled antibody in the host. The binding protein can be labeled with any moiety detectable in an animal, regardless of whether it is by nuclear magnetic resonance, radiation, or other detection means known in the art. 【0165】 The binding protein can also be used to activate cells, target tumors, neutralize cytokine activity, neutralize viral infections, combine multiple signaling events, and treat cancer, arthritis, and / or inflammatory disorders. For example, in some embodiments, the binding protein is 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), CCL8 (also known as mcp-2), CCL11 (also known as eosinophil chemotactic factor), 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 eosinophil chemotactic factor-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, the 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, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2R beta, 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 the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, 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) that specifically binds to one, two, or three antigen targets selected therefrom. In some embodiments, one or more of the above antigen targets are human antigen targets., 【0166】 In some embodiments, the binding proteins of the present disclosure are administered to a patient in need thereof for treating or preventing cancer. For example, in some embodiments, the binding protein comprises one 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 (e.g., 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 human. 【0167】 In some embodiments, the binding proteins of the present disclosure are administered to a patient in need thereof for treating or preventing an inflammatory disease or disorder. In some embodiments, the binding protein comprises three antigen-binding sites that specifically bind to cytokine target proteins selected from IL-4, IL-13, and TNFa, respectively. In some embodiments, the binding protein is co-administered with an anti-inflammatory agent. In some embodiments, the patient is human. 【0168】 The present disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting the level of a target antigen in a biological sample. Such reagents can include a detectable label, a blocking serum, positive and negative control samples, and a detection reagent. In some embodiments, the kit includes a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and / or host cells described herein. In some embodiments, the kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials such as glass or plastic. The container contains the composition itself or a composition combined with another composition effective to treat, prevent, and / or diagnose a condition and may have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper through which a hypodermic needle can penetrate). In some embodiments, the label or package insert indicates that the composition is to be used for the prevention, diagnosis, and / or treatment of a selected condition. Alternatively, or additionally, the manufactured product or kit may further include a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. Further, other materials desirable from a commercial and user perspective may be included, including other buffers, diluents, filters, needles, and syringes. 【0169】 Therapeutic Compositions Containing Multivalent Binding Proteins and Their Administration A therapeutic composition or pharmaceutical composition containing a binding protein is within the scope of the present disclosure. Such a therapeutic composition or pharmaceutical composition can include a therapeutically effective amount of a binding protein, or a binding protein-drug conjugate, mixed with a pharmaceutically or physiologically acceptable formulation agent selected to be compatible with the method of administration. 【0170】 The acceptable formulation materials are preferably non-toxic to the recipient at the dosages and concentrations used. 【0171】The pharmaceutical composition may include formulation materials for modifying, maintaining, or retaining, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorptivity, or permeability of the composition.Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), coloring agents, flavoring agents 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, methyl paraben, propyl paraben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronic; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, or mannitol sorbitol etc.), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants (see, e.g., Remington’s Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990) and subsequent editions incorporated herein by reference for all purposes), but are not limited thereto. 【0172】 The optimal pharmaceutical composition is determined by one skilled in the art, for example, according to the intended route of administration, delivery form, and desired dosage. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein. 【0173】 The main vehicle or carrier in the pharmaceutical composition can, by nature, be either aqueous or non-aqueous. For example, a vehicle or carrier suitable for injection can be water, an aqueous saline solution, or artificial cerebrospinal fluid, although other materials common in parenteral compositions can optionally be supplemented. Further exemplary vehicles are neutral buffered saline, or saline mixed with serum albumin. Other exemplary pharmaceutical compositions include a Tris buffer at about pH 7.0 - 8.5, or an acetate buffer at about pH 4.0 - 5.5, and may further include sorbitol or a suitable alternative. In one embodiment of the present disclosure, the binding protein composition can be prepared for storage by mixing a selected composition having a desired degree of purity, in the form of a lyophilized cake or an aqueous solution, with an optional formulation agent. Further, the binding protein can be formulated as a lyophilized product using a suitable excipient such as sucrose. 