Enhanced Protein Composition

Abstract

The present invention teaches isolated polypeptides, e.g., polypeptides capable of binding to themselves or other polypeptides, including diagnostic, prognostic, and therapeutic polypeptides, which comprise an exposed carboxy terminus ("C-terminus") fused to a capping domain (e.g., a His capping domain, (His)6 (i.e., HHHHHH (SEQ ID NO:26)), SLSLSPGK (SEQ ID NO:36), AS, or TVAPTESS (SEQ ID NO:37)). The present invention further teaches nucleic acids and / or expression vectors encoding the polypeptides, cells containing the polypeptides, nucleic acids, and / or expression vectors, and compositions comprising the polypeptides. Methods of making the polypeptides and methods of using the polypeptides are also taught.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 303,921 (filed January 27, 2022), which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) This application contains a computer readable sequence listing submitted herewith in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted herewith is named "14620-547-228_SEQ_LISTING.xml", was created on January 26, 2023, and is 67,678 bytes in size.

[0003] FIELD OF THE INVENTION The present invention teaches, inter alia, isolated polypeptides, such as polypeptides capable of binding to themselves or other polypeptides or molecules, including diagnostic, prognostic, and therapeutic polypeptides, which comprise an exposed carboxy terminus ("C-terminus") fused to a capping domain (e.g., a His capping domain, (His)6 (i.e., HHHHHH (SEQ ID NO:26)), SLSLSPGK (SEQ ID NO:36), AS, or TVAPTESS (SEQ ID NO:37)). The present invention further teaches nucleic acids and / or expression vectors encoding the polypeptides, cells containing the polypeptides, nucleic acids, and / or expression vectors, and compositions comprising the polypeptides. Methods of making the polypeptides and methods of using the polypeptides are also taught. [Background technology]

[0004] Polypeptide interactions with other molecules, such as other polypeptides, are well known. (See, e.g., Titeca, Kevin; Lemmens, Irma; Tavernier, Jan; Eyckerman, Sven (29 June 2018), "Discovering cellular protein-protein interactions: Technological strategies and opportunities," Mass Spectrometry Reviews. 38(1):79-111. doi:10.1002 / mas.21574. ISSN 0277-7037. PMID 29957823; and Herce HD, Deng W, Helma J, Leonhardt H, Cardoso MC (2013), "Visualization and targeted disruption of protein interactions in living cells," Nature Communications. 4:2660.) Such interacting polypeptides can exist in vitro and in vivo. (See, e.g., Jones S, Thornton JM (January 1996), "Principles of protein-protein interactions," Proceedings of the National Academy of Sciences of the United States of America. 93(1):13-20. Bibcode: 1996PNAS...93...13J. doi:10.1073 / pnas.93.1.13. PMC 40170. PMID 8552589.) Examples of polypeptides that interact with other molecules are polypeptides that interact with proteins, including antigen-binding proteins such as antibodies.(See, e.g., Curr Drug Metab. 2011 May;12(4):313-328, "Characterization of Drug Interactions with Serum Proteins by Using High-Performance Affinity Chromatography," David S. Hage, Jeanethe Anguizola, Omar Barnaby, Abby Jackson, Michelle J. Yoo, Efthimia Papastavros, Erika Pfaunmiller, Matt Sobansky, and Zenghan Tong.) Proteins have been reported to bind to isolated polypeptides and, in some cases, induce anti-polypeptide antibody responses, which may affect their use, including therapeutic use. (See, e.g., "Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography," Ryan Matsuda, So-Hwang Kye, Jeanethe Anguizola and David S. Hage, Reviews in Analytical Chemistry.) Summary of the Invention

[0005] Against this background, the inventors of the present invention have discovered methods and compositions for preventing or ameliorating the interaction of polypeptides with molecules.

[0006] In certain aspects, provided herein are isolated polypeptides comprising one or more target binding regions and an exposed C-terminus fused to a cap domain, wherein the isolated polypeptide exhibits reduced interaction with a reference compared to the interaction of an isolated polypeptide lacking the cap domain with the reference. The cap domain is heterologous to the exposed C-terminus and comprises one or more amino acid residues (e.g., histidine). In some embodiments, the cap domain consists of SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37), or a His cap domain. In some embodiments, the biological material comprises serum. In certain embodiments, the biological material is human serum. In some embodiments, the biological material comprises plasma. In certain embodiments, the biological material is human plasma. In some embodiments, the biological material comprises serum and plasma (e.g., human serum and human plasma).

[0007] In one aspect, provided herein is an isolated polypeptide comprising one or more target binding regions and an exposed C-terminus fused to a His cap domain, wherein the isolated polypeptide exhibits reduced interaction with a reference compared to the interaction of the reference with an isolated polypeptide lacking the His cap domain. In some embodiments, the reference is a molecule present in a biological material. In some embodiments, the biological material comprises serum. In certain embodiments, the biological material is human serum. In some embodiments, the biological material comprises plasma. In certain embodiments, the biological material is human plasma. In some embodiments, the biological material comprises serum and plasma (e.g., human serum and human plasma).

[0008] In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the anti-drug antibody is pre-existing in the subject's serum and / or plasma. In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope.

[0009] In some embodiments, one or more target binding regions comprise an antibody variable domain or antigen-binding fragment thereof. In some embodiments, one or more target binding regions comprise a light chain antibody variable domain or antigen-binding fragment thereof. In some embodiments, one or more target binding regions comprise a heavy antibody variable domain or antigen-binding fragment thereof.

[0010] In some embodiments, the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the His cap domain consists of a single histidine residue. In some embodiments, the His cap domain consists of two histidine residues. In some embodiments, the His cap domain consists of three histidine residues. In some embodiments, the His cap domain consists of four histidine residues (SEQ ID NO: 27). In some embodiments, the His cap domain consists of five histidine residues (SEQ ID NO: 28).

[0011] In some embodiments, the exposed C-terminus consists of the amino acid sequence of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), or VEIKRT (SEQ ID NO:24). In some embodiments, the exposed C-terminus consists of the amino acid sequence of TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), or TQLTVL (SEQ ID NO:42). In some embodiments, the exposed C-terminus consists of the amino acid sequence of TKLTVL (SEQ ID NO:39), TKVTVL (SEQ ID NO:38), NGLVYAG (SEQ ID NO:43), NGLLYAG (SEQ ID NO:44), or TRLTVL (SEQ ID NO:45). In some embodiments, the exposed C-terminus consists of the amino acid sequence provided in Table 1.

[0012] In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15). In some embodiments, the exposed C-terminus fused to the His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20).

[0013] In some embodiments, the exposed C-terminus fused to a His cap domain consists of amino acids TKVTVL(His)n (SEQ ID NO:46), TKLTVL(His)n (SEQ ID NO:47), TQLIIL(His)n (SEQ ID NO:48), TELTVL(His)n (SEQ ID NO:49), or TQLTVL(His)n (SEQ ID NO:50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to a His cap domain consists of amino acids TKLTVL(His)n (SEQ ID NO:47), TKVTVL(His)n (SEQ ID NO:46), NGLVYAG(His)n (SEQ ID NO:51), NGLLYAG(His)n (SEQ ID NO:52), or TRLTVL(His)n (SEQ ID NO:53), where n is 1, 2, 3, 4, or 5.

[0014] In some embodiments, the interaction with the reference is determined by immunoassay. In some embodiments, the interaction with the reference is assessed in vitro. In some embodiments, the interaction with the reference is assessed in vivo.

[0015] In another aspect, provided herein is an isolated polypeptide comprising one or more target binding regions and an exposed C-terminus fused to a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), wherein the isolated polypeptide exhibits reduced aggregation upon incubation with a biological material compared to the aggregation exhibited by an isolated polypeptide lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) upon incubation with a biological material.

[0016] In some embodiments, incubation with the biological material comprises incubation at about 37°C for about 5 to about 14 days. In some embodiments, incubation with the biological material comprises incubation at about 40°C for about 7 to about 14 days. In some embodiments, incubation with the biological material comprises incubation at about 40°C for about 2 to about 7 days. In some embodiments, incubation with the biological material comprises incubation at about 40°C for about 2 to about 5 days. In some embodiments, incubation with the biological material comprises incubation at about 4°C for about 7 to about 14 days. In some embodiments, incubation with the biological material comprises incubation at about 25°C for about 7 to about 14 days. In some embodiments, incubation with the biological material comprises incubation at about 25°C for about 2 to about 7 days. In some embodiments, incubation with the biological material comprises incubation at about 25°C for about 2 to about 5 days.

[0017] In some embodiments, the biological material comprises an anti-drug antibody. In some embodiments, the biological material comprises an anti-drug antibody pre-existing in the serum and / or plasma of a subject (e.g., a human subject). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the one or more target binding regions comprise an antibody variable domain or antigen-binding fragment thereof. In some embodiments, the one or more target binding regions comprise a light chain antibody variable domain or antigen-binding fragment thereof. In some embodiments, the one or more target binding regions comprise a heavy antibody variable domain or antigen-binding fragment thereof.

[0018] In some embodiments, the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the His cap domain consists of a single histidine residue. In some embodiments, the His cap domain consists of two histidine residues. In some embodiments, the His cap domain consists of three histidine residues. In some embodiments, the His cap domain consists of four histidine residues (SEQ ID NO: 27). In some embodiments, the His cap domain consists of five histidine residues (SEQ ID NO: 28).

[0019] In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15). In some embodiments, the exposed C-terminus fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20). In some embodiments, the exposed C-terminus fused to a His capping domain consists of the amino acids TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to the His cap domain consists of the amino acids TKLTVL(His)n (SEQ ID NO: 47), TKVTVL(His)n (SEQ ID NO: 46), NGLVYAG(His)n (SEQ ID NO: 51), NGLLYAG(His)n (SEQ ID NO: 52), or TRLTVL(His)n (SEQ ID NO: 53), where n is 1, 2, 3, 4, or 5.

[0020] In some embodiments, interactions with biological materials are assessed by size exclusion chromatography.

[0021] In another aspect, provided herein is an isolated polypeptide comprising one or more target binding regions and an exposed C-terminus fused to a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), wherein the isolated polypeptide exhibits reduced self-aggregation compared to the self-aggregation exhibited by an isolated polypeptide lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) under the same conditions.

[0022] In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, one or more target binding regions comprise an antibody variable domain or antigen-binding fragment thereof. In some embodiments, one or more target binding regions comprise a light chain antibody variable domain or antigen-binding fragment thereof. In some embodiments, one or more target binding regions comprise a heavy antibody variable domain or antigen-binding fragment thereof.

[0023] In some embodiments, the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the His cap domain consists of a single histidine residue. In some embodiments, the His cap domain consists of two histidine residues. In some embodiments, the His cap domain consists of three histidine residues. In some embodiments, the His cap domain consists of four histidine residues (SEQ ID NO: 27). In some embodiments, the His cap domain consists of five histidine residues (SEQ ID NO: 28).

[0024] In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15). In some embodiments, the exposed C-terminus fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20). In some embodiments, the exposed C-terminus fused to a His capping domain consists of the amino acids TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to the His cap domain consists of the amino acids TKLTVL(His)n (SEQ ID NO: 47), TKVTVL(His)n (SEQ ID NO: 46), NGLVYAG(His)n (SEQ ID NO: 51), NGLLYAG(His)n (SEQ ID NO: 52), or TRLTVL(His)n (SEQ ID NO: 53), where n is 1, 2, 3, 4, or 5.

[0025] In some embodiments, autoaggregation is reduced in a high concentration liquid formulation (HCLF). In some embodiments, the HCLF concentration comprises about 50 mg / mL to about 150 mg / mL of the isolated polypeptide. In some embodiments, autoaggregation is reduced under heat stress. In some embodiments, the heat stress comprises incubation at 40°C for about 2 weeks. In some embodiments, autoaggregation is assessed by size exclusion chromatography.

