Multispecific binding proteins and methods for developing same - Patents.com

Abstract

Multispecific binding proteins that bind to a first antigen and a second antigen, and methods for purifying the multispecific binding proteins.

Description

[Technical field]

[0001] The present invention is in the field of medicine, in particular in the field of multispecific binding proteins (such as bispecific antibodies and trispecific binding proteins) for use in the treatment of disease and methods for developing same. [Background technology]

[0002] Multispecific binding proteins are polypeptides that contain multiple different antigen binding domains.Multiple forms of multispecific binding proteins have been disclosed, such as those described in WO2001077342, WO2007110205, WO2008024188, WO2009089004, WO2012135345 and WO2016118742, and have even been tested for the treatment of various autoimmune diseases, cancers, infectious diseases and cardiovascular diseases.Multispecific binding proteins offer patients the potential for enhanced therapeutic efficacy, potential for cost reduction and improved convenience, for example by targeting multiple antigens, but their development as therapeutic candidates has been limited.

[0003] A factor limiting the progress of multispecific binding proteins is the complexity of assembling, producing and purifying these molecules. For example, the production of bispecific molecules requires not only the proper assembly of different antigen-binding domains, but also the assembly of different antigen-binding domains into a single molecule. Often, during recombinant expression of multispecific binding proteins, a mixture of non-target molecules (e.g., monospecific proteins, single-chain pairs, etc.) is expressed. The multispecific binding protein of interest needs to be purified not only from the expression medium, but also from the mixture of non-target molecules. The formation of non-target molecules and the need for further purification steps leads to a decrease in the yield of the multispecific binding protein of interest and an increase in the overall production cost. Summary of the Invention [Problem to be solved by the invention]

[0004] Attempts to facilitate the development and purification of multispecific binding proteins have been disclosed, for example, as described in WO20100151792, WO2013088259, and WO2013136186. However, these disclosures have proven limited in addressing the multispecific binding protein development problem. For example, in some instances, these disclosures exhibit one or more of impaired effector function, increased immunogenicity concerns, altered assembly, altered affinity, and / or reduced pharmacokinetic properties such as half-life. In some instances, the applicability of the disclosures is limited to specific molecules and / or formats. Therefore, there remains a need for improved multispecific binding proteins and methods of developing the same, facilitating the development of multispecific binding proteins without altered stability or affinity and without unacceptable immunogenicity. [Means for solving the problem]

[0005] Thus, the present disclosure addresses one or more of the above needs by providing improved multispecific binding proteins and methods for developing same. Embodiments of the multispecific binding proteins and methods of the present disclosure provide improved purification of the multispecific binding protein of interest, preserve and / or improve molecular assembly, reduce protein aggregation, and increase physical stability without increasing immunogenicity risk, altered effector function, and / or altered pharmacokinetic properties. Additionally, embodiments of the present disclosure preserve the affinity of the multispecific binding protein and reduce or eliminate undesired binding of kappa light chains to purification reagents. Additionally, embodiments of the present disclosure do not add time and cost to the overall purification or development process.

[0006] Thus, in certain embodiments, the disclosure provides multispecific binding proteins that bind to a first antigen and a second antigen. According to some embodiments, there is provided a multispecific binding protein that binds to a first antigen and a second antigen, said multispecific binding protein comprising a first antigen-binding domain comprising a first light chain Fab region and a first heavy chain Fab region, said first light chain Fab region being a kappa light chain and comprising the following: a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid residue 199 (EU numbering); a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an alanine at amino acid residue 109 (EU numbering); a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an aspartic acid at amino acid residue 110 (EU numbering); a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an aspartic acid at amino acid residue 109 (EU numbering). alanine, and aspartic acid at amino acid residue 110 (EU numbering); aspartic acid at amino acid residue 110 (EU numbering) and lysine at amino acid residue 143 (EU numbering); aspartic acid at amino acid residue 110 (EU numbering), lysine at amino acid residue 143 (EU numbering) and alanine at amino acid residue 109 (EU numbering); aspartic acid at amino acid residue 110 (EU numbering) and lysine at amino acid residue 199 (EU numbering); aspartic acid at amino acid residue 110 (EU numbering), lysine at amino acid residue 199 (EU numbering), and alanine at amino acid residue 109 (EU numbering); alanine at amino acid residue 109 (EU numbering) and lysine at amino acid residue 143 (EU numbering); alanine at amino acid residue 109 (EU numbering) and lysine at amino acid residue 199 (EU numbering);or an alanine at amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110 (EU numbering), and wherein the multispecific binding protein comprises a second antigen-binding domain comprising a second light chain Fab region and a second heavy chain Fab region, wherein the first antigen-binding domain binds to the first antigen and the second antigen-binding domain binds to the second antigen. According to some such embodiments, if the first light chain Fab region comprises a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), then the second light chain Fab region does not comprise a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid residue 199 (EU numbering); if the first light chain Fab region comprises a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an alanine at amino acid residue 109 (EU numbering), then the second light chain Fab region does not comprise a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an alanine at amino acid residue 109 (EU numbering); said second light chain Fab region does not contain a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an aspartic acid at amino acid residue 110 (EU numbering); said second light chain Fab region does not contain a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an aspartic acid at amino acid residue 110 (EU numbering); said second light chain Fab region does not contain a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an alanine at amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110 (EU numbering);if the first light chain Fab region comprises an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino acid residue 143 (EU numbering), the second light chain Fab region does not comprise an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino acid residue 143 (EU numbering); if the first light chain Fab region comprises an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino acid residue 143 (EU numbering) and an alanine at amino acid residue 109 (EU numbering), the second light chain Fab region does not comprise an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino acid residue 143 (EU numbering) and an alanine at amino acid residue 109 (EU numbering); when said first light chain Fab region comprises an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino acid residue 199 (EU numbering) and an alanine at amino acid residue 109 (EU numbering), said second light chain Fab region does not comprise an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino acid residue 199 (EU numbering) and an alanine at amino acid residue 109 (EU numbering); when said first light chain Fab region comprises an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue 143 (EU numbering), said second light chain Fab region does not comprise an alanine at amino acid residue 109 and a lysine at amino acid residue 143 (EU numbering); If the first light chain Fab region comprises an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), then the second light chain Fab region does not comprise an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue 199 (EU numbering); and if the first light chain Fab region comprises an alanine at amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110 (EU numbering), then the second light chain Fab region does not comprise an alanine at amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110 (EU numbering).