【0174】 The pharmaceutical compositions of the present disclosure can be selected for parenteral delivery or subcutaneous delivery. Alternatively, the composition can be selected for delivery via inhalation or via the gastrointestinal tract such as orally. The preparation of such pharmaceutically acceptable compositions is within the scope of the art. 【0175】 The formulation ingredients are present at concentrations acceptable at the site of administration. For example, buffers are used to maintain the composition within a physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8. 【0176】 When parenteral administration is contemplated, the therapeutic composition to be used can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable vehicle. A particularly preferred vehicle for parenteral injection is sterile distilled water, in which the binding protein is formulated as a sterile isotonic solution and appropriately preserved. Still another preparation may involve formulating the desired molecule with a substance that provides for controlled or sustained release of the product and that can be delivered by depot injection, such as injectable microspheres, biodegradable particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes. Hyaluronic acid can also be used, which may have the effect of enhancing the duration of circulation. Another preferred means for introducing the desired molecule is an implantable drug delivery device. 【0177】 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. Also, an inhalation solution of the binding protein can be formulated with a propellant for aerosol delivery. In still another embodiment, the solution can be nebulized. 【0178】 It is also contemplated that certain formulations can be administered orally. In one embodiment of the present disclosure, the binding protein so administered can be formulated with or without such carriers customarily used in the formulation of solid dosage forms such as tablets and capsules. For example, in a capsule, it can be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the binding protein. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders can also be used. 【0179】 Another pharmaceutical composition can comprise an effective amount of the binding protein in a mixture with a non-toxic excipient suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another suitable vehicle, a solution can be prepared in unit dosage form. Suitable excipients include calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc, and other inert diluents, but are not limited thereto. 【0180】 Pharmaceutical compositions of further aspects of the present disclosure, including those comprising a binding protein in a sustained release or controlled release formulation, will be apparent to those skilled in the art. Various other sustained or controlled release means, such as liposome carriers, biodegradable microparticles or porous beads, and techniques for formulating depot injections are also known to those skilled in the art. Further examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles such as films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. 【0181】 Pharmaceutical compositions for in vivo administration typically must be sterile. Sterilization can be achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, sterilization by this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are generally placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper through which a hypodermic needle can penetrate. 【0182】 When the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as a dry powder or lyophilized powder. Such formulations can be stored either in a form ready for immediate use or in a form that requires reconstitution before administration (e.g., lyophilization). 【0183】 The present disclosure also encompasses kits for making single-dose administration units. The kits can each contain both a first container having the dried protein and a second container having an aqueous formulation. Also included within the scope of the present disclosure are kits containing single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyosyringes). 【0184】 The therapeutically effective amount of the binding protein pharmaceutical composition used in treatment depends, for example, on the therapeutic context and purpose. Thus, the appropriate dosage level for treatment will vary in part depending on the molecule being delivered, the efficacy of using the binding protein, the route of administration, and the size (body weight, body surface area, or organ size) and condition (age and general health status) of the patient, which will be understood by those skilled in the art. Accordingly, in order to obtain an optimal therapeutic effect, the clinician can titrate the dosage and change the route of administration. 【0185】 The frequency of administration depends on the pharmacokinetic parameters of the binding protein in the formulation used. Typically, the clinician administers the composition until a dosage that provides the desired effect is reached. Thus, the composition can be administered over time as a single dose, multiple doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion by an implantable device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of the work routinely performed by those skilled in the art. By using appropriate dose-response data, the appropriate dosage can be determined. 【0186】 The route of administration of the pharmaceutical composition follows known methods, for example, oral; injection by intravenous, intraperitoneal, intracerebral (parenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; sustained release systems; or implantable devices. If desired, the composition may be administered by bolus injection, or continuously by infusion, or by an implantable device. 【0187】 The composition can also be administered locally by implanting a membrane, sponge, or other suitable material in which the desired molecule is adsorbed or encapsulated. When an implantable device is used, the device can be implanted in any suitable tissue or organ, and the desired molecule can be delivered by diffusion, sustained release bolus, or continuous administration. 