[0026] In another aspect, provided herein is an isolated protein, the isolated protein comprising: (a) a first polypeptide comprising a target binding region and an exposed C-terminus fused to a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO:36), or TVAPTESS (SEQ ID NO:37)); and (b) a second polypeptide comprising the target binding region, wherein the isolated protein exhibits reduced interaction with a reference compared to the interaction of the reference with an isolated protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO:36), or TVAPTESS (SEQ ID NO:37)). In some embodiments, the presence of the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO:36), or TVAPTESS (SEQ ID NO:37)) results in reduced neutralization of the protein compared to neutralization of the same protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO:36), or TVAPTESS (SEQ ID NO:37)). In some embodiments, the presence of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) results in improved safety of the protein compared to the safety of a reference of the same protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In some embodiments, the presence of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) results in improved pharmacokinetics of the protein compared to the pharmacokinetics of a reference of the same protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)).In some embodiments, the presence of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) results in a reduced interaction between the protein and an anti-drug antibody compared to the interaction with a reference of the same protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)).

[0027] In some embodiments, the exposed C-terminus of the first polypeptide comprises an anti-drug antibody epitope. In some embodiments, the exposed C-terminus of the second polypeptide comprises an anti-drug antibody epitope. In some embodiments, the second polypeptide comprises an exposed C-terminus fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)).

[0028] In some embodiments, the target binding regions of the first and second polypeptides are capable of binding to different targets. In some embodiments, the target binding region of the first polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the second polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the first polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the second polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the first polypeptide comprises a heavy chain antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the second polypeptide comprises a heavy chain antibody variable domain or an antigen-binding fragment thereof.

[0029] In some embodiments, the cap domain of a first polypeptide is a His cap domain, or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In certain embodiments, the cap domain of a first polypeptide consists of a His cap domain. In some embodiments, the cap domain of a second polypeptide is a His cap domain, or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In certain embodiments, the cap domain of a second polypeptide consists of a His cap domain. In some embodiments, the His cap domain of a first polypeptide consists of a single histidine residue. In some embodiments, the His cap domain of a second polypeptide consists of a single histidine residue. In some embodiments, the His cap domain of a first polypeptide consists of two histidine residues. In some embodiments, the His cap domain of a second polypeptide consists of two histidine residues. In some embodiments, the His cap domain of a first polypeptide consists of three histidine residues. In some embodiments, the His cap domain of the second polypeptide consists of three histidine residues. In some embodiments, the His cap domain of the first polypeptide consists of four histidine residues (SEQ ID NO:27). In some embodiments, the His cap domain of the second polypeptide consists of four histidine residues (SEQ ID NO:27). In some embodiments, the His cap domain of the first polypeptide consists of five histidine residues (SEQ ID NO:28). In some embodiments, the His cap domain of the second polypeptide consists of five histidine residues (SEQ ID NO:28).

[0030] In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15). In some embodiments, the exposed C-terminus fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20). In some embodiments, the exposed C-terminus fused to a His capping domain consists of the amino acids TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to the His cap domain consists of the amino acids TKLTVL(His)n (SEQ ID NO: 47), TKVTVL(His)n (SEQ ID NO: 46), NGLVYAG(His)n (SEQ ID NO: 51), NGLLYAG(His)n (SEQ ID NO: 52), or TRLTVL(His)n (SEQ ID NO: 53), where n is 1, 2, 3, 4, or 5.

[0031] In some embodiments, the isolated protein further comprises a third polypeptide comprising a target binding region. In some embodiments, the third polypeptide comprises an exposed C-terminus fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In some embodiments, the third polypeptide is capable of binding to a different target than the first polypeptide, the second polypeptide, or both. In some embodiments, the capping domain is a His capping domain.

[0032] In some embodiments, the exposed C-terminus of the third polypeptide comprises an anti-drug antibody epitope. In some embodiments, the target binding region of the third polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the third polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the third polypeptide comprises a heavy chain antibody variable domain or an antigen-binding fragment thereof.

[0033] In some embodiments, the His capping domain of the third polypeptide consists of a single histidine residue. In some embodiments, the His capping domain of the third polypeptide consists of two histidine residues. In some embodiments, the His capping domain of the third polypeptide consists of three histidine residues. In some embodiments, the His capping domain of the third polypeptide consists of four histidine residues (SEQ ID NO: 27). In some embodiments, the His capping domain of the third polypeptide consists of five histidine residues (SEQ ID NO: 28).

[0034] In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO:11), VEIKRHH (SEQ ID NO:12), VEIKRHHH (SEQ ID NO:13), VEIKRHHHH (SEQ ID NO:14), and VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO:16), VEIKRTHH (SEQ ID NO:17), VEIKRTHHH (SEQ ID NO:18), VEIKRTHHHH (SEQ ID NO:19), and VEIKRTHHHHH (SEQ ID NO:20). In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of amino acids TKVTVL(His)n (SEQ ID NO:46), TKLTVL(His)n (SEQ ID NO:47), TQLIIL(His)n (SEQ ID NO:48), TELTVL(His)n (SEQ ID NO:49), or TQLTVL(His)n (SEQ ID NO:50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of amino acids TKLTVL(His)n (SEQ ID NO:47), TKVTVL(His)n (SEQ ID NO:46), NGLVYAG(His)n (SEQ ID NO:51), NGLLYAG(His)n (SEQ ID NO:52), or TRLTVL(His)n (SEQ ID NO:53), where n is 1, 2, 3, 4, or 5.

[0035] In some embodiments, the isolated protein is bispecific. In some embodiments, the isolated protein is multispecific. In some embodiments, the isolated protein is multispecific, such as trispecific. In some embodiments, the isolated protein is a heterodimer.

[0036] In another aspect, provided herein is an isolated nucleic acid sequence comprising a nucleotide sequence that encodes a polypeptide provided herein or a protein provided herein.

[0037] In another aspect, provided herein is an isolated cell that expresses a polypeptide provided herein or a protein provided herein.

[0038] In another aspect, provided herein is an expression vector comprising a nucleotide sequence encoding a polypeptide provided herein, or a protein provided herein.

[0039] In another aspect, provided herein is an isolated cell comprising a nucleotide sequence encoding a polypeptide provided herein, or a protein provided herein, or an expression vector comprising a nucleotide sequence encoding a polypeptide provided herein, or a protein provided herein.

[0040] In another aspect, provided herein are methods for producing a polypeptide provided herein or a protein provided herein, comprising culturing a cell provided herein. In some embodiments, the method further comprises isolating the polypeptide or protein.

[0041] In a further aspect, provided herein are bispecific or multispecific antibodies comprising one or more target binding regions and an exposed C-terminus fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), wherein the antibody exhibits reduced interaction with an anti-drug antibody compared to the interaction of an antibody lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In some embodiments, the anti-drug antibody is pre-existing in the serum and / or plasma of a subject (e.g., a human subject). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the antibody exhibits reduced interaction with an anti-drug antibody in an immunoassay compared to the interaction of an antibody lacking the His capping domain with the anti-drug antibody. In some embodiments, the one or more target binding regions comprise at least one antibody variable domain. In some embodiments, the at least one antibody variable domain comprises a heavy chain variable domain. In some embodiments, at least one antibody variable domain comprises a light chain variable domain.

[0042] In some embodiments, the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the His cap domain consists of a single histidine residue. In some embodiments, the His cap domain consists of two histidine residues. In some embodiments, the His cap domain consists of three histidine residues. In some embodiments, the His cap domain consists of four histidine residues (SEQ ID NO: 27). In some embodiments, the His cap domain consists of five histidine residues (SEQ ID NO: 28).

[0043] In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15). In some embodiments, the exposed C-terminus fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20). In some embodiments, the exposed C-terminus fused to a His capping domain consists of the amino acids TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to the His cap domain consists of the amino acids TKLTVL(His)n (SEQ ID NO: 47), TKVTVL(His)n (SEQ ID NO: 46), NGLVYAG(His)n (SEQ ID NO: 51), NGLLYAG(His)n (SEQ ID NO: 52), or TRLTVL(His)n (SEQ ID NO: 53), where n is 1, 2, 3, 4, or 5.

[0044] In some embodiments, the multispecific antibody comprises a trispecific antibody.

[0045] In another aspect, provided herein are nucleic acid sequences comprising nucleotide sequences encoding the antibodies provided herein.

[0046] In another aspect, provided herein is an expression vector comprising a nucleic acid sequence comprising a nucleotide sequence encoding an antibody provided herein.

[0047] In another aspect, provided herein is an isolated cell comprising a nucleotide sequence encoding an antibody provided herein, or an expression vector comprising a nucleic acid sequence comprising a nucleotide sequence encoding an antibody provided herein.

[0048] In another aspect, provided herein is an isolated cell that expresses an antibody provided herein.

[0049] In another aspect, provided herein are methods for producing the antibodies provided herein, comprising culturing a cell provided herein. In some aspects, the method further comprises isolating the antibody.

[0050] In another aspect, provided herein are methods for reducing interactions between an isolated polypeptide and a reference, wherein the isolated polypeptide comprises one or more target binding regions and an exposed C-terminus, and the method comprises fusing a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to the end of the exposed C-terminus. In some embodiments, the reference is a molecule present in a biological material. In some embodiments, the biological material comprises serum. In certain embodiments, the biological material is human serum. In some embodiments, the biological material comprises plasma. In certain embodiments, the biological material is human plasma.

[0051] In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the reference comprises an anti-drug antibody pre-existing in the serum and / or plasma of a subject (e.g., a human subject). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope.

[0052] In some embodiments, one or more target binding regions comprise an antibody variable domain or antigen-binding fragment thereof. In some embodiments, one or more target binding regions comprise a light chain antibody variable domain or antigen-binding fragment thereof. In some embodiments, one or more target binding regions comprise a heavy antibody variable domain or antigen-binding fragment thereof.

[0053] In some embodiments, the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the His cap domain consists of a single histidine residue. In some embodiments, the His cap domain consists of two histidine residues. In some embodiments, the His cap domain consists of three histidine residues. In some embodiments, the His cap domain consists of four histidine residues (SEQ ID NO: 27). In some embodiments, the His cap domain consists of five histidine residues (SEQ ID NO: 28).

[0054] In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15). In some embodiments, the exposed C-terminus fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20). In some embodiments, the exposed C-terminus fused to a His capping domain consists of the amino acids TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to the His cap domain consists of the amino acids TKLTVL(His)n (SEQ ID NO: 47), TKVTVL(His)n (SEQ ID NO: 46), NGLVYAG(His)n (SEQ ID NO: 51), NGLLYAG(His)n (SEQ ID NO: 52), or TRLTVL(His)n (SEQ ID NO: 53), where n is 1, 2, 3, 4, or 5.

[0055] In some embodiments, the interaction with the reference is determined by immunoassay. In some embodiments, the interaction with the reference is assessed in vitro. In some embodiments, the interaction with the reference is assessed in vivo.

[0056] In another aspect, provided herein are methods for reducing aggregation of an isolated polypeptide in a biological material, wherein the isolated polypeptide comprises one or more target binding regions and an exposed C-terminus, the method comprising fusing a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to the end of the exposed C-terminus. In some embodiments, the biological material comprises serum and / or plasma. In some embodiments, the biological material is human serum. In some embodiments, the biological material is human plasma.