[0007] According to some embodiments, the multispecific binding protein comprises a first antigen binding domain comprising a first light chain Fab region and a first heavy chain Fab region, where the first light chain Fab region is a kappa light chain and comprises a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), and the multispecific binding protein comprises a second antigen binding domain comprising a second light chain Fab region and a second heavy chain Fab region, where the first antigen binding domain binds to the first antigen and the second antigen binding domain binds to the second antigen. In some embodiments, the first light chain Fab region further comprises an aspartic acid at amino acid residue 110 (EU numbering). In some embodiments, the first light chain Fab region further comprises an alanine at amino acid residue 109 (EU numbering).

[0008] According to further embodiments of the multispecific binding proteins provided herein, the multispecific binding protein comprises a first antigen-binding domain comprising a first light chain Fab region and a first heavy chain Fab region, wherein said first light chain Fab region is a kappa light chain and comprises an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino acid residue 143 (EU numbering), and said multispecific binding protein comprises a second antigen-binding domain comprising a second light chain Fab region and a second heavy chain Fab region, wherein said first antigen-binding domain binds to said first antigen and said second antigen-binding domain binds to said second antigen. In some embodiments, said first light chain Fab region further comprises an alanine at amino acid residue 109 (EU numbering).

[0009] According to embodiments of the multispecific binding proteins provided herein, the multispecific binding protein comprises a first antigen-binding domain comprising a first light chain Fab region and a first heavy chain Fab region, wherein said first light chain Fab region is a kappa light chain and comprises an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), and said multispecific binding protein comprises a second antigen-binding domain comprising a second light chain Fab region and a second heavy chain Fab region, wherein said first antigen-binding domain binds to said first antigen and said second antigen-binding domain binds to said second antigen. In some embodiments, said first light chain Fab region further comprises an alanine at amino acid residue 109 (EU numbering).

[0010] According to embodiments of the multispecific binding proteins provided herein, the multispecific binding protein comprises a first antigen-binding domain comprising a first light chain Fab region and a first heavy chain Fab region, where said first light chain Fab region is a kappa light chain and comprises an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue 143 (EU numbering), and said multispecific binding protein comprises a second antigen-binding domain comprising a second light chain Fab region and a second heavy chain Fab region, where said first antigen-binding domain binds to said first antigen and said second antigen-binding domain binds to said second antigen.

[0011] According to some embodiments of the multispecific binding proteins provided herein, the multispecific binding protein comprises a first antigen-binding domain comprising a first light chain Fab region and a first heavy chain Fab region, where said first light chain Fab region is a kappa light chain and comprises an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), and said multispecific binding protein comprises a second antigen-binding domain comprising a second light chain Fab region and a second heavy chain Fab region, where said first antigen-binding domain binds to said first antigen and said second antigen-binding domain binds to said second antigen.

[0012] According to further embodiments of the multispecific binding protein provided herein, the multispecific binding protein comprises a first antigen binding domain comprising a first light chain Fab region and a first heavy chain Fab region, where said first light chain Fab region is a kappa light chain and comprises an alanine at amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110 (EU numbering), and said multispecific binding protein comprises a second antigen binding domain comprising a second light chain Fab region and a second heavy chain Fab region, where said first antigen binding domain binds to said first antigen and said second antigen binding domain binds to said second antigen.

[0013] According to some embodiments of the multispecific binding proteins of the present disclosure, the first antigen-binding domain of the multispecific binding protein further comprises a first heavy chain Fc region. In further embodiments of the multispecific binding proteins of the present disclosure, the first heavy chain Fc region comprises a human IgG1, human IgG2 or human IgG4 constant region. In some embodiments of the multispecific binding proteins of the present disclosure, the second antigen-binding domain further comprises a second heavy chain Fc region. In further embodiments of the multispecific binding proteins of the present disclosure, the second heavy chain Fc region comprises a human IgG1, human IgG2 or human IgG4 constant region. In some embodiments of the multispecific binding proteins of the present disclosure, the first heavy chain Fc region comprises an arginine at amino acid residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU numbering). According to some embodiments of the multispecific binding proteins of the present disclosure, the second heavy chain Fc region comprises an arginine at amino acid residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU numbering). In some embodiments of the multispecific binding proteins of the present disclosure, the first and second heavy chain Fc regions both comprise a human IgG1 constant region; both comprise a human IgG2 constant region; or both comprise a human IgG4 constant region. In further embodiments of the multispecific binding proteins of the present disclosure, the first and second heavy chain Fc regions both comprise an arginine at amino acid residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU numbering). According to some embodiments of the multispecific binding proteins of the present disclosure, the second light chain Fab region does not include an alanine at amino acid residue 109; does not include an aspartic acid at amino acid residue 110; does not include a lysine at amino acid residue 143; or does not include a lysine at amino acid residue 199. In some embodiments of the multispecific binding proteins of the disclosure, the second light chain Fab region does not contain an alanine at amino acid residue 109; does not contain an aspartic acid at amino acid residue 110; does not contain a lysine at amino acid residue 143; or does not contain a lysine at amino acid residue 199.According to some embodiments of the multispecific binding proteins of the present disclosure, the second light chain Fab region is a kappa light chain.Further, according to some embodiments of the multispecific binding proteins of the present disclosure, the second light chain Fab region is a lambda light chain.

[0014] According to some embodiments, the multispecific binding protein comprises a bispecific binding protein. According to some such embodiments, the bispecific binding protein is an immunoglobulin heteromab. In some more specific embodiments, the immunoglobulin heteromab is an IgG heteromab. According to further embodiments, the multispecific binding protein comprises a multispecific binding protein.

[0015] Additionally, embodiments of the present disclosure also provide pharmaceutical compositions comprising a multispecific binding protein of the present disclosure and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0016] According to further embodiments of the present disclosure, a method for purifying a multispecific binding protein of the present disclosure is provided. According to some such embodiments, the method comprises the steps of: introducing a first light chain Fab region comprising a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid residue 199 (EU numbering) into the first antigen-binding domain, where the first light chain Fab region is a kappa light chain; expressing the multispecific binding protein, where the first antigen-binding domain is assembled with the second antigen-binding domain; subjecting the multispecific binding protein to an affinity chromatography column; and recovering the purified multispecific binding protein. According to some such embodiments, the introducing step further comprises introducing an alanine at amino acid residue 109 (EU numbering) into the first antigen-binding domain. In some embodiments, the introducing step further comprises introducing an aspartic acid at amino acid residue 110 (EU numbering) into the first antigen-binding domain.