【0188】 In some embodiments, the present disclosure relates to methods of preventing and / or treating a proliferative disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins described herein. In some embodiments, the patient is human. In some embodiments, the at least one binding protein is administered in combination with one or more anti-cancer therapies (e.g., any anti-cancer therapy known in the art). In some embodiments, the at least one binding protein is administered prior to one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered simultaneously with one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered after one or more anti-retroviral therapies. 【0189】 In some embodiments, the present disclosure relates to methods of 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 of the binding proteins described herein. In some embodiments, the patient is human. In some embodiments, the at least one binding protein is administered in combination with one or more anti-inflammatory therapies (e.g., any anti-inflammatory therapy known in the art). In some embodiments, the at least one binding protein is administered prior to the one or more anti-inflammatory therapies. In some embodiments, the at least one binding protein is administered simultaneously with the one or more anti-inflammatory therapies. In some embodiments, the at least one binding protein is administered after the one or more anti-inflammatory therapies. 【0190】 Without limiting the present disclosure, numerous embodiments of the present disclosure are described below for purposes of illustration. 【0191】 Exemplary embodiments The present invention provides the following exemplary embodiments. 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 having a structure represented by the following formula: VH1-CH1 [I], a first light-chain polypeptide chain having a structure represented by the following formula: VL1-CL1 [II], a second heavy-chain polypeptide having a structure represented by the following formula: VH2-CH1 [III], and a second light-chain polypeptide chain having a structure represented by the following formula: VL2-CL2 [IV] wherein: In the formula, VL1 is a first immunoglobulin light-chain variable domain; VL2 is a second immunoglobulin light-chain variable domain, and VL2 is a κ1, κ3, or κ4 subtype light-chain variable domain. CL1 is the first immunoglobulin light chain constant domain, and 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 an immunoglobulin heavy chain constant domain; CL1 contains one or more amino acid substitutions, which reduce the binding to KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, which reduce the binding to protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions, A multivalent binding protein in which 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 that form two antigen-binding domains; the four polypeptide chains are A first heavy chain polypeptide having a structure represented by the following formula: VH1-CH1-CH2-CH3 [I], A first light chain polypeptide chain having a structure represented by the following formula: VL1-CL1 [II], A second heavy chain polypeptide having a structure represented by the following formula: VH2-CH1-CH2-CH3 [III], And a second light chain polypeptide chain having a structure represented by the following formula: VL2-CL2 [IV] comprising; wherein, 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 the immunoglobulin CH2 heavy chain constant domain; CH3 is the immunoglobulin CH3 heavy chain constant domain; CL1 contains one or more amino acid substitutions, which reduce the binding to KappaSelect chromatography material compared to CL1 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, which reduce the binding to protein L chromatography material compared to VL2 that does not contain one or more amino acid substitutions, A multivalent binding protein in which 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 according to embodiment 1 or embodiment 2, wherein the binding of CL1 containing one or more substitutions to KappaSelect is reduced by about 90% compared to CL1 that does not contain one or more amino acid substitutions. 4. The multivalent binding protein according to any one of embodiments 1 to 3, wherein the binding of VL2 containing one or more substitutions to the protein L chromatography material is reduced by about 90% compared to VL2 that does not contain one or more amino acid substitutions. 5. The multivalent binding protein according to any one of embodiments 1 to 4, wherein one or more amino acid substitutions in CL1 containing one or more substitutions are at positions corresponding to 109, 110 or 199, and the numbering is according to the EU index. 6. The multivalent binding protein according to embodiment 5, wherein one or more amino acid substitutions in CL1 containing one or more substitutions are T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution, and the amino acid numbering is according to the EU index. 7. One or more amino acid substitutions in CL1 that include one or more substitutions are at positions corresponding to 109, 198, 199, or 202, numbered according to the EU index, the multivalent binding protein according to any one of Embodiments 1 to 4. 8. One or more amino acid substitutions in CL1 that include one or more substitutions are H198R substitution, Q199W substitution, or T109A - S202R substitution, and the amino acid numbering is according to the EU index, the multivalent binding protein according to Embodiment 7. 9. One or more amino acid substitutions in VL2 that include one or more substitutions are framework amino acid substitutions, the multivalent binding protein according to any one of Embodiments 1 to 8. 10. One or more amino acid substitutions in VL2 that include one or more substitutions are at positions corresponding to 12 or 18, numbered according to Kabat, the multivalent binding protein according to any one of Embodiments 1 to 9. 11. One or more amino acid substitutions in VL2 that include one or more substitutions are S12P substitution, R18P substitution, R18Q substitution, S12P - R18P substitution, or S12P - R18Q substitution, numbered according to Kabat, the multivalent binding protein according to Embodiment 10. 