[0057] In some embodiments, incubation with the biological material comprises incubation at about 37°C for about 5 to about 14 days. In some embodiments, incubation with the biological material comprises incubation at 37°C for about 2 to about 7 days. In some embodiments, incubation with the biological material comprises incubation at 37°C for about 2 to about 5 days. In some embodiments, incubation with the biological material comprises incubation at about 40°C for about 7 to about 14 days. In some embodiments, incubation with the biological material comprises incubation at 40°C for about 2 to about 7 days. In some embodiments, incubation with the biological material comprises incubation at 40°C for about 2 to about 5 days. In some embodiments, incubation with the biological material comprises incubation at about 4°C for about 7 to about 14 days. In some embodiments, incubation with the biological material comprises incubation at 4°C for about 2 to about 7 days. In some embodiments, incubation with the biological material comprises incubation at 4°C for about 2 to about 5 days. In some embodiments, incubation with the biological material comprises incubation at about 25°C for about 7 to about 14 days. In some embodiments, incubation with the biological material comprises incubation at 25°C for about 2 to about 7 days. In some embodiments, incubation with the biological material comprises incubation at 25°C for about 2 to about 5 days.

[0058] In some embodiments, the biological material comprises an anti-drug antibody. In some embodiments, the anti-drug antibody is pre-existing in the serum and / or plasma of a subject (e.g., a human subject). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the one or more target binding regions comprise an antibody variable domain or antigen-binding fragment thereof. In some embodiments, the one or more target binding regions comprise a light chain antibody variable domain or antigen-binding fragment thereof. In some embodiments, the one or more target binding regions comprise a heavy antibody variable domain or antigen-binding fragment thereof.

[0059] In some embodiments, the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the His cap domain consists of a single histidine residue. In some embodiments, the His cap domain consists of two histidine residues. In some embodiments, the His cap domain consists of three histidine residues. In some embodiments, the His cap domain consists of four histidine residues (SEQ ID NO: 27). In some embodiments, the His cap domain consists of five histidine residues (SEQ ID NO: 28).

[0060] In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15). In some embodiments, the exposed C-terminus fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20). In some embodiments, the exposed C-terminus fused to a His capping domain consists of the amino acids TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to the His cap domain consists of the amino acids TKLTVL(His)n (SEQ ID NO: 47), TKVTVL(His)n (SEQ ID NO: 46), NGLVYAG(His)n (SEQ ID NO: 51), NGLLYAG(His)n (SEQ ID NO: 52), or TRLTVL(His)n (SEQ ID NO: 53), where n is 1, 2, 3, 4, or 5.

[0061] In some embodiments, interactions with biological materials are assessed by size exclusion chromatography.

[0062] In another aspect, provided herein is a method for reducing self-aggregation of an isolated polypeptide, wherein the isolated polypeptide comprises one or more target binding regions and an exposed C-terminus, the method comprising fusing a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to the end of the exposed C-terminus.

[0063] In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, one or more target binding regions comprise an antibody variable domain or antigen-binding fragment thereof. In some embodiments, one or more target binding regions comprise a light chain antibody variable domain or antigen-binding fragment thereof. In some embodiments, one or more target binding regions comprise a heavy antibody variable domain or antigen-binding fragment thereof.

[0064] In some embodiments, the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the His cap domain consists of a single histidine residue. In some embodiments, the His cap domain consists of two histidine residues. In some embodiments, the His cap domain consists of three histidine residues. In some embodiments, the His cap domain consists of four histidine residues (SEQ ID NO: 27). In some embodiments, the His cap domain consists of five histidine residues (SEQ ID NO: 28).

[0065] In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15). In some embodiments, the exposed C-terminus fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20). In some embodiments, the exposed C-terminus fused to a His capping domain consists of the amino acids TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to the His cap domain consists of the amino acids TKLTVL(His)n (SEQ ID NO: 47), TKVTVL(His)n (SEQ ID NO: 46), NGLVYAG(His)n (SEQ ID NO: 51), NGLLYAG(His)n (SEQ ID NO: 52), or TRLTVL(His)n (SEQ ID NO: 53), where n is 1, 2, 3, 4, or 5.

[0066] In some embodiments, autoaggregation is reduced in a high concentration liquid formulation (HCLF). In some embodiments, the HCLF concentration comprises about 50 mg / mL to about 150 mg / mL of the isolated polypeptide. In some embodiments, autoaggregation is reduced under heat stress. In some embodiments, the heat stress comprises incubation at 40°C for about 2 weeks. In some embodiments, autoaggregation is assessed by size exclusion chromatography.

[0067] In another aspect, provided herein are methods for reducing interactions between an isolated protein and a reference, the isolated protein comprising (a) a first polypeptide comprising a target binding region and an exposed C-terminus; and (b) a second polypeptide comprising the target binding region, wherein the method comprises fusing a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to the exposed C-terminus. In some embodiments, the exposed C-terminus of the first polypeptide comprises an anti-drug antibody epitope. In some embodiments, the exposed C-terminus of the second polypeptide comprises an anti-drug antibody epitope. In some embodiments, the second polypeptide comprises an exposed C-terminus, and the method further comprises fusing a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to the exposed C-terminus of the second polypeptide. In some embodiments, the target binding regions of the first and second polypeptides are capable of binding to different targets.

[0068] In some embodiments, the target binding region of the first polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the second polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the first polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the second polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the first polypeptide comprises a heavy chain antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the second polypeptide comprises a heavy chain antibody variable domain or an antigen-binding fragment thereof.

[0069] In some embodiments, the cap domain of the first polypeptide is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37). In some embodiments, the cap domain of the second polypeptide is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37). In some embodiments, the His cap domain of the first polypeptide consists of a single histidine residue. In some embodiments, the His cap domain of the second polypeptide consists of a single histidine residue. In some embodiments, the His cap domain of the first polypeptide consists of two histidine residues. In some embodiments, the His cap domain of the second polypeptide consists of two histidine residues. In some embodiments, the His cap domain of the first polypeptide consists of three histidine residues. In some embodiments, the His cap domain of the second polypeptide consists of three histidine residues. In some embodiments, the His cap domain of the first polypeptide consists of four histidine residues (SEQ ID NO:27). In some embodiments, the His cap domain of the second polypeptide consists of four histidine residues (SEQ ID NO:27). In some embodiments, the first polypeptide consists of five histidine residues (SEQ ID NO:28). In some embodiments, the His cap domain of the second polypeptide consists of five histidine residues (SEQ ID NO:28).

[0070] In some embodiments, the exposed C-terminus of a first polypeptide fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus of a first polypeptide fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus of a first polypeptide fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO:11), VEIKRHH (SEQ ID NO:12), VEIKRHHH (SEQ ID NO:13), VEIKRHHHH (SEQ ID NO:14), and VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the exposed C-terminus of a first polypeptide fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO:16), VEIKRTHH (SEQ ID NO:17), VEIKRTHHH (SEQ ID NO:18), VEIKRTHHHH (SEQ ID NO:19), and VEIKRTHHHHH (SEQ ID NO:20). In some embodiments, the exposed C-terminus of a first polypeptide fused to a His cap domain consists of amino acids TKVTVL(His)n (SEQ ID NO:46), TKLTVL(His)n (SEQ ID NO:47), TQLIIL(His)n (SEQ ID NO:48), TELTVL(His)n (SEQ ID NO:49), or TQLTVL(His)n (SEQ ID NO:50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus of a first polypeptide fused to a His cap domain consists of amino acids TKLTVL(His)n (SEQ ID NO:47), TKVTVL(His)n (SEQ ID NO:46), NGLVYAG(His)n (SEQ ID NO:51), NGLLYAG(His)n (SEQ ID NO:52), or TRLTVL(His)n (SEQ ID NO:53), where n is 1, 2, 3, 4, or 5.

[0071] In some embodiments, the exposed C-terminus of a second polypeptide fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus of a second polypeptide fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus of a second polypeptide fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO:11), VEIKRHH (SEQ ID NO:12), VEIKRHHH (SEQ ID NO:13), VEIKRHHHH (SEQ ID NO:14), and VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the exposed C-terminus of a second polypeptide fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO:16), VEIKRTHH (SEQ ID NO:17), VEIKRTHHH (SEQ ID NO:18), VEIKRTHHHH (SEQ ID NO:19), and VEIKRTHHHHH (SEQ ID NO:20). In some embodiments, the exposed C-terminus of a second polypeptide fused to a His cap domain consists of amino acids TKVTVL(His)n (SEQ ID NO:46), TKLTVL(His)n (SEQ ID NO:47), TQLIIL(His)n (SEQ ID NO:48), TELTVL(His)n (SEQ ID NO:49), or TQLTVL(His)n (SEQ ID NO:50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus of a second polypeptide fused to a His cap domain consists of amino acids TKLTVL(His)n (SEQ ID NO:47), TKVTVL(His)n (SEQ ID NO:46), NGLVYAG(His)n (SEQ ID NO:51), NGLLYAG(His)n (SEQ ID NO:52), or TRLTVL(His)n (SEQ ID NO:53), where n is 1, 2, 3, 4, or 5.

[0072] In some embodiments, the isolated protein further comprises a third polypeptide comprising a target binding region. In some embodiments, the third polypeptide comprises an exposed C-terminus, and the method further comprises fusing a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to the end of the exposed C-terminus of the third polypeptide. In some embodiments, the third polypeptide is capable of binding to a target different from the first polypeptide, the second polypeptide, or both. In some embodiments, the exposed C-terminus of the third polypeptide comprises an anti-drug antibody epitope. In some embodiments, the target binding region of the third polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the third polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the target binding region of the third polypeptide comprises a heavy chain antibody variable domain or an antigen-binding fragment thereof.

[0073] In some embodiments, the capping domain of the third polypeptide is a His capping domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37). In some embodiments, the His capping domain of the third polypeptide consists of a single histidine residue. In some embodiments, the His capping domain of the third polypeptide consists of two histidine residues. In some embodiments, the His capping domain of the third polypeptide consists of three histidine residues. In some embodiments, the His capping domain of the third polypeptide consists of four histidine residues (SEQ ID NO:27). In some embodiments, the His capping domain of the third polypeptide consists of five histidine residues (SEQ ID NO:28).

[0074] In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5). In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10). In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO:11), VEIKRHH (SEQ ID NO:12), VEIKRHHH (SEQ ID NO:13), VEIKRHHHH (SEQ ID NO:14), and VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO:16), VEIKRTHH (SEQ ID NO:17), VEIKRTHHH (SEQ ID NO:18), VEIKRTHHHH (SEQ ID NO:19), and VEIKRTHHHHH (SEQ ID NO:20). In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of amino acids TKVTVL(His)n (SEQ ID NO:46), TKLTVL(His)n (SEQ ID NO:47), TQLIIL(His)n (SEQ ID NO:48), TELTVL(His)n (SEQ ID NO:49), or TQLTVL(His)n (SEQ ID NO:50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus of a third polypeptide fused to a His capping domain consists of amino acids TKLTVL(His)n (SEQ ID NO:47), TKVTVL(His)n (SEQ ID NO:46), NGLVYAG(His)n (SEQ ID NO:51), NGLLYAG(His)n (SEQ ID NO:52), or TRLTVL(His)n (SEQ ID NO:53), where n is 1, 2, 3, 4, or 5.

[0075] In some embodiments, the isolated protein is bispecific. In some embodiments, the isolated protein is multispecific. In some embodiments, the isolated protein is trispecific. In some embodiments, the isolated protein is a heterodimer.

[0076] In another aspect, provided herein are capping means (e.g., histidine capping means) for reducing interaction between an isolated polypeptide and a reference, wherein the isolated polypeptide comprises one or more target binding regions and an exposed C-terminus. In some embodiments, the one or more target binding regions comprise an antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the reference is a molecule present in biological material. In some embodiments, the biological material comprises serum and / or plasma. In some embodiments, the biological material comprises serum (e.g., human serum). In some embodiments, the biological material comprises plasma (e.g., human plasma). In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the reference comprises an anti-drug antibody pre-existing in the serum and / or plasma of a subject (e.g., a human subject). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), and TQLTVL (SEQ ID NO:42). In some embodiments, the exposed C-terminus consists of an amino acid sequence provided in Table 1.