[0017] Further embodiments of methods of purifying a multispecific binding protein of the present disclosure are provided, wherein the method comprises the steps of: introducing into said first antigen-binding domain a first light chain Fab region comprising an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino acid residue 143 (EU numbering), wherein said first light chain Fab region is a kappa light chain; expressing said multispecific binding protein, wherein said first antigen-binding domain is assembled with said second antigen-binding domain; subjecting said multispecific binding protein to an affinity chromatography column; and recovering the purified multispecific binding protein. According to some embodiments, said introducing step further comprises introducing into said first antigen-binding domain an alanine at amino acid residue 109 (EU numbering).

[0018] Further embodiments of the method of purifying a multispecific binding protein of the present disclosure are provided, wherein the method comprises the steps of: introducing into said first antigen-binding domain a first light chain Fab region comprising an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), wherein said first light chain Fab region is a kappa light chain; expressing said multispecific binding protein, wherein said first antigen-binding domain is assembled with said second antigen-binding domain; subjecting said multispecific binding protein to an affinity chromatography column; and recovering the purified multispecific binding protein. According to some embodiments, said introducing step further comprises introducing into said first antigen-binding domain an alanine at amino acid residue 109 (EU numbering).

[0019] According to further embodiments, there is provided a method of purifying a multispecific binding protein of the present disclosure, comprising the steps of: introducing into said first antigen-binding domain a first light chain Fab region comprising an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue 143 (EU numbering), wherein said first light chain Fab region is a kappa light chain; expressing said multispecific binding protein, wherein said first antigen-binding domain is assembled with said second antigen-binding domain; applying said multispecific binding protein to an affinity chromatography column; and recovering the purified multispecific binding protein.

[0020] Further embodiments of methods of purifying a multispecific binding protein of the disclosure are provided, wherein the method comprises the steps of: introducing into said first antigen-binding domain a first light chain Fab region comprising an alanine at amino acid residue 109 (EU numbering) and a lysine at amino acid residue 199 (EU numbering), wherein said first light chain Fab region is a kappa light chain; expressing said multispecific binding protein, wherein said first antigen-binding domain is assembled with said second antigen-binding domain; applying said multispecific binding protein to an affinity chromatography column; and recovering the purified multispecific binding protein.

[0021] Further embodiments of methods of purifying a multispecific binding protein of the disclosure are provided, wherein the method comprises the steps of: introducing into said first antigen-binding domain a first light chain Fab region comprising an alanine at amino acid residue 109 (EU numbering) and an aspartic acid at amino acid residue 110 (EU numbering), wherein said first light chain Fab region is a kappa light chain; expressing said multispecific binding protein, wherein said first antigen-binding domain is assembled with said second antigen-binding domain; applying said multispecific binding protein to an affinity chromatography column; and recovering the purified multispecific binding protein.

[0022] According to some embodiments of the method of purifying a multispecific binding protein of the present disclosure, the introducing step further comprises introducing a first heavy chain Fc region to the first antigen-binding domain. In further embodiments of the method of the present disclosure, the first heavy chain Fc region comprises a human IgG1, human IgG2 or human IgG4 constant region. In some embodiments of the method of the present disclosure, the introducing step further comprises introducing a second heavy chain Fc region to the second antigen-binding domain. In further embodiments of the method of the present disclosure, the second heavy chain Fc region comprises a human IgG1, human IgG2 or human IgG4 constant region. According to some embodiments of the method of the present disclosure, the introducing step further comprises introducing an arginine at amino acid residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU numbering) to the first heavy chain Fc region. In some embodiments of the methods of the present disclosure, the introducing step further comprises introducing an arginine at amino acid residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU numbering) into the second heavy chain Fc region. According to some embodiments of the methods of the present disclosure, the first heavy chain Fc region and the second heavy chain Fc region both comprise a human IgG1 constant region; both comprise a human IgG2 constant region; or both comprise a human IgG4 constant region. In some embodiments of the methods of the present disclosure, the introducing step further comprises introducing an arginine at amino acid residue 311 (EU numbering) and a glutamic acid at amino acid residue 317 (EU numbering) into both the first heavy chain Fc region and the second heavy chain Fc region. In some embodiments of the disclosed methods, the second light chain Fab region does not contain an alanine at amino acid residue 109; does not contain an aspartic acid at amino acid residue 110; does not contain a lysine at amino acid residue 143; or does not contain a lysine at amino acid residue 199. In some embodiments of the disclosed methods, the second light chain Fab region does not contain an alanine at amino acid residue 109; does not contain an aspartic acid at amino acid residue 110; does not contain a lysine at amino acid residue 143; and does not contain a lysine at amino acid residue 199.In some embodiments of the methods of the present disclosure, the second light chain Fab region is a kappa light chain. In further embodiments of the methods of the present disclosure, the second light chain Fab region is a lambda light chain.

[0023] According to further embodiments of the disclosed method, the affinity chromatography column comprises a kappa affinity ligand. In some embodiments of the disclosed method, the affinity chromatography column comprises a lambda affinity ligand. According to some embodiments of the disclosed method, the affinity chromatography column comprises Protein A. In some embodiments of the disclosed method, the second light chain Fab region binds to the affinity chromatography column with a higher affinity than the first light chain Fab region. In further embodiments of the disclosed method, the first light chain Fab region does not bind to the affinity chromatography column.

[0024] According to further embodiments of the method of purifying a multispecific binding protein of the present disclosure, the method further comprises the following steps: after recovering the purified multispecific binding protein, subjecting the purified multispecific binding protein to a second affinity chromatography column; and after subjecting the purified multispecific binding protein to the second affinity chromatography column, subjecting the purified multispecific binding protein to a second affinity chromatography column, recovering the purified multispecific binding protein. According to some embodiments, the second affinity chromatography column comprises a kappa affinity ligand. In some embodiments, the second affinity chromatography column comprises a lambda affinity ligand. According to some embodiments, the second affinity chromatography column comprises Protein A. In some further embodiments, the second light chain Fab region binds to the second affinity chromatography column with a higher affinity than the first light chain Fab region. Furthermore, in some embodiments, the first light chain Fab region does not bind to the second affinity chromatography column.