12. The CH3 domain of the first heavy - chain polypeptide and / or the CH3 domain of the second heavy - chain polypeptide is the CH3 domain of human IgG1 or IgG4, the multivalent binding protein according to any one of Embodiments 2 to 11. 13. The CH3 domain of the first heavy - chain polypeptide includes amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, numbered according to the EU index, and the amino acid substitutions are S354C and T366W; the CH3 domain of the second heavy - chain polypeptide includes amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, numbered according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V, the multivalent binding protein according to any one of Embodiments 2 to 12. 14. The CH3 domain of the first heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1, numbered according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V; the CH3 domain of the second heavy chain polypeptide contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1, numbered according to the EU index, and the amino acid substitutions are S354C and T366W, the multivalent binding protein according to any one of Embodiments 2 to 13. 15. The CH3 of the second heavy chain polypeptide contains one or more amino acid substitutions that reduce the binding to protein A, the multivalent binding protein according to Embodiment 13. 16. The CH3 of the first heavy chain polypeptide contains one or more amino acid substitutions that reduce the binding to protein A, the multivalent binding protein according to Embodiment 14. 17. The one or more amino acid substitutions that reduce the binding to protein A are amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1, numbered according to the EU index, the multivalent binding protein according to Claim 15 or 16. 18. The amino acid substitutions are H435R and Y436F, and the amino acid numbering is according to the EU index, the multivalent binding protein according to Claim 17. 19. 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, the multivalent binding protein according to any one of Claims 2 to 18. 20. The first heavy chain polypeptide is derived from a different species than the second heavy chain polypeptide, the multivalent binding protein according to any one of Claims 2 to 19. 21. 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, the multivalent binding protein according to any one of Claims 2 to 11, 19, or 20. 22. The multivalent binding protein according to any one of claims 2 to 11, wherein the first heavy chain polypeptide and the second heavy chain polypeptide each contain the CH3 domain of IgG4. 23. The multivalent binding protein according to claim 22, wherein the first heavy chain polypeptide contains a K409R amino acid substitution, the second heavy chain polypeptide contains an F405L amino acid substitution, and the numbering is according to the EU index. 24. The multivalent binding protein according to any one of claims 1 to 23, which is a bispecific antigen-binding protein. 25. The multivalent binding protein according to any one of claims 1 to 24, wherein the first antigen-binding domain and the second antigen-binding domain bind to different antigens. 26. The multivalent binding protein according to any one of claims 2 to 18, wherein the first heavy chain polypeptide chain has a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3-L-VL3 [Ia] and the second heavy chain polypeptide has a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIa] and 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; VH3 and VL3 associate to form a 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 has a structure represented by the following formula: VH1-CH1-CH2-CH3-VH3 [Ib] and the second heavy chain polypeptide has a structure represented by the following formula: VH2-CH1-CH2-CH3 [IIIb] and wherein VH3 is a multivalent binding protein that is the third immunoglobulin heavy chain variable domain. 28. The multivalent binding protein according to claim 26, wherein VL3 comprises one or more amino acid substitutions, thereby reducing binding to protein L chromatography material as compared to VL3 that does not comprise one or more amino acid substitutions. 29. The multivalent binding protein according to claim 26, wherein VL3 is a λ subtype immunoglobulin light chain variable domain or a κ2 immunoglobulin light chain variable domain. 30. The multivalent binding protein according to any one of claims 26 to 29, which is bispecific or trispecific. 31. The multivalent binding protein according to any one of claims 26 to 30, wherein the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain bind to two or three different antigens. 32. The multivalent binding protein according to any one of claims 26 to 30, wherein 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. 33. The multivalent binding protein according to any one of claims 26 to 30, wherein 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. 34. A multivalent binding protein comprising four polypeptide chains that form three antigen binding domains; 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 chain polypeptide chain comprising a structure represented by the following formula: VL2-L1-VL1-L2-CL1 [II], a second heavy chain polypeptide comprising a structure represented by the following formula: VH3-CH1 [III], and a second light chain polypeptide chain comprising a structure represented by the following formula: VL3-CL2 [IV] and In the formula, 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; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form an intersected light chain-heavy chain pair; VH1 and VL1 associate to form a first antigen-binding domain, VH2 and VL2 associate to form a second antigen-binding domain, and VH3 and VL3 associate to form a third antigen-binding domain; a) CL1 contains one or more amino acid substitutions, whereby the binding to the KappaSelect chromatography material is reduced as compared to CL1 that does not contain one or more amino acid substitutions, and VL3 contains one or more amino acid substitutions, whereby the binding to the Protein L chromatography material is reduced as compared to VL3 that does not contain one or more amino acid substitutions; b) CL2 contains one or more amino acid substitutions, whereby the binding to the KappaSelect chromatography material is reduced as compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, whereby the binding to the Protein L chromatography material is reduced as compared to VL1 and VL2 that do not contain one or more amino acid substitutions; c) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, VL1 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions, VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain; or d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to the KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing its binding to the Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions, VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype immunoglobulin light chain variable domain, a multivalent binding protein. 