[0077] In another aspect, provided herein is a capping means (e.g., histidine capping means) for reducing interaction between an isolated protein and a reference, wherein the isolated protein comprises (a) a first polypeptide comprising a target binding region and an exposed C-terminus, and (b) a second polypeptide comprising a target binding region. In some embodiments, the second polypeptide comprises an exposed C-terminus. In some embodiments, the target binding regions of the first and second polypeptides are capable of binding to different targets. In some embodiments, the isolated protein further comprises a third polypeptide comprising a target binding region. In some embodiments, the third polypeptide comprises an exposed C-terminus. In some embodiments, the third polypeptide is capable of binding to a different target than the first polypeptide, the second polypeptide, or both. In some embodiments, the isolated protein is a heterodimer. In some embodiments, the isolated protein is bispecific or multispecific. In certain embodiments, the multispecific protein is trispecific. In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), and VEIKRT (SEQ ID NO:24). In some embodiments, the reference comprises a molecule in biological material. In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the anti-drug antibody is pre-existing in the serum and / or plasma of a subject (e.g., a human subject).

[0078] In another aspect, provided herein are capping means (e.g., histidine capping means) for reducing interactions between a bispecific or multispecific antibody and a reference, wherein the bispecific or multispecific antibody comprises one or more target binding regions and an exposed C-terminus. In some embodiments, the one or more target binding regions comprise at least one antibody variable domain. In some embodiments, at least one antibody variable domain comprises a heavy chain variable domain. In some embodiments, at least one antibody variable domain comprises a light chain variable domain. In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the multispecific antibody comprises a trispecific antibody. In some embodiments, the reference comprises a molecule in biological material. In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the anti-drug antibody is pre-existing in the serum and / or plasma of a subject (e.g., a human subject).

[0079] In another aspect, provided herein are isolated polypeptides and capping means (e.g., histidine capping means) for reducing interaction between the isolated polypeptide and a reference, wherein the isolated polypeptide comprises one or more target binding regions and an exposed C-terminus. In some embodiments, the one or more target binding regions comprise an antibody variable domain or an antigen-binding fragment thereof. In some embodiments, the reference is a molecule present in biological material. In some embodiments, the biological material comprises serum and / or plasma. In some embodiments, the biological material comprises serum. In some embodiments, the biological material comprises plasma. In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the anti-drug antibody is pre-existing in the serum and / or plasma of a subject (e.g., a human subject). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), and TQLTVL (SEQ ID NO:42). In some embodiments, the exposed C-terminus consists of an amino acid sequence provided in Table 1.

[0080] In another aspect, provided herein are isolated proteins and capping means (e.g., histidine capping means) for reducing interaction between the isolated protein and a reference, wherein the isolated protein comprises (a) a first polypeptide comprising a target binding region and an exposed C-terminus; and (b) a second polypeptide comprising a target binding region. In some embodiments, the second polypeptide comprises an exposed C-terminus. In some embodiments, the target binding regions of the first and second polypeptides are capable of binding to different targets. In some embodiments, the isolated protein further comprises a third polypeptide comprising a target binding region. In some embodiments, the third polypeptide comprises an exposed C-terminus. In some embodiments, the third polypeptide is capable of binding to a different target than the first polypeptide, the second polypeptide, or both. In some embodiments, the isolated protein is a heterodimer. In some embodiments, the isolated protein is bispecific or multispecific. In some embodiments, the multispecific protein is trispecific. In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the reference comprises a molecule in a biological material. In some embodiments, the biological material comprises serum and / or plasma. In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the anti-drug antibody is pre-existing in the serum and / or plasma of a subject (e.g., a human subject).

[0081] In another aspect, provided herein are bispecific or multispecific antibodies and capping means (e.g., histidine capping means) for reducing interaction between an isolated protein and a reference, wherein the bispecific or multispecific antibody comprises one or more target binding regions and an exposed C-terminus. In some embodiments, the one or more target binding regions comprise at least one antibody variable domain. In some embodiments, at least one antibody variable domain comprises a heavy chain variable domain. In some embodiments, at least one antibody variable domain comprises a light chain variable domain. In some embodiments, the isolated protein is a heterodimer. In some embodiments, the multispecific antibody comprises a trispecific antibody. In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the reference comprises a molecule in biological material. In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the anti-drug antibody is pre-existing in the serum and / or plasma of a subject (e.g., a human subject).

[0082] In one embodiment, provided herein is an isolated protein for use in a diagnostic assay, comprising a target binding region and an exposed C-terminus fused to a capping domain, wherein the capping domain consists of a His capping domain, or the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In an embodiment, the protein is an antibody (e.g., a bispecific or multispecific antibody). In some embodiments, the antibody is directed against glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin, and telomerase, prostate-carcinoma tumor antigen-1 (PSA), and / or prostate-associated antigens. In some embodiments, the antibody binds to an antigen of a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the protein is used in a diagnostic assay to detect the presence of a molecule in a biological material. In some embodiments, the molecule is an antigen. In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In some embodiments, the diagnostic assay is performed in vitro or in vivo. In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope.In some embodiments, the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO: 21), VEIK (SEQ ID NO: 22), VEIKR (SEQ ID NO: 23), VEIKRT (SEQ ID NO: 24), TKVTVL (SEQ ID NO: 38), TKLTVL (SEQ ID NO: 39), TQLIIL (SEQ ID NO: 40), TELTVL (SEQ ID NO: 41), and TQLTVL (SEQ ID NO: 42).

[0083] In one embodiment, provided herein is an isolated therapeutic protein for use in therapy, comprising a target binding region and an exposed C-terminus fused to a capping domain, wherein the capping domain consists of a His capping domain or the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the protein is a cytokine or an antibody (e.g., a bispecific or multispecific antibody). In some embodiments, the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17. In some embodiments, the antibody binds to a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin. In some embodiments, the antibody binds to an antigen of a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), and TQLTVL (SEQ ID NO:42).

[0084] In one embodiment, provided herein is an isolated therapeutic protein comprising a target binding region and an exposed C-terminus fused to a capping domain, wherein the exposed C-terminus consists of the amino acid sequence VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), or TQLTVL (SEQ ID NO:42), and wherein the capping domain consists of a His capping domain, or the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37). In some embodiments, the protein is an antibody (e.g., a bispecific or multispecific antibody). In some embodiments, the antibody binds to a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin. In some embodiments, the antibody binds to an antigen of a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite.

[0085] In one embodiment, provided herein is a therapeutic bispecific or multispecific (e.g., trispecific) antibody for use in therapy, comprising two or more target binding regions and an exposed C-terminus fused to a capping domain, wherein the capping domain is a His capping domain or the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the two or more target binding regions bind to two or more antigens. In some embodiments, the at least one antigen is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin. In some embodiments, the antibody binds to an antigen of a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. The exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), or TQLTVL (SEQ ID NO:42).

[0086] In another aspect, provided herein is a pharmaceutical composition for use in therapy comprising a protein (e.g., a bispecific or multispecific antibody) comprising an exposed C-terminus and a cap domain, and a pharmaceutically acceptable excipient. In one embodiment, provided herein is a pharmaceutical composition for use in therapy comprising a protein described herein, a bispecific antibody described herein, or a multispecific (e.g., trispecific) antibody described herein, and a pharmaceutically acceptable excipient.

[0087] In another aspect, provided herein is a pharmaceutical composition for use in therapy, comprising a protein (e.g., a bispecific or multispecific antibody) comprising an exposed C-terminus and a cap domain, and a pharmaceutically acceptable excipient. In another embodiment, provided herein is a pharmaceutical composition for use in a diagnostic assay, comprising a protein described herein, a bispecific antibody described herein, or a multispecific (e.g., trispecific) antibody described herein, and a pharmaceutically acceptable excipient.

[0088] In another aspect, provided herein is the use of a cap domain to reduce interactions between an anti-drug antibody and a protein, wherein the protein comprises one or more target binding regions and an exposed C-terminus. In certain embodiments, the cap domain is a His cap domain or consists of the amino acid sequence SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the cap domain is a His cap domain consisting of 1, 2, 3, 4, or 5 histidines.

[0089] In another aspect, provided herein is the use of a cap domain to reduce aggregation in a biological material, wherein the protein comprises one or more target binding regions and an exposed C-terminus. In certain embodiments, the cap domain is a His cap domain or consists of the amino acid sequence SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the cap domain is a His cap domain consisting of 1, 2, 3, 4, or 5 histidines.

[0090] In another aspect, provided herein is the use of a cap domain to reduce self-aggregation, wherein the protein comprises one or more target binding regions and an exposed C-terminus. In certain embodiments, the cap domain consists of a His cap domain, or consists of the amino acid sequence SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. In some embodiments, the cap domain is a His cap domain consisting of 1, 2, 3, 4, or 5 histidines.

[0091] In one embodiment, provided herein is an isolated protein comprising a target binding region and an exposed C-terminus fused to a cap domain, wherein the cap domain consists of a His cap domain or the amino acid sequence of HHHHHH (SEQ ID NO:26), SLSLSPGK (SEQ ID NO:36), or AS, TVAPTESS (SEQ ID NO:37). In some embodiments, the protein exhibits reduced interaction with anti-drug antibodies in biological materials. In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In some embodiments, the exposed C-terminus comprises an anti-drug antibody epitope. The exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), or TQLTVL (SEQ ID NO:42). In some embodiments, the assay is for use in a therapeutic or diagnostic assay. [Brief explanation of the drawings]

[0092] The foregoing Summary of the Invention, as well as the following Detailed Description of the embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1] An exemplary antibody structure comprising an Fc domain and two scFvs (e.g., an Fc-scFv antibody) is shown, suitable for use in the present invention (e.g., addition of one or more cap domains). [Figure 2] Exemplary antibody structures comprising an Fc domain and an scFv heterodimerized with an Fc domain (e.g., an Fc-scFv x Fc antibody) are shown and are suitable for use in the present invention (e.g., by addition of one or more cap domains). [Figure 3]Exemplary triabody structures comprising scFv and scFv heterodimerized with heavy chain and Fc domains (e.g., HC-scFv x Fc-scFv triabodies) are shown and suitable for use in the present invention (e.g., addition of one or more cap domains). [Figure 4] An exemplary antibody structure comprising a heavy chain heterodimerized with an Fc and two scFvs (e.g., Hc×scFv-Fc-scFv) is shown, suitable for use in the present invention (e.g., by addition of one or more cap domains). [Figure 5] Exemplary bispecific antibody structures comprising an Fc domain and two scFvs heterodimerized with Fc and two scFvs (e.g., scFv-Fc-scFv bispecific antibodies) are shown and suitable for use in the present invention (e.g., by addition of one or more cap domains). [Figure 6] Exemplary antibody structures comprising variable heavy domain-Fc domain fusions (e.g., Fc-VHH fusions) are shown and suitable for use in the present invention (e.g., addition of one or more cap domains). [Figure 7] Exemplary multispecific antibody structures comprising an Fc domain, an scFv, and a variable heavy domain heterodimerized with a heavy and variable heavy domain (e.g., scFv-Fc-VHH×HC-VHH) are shown and are suitable for use in the present invention (e.g., with the addition of one or more cap domains). [Figure 8] An exemplary multispecific antibody (e.g., triabody) structure comprising a heavy chain heterodimerized with an Fc domain and two scFvs (e.g., Hc×scFv-Fc-scFv) is shown, suitable for use in the present invention (e.g., by addition of one or more cap domains). [Figure 9A] Exemplary trispecific antibodies are shown without (FIG. 9A) or with (FIG. 9B) a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) at the C-terminus of the antigen binding region of the trispecific antibody. [Figure 9B]Exemplary trispecific antibodies are shown without (FIG. 9A) or with (FIG. 9B) a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) at the C-terminus of the antigen binding region of the trispecific antibody. [Figure 10A] 1 shows an exemplary trispecific antibody having a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) at the C-terminus of the antigen binding region of the trispecific antibody. [Figure 10B] 1 shows an exemplary trispecific antibody having a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) at the C-terminus of the antigen binding region of the trispecific antibody. DETAILED DESCRIPTION OF THE INVENTION

[0093] The present disclosure relates, in part, to proteins, such as therapeutic proteins (e.g., hormones, cytokines, enzymes, fusion proteins, or antibodies or other antigen-binding proteins), modified at the exposed C-terminus of the protein with a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In certain aspects, the disclosure relates to isolated proteins comprising one or more target binding regions and an exposed C-terminus fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In certain embodiments, such proteins exhibit reduced interaction with a reference compared to the interaction of the reference with a protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In another specific embodiment, such proteins exhibit reduced aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation with a biological material by a protein lacking a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In another specific embodiment, such proteins exhibit reduced self-aggregation compared to the self-aggregation exhibited by a protein lacking a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)).