[0025] According to further embodiments, the present disclosure provides a method for producing a multispecific binding protein of the present disclosure. In some such embodiments, such methods include a multispecific binding protein of the present disclosure prepared according to a process, wherein the process comprises the steps of: culturing a host cell comprising a polynucleotide sequence encoding a first antigen binding domain and a second antigen binding domain of the present disclosure under conditions such that the multispecific binding protein is expressed; and recovering the multispecific binding protein of the present disclosure from the host cell. According to some embodiments, the polynucleotide sequence comprises a single vector encoding the first antigen binding domain and the second antigen binding domain. According to further embodiments, the polynucleotide sequence comprises a first vector encoding the first antigen binding domain and a second vector comprising the second antigen binding domain. In some embodiments, the method of the present disclosure further comprises the steps of: subjecting the recovered multispecific binding protein to an affinity chromatography column; and recovering the purified multispecific binding protein. In some embodiments, the affinity chromatography column comprises Protein A. In some embodiments, the affinity chromatography column comprises a kappa affinity ligand. In some embodiments, the affinity chromatography column comprises a lambda affinity ligand. According to some embodiments, the first antigen-binding domain comprises a first light chain Fab region and the second antigen-binding domain comprises a second light chain Fab region, the second light chain Fab region binding to the affinity chromatography column with a higher affinity than the first light chain Fab region. In some embodiments, the first light chain Fab region does not bind to the affinity chromatography column.According to further embodiments, the method of the disclosure further comprises the steps of: after recovering the purified multispecific binding protein, subjecting the purified multispecific binding protein to a second affinity chromatography column; and after subjecting the purified multispecific binding protein to the second affinity chromatography column, recovering the purified multispecific binding protein. In some embodiments, the second affinity chromatography column comprises Protein A. In some embodiments, the second affinity chromatography column comprises a kappa affinity ligand. In some embodiments, the second affinity chromatography column comprises a lambda affinity ligand. According to some embodiments, the first antigen binding domain comprises a first light chain Fab region, and the second antigen binding domain comprises a second light chain Fab region, and the second light chain Fab region binds to the second affinity chromatography column with a higher affinity than the first light chain Fab region. In some embodiments, the first light chain Fab region does not bind to the second affinity chromatography column.

[0026] In further embodiments, the disclosure provides multispecific binding proteins for use in therapy. In some embodiments, the disclosure provides multispecific binding proteins for use in the treatment of a medical condition. In some such embodiments, the medical condition is one of cancer, cardiovascular disease, autoimmune disease, or neurodegenerative disease.

[0027] In further embodiments, the disclosure provides a multispecific binding protein for use in the manufacture of a medicament. In some embodiments, the disclosure provides a multispecific binding protein for use in the manufacture of a medicament for treatment. In further embodiments, the disclosure provides a multispecific binding protein for use in the manufacture of a medicament for the treatment of a medical condition. In some such embodiments, the medical condition is one of cancer, cardiovascular disease, autoimmune disease, or neurodegenerative disease.

[0028] The term "multispecific binding protein" as used herein refers to a molecule having two or more different antigen binding domains. Multispecific binding proteins of the present disclosure bind to two or more different antigens or two or more different epitopes of the same antigen. Multispecific binding protein embodiments of the present disclosure include bispecific antibodies, as well as trispecific or tetraspecific binding molecules known in the art, as well as single chain multispecific binding molecules such as bispecific antibodies. Multispecific binding proteins of the present disclosure may vary in size and shape and may include multiple formats known in the art.

[0029] The "antigen-binding domain" referred to herein refers to the portion of a multispecific binding protein that contains the amino acid residues that interact with and confer specificity to the respective antigen. The antigen-binding domain of the multispecific binding protein of the present disclosure includes a light chain Fab region and a heavy chain Fab region. Both the heavy chain and the light chain Fab region contain variable portions at the amino terminus that contain CDRs interspersed with more conserved regions (called framework regions). Both the heavy chain and the light chain Fab region also contain conserved regions (e.g., CL of the light chain Fab region and CH1 of the heavy chain Fab region, as known in the art). The light chain Fab region is classified as kappa or lambda, as known in the art.

[0030] Some embodiments of the multispecific binding proteins of the present disclosure include a heavy chain Fc region linked at the carboxy terminus of a heavy chain Fab region (e.g., to form a heavy chain as known in the art). The heavy chain Fc region of the present disclosure is classified as gamma, which defines the isotype of the heavy chain as IgG and one of the subclasses IgG1, IgG2, IgG3, or IgG4. The heavy chain Fc region may further provide effector functions (known in the art) for the multispecific binding protein.

[0031] According to some particular embodiments, the multispecific binding proteins of the present disclosure comprise IgG heteromab molecules, or fragments thereof. As known in the art, IgG heteromab molecules comprise a typical Fab structure and an IgG structure (one Fab "arm", i.e., antigen-binding domain, binds to a first antigen, and the other Fab "arm", i.e., antigen-binding domain, binds to a second antigen).

[0032] The art-recognized term "EU numbering" refers to a system for numbering amino acid residues in immunoglobulin molecules. EU numbering is described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991); Edelman, GM, et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); and http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs. The term "Kabat numbering" is art-recognized as referring to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions (see, e.g., Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). The term "North numbering" refers to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions, and is based, at least in part, on affinity propagation clustering using multiple crystal structures, as described in North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011).

[0033] The term "affinity chromatography" as used herein refers to a chromatographic method that separates biochemical mixtures (e.g., multispecific binding proteins and undesired biomolecular species) based on specific and reversible interactions between biomolecules. Representative embodiments of affinity chromatography include Protein A affinity columns, kappa affinity ligand chromatography (e.g., CaptureSelect™, KappaXL™, KappaSelect™, KappaXP™, etc.), or lambda affinity ligand chromatography.

[0034] A "parent" or "parental" molecule as referred to herein is a molecule that is encoded by an amino acid sequence used to prepare one of the exemplary embodiments described herein, e.g., through amino acid substitutions and structural changes. A parent molecule may include, for example, a murine antibody, or a fragment thereof, or a binding protein obtained, for example, by phage display or a transgenic non-human animal.

[0035] The multispecific binding proteins of the disclosure may be incorporated into pharmaceutical compositions comprising the multispecific binding proteins of the disclosure and one or more pharma- ceutically acceptable carriers and / or diluents, which can be prepared by methods well known in the art (e.g., Remington, The Science and Practice of Pharmacy, 22nd Edition, Loyd V., Ed., Pharmaceutical Press, 2012, which provides an overview of formulation techniques generally known to those of skill in the art). Carriers suitable for pharmaceutical compositions include any material that, when combined with the multispecific binding protein, retains the activity of the molecule and is non-reactive with the patient's immune system.