35. A multivalent binding protein comprising four polypeptide chains that form three antigen-binding domains; the four polypeptide chains A first heavy chain polypeptide having a structure represented by the following formula: VH1-L3-VH2-L4-CH1-CH2-CH3 [Ia], A first light chain polypeptide chain having a structure represented by the following formula: VL2-L1-VL1-L2-CL1 [II], A second heavy chain polypeptide having a structure represented by the following formula: VH3-CH1-CH2-CH3 [IIIa], And a second light chain polypeptide chain having a structure represented by the following formula: VL3-CL2 [IV] comprising 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 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; L1, L2, L3, and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form an intersected light chain-heavy chain pair; VH1 and VL1 associate to form a first antigen-binding domain, VH2 and VL2 associate to form a second antigen-binding domain, and VH3 and VL3 associate to form a third antigen-binding domain; a) CL1 contains one or more amino acid substitutions, whereby the binding to KappaSelect chromatography material is reduced compared to CL1 that does not contain one or more amino acid substitutions, and VL3 contains one or more amino acid substitutions, whereby the binding to protein L chromatography material is reduced compared to VL3 that does not contain one or more amino acid substitutions; b) CL2 contains one or more amino acid substitutions, whereby the binding to KappaSelect chromatography material is reduced compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, whereby the binding to protein L chromatography material is reduced compared to VL1 and VL2 that do not contain one or more amino acid substitutions; c) CL2 contains one or more amino acid substitutions, thereby reducing its binding to KappaSelect chromatography material compared to CL2 without any amino acid substitutions, VL1 contains one or more amino acid substitutions, thereby reducing its binding to Protein L chromatography material compared to VL1 without any amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype light chain variable immunoglobulin domain; or d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to KappaSelect chromatography material compared to CL2 without any amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing its binding to Protein L chromatography material compared to VL2 without any amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa2 subtype light chain variable immunoglobulin domain, a multivalent binding protein. 36. The binding protein is triple specific and can specifically bind to three different antigen targets, the multivalent binding protein according to embodiment 34 or 35. 37. At least one of L1, L2, L3 and / or L4 is independently 0 amino acids in length, the multivalent binding protein according to any one of embodiments 34 to 36. 38. L1, L2, L3 and / or L4 are each independently at least 1 amino acid in length, the multivalent binding protein according to any one of embodiments 34 to 36. 39. The multivalent binding protein according to embodiment 34 or 35, wherein the second heavy chain polypeptide has a structure represented by the following formula: VH3-L5-VH4-L6-CH1 [IIIb] and the second light chain polypeptide chain has a structure represented by the following formula: VL4-L7-VL3-L8-CL2 [IVa] and wherein 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; The polypeptide of Formula IIIa and the polypeptide of Formula IVa form an intersected light chain - heavy chain pair; VH4 and VL4 associate to form the fourth antigen - binding domain; a) CL1 contains one or more amino acid substitutions, whereby compared to CL1 that does not contain one or more amino acid substitutions, the binding to KappaSelect chromatography material is reduced, and VL3 and VL4 each contain one or more amino acid substitutions, whereby compared to VL3 and VL4 that do not contain one or more amino acid substitutions, the binding to Protein L chromatography material is reduced; b) CL1 contains one or more amino acid substitutions, whereby compared to CL1 that does not contain one or more amino acid substitutions, the binding to KappaSelect chromatography material is reduced, VL3 contains one or more amino acid substitutions, whereby compared to VL3 that does not contain one or more amino acid substitutions, the binding to Protein L chromatography material is reduced, and VL4 is a λ - subtype immunoglobulin light chain variable domain or a κ2 - subtype immunoglobulin light chain variable domain; c) CL1 contains one or more amino acid substitutions, whereby compared to CL1 that does not contain one or more amino acid substitutions, the binding to KappaSelect chromatography material is reduced, VL4 contains one or more amino acid substitutions, whereby compared to VL4 that does not contain one or more amino acid substitutions, the binding to Protein L chromatography material is reduced, and VL3 is a λ - subtype immunoglobulin light chain variable domain or a κ2 - subtype immunoglobulin light chain variable domain; d) CL2 contains one or more amino acid substitutions, thereby reducing its binding to KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, and