[0094] In certain aspects, the present disclosure relates to proteins, such as therapeutic proteins (e.g., hormones, cytokines, enzymes, fusion proteins, or antibodies or other antigen binding proteins) modified with a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) that reduce or prevent anti-drug antibody (ADA) responses. ... nFor proteins, such as therapeutic proteins, having the sequence (SEQ ID NO: 25), n is 1, 2, 3, 4, or 5, and (His) n The presence of (SEQ ID NO: 25) reduces or prevents the ADA response.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein. For purposes of interpreting this specification, the following explanations of terms shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any explanation of a given term conflicts with any document incorporated herein by reference, the explanation of the term set forth below shall control.

[0096] As used herein, the abbreviations for genetically encoded amino acids are conventional and are as follows:

[0097] [Table 1]

[0098] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0099] As used herein, the terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0100] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0101] As used herein, the term "anti-drug antibody" or "ADA" refers to an antibody capable of interacting with a protein, e.g., a therapeutic protein (e.g., an antibody or another antigen-binding protein). In certain embodiments, the anti-drug antibody is pre-existing in the serum of a subject (e.g., a human subject). In another specific embodiment, the anti-drug antibody is pre-existing in the plasma of a subject (e.g., a human subject). In certain embodiments, the anti-drug antibody binds to or cross-reacts with a polypeptide (e.g., an antigen-binding protein, e.g., an antibody) or other protein comprising one or more target-binding regions, and neutralizes the polypeptide or other protein. In certain embodiments, the anti-drug antibody alters the pharmacokinetics and / or safety profile of a polypeptide (e.g., an antigen-binding protein, e.g., an antibody) or other protein comprising one or more target-binding regions. In some embodiments, the anti-drug antibody inhibits the function of a polypeptide (e.g., an antigen-binding protein, e.g., an antibody) or other protein (e.g., a therapeutic protein) comprising one or more target-binding regions. In certain embodiments, the anti-drug antibody interferes with the binding of a polypeptide (e.g., an antigen binding protein, such as an antibody) comprising one or more target binding regions to one or more targets (e.g., one or more antigens).

[0102] As used herein, the term "target binding region" refers to a plurality of contiguous or non-contiguous amino acid residues in a polypeptide that are capable of binding to a target, e.g., an antigen, in the presence of the target. In certain embodiments, the target binding region comprises an antigen binding region.

[0103] As used herein, the term "exposed C-terminus" refers to the C-terminus of a polypeptide (e.g., an antibody, e.g., a bispecific, trispecific, or other multispecific antibody), where a polypeptide having such a C-terminus lacks a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) and exhibits: (1) greater interaction with a reference, (2) greater aggregation with a biological material, (3) greater self-aggregation, or a combination thereof, than the same polypeptide having a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In certain embodiments, an exposed C-terminus is present only in non-naturally occurring polypeptides (e.g., antibodies, e.g., bispecific, trispecific, or other multispecific antibodies). In certain embodiments, an exposed C-terminus is a sequence that is typically buried, i.e., not otherwise exposed, in naturally occurring polypeptides (e.g., standard immunoglobulin constructs). In some embodiments, the exposed C-terminus comprises a C-terminal anti-drug antibody epitope. In some embodiments, the exposed C-terminus comprises the 4-6 C-terminal amino acid residues of a kappa light chain, a lambda light chain, or a variable heavy chain region of any species found, for example, at https: / / www.imgt.org / IMGTrepertoire / Proteins / index.php#B. In some embodiments, the exposed C-terminus consists of the 4-6 C-terminal amino acid residues of a kappa light chain, a lambda light chain, or a variable heavy chain region of any species found, for example, at https: / / www.imgt.org / IMGTrepertoire / Proteins / index.php#B. In some embodiments, the species is human, monkey, chicken, or bovine. In some embodiments, the exposed C-terminus comprises the 4-6 C-terminal amino acid residues of a sequence found under IGKJ, IGLJ, or IGHJ at https: / / www.imgt.org / IMGTrepertoire / Proteins / index.php#B.In some embodiments, the exposed C-terminus comprises 4 to 6 C-terminal amino acid residues of the sequence found under IGKJ, IGLJ, or IGHJ at https: / / www.imgt.org / IMGTrepertoire / Proteins / index.php#B. In some embodiments, the exposed C-terminus consists of one of the C-terminal sequences provided in Table 1. In some embodiments, the exposed C-terminus consists of 4 to 6 C-terminal amino acid residues of the sequence found under IGKJ, IGLJ, or IGHJ at https: / / www.imgt.org / IMGTrepertoire / Proteins / index.php#B. In some embodiments, the exposed C-terminus consists of one of the C-terminal sequences provided in Table 1.

[0104] [Table 2]

[0105] As used herein, the term "non-naturally occurring C-terminus" generally refers to a C-terminus that is not normally present as the C-terminus of a polypeptide or protein found in nature, such as a therapeutic or diagnostic protein (e.g., an antigen-binding protein). An example of a non-naturally occurring C-terminus of a protein is the C-terminus of a protein (e.g., an antigen-binding domain) that has been engineered or modified from its wild-type sequence. In certain embodiments, a non-naturally occurring C-terminus of a protein is a C-terminus of a protein that is not generally naturally exposed due to interaction with a reference (e.g., a biological agent or ADA). In certain embodiments, a non-naturally occurring C-terminus is an exposed C-terminus.

[0106] As used herein, the term "at the end," when used in the context of an exposed C-terminus, refers to the exposed C-terminus or the last amino acid residue of a non-naturally occurring C-terminus or C-terminal ADA epitope. For example, in some embodiments, an exposed C-terminus terminating in the amino acid sequence VTVSS (SEQ ID NO:21) and having a His capping domain fused to the end of the C-terminus may result in an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO:1), VTVSSHH (SEQ ID NO:2), VTVSSHHH (SEQ ID NO:3), VTVSSHHHH (SEQ ID NO:4), and VTVSSHHHHH (SEQ ID NO:5). Similarly, in some embodiments, an exposed C-terminus terminating in the amino acid sequence VEIK (SEQ ID NO:22) and having a His capping domain fused to the end of the C-terminus may result in an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO:6), VEIKHH (SEQ ID NO:7), VEIKHHH (SEQ ID NO:8), VEIKHHHH (SEQ ID NO:9), and VEIKHHHHH (SEQ ID NO:10). In other embodiments, an exposed C-terminus terminating in the amino acid sequence VEIKR (SEQ ID NO:23) and having a His capping domain fused to the C-terminus may result in an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO:11), VEIKRHH (SEQ ID NO:12), VEIKRHHH (SEQ ID NO:13), VEIKRHHHH (SEQ ID NO:14), and VEIKRHHHHH (SEQ ID NO:15). In further embodiments, an exposed C-terminus terminating in the amino acid sequence VEIKRT (SEQ ID NO:24) and having a His capping domain fused to the C-terminus may result in an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO:16), VEIKRTHH (SEQ ID NO:17), VEIKRTHHH (SEQ ID NO:18), VEIKRTHHHH (SEQ ID NO:19), and VEIKRTHHHHH (SEQ ID NO:20).In some embodiments, an exposed C-terminus terminating in the amino acid sequence TKVTVL (SEQ ID NO: 38), TKLTVL (SEQ ID NO: 39), TQLIIL (SEQ ID NO: 40), TELTVL (SEQ ID NO: 41), or TQLTVL (SEQ ID NO: 42) and having a His cap domain fused to the C-terminal end can result in the amino acid sequence TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5. In some embodiments, an exposed C-terminus terminating in the amino acid sequence TKLTVL (SEQ ID NO: 39), TKVTVL (SEQ ID NO: 38), NGLVYAG (SEQ ID NO: 43), NGLLYAG (SEQ ID NO: 44), or TRLTVL (SEQ ID NO: 45) and having a His cap domain fused to the C-terminal end can result in the amino acid sequence TKLTVL(His)n (SEQ ID NO: 47), TKVTVL(His)n (SEQ ID NO: 46), NGLVYAG(His)n (SEQ ID NO: 51), NGLLYAG(His)n (SEQ ID NO: 52), or TRLTVL(His)n (SEQ ID NO: 53), where n is 1, 2, 3, 4, or 5.

[0107] As used herein, the term "cap domain" refers to amino acid residues that are not part of the exposed C-terminus of a polypeptide (e.g., an antibody, such as a bispecific, trispecific, or other multispecific antibody). In other words, the cap domain is heterologous to the exposed C-terminus of a polypeptide (e.g., an antibody, such as a bispecific, trispecific, or other multispecific antibody). In certain embodiments, the cap domain consists of (His)6 (i.e., HHHHHH (SEQ ID NO:26)), SLSLSPGK (SEQ ID NO:36), AS, TVAPTESS (SEQ ID NO:37), or a His cap domain. In some embodiments, the cap domain does not consist of (His)6 (i.e., HHHHHH (SEQ ID NO:26)) or AS. In certain embodiments, the cap domain consists of a His cap domain. In certain embodiments, the cap domain does not interfere with one, two, more than two, or all of the functions of the protein as assessed by techniques described herein or known to those of skill in the art. In certain embodiments, the cap domain does not interfere with the structure of the protein as assessed by techniques described herein or known to those of skill in the art.

[0108] As used herein, the term "capping means" refers to the presence or addition of an amino acid sequence consisting of (His)6 (i.e., HHHHHH (SEQ ID NO:26)), SLSLSPGK (SEQ ID NO:36), AS, or TVAPTESS (SEQ ID NO:37), or a His capping domain. In certain embodiments, capping means refers to the presence or addition of an amino acid sequence consisting of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37), or a His capping domain. In some embodiments, capping means does not refer to the presence or addition of an amino acid sequence consisting of (His)6 (i.e., HHHHHH (SEQ ID NO:26)) or an AS amino acid sequence.

[0109] As used herein, the term "His capping domain" refers to one or more histidine amino acid residues that are not part of the exposed C-terminus of a polypeptide (e.g., an antibody, such as a bispecific, trispecific, or other multispecific antibody). In other words, the His capping domain is heterologous to the exposed C-terminus of a polypeptide (e.g., an antibody, such as a bispecific, trispecific, or other multispecific antibody). In certain embodiments, the His capping domain is (His) n (SEQ ID NO:25), where n is 1, 2, 3, 4, or 5. In another particular embodiment, the His capping domain consists of a single His. As used herein, a His capping domain does not contain (His)6 (i.e., HHHHHH (SEQ ID NO:26)). In particular embodiments, the His capping domain does not interfere with one, two, more than two, or all of the functions of the protein, as assessed by the techniques described herein or known to those of skill in the art. In particular embodiments, the His capping domain does not interfere with the structure of the protein, as assessed by the techniques described herein or known to those of skill in the art.

[0110] In view of the present disclosure, one of skill in the art would understand that a "His capping means" is a His capping means wherein n is 1, 2, 3, 4, or 5 (His) n (SEQ ID NO: 25). In certain embodiments, the His capping means consists of a single His. Thus, the His capping means does not contain (His)6 (i.e., HHHHHH (SEQ ID NO: 26)).