[0036] Expression vectors capable of inducing the expression of operably linked genes are well known in the art. The expression vector can encode a signal peptide that facilitates the secretion of a polypeptide from a host cell. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Each of the expression peptides can be expressed independently from different promoters operably linked in one vector, or from different promoters operably linked in multiple vectors. Expression vectors are generally replicable in the host organism as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selection marker (e.g., tetracycline, neomycin, and dihydrofolate reductase) to allow detection of cells transformed with the DNA sequence of interest.

[0037] A host cell refers to a cell that is stably or transiently transfected, transformed, transduced or infected with one or more expression vectors that express one or more polypeptide chains of the multispecific binding protein of the present disclosure. Creation and isolation of host cell lines that produce the binding proteins of the present disclosure can be accomplished using standard techniques known in the art. Mammalian cells are preferred host cells for expression of the multispecific binding proteins of the present disclosure. Particular mammalian cells include HEK 293, NS0, DG-44, and CHO. Preferably, the binding protein is secreted into the medium in which the host cells are cultured and from which the binding protein can be recovered or purified, for example, using conventional techniques. For example, the medium may be applied to and eluted from a Protein A affinity chromatography column and / or a kappa or lambda affinity ligand chromatography column. Undesirable biomolecular species, such as soluble aggregates and multimers, may be efficiently removed by common techniques such as size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product may be immediately frozen, refrigerated, or lyophilized, e.g., at −70° C. A variety of protein purification methods may be utilized, such methods being known in the art and described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994). EXAMPLES

[0038] Expression and purification of exemplary multispecific binding proteins Exemplary multispecific binding proteins of the present disclosure, including those in the form of an IgG heteromab with a first antigen-binding domain that binds cMet and a second antigen-binding domain that binds BHA10, may be expressed and purified essentially as follows: Briefly, the first light and heavy chain Fab regions are cloned into an expression vector (such as the pEHG1 and pEHK expression vectors, which contain the human G1 allotype constant region and the human kappa light chain constant region, respectively). Both vectors contain a mouse kappa leader sequence to facilitate secretion (WO2014 / 150973 A1; Lewis SM, et al., 2014 Nat. Biotechnol. 32, 191-8).

[0039] Amino acid residue changes may be introduced into the binding domain by methods known in the art (such as the light chain, Quickchange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.), codon-optimized coding regions synthesized de novo (in a single or separate vector), and the like). The EU numbering convention may be used to determine the position of the mutation.

[0040] Exemplary modified kappa light chain Fab and heavy chain Fc region formats of the present disclosure are set forth in Tables 1a and 1b, respectively (amino acid modifications are numbered according to EU numbering). [Table 1] [Table 2]

[0041] Various combinations of IgG heteromab embodiments, including the heavy and light chain formats of Tables 1a and 1b, are described in Table 2. Exemplary IgG heteromab include a first antigen-binding domain that binds cMet and a second antigen-binding domain that binds BHA10; or a first antigen-binding domain that binds PD-1 and a second antigen-binding domain that binds Tigit. Exemplary IgG heteromab of Table 2 include various combinations of modified heavy and light chain formats (of Tables 1a and 1b), including first and second antigen-binding domains, respectively, to evaluate the effect, if any, on expression, assembly and purification based on the orientation of the format. Parent monoclonal antibodies and IgG1 heteromab molecules (e.g., molecules that do not include modified light or heavy chain formats described in Tables 1a and 1b) are also evaluated as controls.

[0042] Suitable host cells (such as CHO) are transiently transfected with an expression system to secrete the exemplary IgG heteromab of Table 2. The exemplary IgG heteromab is detected by 280 nm absorbance in the purified medium into which the exemplary IgG heteromab is secreted. As shown in Table 2, the expression levels of the modified kappa light chain Fab antibody format and the modified heavy chain Fc antibody format of Table 1 are comparable to the respective parent antibodies. Thus, the modifications according to the formats of Tables 1a and 1b did not negatively affect the expression levels and in some cases improved the expression titers. [Table 3] [Table 4]

[0043] Comparison of % flow-through and % elution of exemplary modified kappa light chain Fab region formats on a Kappa XL column The multispecific binding proteins of the disclosure in the purified medium or recovered from Protein A purification may be subjected to a second purification step using a CaptureSelect™ Kappa XL (Thermo Fisher Cat. No. 494321001) prepacked affinity column. Briefly, the multispecific binding proteins of the disclosure recovered from Protein A purification are applied to a Kappa XL affinity column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (PBS) at pH 7.4. The column is then washed to remove non-specifically bound components. The bound multispecific binding proteins are eluted, for example, by a pH gradient, such as 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0. Fractions of bound proteins are detected, for example, by UV absorbance or SDS-PAGE, and then pooled.

[0044] The % flow-through (%FT) and % elution of exemplary heteromab of the present disclosure following Kappa XL column purification essentially as described herein are evaluated. Briefly, various antibody formats (as described in Table 3) are run through a Kappa XL column. The flow-through material is considered to be comprised of impurities (e.g., homodimers, or misassembled antibodies, etc.), while the Kappa XL ligand-bound material (eluted material) is considered to be correctly assembled bispecific antibodies.

[0045] Table 3 shows that the cMet parent mAb with DKK format for both light chain Fab regions abolished binding to the Kappa XL column, therefore 100% of the antibody was recovered in the flow-through, preventing differentiation of the desired antibody species from the undesired antibody species. Similarly, the flow-through percentages for the cMet parent mAb and the cMet-BAH10 IgG1 heteromab were 2.8% and 5.5%, respectively, indicating that the majority of the antibody bound to the Kappa XL column, thus preventing differentiation of the desired antibody species from the undesired antibody species. In contrast, the results in Table 3 show that the cMet-BAH10 IgG1 heteromab with DKK format only for the cMet light chain Fab region resulted in 29.3% of the flow-through species and 70.7% of the eluted multispecific binding protein species, meaning that the DKK format of the kappa light chain Fab region allows selective discrimination and separation of the desired multispecific binding protein species from the undesired species.

[0046] Furthermore, Table 3 shows that the formats of DKK and ADK kappa light chain Fab region on the PD-1 light chain Fab region (of PD-1-Tigit IgG1 heteromab) result in 80.72% and 78.24% eluted species, respectively, therefore implying that both formats selectively distinguish the eluted species of interest from the undesired (flow-through) species of the multispecific binding protein. [Table 5]

[0047] Taken together, the results show that the kappa light chain Fab region format of Table 1a, when expressed in only one light chain Fab region of an IgG heteromab, efficiently distinguishes the multispecific binding protein of interest from undesired species and therefore allows for efficient separation and purification of the binding protein of interest.