VL1 and VL2 each contain one or more amino acid substitutions, thereby reducing their binding to Protein L chromatography material as compared to VL1 and VL2 that do not contain one or more amino acid substitutions; e) CL2 contains one or more amino acid substitutions, thereby reducing its binding to KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, VL1 contains one or more amino acid substitutions, thereby reducing its binding to Protein L chromatography material as compared to VL1 that does not contain one or more amino acid substitutions, and VL2 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain; or f) CL2 contains one or more amino acid substitutions, thereby reducing its binding to KappaSelect chromatography material as compared to CL2 that does not contain one or more amino acid substitutions, VL2 contains one or more amino acid substitutions, thereby reducing its binding to Protein L chromatography material as compared to VL2 that does not contain one or more amino acid substitutions, and VL1 is a lambda subtype immunoglobulin light chain variable domain or a kappa 2 subtype immunoglobulin light chain variable domain, the multivalent binding protein. 40. The multivalent binding protein according to embodiment 39, wherein the second heavy chain polypeptide has a structure represented by the following formula: VH3-L5-VH4-L6-CH1-CH2-CH3 [IIIc] comprising wherein CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain, the multivalent binding protein. 41. The multivalent binding protein according to embodiment 39 or 40, wherein the binding protein is quad-specific and can specifically bind to four different antigen targets. 42. The multivalent binding protein according to any one of embodiments 39 to 41, wherein at least one of L1, L2, L3, L4, L5, L6, L7, and / or L8 is independently 0 amino acids in length. 43. The multivalent binding protein according to any one of embodiments 39 to 41, wherein L1, L2, L3, L4, L5, L6, L7, and / or L8 are each independently at least 1 amino acid in length. 44. The multivalent binding protein according to any one of embodiments 34 to 43, wherein the binding of CL1 or CL2 containing one or more amino acid substitutions to the KappaSelect chromatography material is reduced by about 90% compared to CL1 or CL2 without one or more amino acid substitutions. 45. The multivalent binding protein according to any one of embodiments 34 to 44, wherein the binding of VL1, VL2, VL3, and / or VL4 containing one or more substitutions to the protein L chromatography material is reduced by about 90% compared to VL1, VL2, VL3, and / or VL4 without one or more amino acid substitutions. 46. The multivalent binding protein according to any one of embodiments 34 to 45, wherein one or more amino acid substitutions in CL1 or CL2 are at positions corresponding to 109, 110, or 199, and the numbering is according to the EU index. 47. The multivalent binding protein according to embodiment 46, wherein one or more amino acid substitutions in CL1 or CL2 containing one or more amino acid substitutions are T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution, and the amino acid numbering is according to the EU index. 48. The multivalent binding protein according to any one of embodiments 34 to 45, wherein one or more amino acid substitutions in CL1 or CL2 containing one or more substitutions are at positions corresponding to 109, 198, 199, or 202, and the numbering is according to the EU index. 49. The multivalent binding protein according to embodiment 48, wherein one or more amino acid substitutions in CL1 or CL2 containing one or more substitutions are H198R substitution, Q199W substitution, or T109A-S202R substitution, and the amino acid numbering is according to the EU index. 50. The multivalent binding protein according to any one of embodiments 34 to 49, wherein one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4, including one or more substitutions, are framework amino acid substitutions. 51. The multivalent binding protein according to any one of embodiments 34 to 50, wherein one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4, including one or more substitutions, are at positions corresponding to 12 or 18, numbered according to Kabat. 52. The multivalent binding protein according to embodiment 51, wherein one or more amino acid substitutions in VL1, VL2, VL3 and / or VL4, including one or more substitutions, are S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution, numbered according to Kabat. 53. The multivalent binding protein according to any one of embodiments 35 to 38 and 40 to 52, wherein the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is the CH3 domain of human IgG1 or IgG4. 54. The CH3 domain of the first heavy chain polypeptide conta...

Claims

[Claim 1] A polyvalent binding protein comprising four polypeptide chains that form three antigen-binding domains; the four polypeptide chains are The first heavy chain polypeptide, comprising the structure represented by the following equation: VH 1 -L 3 -VH 2 -L 4 -CH1-CH2-CH3 [Ia]、 A first light polypeptide chain containing the structure represented by the following equation: 67 2 -8 1 -68 1 -8 2 - K1 1 [A] A second heavy chain polypeptide containing the structure represented by the following equation: VH 3 -CH1-CH2-CH3 [III]]、 And a second light polypeptide chain having a structure represented by the following formula: VL 3 -CL 2 [IV] Includes, During the ceremony, VL 1 This is the first immunoglobulin light chain variable domain; VL 2 This is the second immunoglobulin light chain variable domain; VL 3 This is the third immunoglobulin light chain variable domain; VH 1 This is the first immunoglobulin heavy chain variable domain; VH 2 This is the second immunoglobulin heavy chain variable domain; VH 3 This is the third immunoglobulin heavy chain variable domain; CL 1 This is the first constant domain of the immunoglobulin light chain; CL 2 This is the second constant domain of the immunoglobulin light chain; CH1 is the constant domain of the immunoglobulin CH1 heavy chain; CH2 is the constant domain of the immunoglobulin CH2 heavy chain; CH3 is the