[0111] As used herein, the term "anti-drug antibody epitope" refers to a plurality of contiguous or non-contiguous amino acids recognized by ADA. In certain embodiments, an anti-drug antibody epitope is a neoepitope or neoepitope-like structure at the C-terminus of a polypeptide (e.g., an antibody) or other protein that contains one or more target binding regions. In certain embodiments, an anti-drug antibody epitope is present only in a non-naturally occurring antigen-binding protein (e.g., an antibody) or other protein.

[0112] As used herein, the term "reference" refers to a substance or molecule to which a polypeptide (e.g., an antibody, such as a bispecific, trispecific, or other multispecific antibody) comprising an exposed C-terminus binds. In certain embodiments, the reference contacts the exposed C-terminus when bound to the polypeptide. For example, in certain embodiments, the reference comprises an antigen-binding region, e.g., an anti-drug antibody, and the reference binds to an epitope of the polypeptide comprising the exposed C-terminus.

[0113] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses, for example, monoclonal antibodies (including agonist, antagonist, neutralizing, and full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific and trispecific antibodies, so long as they exhibit the desired biological activity), fusion molecules such as those described below (e.g., IgG-scFv, scFv-Fc-scFv, VHH-Fc, Fc-VHH, Fc-scFv, HC-VHH, scFv-Fc-VHH, HC-scFv), single-chain antibodies, and fragments thereof (e.g., domain antibodies). Antibodies may be human, humanized, chimeric, and / or affinity matured, and may also be antibodies from other species, such as mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997).

[0114] Antibodies also include, but are not limited to, synthetic antibodies, nanobodies, recombinantly produced antibodies, antibodies derived from Camelidae species (e.g., llamas and alpacas) or their humanized variants, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above. These fragments refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which they are derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific and bispecific Fvs), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen-binding domains or molecules containing an antigen-binding site that binds to an antigen (e.g., one or more complementarity-determining regions (CDRs) of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibody can be an agonist antibody or an antagonist antibody. The antibody may be neither an agonist nor an antagonist.

[0115] In some embodiments, the antibodies may have modifications, including cross-linking agents, glycosylation, conjugated drugs, or thio-modified thiol linkages.

[0116] As used herein, the term "antigen" has its ordinary meaning in the art. An "antigen" includes a structure to which an antigen-binding protein (e.g., an antibody) can bind. An antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, an antigen is a polypeptide. In particular embodiments, an antigen is associated with a cell, e.g., present on or within a cell. In some embodiments, an antigen is associated with a cancer cell, e.g., present on or within a cancer cell. In certain embodiments, an antigen is associated with a pathogen, such as a virus, bacterium, fungus, or parasite.

[0117] As used herein, the term "antigen-binding protein" refers to a protein that binds to an antigen. An antibody is an example of an antigen-binding protein. Antigen-binding proteins include, but are not limited to, single-chain antibodies, nanobodies, multi-domain antibodies, scFvs, Fabs, and diabodies.

[0118] The term "binding" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site, e.g., the antigen-binding site of an antibody, and a single epitope on a target molecule, such as an antigen, is the affinity of the antibody for that epitope. The dissociation rate (k off ) and association rate (k on ) and the ratio (k off / k on ) is the dissociation constant K Dand has an inverse relationship with affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different complexes of antibody and antigen. on and k off The dissociation constant K of the antibodies provided herein depends on both D Affinity can be determined using any of the methods provided herein or any other method known to those skilled in the art. The affinity at one binding site does not necessarily reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, comes into contact with an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site. The strength of multiple interactions between such a multivalent antibody and an antigen is called avidity.

[0119] Antigen-binding proteins that bind, or specifically bind, to an antigen can be identified, for example, by immunoassays (e.g., ELISA, radioimmunoassays, and electrochemiluminescence immunoassays), Octet®, surface plasmon resonance (e.g., Biacore®), or other techniques known to those of skill in the art. In some embodiments, a binding protein specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens as determined using experimental techniques such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). Typically, a specific or selective response is at least two times the background signal or noise, and may exceed ten times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent of binding of an antigen binding protein to a "non-target" protein is less than about 10% of the binding of the antigen binding protein to its particular target antigen, as determined, for example, by fluorescence activated cell sorting (FACS) analysis or RIA. Antigen binding proteins that bind to antigens may be useful, for example, as antigen-targeted therapeutic and / or diagnostic agents. In certain embodiments, the antigen binding protein has a dissociation constant (K) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. D In certain embodiments, the antigen binding protein binds to an epitope of an antigen that is conserved among antigens of different species.

[0120] In certain embodiments, antigen-binding proteins may comprise "chimeric" sequences in which a portion of the heavy and / or light chain is identical to or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567, and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may include humanized sequences.

[0121] In certain embodiments, antigen-binding proteins may comprise portions of "humanized" forms of non-human (e.g., camelid, murine, non-human primate) antibodies containing sequences from a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced by residues from corresponding CDRs of a non-human species (e.g., donor antibody) such as camel, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. The heavy or light chains of humanized antibodies can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol., 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0122] In certain embodiments, an antigen-binding protein can comprise a portion of a "fully human antibody" or "human antibody," which terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. The antigen-binding protein can comprise an antibody sequence. In specific embodiments, these terms refer to an antibody comprising variable and constant regions of human origin. A "fully human" antibody can also encompass, in certain embodiments, an antibody that binds a polypeptide and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies having variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or has been produced using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao, et al., Nature Protocols, 1:755-68 (2006)).Furthermore, human monoclonal antibodies can be prepared using the methods described in Cole, et al., Monoclonal Antibodies and Cancer Therapy 77 (1985), Boerner et al., J. Immunol. 147(1):86-95 (1991), and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li, et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006) (regarding human antibodies generated by human B-cell hybridoma technology).

[0123] In certain embodiments, an antigen binding protein may comprise a portion of a "recombinant human antibody," which phrase includes human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector introduced into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice or cows) that are transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0124] In certain embodiments, an antigen-binding protein can comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, where the individual antibodies comprising the population are identical except for minor naturally occurring mutations and well-known post-translational modifications, such as amino acid isomerization and deamidation, oxidation of methionine, and deamidation of asparagine and glutamine, and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to a particular method for producing the antibody. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0125] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). In the case of IgG, the four-chain unit generally has a size of approximately 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. The structure and properties of different classes of antibodies are described, for example, in Basic and Clinical Immunology 71 (Stites, et al. eds., 8th ed. 1994) and Immunobiology (Janeway, et al. eds., 5th ed. 1995). th ed. 2001).

[0126] As used herein, the terms "Fab" and "Fab region" have their usual meanings in the art. Typically, the term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. Conventional IgG usually contains two Fab regions, each of which is present in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions in the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, while the VL and CL regions can be on separate polypeptides, as in the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be present on the same polypeptide and oriented in a different order, as described in more detail in the following sections.

[0127] As used herein, the terms "variable region" and "variable domain" in the context of antibodies have their usual meaning in the art. Typically, the terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody's light or heavy chain, located generally at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, and are used to determine the binding and specificity of each particular antibody for its particular antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region vary significantly in sequence among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, variability is not uniform across the 110-amino acid span of the variable region. Instead, V regions consist of less variable (e.g., relatively invariant) stretches of approximately 15–30 amino acids called framework regions (FRs), separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions," each approximately 9–12 amino acids in length. The variable regions of the heavy and light chains each contain four FRs, which largely adopt a β-sheet structure and are connected by three hypervariable regions, which form loops that connect the β-sheet structure and, in some cases, form part of it. The hypervariable regions within each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding the antibody to an antigen but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary significantly in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0128] The terms "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the antibody compilation of Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the sequence of that antibody with the "standard" Kabat numbered sequence at the regions of homology. The Kabat numbering system is generally used when referring to residues in the variable domain (from about residues 1 to 107 in the light chain, and from about residues 1 to 113 in the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0129] As used herein, the term "heavy chain" in the context of an antibody has its conventional meaning in the art. Typically, the term "heavy chain" when used with respect to an antibody refers to a polypeptide chain of about 50 to 70 kDa, the amino-terminal portion of which contains a variable region of about 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five distinct types (e.g., isotypes), designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains vary in size, with α, δ, and γ containing approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these distinct types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM (which also include the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4). The sequences of heavy chains from various species are known in the art (see, e.g., IMGT®, the international ImMunoGeneTics information system®, imgt.org).

[0130] As used herein, the term "light chain" in the context of an antibody has its usual meaning in the art. Typically, the term "light chain" when used with respect to an antibody refers to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. The sequences of light chains from various species are known in the art (see, e.g., IMGT®, the international ImMunoGeneTics information system®, imgt.org). As used herein, the terms "constant region" or "constant domain" in the context of an antibody have their usual meaning in the art. Typically, the term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding the antibody to an antigen, but that exhibit various effector functions, such as interaction with Fc receptors. Typically, the term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable region, which contains the antigen-binding site. The constant region can include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0131] As used herein, the term "framework" or "FR" in the context of an antibody has its conventional meaning in the art. Typically, the term "framework" or "FR" refers to variable region residues that flank the CDRs. FR residues are present, for example, in chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region or CDR residues.

[0132] As used herein, the term "Fc region" of an antibody has its conventional meaning in the art. Typically, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is often defined to stretch from the amino acid residue at position Cys226, or from the amino acid residue at position Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. A "functional Fc region" possesses the "effector functions" of a native-sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region comprises at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0133] In one embodiment, homology is determined by sequence similarity. Modern protein sequence databases are very comprehensive. Widely used similarity search programs, such as BLAST (Altschul et al. (1997); units 3.3 and 3.4), PSI-BLAST (Altschul et al., 1997), SSEARCH (Smith and Waterman (1981); Pearson (1991), unit 3.10), FASTA (Pearson and Lipman (1988) unit 3.9), and HMMER3 (Johnson et al., 2010), generate accurate statistical estimates and ensure that protein sequences that share significant similarity also have similar structures.

[0134] The term "specificity" in the context of an antibody or other antibody-binding protein refers to the selective recognition of the antigen-binding protein for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. As used herein, the term "multispecific" refers to an antigen-binding protein having two or more antigen-binding sites, at least two of which bind different antigens. "Bispecific" as used herein refers to an antigen-binding protein having two different antigen-binding specificities. As used herein, the term "monospecific" antibody refers to an antigen-binding protein having one or more binding sites, each of which binds to the same antigen.

[0135] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also refers to amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes polypeptides containing one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can occur as a single chain or as two or more related chains.

[0136] "Polynucleotide," "nucleotide sequence," or "nucleic acid sequence," as used interchangeably herein, refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, generally single-stranded, synthetic polynucleotide, generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally fully applicable to oligonucleotides. Cells that produce the antigen-binding proteins of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end. The left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction. The region of the sequence on the DNA strand that has the same sequence as the RNA transcript at the 5' to 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the sequence on the DNA strand that has the same sequence as the RNA transcript at the 3' to 3' end of the RNA transcript is referred to as the "downstream sequence."

[0137] An "isolated" polynucleotide, nucleotide sequence, nucleic acid molecule, or nucleic acid sequence is a nucleic acid sequence, e.g., an RNA sequence, a DNA sequence, or a sequence containing a mixture of nucleic acids, that is substantially separated from other RNA sequences, genomic DNA sequences, and proteins or complexes, such as ribosomes and polymerases, that are naturally associated with the naturally occurring sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Furthermore, an "isolated" polynucleotide, nucleotide sequence, nucleic acid molecule, or nucleic acid sequence, e.g., a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In certain embodiments, one or more polynucleotides, nucleotide sequences, nucleic acid molecules, or nucleic acid sequences encoding antibodies or other antigen-binding proteins described herein are isolated or purified. This term encompasses polynucleotides, nucleotide sequences, nucleic acid molecules, and nucleic acid sequences that are removed from their naturally occurring environment, including recombinant or cloned DNA isolates, and chemically synthesized analogs, or analogs biologically synthesized by heterologous systems. A substantially pure molecule can include isolated forms of the molecule. Specifically, an "isolated" polynucleotide, nucleotide sequence, nucleic acid molecule, or nucleic acid sequence encoding an antibody or antigen-binding protein described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the production environment.