[0048] A purified, multispecific binding protein that binds Protein A and KappaXL ligands. Protein A binding analysis Binding to Protein A ligands of the heavy chain Fc region format of Table 1b for the exemplified IgG heteromab may be assessed by ELISA. Briefly, 96-well flat-bottom Elisa plates are coated with 2ug / mL goat anti-human kappa protein, 100ul / well, and incubated overnight at 4°C. The next day, the plates are washed 3 times with wash buffer (0.05% PBS-Tween20 (PBS-T)) and blocked with blocking buffer (casein, 200μL / well) for 1 hour at room temperature (RT). The plates are washed 3 times with wash buffer and binding proteins (shown in Table 4) are added to each well at 10μg / mL, serially diluted 1:3 in PBS-T in a volume of 100uL / well. The plates are incubated at RT for 1 hour and washed 3 times with wash buffer. Biotin-Protein A at 0.5ug / ml is added at 100uL / well, the plate is incubated for 1 hour at RT, washed 3 times, and 100uL / well of streptavidin-labeled alkaline phosphatase (SA-AP) is added to each well. The plate is incubated for 30 minutes at RT. The plate is then washed 3 times and 100uL / well of p-nitrophenyl phosphate disodium salt (PNAP) (Thermo Fisher Scientific) substrate is added. The reaction is stopped and absorbance is measured using a colorimetric microplate reader set at 405nm. The results are shown in Table 4. [Table 6]

[0049] The results show that the heavy chain Fc region RE format, when expressed as part of only the cMet heavy chain Fc region of the cMet-BAH10 IgG1 heteromab, reduces binding to Protein A by approximately 1.2-fold compared to the cMet-BAH10 IgG1 heteromab. When the heavy chain Fc region RE format is expressed as part of both heavy chain Fc regions, it reduces binding affinity to Protein A by approximately 2-fold compared to the parental mAb. This data indicates that the heavy chain Fc region RE format allows for elution of the binding molecule of interest at higher pH and discrimination from undesirable or contaminating species.

[0050] Kappa XL binding analysis Binding to Kappa XL ligands of the light chain Fab region format of Table 1a for the exemplified IgG heteromab may be assessed by ELISA. Briefly, 96-well flat-bottom ELISA plates are coated with goat anti-human IgG protein at 2ug / mL, 100ul / well, and incubated overnight at 4°C. The next day, plates are washed 3 times with wash buffer (0.05% PBS-Tween20) and blocked with blocking buffer (casein, 200μL / well) for 1 hour at room temperature (RT). Plates are washed 3 times with wash buffer and binding proteins (shown in Table 5) are added at 10μg / mL and serially diluted 1:3 in DPBS (Dulbecco's HyClone) at 100uL / well. Plates are incubated 1 hour at RT, washed 3 times with wash buffer, and biotin-KappaXL is added at 1ug / ml, 100uL / well. The plate is then incubated for 1 hour at RT, washed 3 times, and 100 uL / well of SA-AP is added to each well and incubated for 30 minutes at RT. The plate is then washed 3 times and 100 uL / well of PNAP substrate is added. The reaction is stopped and absorbance is measured using a colorimetric microplate reader set at 405 nm. The results are shown in Table 5. [Table 7]

[0051] The results in Table 5 demonstrate that both the cMet-BAH10 IgG heteromab with the DKK format as part of the cMet light chain Fab region only (2.50 nm) and the cMet-BAH10 IgG heteromab with the ADK format as part of the cMet light chain Fab region only (1.27 nM) show lower binding affinity to the Kappa XL ligand compared to the cMet-BAH10 IgG1 heteromab without the light chain Fab region format in Table 1a (0.57 nM). The cMet parent mAb with the DKK format expressed as part of both LCs showed no detectable binding to the Kappa XL ligand. These results demonstrated that both the DKK and ADK light chain Fab region formats, when incorporated onto a single "arm" of a multispecific binding protein, reduce the binding affinity to the Kappa XL ligand and allow for the removal of undesirable or contaminating species (e.g., in the flow-through). Note that this advantage may be further enhanced for multispecific binding proteins designed such that the light chain Fab region format of Table 1a is included only in the highly expressed "arms" of the binding protein.

[0052] Purification, identity and heterogeneity analysis of purified antibodies Multispecific binding proteins containing the heavy chain Fc region and / or light chain Fab region formats of Table 1 are subjected to Protein A (Step 1) purification followed by Kappa XL (Step 2) purification. Flow-through and eluted material are analyzed for purity, identity and heterogeneity by standard techniques such as size exclusion chromatography (SEC), capillary electrophoresis (lab chip NR ceSDS), high performance liquid chromatography (HIC-HPLC) and intact mass spectrometry. SEC is used to analyze samples for high molecular weight (HMW) percentage, front shoulder percentage, main peak percentage and low molecular weight (LMW) percentage. Percentages are calculated by Empower analysis of the chromatographs using the ratio of AUC of the peak eluting before the monomeric peak to total AUC. NR ceSDS is used to quantify the concentration of total Ab (%) and ½ Ab (%) in the purified material. The binding protein formats evaluated at each step are listed in Tables 6, 7 and 8. [Table 8]

[0053] As shown in Table 6, step 1 (Protein A purification) showed that the kappa light chain Fab region format in Table 1a did not negatively affect the HMW species compared to the control (PD-1-TIGIT IgG1 heteromab), which means that the light chain Fab region format in Table 1a did not negatively affect the assembly of the multispecific binding protein. Furthermore, the main peak of the light chain Fab region format in Table 1a was comparable to the control (PD-1-TIGIT IgG1 heteromab), and no significant difference was observed between the duplex and triplex formats of the light chain Fab region in Table 1a. [Table 9]

[0054] As shown in Table 7, the step 2 (Kappa XL purification) elution profile indicates that the purity of the main peak was increased to greater than 95% for all kappa light chain Fab region formats of Table 1a, compared to approximately 83% for each binding protein lacking the kappa light chain Fab region format of Table 1a disclosed herein. The results further indicate that the impurity represented by the LMW peak was reduced to less than 1.0% for the kappa light chain Fab region format of Table 1a, compared to 7.7% for each binding protein lacking the kappa light chain Fab region format of Table 1a. Similarly, the NR ceSDS profile indicates that the amount of all binding proteins containing the kappa light chain Fab region format of Table 1a was greater than 94%, while the binding protein lacking the kappa light chain Fab region format of Table 1a was only 80.2%. Furthermore, less than 4% of the binding proteins contained the kappa light chain Fab region format of Table 1a, whereas 17.8% of the binding proteins lacked the kappa light chain Fab region format of Table 1a. No significant differences in elution profiles were observed between the different binding proteins containing the various kappa light chain Fab region formats of Table 1a. [Table 10]

[0055] Table 8 shows that the undesired impurities (Front and LMW %) were efficiently separated in the Kappa XL flow-through and the desired multispecific binding proteins, including the formats in Table 1, were enriched in the elution. No unmodified control was observed in the flow-through profile, indicating that all unmodified heteromab bound to the Kappa XL column.