constant domain of the immunoglobulin CH3 heavy chain; L 1 , L 2 , L 3 and L 4 It is an amino acid linker; The polypeptide of formula I and the polypeptide of formula II form a crossed light-chain-heavy-chain pair; VH 1 and VL 1 These two combine to form the first antigen-binding domain, VH 2 and VL 2 These two combine to form a second antigen-binding domain, VH 3 and VL 3 These two combine to form a third antigen-binding domain; a) CL 1 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 3 This includes one or more amino acid substitutions selected from S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions (where numbering is by Kabat); b) CL 2 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 1 and VL 2 This includes one or more amino acid substitutions selected from S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions (where numbering is by Kabat); c) CL 2 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 1 This includes one or more amino acid substitutions selected from S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution (where the numbering is by Kabat), and VL 2 is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain; or d) CL 2 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 2 This includes one or more amino acid substitutions selected from S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution (where the numbering is by Kabat), and VL 1 This is a polyvalently bound protein that is either a λ subtype immunoglobulin light chain variable domain or a κ2 subtype light chain variable immunoglobulin domain. [Claim 2] The polyvalent binding protein according to claim 1, wherein the binding protein is triply specific and can specifically bind to three different antigen targets. [Claim 3] (1) L 1 , L 2 , L 3 and / or L 4 At least one of them is independently 0 amino acid length; or (2) L 1 , L 2 , L 3 and / or L 4 The polyvalent binding protein according to claim 1, wherein each of them is independently at least one amino acid in length. [Claim 4] A polyvalent binding protein according to claim 1, The second heavy chain polypeptide has the following structure: VH 3 -L 5 -VH 4 -L 6 -CH1 [IIIｬ] Includes, The second light chain polypeptide chain has the structure represented by the following equation: VL 4 -L 7 -VL 3 -L 8 -CL 2 [IVa] Includes, During the ceremony, VL 4 This is the fourth immunoglobulin light chain variable domain; VH 4 This is the fourth immunoglobulin heavy chain variable domain; L 5 , L 6 , L 7 and L 8 It is an amino acid linker; The polypeptide of formula IIIa and the polypeptide of formula IVa form a crossed light-chain-heavy-chain pair; VH 4 and VL 4 These two combine to form a fourth antigen-binding domain; a) CL 1 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 3 and VL 4 This includes one or more amino acid substitutions selected from S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions (where numbering is by Kabat); b) CL 1 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 3 This includes one or more amino acid substitutions selected from S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution (where the numbering is by Kabat), and VL 4 This is the λ subtype immunoglobulin light chain variable domain or the κ2 subtype immunoglobulin light chain variable domain; c) CL 1 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 4 This includes one or more amino acid substitutions selected from S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution (where the numbering is by Kabat), and VL 3 This is the λ subtype immunoglobulin light chain variable domain or the κ2 subtype immunoglobulin light chain variable domain; d) CL 2 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 1 and VL 2 This includes one or more amino acid substitutions selected from S12P substitutions, R18P substitutions, R18Q substitutions, S12P-R18P substitutions, or S12P-R18Q substitutions (where numbering is by Kabat); e) CL 2 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where amino acid numbering is according to the EU index), or (ii) H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), and VL 1 This includes one or more amino acid substitutions selected from S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution (where the numbering is by Kabat), and VL 2 is the λ subtype immunoglobulin light chain variable domain or the κ2 subtype immunoglobulin light chain variable domain; or f) CL 2 (i) T109A substitution, V110D substitution, Q199K substitution, T109A-V110D substitution, or T109A-V110D-Q199K substitution (where the amino acid (Numbering is according to the EU index), or (ii) containing one or more amino acid substitutions selected from H198R substitution, Q199W substitution, or T109A-S202R substitution (where amino acid numbering is according to the EU index), VL 2 This includes one or more amino acid substitutions selected from S12P substitution, R18P substitution, R18Q substitution, S12P-R18P substitution, or S12P-R18Q substitution (where the numbering is by Kabat), and VL 1 This is a λ subtype immunoglobulin light chain variable domain or a κ2 subtype immunoglobulin light chain variable domain. Depending on the circumstances, (1) The second heavy chain polypeptide has the structure represented by the following formula: VH 3 -L 5 -VH 4 -L 6 -CH1-CH2-CH3 [IIIc] Includes, During the ceremony, CH2 is the constant domain of the immunoglobulin CH2 heavy chain. CH3 is the constant domain of the immunoglobulin CH3 heavy chain; and / or (2) The binding protein is a polyvalent binding protein that is quadruple specific and can specifically bind to four different antigen targets. [Claim 5] (1) At least one of L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 and / or L 8 has a length of 0 amino acids independently; or (2) L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 and / or L 8 has a length of at least 1 amino acid independently, the multivalent binding protein according to claim 4. [Claim 6] The polyvalent binding protein according to claim 1, wherein the CH3 domain of the first heavy chain polypeptide and / or the CH3 domain of the second heavy chain polypeptide is the CH3 domain