[0138] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes nucleotide sequences that are degenerate versions of each other and all nucleotide sequences that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns to the extent that in some cases the nucleotide sequence encoding the protein may contain introns.

[0139] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid encoding an antigen-binding protein (e.g., an antibody) described herein, to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain operable selection sequences or markers capable of stable integration into a host cell chromosome. In addition, a vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included provide, for example, resistance to antibiotics or toxins, complement deficiencies in auxiliary auxotrophies, or supply critical nutrients absent from the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more nucleic acid molecules are coexpressed (e.g., both the heavy and light chains of an antibody, or antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector or into separate expression vectors. For expression in a single vector, the encoding nucleic acids can be operably linked to a common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or amplification of mRNA by polymerase chain reaction (PCR), immunoblotting for expression of gene products, or other suitable analytical methods for testing expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules will be expressed in amounts sufficient to produce the desired product, and will further understand that expression levels can be optimized to obtain sufficient expression using methods well known to those skilled in the art.

[0140] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly for use in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0141] "Excipient" refers to a material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, encapsulating material, etc. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, wetting agents, lubricants, fragrances, preservatives, propellants, release agents, bactericides, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0142] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; proteins such as low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0143] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005, Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009, Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007, Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0144] In some embodiments, the excipient may be a sterile liquid, such as water or oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid excipients, particularly for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, or the like. If desired, the composition may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like. Oral composition containing formulations can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0145] A composition containing a pharmaceutical compound can include, for example, an antigen binding protein (eg, an antibody) in isolated or purified form, together with a suitable amount of excipients.

[0146] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a polypeptide (e.g., an antigen binding protein, such as an antibody), agent (e.g., a therapeutic protein or other agent), or pharmaceutical composition described herein sufficient to bring about a desired result.

[0147] The terms "subject" and "patient" may be used interchangeably herein to refer to an animal. As used herein, in certain embodiments, a subject is a mammal, either non-primate or primate (e.g., human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, at risk of developing a disease or disorder. In certain embodiments, a subject is a non-human animal, such as a pet or livestock animal (e.g., a dog, cat, cow, pig, horse, donkey, etc.).

[0148] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0149] The practice of the embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology that are within the skill of those in the art, and such techniques are fully described in the literature. Examples of particularly suitable texts for reference include: Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Glover, ed., DNA Cloning, Volumes I and II (1985); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, NY, 2nd ed. 1987); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed.2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds.,2d ed.2010).

[0150] To assist the reader of this application, the description of the specification is divided into various paragraphs or sections or directed to various embodiments of the application. These separations should not be considered as separating one paragraph or section or embodiment entity from another paragraph or section or embodiment entity. To the contrary, those skilled in the art will understand that the present description has broad application and encompasses all combinations of the various sections, paragraphs, and sentences that may be contemplated. The discussion of any embodiments is intended to be merely exemplary and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples. The present application contemplates the use of any of the applicable components in any combination, regardless of whether a specific combination is explicitly described.

[0151] Protein Provided herein are proteins (e.g., antibodies or other antigen binding proteins) that comprise an exposed C-terminus fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In certain embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antigen binding protein. In some embodiments, the antigen binding protein is any in which an exposed C-terminus, e.g., a VHH or scFv / spFv, is present at the C-terminus of the protein, e.g., an antibody or immunoglobulin superfamily domain of interest. In some embodiments, the antibody has a configuration as shown in any one of Figures 1-9A.

[0152] A cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) fused to the exposed C-terminus of a polypeptide can be achieved by any means known in the art. In some embodiments, the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) is linked directly to the exposed C-terminus of the polypeptide. In some embodiments, the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) is conjugated to the exposed C-terminus of the polypeptide.

[0153] In certain aspects, provided herein are proteins comprising a capping domain, as described in Examples 8.1 and / or 8.2 (below). In certain embodiments, the capped proteins provided herein have one or more of the properties of the capped trispecific antibodies described in Examples 8.1 and / or 8.2 (below). In certain embodiments, the protein is a capped trispecific antibody described in Examples 8.1 and / or 8.2 (below).

[0154] In one aspect, provided herein is a polypeptide comprising one or more target binding regions (e.g., two, three, or more target binding regions) and an exposed C-terminus fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In some embodiments, the one or more target binding regions comprise an antibody variable domain (e.g., a heavy chain variable domain or a light chain variable domain), or an antigen-binding fragment thereof. In some embodiments, the capping domain consists of a His capping domain, or the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In certain embodiments, the His capping domain is (His) n(SEQ ID NO:25), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO:1), VTVSSHH (SEQ ID NO:2), VTVSSHHH (SEQ ID NO:3), VTVSSHHHH (SEQ ID NO:4), and VTVSSHHHHH (SEQ ID NO:5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO:6), VEIKHH (SEQ ID NO:7), VEIKHHH (SEQ ID NO:8), VEIKHHHH (SEQ ID NO:9), and VEIKHHHHH (SEQ ID NO:10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO:11), VEIKRHH (SEQ ID NO:12), VEIKRHHH (SEQ ID NO:13), VEIKRHHHH (SEQ ID NO:14), and VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO:16), VEIKRTHH (SEQ ID NO:17), VEIKRTHHH (SEQ ID NO:18), VEIKRTHHHH (SEQ ID NO:19), and VEIKRTHHHHH (SEQ ID NO:20). In some embodiments, the exposed C-terminus fused to a His cap domain consists of amino acids TKVTVL(His)n (SEQ ID NO:46), TKLTVL(His)n (SEQ ID NO:47), TQLIIL(His)n (SEQ ID NO:48), TELTVL(His)n (SEQ ID NO:49), or TQLTVL(His)n (SEQ ID NO:50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to a His cap domain consists of amino acids TKLTVL(His)n (SEQ ID NO:47), TKVTVL(His)n (SEQ ID NO:46), NGLVYAG(His)n (SEQ ID NO:51), NGLLYAG(His)n (SEQ ID NO:52), or TRLTVL(His)n (SEQ ID NO:53), where n is 1, 2, 3, 4, or 5. In certain embodiments, the polypeptide is isolated.

[0155] In another aspect, provided herein is a protein comprising: (a) a first polypeptide comprising a target binding region and an exposed C-terminus fused to a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)); and (b) a second polypeptide comprising the target binding region. In some embodiments, the second polypeptide comprises an exposed C-terminus fused to a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In some embodiments, the target binding regions of the first and second polypeptides are capable of binding to different targets. In certain embodiments, the protein further comprises a third polypeptide comprising a target binding region. In some embodiments, the third polypeptide comprises an exposed C-terminus fused to a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In some embodiments, the third polypeptide is capable of binding to a different target than the first polypeptide, the second polypeptide, or both. In some embodiments, the target binding region comprises an antibody variable domain (e.g., a heavy chain variable domain or a light chain variable domain), or an antigen-binding fragment thereof. In some embodiments, the capping domain consists of a His capping domain or the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37). In certain embodiments, the His capping domain consists of (His) n(SEQ ID NO:25), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO:1), VTVSSHH (SEQ ID NO:2), VTVSSHHH (SEQ ID NO:3), VTVSSHHHH (SEQ ID NO:4), and VTVSSHHHHH (SEQ ID NO:5). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO:6), VEIKHH (SEQ ID NO:7), VEIKHHH (SEQ ID NO:8), VEIKHHHH (SEQ ID NO:9), and VEIKHHHHH (SEQ ID NO:10). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO:11), VEIKRHH (SEQ ID NO:12), VEIKRHHH (SEQ ID NO:13), VEIKRHHHH (SEQ ID NO:14), and VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the exposed C-terminus fused to a His cap domain consists of an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO:16), VEIKRTHH (SEQ ID NO:17), VEIKRTHHH (SEQ ID NO:18), VEIKRTHHHH (SEQ ID NO:19), and VEIKRTHHHHH (SEQ ID NO:20). In some embodiments, the exposed C-terminus fused to a His cap domain consists of amino acids TKVTVL(His)n (SEQ ID NO:46), TKLTVL(His)n (SEQ ID NO:47), TQLIIL(His)n (SEQ ID NO:48), TELTVL(His)n (SEQ ID NO:49), or TQLTVL(His)n (SEQ ID NO:50), where n is 1, 2, 3, 4, or 5. In some embodiments, the exposed C-terminus fused to a His cap domain consists of amino acids TKLTVL(His)n (SEQ ID NO:47), TKVTVL(His)n (SEQ ID NO:46), NGLVYAG(His)n (SEQ ID NO:51), NGLLYAG(His)n (SEQ ID NO:52), or TRLTVL(His)n (SEQ ID NO:53), where n is 1, 2, 3, 4, or 5. In some embodiments, the protein is trispecific.In some embodiments, the isolated protein is multispecific. In certain embodiments, the protein is isolated.

[0156] In another aspect, provided herein is a protein comprising an antigen-binding region of an antibody, the antigen-binding region comprising an exposed C-terminus and a (His) n (SEQ ID NO:25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein are proteins (e.g., antigen binding proteins) comprising an antibody variable domain, wherein the antibody variable domain comprises an exposed C-terminus and a terminal end of the exposed C-terminus of the antibody variable domain (His) n (SEQ ID NO:25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein are proteins comprising an antibody fragment, wherein the antibody fragment comprises an exposed C-terminus and a terminal (His) at the exposed C-terminus of the antibody fragment. n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In some embodiments, (His) n In the sequence, n is 1. In one embodiment, (His) n In the sequence, n is 2. In some embodiments, (His) n In the sequence, n is 3. In one embodiment, (His) n The n in the sequence is 4 (SEQ ID NO: 27). In some embodiments, (His) n In the sequence, n is 5 (SEQ ID NO: 28). In certain embodiments, the antibody variable domain is a heavy chain variable domain. In certain embodiments, the heavy chain variable domain is not camelized. In certain embodiments, the exposed C-terminus consists of the amino acid sequence VTVSS (SEQ ID NO: 21), LTVSS (SEQ ID NO: 29), or VTVSA (SEQ ID NO: 30). In certain embodiments, the antibody variable domain is a light chain variable domain. In certain embodiments, the exposed C-terminus consists of the amino acid sequence VEIK (SEQ ID NO: 22), VEIKR (SEQ ID NO: 23), or VEIKRT (SEQ ID NO: 24). In certain embodiments, the protein is isolated.

[0157] In some embodiments, provided herein are bispecific antibodies comprising an antibody variable domain, the antibody variable domain comprising an exposed C-terminus and a (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In one embodiment, provided herein is a multispecific antibody comprising an antibody variable domain, the antibody variable domain comprising an exposed C-terminus and a terminal (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, the bispecific or multispecific antibody is isolated. In some embodiments, the antibody variable domain is a light chain variable domain. In other embodiments, the antibody variable domain is a heavy chain variable domain. In certain embodiments, the heavy chain variable domain is not camelized.

[0158] Heavy chain immunoglobulin variable (V H The C-terminal amino acid sequence of the light chain immunoglobulin variable (V) domain generally has the sequence VTVSS (SEQ ID NO: 21) in humans and VTVSS (SEQ ID NO: 21), LTVSS (SEQ ID NO: 29), or VTVSA (SEQ ID NO: 30) in mice. L The C-terminal amino acid sequence of the V domain generally has the sequence VEIK (SEQ ID NO: 22), VEIKR (SEQ ID NO: 23), or VEIKRT (SEQ ID NO: 24) in humans. H The C-terminal amino acids of the VL domain are buried and not exposed by the first heavy chain constant (CH1) domain, and the C-terminal amino acids of the VL domain are buried and not exposed by the first heavy chain constant (C L ) domain and are not exposed. However, antibody fragments H Domain or V L The C-termini of either or both domains can be engineered to be non-exposed, which allows for the creation of otherwise non-naturally occurring V H Domain or V LThis effectively exposes part of the V domain, resulting in a V with an exposed C-terminus. H Domain and / or V L Antibody fragments containing the domains are capable of eliciting an ADA response.