[0056] The results, presented in Tables 3-8, demonstrate that the modified kappa light chain Fab region format of Table 1a provides robust purification, allowing the multispecific binding protein of interest to be selectively differentiated and separated from undesired species and / or impurities.

[0057] Thermal stability assessment of exemplified multispecific binding proteins The thermal stability of the exemplary multispecific binding proteins provided herein is assessed by differential scanning calorimetry (DSC) after Protein A and Kappa XL purification. The results (unfolding temperature reported as Tm1) are listed in Table 9. [Table 11]

[0058] Table 9 shows that the format of the heavy chain Fc region RE (on both arms of the cMet parent antibody) affected thermal stability, but no effect was observed when the heavy chain Fc region RE format was expressed as part of only one arm of the exemplified IgG1 heteromab. Furthermore, all modified kappa light chain Fab formats exhibited thermal stability comparable to their respective unmodified parent molecules.

[0059] Binding affinity analysis of exemplified multispecific binding proteins The binding affinity of the exemplary multispecific binding proteins described herein is evaluated by ELISA. Briefly, 384-well flat-bottom Elisa plates are coated with 1ug / mL anti-human-Fc protein at 20ul / well and incubated overnight at 4°C. The next day, the plates are washed 3 times with washing buffer (0.05% PBS-Tween20) and blocked with blocking buffer (casein, 60μL / well) for 1 hour at room temperature (RT). The plates are washed 3 times with washing buffer and the binding proteins shown in Table 10 are added at 1μg / mL in DPBS (Dulbecco's HyClone) in triplicate at 20uL / well. The plates are incubated for 1 hour at RT, washed 3 times with washing buffer, and a determined amount of antigen is added at 20uL / well and incubated for 60 minutes at RT. The plates are washed 3 times and NAAP substrate is added at 20uL / well and incubated for 20 minutes. The plate is washed three times, PNPP substrate is added at 20 uL / well, the reaction is stopped, and the absorbance is measured using a colorimetric microplate reader set at 405 nm. The results are shown in Table 10. [Table 12]

[0060] These results, set forth in Table 10, demonstrate that the exemplary multispecific binding proteins comprising the modified kappa light chain Fab region format of Table 1a maintain comparable, and in some instances improved, binding affinity to the target antigens (compared to the unmodified parent multispecific binding protein).

[0061] In silico immunogenicity analysis The immunogenicity of the modified heavy chain Fc region and light chain Fab region formats of the exemplary multispecific binding proteins is analyzed by in silico immunogenicity analysis using Immune Epitope Database Analysis (IEDB). The immunogenicity (IG) score and rarefaction score (frequency of amino acid usage at positions corresponding to the human Ig repertoire) of the antibody sequence are calculated (lower scores indicate less immunogenicity). The results are listed in Tables 11a and 11b. [Table 13] [Table 14]

[0062] The results, presented in Tables 11a and 11b, reveal that the modified light chain Fab region and heavy chain Fc region formats of Tables 1a and 1b exhibit IG and rarity scores comparable to the respective unmodified parent molecules, suggesting that the modified formats do not pose additional immunogenicity risk.

[0063] array SEQ ID NO:1 (Representative cMet HC (Fab region underlined)) [ka]

[0064] SEQ ID NO:2 (representative cMet LC) [ka]

[0065] SEQ ID NO:3 (Representative BAH10 HC (Fab region underlined)) [ka]

[0066] SEQ ID NO:4 (representative BAH10 LC) [ka]

[0067] SEQ ID NO:5 (Representative cMet HC with RE format (RE format underlined and italicized, Fab region underlined)) [ka]

[0068] SEQ ID NO:6 (Representative cMet HC (Fab region underlined)) [ka]

[0069] SEQ ID NO:7 (Representative cMet LC with the format DKK (DKK format is underlined)) [ka]

[0070] SEQ ID NO:8 (Representative cMet HC with RE format (RE format is underlined and italicized, Fab region is underlined)) [ka]

[0071] SEQ ID NO:9 (Representative cMet LC with ADK format (ADK format is underlined)) [ka]

[0072] SEQ ID NO: 10 (Representative PD1 HC (Fab region underlined)) [ka]

[0073] SEQ ID NO: 11 (representative PD1 LC) [ka]

[0074] SEQ ID NO: 12 (Representative TIGIT HC (Fab region underlined)) [ka]

[0075] SEQ ID NO: 13 (representative TIGIT LC) [ka]

[0076] SEQ ID NO: 14 (Representative DKK LC having the format DKK (the format DKK is underlined)) [ka]

[0077] SEQ ID NO: 15 (Representative PD1 LC with ADK format (ADK format is underlined)) [ka]

[0078] SEQ ID NO: 16 (Representative PD1 LC with DK format (DK format is underlined)) [ka]

[0079] SEQ ID NO: 17 (Representative PD1 LC with the format KK (KK format is underlined)) [ka]

Claims

1. 1. A multispecific binding protein that binds a first antigen and a second antigen, the multispecific binding protein comprising: A first antigen-binding domain comprising a first light chain Fab region and a first heavy chain Fab region, wherein the first light chain Fab region has the following mutation in the amino acid sequence of SEQ ID NO: 2, 4, 11, or 13: E143K and Q199K mutations; E143K, Q199K, and V110D mutations; V110D and Q199K mutations; or V110D, Q199K, and T109A mutations; or a human kappa light chain having the amino acid sequence of SEQ ID NO:9; and a second antigen-binding domain comprising a second light chain Fab region and a second heavy chain Fab region; wherein said first antigen-binding domain binds to said first antigen and said second antigen-binding domain binds to said second antigen.