of human IgG1 or IgG4. [Claim 7] (a) The CH3 domain of the first heavy chain polypeptide contains amino acid substitutions at positions corresponding to human IgG1 positions 354 and 366, numbered according to the EU index, the amino acid substitutions being S354C and T366W; the CH3 domain of the second heavy chain polypeptide contains amino acid substitutions at positions corresponding to human IgG1 positions 349, 366, 368, 407, 435 and 436, numbered according to the EU index, the amino acid substitutions being Y349C, T366S, L368A and Y407V; and / or (b) The CH3 domain of the first heavy chain polypeptide contains amino acid substitutions at positions corresponding to human IgG1 positions 349, 366, 368, 407, 435, and 436, numbered according to the EU index, and the amino acid substitutions are Y349C, T366S, L368A, and Y407V; the CH3 domain of the second heavy chain polypeptide contains amino acid substitutions at positions corresponding to human IgG1 positions 354 and 366, numbered according to the EU index, and the amino acid substitutions are S354C and T366W. The polyvalent binding protein according to claim 6. [Claim 8] The polyvalent binding protein according to claim 7, wherein the CH3 of the second heavy chain polypeptide or the CH3 of the first heavy chain polypeptide comprises one or more amino acid substitutions that reduce binding to protein A. [Claim 9] The polyvalent binding protein according to claim 8, wherein one or more amino acid substitutions that reduce binding to protein A are amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1, numbered according to the EU index, and optionally, the amino acid substitutions are H435R and Y436F, numbered according to the EU index. [Claim 10] The polyvalent binding protein according to claim 1, wherein the polyvalent binding protein is a multispecific antibody or an antigen-binding fragment thereof. [Claim 11] A polynucleotide encoding a polyvalent binding protein according to any one of claims 1 to 10. [Claim 12] A vector comprising the polynucleotide described in claim 11. [Claim 13] A host cell comprising the polynucleotide described in claim 11. [Claim 14] A method for producing a polyvalent binding protein, comprising culturing a host cell according to claim 13 so as to produce the binding protein, and optionally further comprising recovering the binding protein from the host cell. [Claim 15] A pharmaceutical composition comprising a polyvalent binding protein according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier. [Claim 16] A method for purifying a polyvalently bound protein according to any one of claims 1 to 10, (1) Subjecting a composition containing the polyvalent binding protein to protein L chromatography in binding and elution mode to produce a protein L eluate, and subjecting the protein L eluate to KappaSelect chromatography in binding and elution mode to produce a KappaSelect eluate, wherein the KappaSelect eluate contains the polyvalent binding protein and is essentially free of mispaired polypeptides, and optionally the polyvalent binding protein in the KappaSelect eluate is at least 85% pure, at least 90% pure, or at least 95% pure, and / or less than 15%, less than 10%, or less than 5% of the polypeptides in the KappaSelect eluate are mispaired polypeptides; or (2) The composition comprising subjecting the composition containing the polyvalent binding protein and the mispaired antibody to KappaSelect chromatography in binding and elution chromatography to produce a KappaSelect eluate, and subjecting the KappaSelect eluate to protein L chromatography in binding and elution mode to produce a protein L eluate, wherein the protein L eluate contains the polyvalent binding protein and is essentially free of the mispaired polypeptide, and optionally the polyvalent binding protein in the protein L eluate is at least 85% pure, at least 90% pure, or at least 95% pure, and / or less than 15%, less than 10%, or less than 5% of the polypeptide in the protein L eluate is the mispaired polypeptide; or (3) The method comprises subjecting the composition containing the polyvalent binding protein to protein A chromatography in binding and elution mode to produce a protein A eluate, subjecting the protein A eluate to protein L chromatography in binding and elution mode to produce a protein L eluate, and subjecting the protein L eluate to KappaSelect chromatography in binding and elution mode to produce a KappaSelect eluate, wherein the KappaSelect eluate contains the polyvalent binding protein and is essentially free of mispaired polypeptides, and optionally the polyvalent binding protein in the KappaSelect eluate is at least 85% pure, at least 90% pure, or at least 95% pure, and / or less than 15%, less than 10%, or less than 5% of the polypeptides in the KappaSelect eluate are mispaired polypeptides; or (4) Subject the composition containing the polyvalent binding protein to protein A chromatography in binding and elution mode to produce protein A eluate, and subject the protein A eluate to KappaSelect chromatography in binding and elution mode to produce KappaS A method comprising generating an elect eluate, and subjecting the protein KappaSelect eluate to protein L chromatography in binding and elution modes to generate a protein L eluate, wherein the L eluate contains the polyvalent binding protein and is essentially free of mispaired polypeptides, and optionally the polyvalent binding protein in the protein L eluate is at least 85% pure, at least 90% pure, or at least 95% pure, and / or less than 15%, less than 10%, or less than 5% of the polypeptides in the protein L eluate are mispaired polypeptides. [Claim 17] (a) The composition containing the polyvalent binding protein is filtered before chromatography; and / or (b) The process further comprises a polishing step after the KappaSelect or Protein L chromatography, wherein the polishing step is size exclusion chromatography; and / or (c) The composition comprising the polyvalent binding protein is combined with a pharmaceutically acceptable carrier. The method according to claim 16.

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