2. The second light chain Fab region comprises the following mutation in the amino acid sequence of SEQ ID NO: 2, 4, 11, or 13: E143K and Q199K mutations; E143K, Q199K, and V110D mutations; V110D and Q199K mutations; or V110D, Q199K, and T109A mutations; or a human kappa light chain having the amino acid sequence of SEQ ID NO:9, with the proviso that if the first light chain Fab region comprises the mutations E143K and Q199K, then the second light chain Fab region does not contain a lysine at amino acid residue 143 (EU numbering) and a lysine at amino acid residue 199 (EU numbering); if the first light chain Fab region comprises the mutations E143K, Q199K, and V110D, then the second light chain Fab region does not contain a lysine at amino acid residue 143 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an aspartic acid at amino acid residue 110 (EU numbering); if the first light chain Fab region comprises the mutations V110D and Q199K, then the second light chain Fab region does not comprise an aspartic acid at amino acid residue 110 (EU numbering) and a lysine at amino acid residue 199 (EU numbering); and if said first light chain Fab region comprises the mutations V110D, Q199K, and T109A, said second light chain Fab region does not contain an aspartic acid at amino acid residue 110 (EU numbering), a lysine at amino acid residue 199 (EU numbering), and an alanine at amino acid residue 109 (EU numbering); The multispecific binding protein of claim 1.

3. 2. The multispecific binding protein of claim 1 , wherein the first light chain Fab region comprises the following mutations: E143K and Q199K.

4. 4. The multispecific binding protein of claim 3, wherein the first light chain Fab region further comprises a V110D mutation.

5. 2. The multispecific binding protein of claim 1 , wherein the first light chain Fab region comprises the mutations V110D and Q199K.

6. 6. The multispecific binding protein of claim 5, wherein the first light chain Fab region further comprises a T109A mutation.

7. 2. The multispecific binding protein of claim 1 , wherein the first antigen binding domain further comprises a first heavy chain Fc region.

8. 8. The multispecific binding protein of claim 7, wherein the first heavy chain Fc region comprises a human IgG1, human IgG2, or human IgG4 constant region.

9. 8. The multispecific binding protein of claim 7, wherein the second antigen binding domain further comprises a second heavy chain Fc region.

10. 10. The multispecific binding protein of claim 9, wherein the second heavy chain Fc region comprises a human IgG1, human IgG2, or human IgG4 constant region.

11. The multispecific binding protein of claim 7 , wherein the first heavy chain is a heavy chain having the amino acid sequence of SEQ ID NO: 1, 3, 6, 10, or 12 with Q311R and K317E mutations introduced therein.

12. The multispecific binding protein of claim 9 , wherein the second heavy chain is a heavy chain having the amino acid sequence of SEQ ID NO: 1, 3, 6, 10, or 12 with Q311R and K317E mutations introduced therein.

13. 10. The multispecific binding protein of claim 9, wherein the first and second heavy chain Fc regions both comprise a human IgG1 constant region; both comprise a human IgG2 constant region; or both comprise a human IgG4 constant region.

14. The multispecific binding protein of claim 9 , wherein both the first and second heavy chains are heavy chains having the amino acid sequence of SEQ ID NO: 1, 3, 6, 10, or 12 with Q311R and K317E mutations introduced therein.

15. 2. The multispecific binding protein of claim 1 , wherein the second light chain Fab region is a kappa light chain.

16. 2. The multispecific binding protein of claim 1 , wherein the second light chain Fab region is a lambda light chain.

17. 1. A method for purifying a multispecific binding protein comprising a first antigen-binding domain that binds to a first antigen, the first antigen-binding domain comprising a first light chain Fab region and a first heavy chain Fab region, and a second antigen-binding domain that binds to a second antigen, the second light chain Fab region and a second heavy chain Fab region, comprising: The first light chain Fab region has the following mutation in the amino acid sequence of SEQ ID NO: 2, 4, 11, or 13: E143K and Q199K mutations; V110D and Q199K mutations; T109A, V110D, and Q199K mutations; or V110D, E143K, and Q199K mutations; or a human kappa light chain having the amino acid sequence of SEQ ID NO:9; The method comprises the steps of: expressing the multispecific binding protein, wherein the first antigen-binding domain is assembled with the second antigen-binding domain; and applying the multispecific binding protein to an affinity chromatography column; and Recovering the purified multispecific binding protein. A method comprising:

18. 18. The method of claim 17, wherein the first antigen-binding domain comprises a first heavy chain Fc region.

19. 20. The method of claim 18, wherein the second antigen binding domain comprises a second heavy chain Fc region.

20. The method according to claim 18, wherein the first heavy chain is a heavy chain having the amino acid sequence of SEQ ID NO: 1, 3, 6, 10, or 12 into which Q311R and K317E mutations have been introduced.

21. The method according to claim 19, wherein the second heavy chain is a heavy chain having the amino acid sequence of SEQ ID NO: 1, 3, 6, 10, or 12 into which Q311R and K317E mutations have been introduced.

22. The method according to claim 19, wherein both the first heavy chain and the second heavy chain are heavy chains having the amino acid sequence of SEQ ID NO: 1, 3, 6, 10, or 12 with Q311R and K317E mutations introduced therein.

23. The method of claim 17, wherein the affinity chromatography column comprises a kappa affinity ligand.

24. The method of claim 17, wherein the affinity chromatography column comprises a lambda affinity ligand.

25. 20. The method of claim 17, wherein the affinity chromatography column comprises Protein A.

26. 18. The method of claim 17, wherein the second light chain Fab region binds to the affinity chromatography column with a higher affinity than the first light chain Fab region.

27. 18. The method of claim 17, wherein the first light chain Fab region does not bind to the affinity chromatography column.

28. Steps below: After recovering the purified multispecific binding protein, subjecting the purified multispecific binding protein to a second affinity chromatography column; and applying the purified multispecific binding protein to a second affinity chromatography column followed by recovering the purified multispecific binding protein.

20. The method of claim 17, further comprising:

29. 30. The method of claim 28, wherein the second affinity chromatography column comprises a kappa affinity ligand.

30. 29. The method of claim 28, wherein the second affinity chromatography column comprises a lambda affinity ligand.

31. 30. The method of claim 28, wherein the second affinity chromatography column comprises Protein A.

32. 29. The method of claim 28, wherein the second light chain Fab region binds to the second affinity chromatography column with a higher affinity than the first light chain Fab region.

33. 29. The method of claim 28, wherein the first light chain Fab region does not bind to the second affinity chromatography column.

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