[0159] Thus, in one aspect, provided herein is a protein (e.g., an antigen binding protein) comprising an antibody heavy chain variable domain, wherein the antibody heavy chain variable domain comprises a C-terminal amino acid sequence, the last 6 to 10 amino acid residues of which are the amino acid sequence VTVSS(H) n (SEQ ID NO:31), where n is 1, 2, 3, 4, or 5. In some embodiments, the C-terminal amino acid sequence consists of VTVSSH (SEQ ID NO:1). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHH (SEQ ID NO:2). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHH (SEQ ID NO:3). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHHH (SEQ ID NO:4). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHHHH (SEQ ID NO:5). In certain embodiments, the protein is isolated.

[0160] In some embodiments, provided herein is a bispecific antibody comprising an antibody heavy chain variable domain, wherein the antibody heavy chain variable domain comprises a C-terminal amino acid sequence, the last 6 to 10 amino acid residues of which are the amino acid sequence VTVSS(H) n(SEQ ID NO:31), where n is 1, 2, 3, 4, or 5. In some embodiments, the C-terminal amino acid sequence consists of VTVSSH (SEQ ID NO:1). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHH (SEQ ID NO:2). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHH (SEQ ID NO:3). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHHH (SEQ ID NO:4). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHHHH (SEQ ID NO:5). In certain embodiments, the bispecific antibody is isolated.

[0161] In certain embodiments, provided herein is a multispecific antibody comprising an antibody heavy chain variable domain, wherein the antibody heavy chain variable domain comprises a C-terminal amino acid sequence, the last 6 to 10 amino acid residues of which are the amino acid sequence VTVSS(H) n (SEQ ID NO:31), where n is 1, 2, 3, 4, or 5. In some embodiments, the C-terminal amino acid sequence consists of VTVSSH (SEQ ID NO:1). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHH (SEQ ID NO:2). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHH (SEQ ID NO:3). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHHH (SEQ ID NO:4). In some embodiments, the C-terminal amino acid sequence consists of VTVSSHHHHH (SEQ ID NO:5). In certain embodiments, the multispecific antibody is isolated. In certain embodiments, the multispecific antibody is trispecific.

[0162] In another aspect, provided herein is a protein (e.g., an antigen binding protein) comprising an antibody light chain variable domain, wherein the antibody light chain variable domain comprises a C-terminal amino acid sequence, the last 5 to 11 amino acid residues of which are the amino acid sequence VEIK(H). n (SEQ ID NO: 32), VEIKR(H) n (SEQ ID NO: 33), or VEIKRT(H) n(SEQ ID NO:34), where n is 1, 2, 3, 4, or 5. In some embodiments, the C-terminal amino acid sequence consists of VEIKH (SEQ ID NO:6). In some embodiments, the C-terminal amino acid sequence consists of VEIKHH (SEQ ID NO:7). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHH (SEQ ID NO:8). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHHH (SEQ ID NO:9). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHHHH (SEQ ID NO:10). In some embodiments, the C-terminal amino acid sequence consists of VEIKRH (SEQ ID NO:11). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHH (SEQ ID NO:12). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHH (SEQ ID NO:13). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHHH (SEQ ID NO:14). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTH (SEQ ID NO: 16). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHH (SEQ ID NO: 17). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHH (SEQ ID NO: 18). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHHH (SEQ ID NO: 19). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHHHH (SEQ ID NO: 20). In certain embodiments, the protein is isolated.

[0163] In certain embodiments, provided herein is a bispecific antibody comprising an antibody light chain variable domain, wherein the antibody light chain variable domain comprises a C-terminal amino acid sequence, the last 5 to 11 amino acid residues of which are the amino acid sequence VEIK(H) n (SEQ ID NO: 32), VEIKR(H) n (SEQ ID NO: 33), or VEIKRT(H) n(SEQ ID NO:34), where n is 1, 2, 3, 4, or 5. In some embodiments, the C-terminal amino acid sequence consists of VEIKH (SEQ ID NO:6). In some embodiments, the C-terminal amino acid sequence consists of VEIKHH (SEQ ID NO:7). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHH (SEQ ID NO:8). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHHH (SEQ ID NO:9). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHHHH (SEQ ID NO:10). In some embodiments, the C-terminal amino acid sequence consists of VEIKRH (SEQ ID NO:11). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHH (SEQ ID NO:12). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHH (SEQ ID NO:13). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHHH (SEQ ID NO:14). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTH (SEQ ID NO: 16). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHH (SEQ ID NO: 17). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHH (SEQ ID NO: 18). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHHH (SEQ ID NO: 19). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHHHH (SEQ ID NO: 20). In certain embodiments, the bispecific antibody is isolated.

[0164] In some embodiments, provided herein is a multispecific antibody (e.g., a triabody) comprising an antibody light chain variable domain, wherein the antibody light chain variable domain comprises a C-terminal amino acid sequence, the last 5 to 11 amino acid residues of which are the amino acid sequence VEIK(H) n (SEQ ID NO: 32), VEIKR(H) n (SEQ ID NO: 33), or VEIKRT(H) n(SEQ ID NO:34), where n is 1, 2, 3, 4, or 5. In some embodiments, the C-terminal amino acid sequence consists of VEIKH (SEQ ID NO:6). In some embodiments, the C-terminal amino acid sequence consists of VEIKHH (SEQ ID NO:7). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHH (SEQ ID NO:8). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHHH (SEQ ID NO:9). In some embodiments, the C-terminal amino acid sequence consists of VEIKHHHHH (SEQ ID NO:10). In some embodiments, the C-terminal amino acid sequence consists of VEIKRH (SEQ ID NO:11). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHH (SEQ ID NO:12). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHH (SEQ ID NO:13). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHHH (SEQ ID NO:14). In some embodiments, the C-terminal amino acid sequence consists of VEIKRHHHHH (SEQ ID NO:15). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTH (SEQ ID NO: 16). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHH (SEQ ID NO: 17). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHH (SEQ ID NO: 18). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHHH (SEQ ID NO: 19). In some embodiments, the C-terminal amino acid sequence consists of VEIKRTHHHHH (SEQ ID NO: 20). In certain embodiments, the multispecific antibody is a trispecific antibody.

[0165] In certain aspects, provided herein are trispecific antibodies as illustrated in Figures 10A and 10B, which comprise an exposed C-terminus fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). For example, in some embodiments, the trispecific antibody comprises an Fc-scFv fusion, and the exposed C-terminus fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) is at the C-terminus of the scFv fusion. In some embodiments, the Fc-scFv fusion further comprises a second scFv having a target binding region distinct from the first scFv. In some embodiments, the Fc-scFv fusion further comprises a Fab. In some embodiments, provided herein are trispecific antibodies comprising two or more exposed C-termini, each fused to a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)). In certain embodiments, the trispecific antibody comprises target binding domains capable of interacting with CD3 on T cells and an antigen present on the surface of cancer cells. See, for example, Table 2 below for antigens present on cancer cells. It should be understood that the antibodies depicted in Figures 10A and 10B are non-limiting and exemplary only. In certain embodiments, the trispecific antibody is isolated.

[0166] In another aspect, provided herein is a protein (e.g., an antigen binding protein) that has a non-naturally occurring C-terminus and a terminal (His) at the non-naturally occurring C-terminus. n (SEQ ID NO:25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein is a protein comprising an antigen-binding region of an antibody, wherein the antigen-binding region comprises a non-naturally occurring C-terminus and a terminal (His) at the non-naturally occurring C-terminus. n(SEQ ID NO:25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein is a protein comprising an antibody variable domain, wherein the antibody variable domain comprises a non-naturally occurring C-terminus and a (His) n (SEQ ID NO:25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein are proteins comprising antibody fragments, wherein the antibody fragments comprise a non-naturally occurring C-terminus and a (His) at the terminal end of the non-naturally occurring C-terminus. n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In some embodiments, (His) n In the sequence, n is 1. In one embodiment, (His) n In the sequence, n is 2. In some embodiments, (His) n In the sequence, n is 3. In one embodiment, (His) n The n in the sequence is 4 (SEQ ID NO: 27). In some embodiments, (His) n The n in the sequence is 5 (SEQ ID NO: 28). In certain embodiments, the protein is isolated.

[0167] In some embodiments, provided herein are bispecific antibodies comprising an antibody variable domain, the antibody variable domain comprising a non-naturally occurring C-terminus and a non-naturally occurring C-terminal end (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In one embodiment, provided herein is a multispecific antibody comprising an antibody variable domain, the antibody variable domain comprising a non-naturally occurring C-terminus and a (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, the antibody variable domain is a light chain variable domain. In other embodiments, the antibody variable domain is a heavy chain variable domain. In certain embodiments, the heavy chain variable domain is not camelized. In certain embodiments, the bispecific or multispecific antibody is isolated.

[0168] In certain embodiments, provided herein are bispecific antibodies comprising an antibody fragment, wherein the antibody fragment comprises a non-naturally occurring C-terminus and a non-naturally occurring C-terminal end (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In some embodiments, provided herein are multispecific antibodies comprising antibody fragments, the antibody fragments comprising a non-naturally occurring C-terminus and a (His) at the terminal end of the non-naturally occurring C-terminus. n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, the bispecific or multispecific antibody is isolated. In certain embodiments, the multispecific antibody is trispecific.

[0169] In another aspect, provided herein is a protein, the protein comprising a C-terminal anti-drug antibody epitope and a (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein is a protein, the protein comprising a C-terminal anti-drug antibody epitope and a (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein is a protein comprising an antigen-binding region of an antibody, the antigen-binding region comprising a C-terminal anti-drug antibody epitope and a (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein is a protein comprising an antibody variable region, wherein the antibody variable region comprises a C-terminal anti-drug antibody epitope and a (His) n(SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein are proteins comprising an antibody fragment, the antibody fragment comprising a C-terminal anti-drug antibody epitope and a (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In some embodiments, (His) n In the sequence, n is 1. In one embodiment, (His) n In the sequence, n is 2. In some embodiments, (His) n In the sequence, n is 3. In one embodiment, (His) n The n in the sequence is 4 (SEQ ID NO: 27). In some embodiments, (His) n The n in the sequence is 5 (SEQ ID NO: 28). In certain embodiments, the protein is isolated.

[0170] In some embodiments, provided herein are bispecific antibodies comprising an antigen-binding region of an antibody, the antigen-binding region comprising a C-terminal anti-drug antibody epitope and a C-terminal anti-drug antibody epitope terminated by (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein are multispecific antibodies (e.g., trispecific antibodies) comprising an antigen-binding region of an antibody, the antigen-binding region comprising a C-terminal anti-drug antibody epitope and (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, the bispecific or multispecific antibody is isolated. In certain embodiments, the multispecific antibody is trispecific.

[0171] In some embodiments, provided herein are bispecific antibodies comprising an antibody variable domain, the antibody variable domain comprising a C-terminal anti-drug antibody epitope and a (His) n(SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein are multispecific antibodies (e.g., trispecific antibodies) comprising an antibody variable domain comprising a C-terminal anti-drug antibody epitope and (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, the antibody variable domain is a light chain variable domain. In other embodiments, the antibody variable domain is a heavy chain variable domain. In certain embodiments, the heavy chain variable domain is not camelized. In certain embodiments, the bispecific or multispecific antibody is isolated. In certain embodiments, the multispecific antibody is trispecific.

[0172] In some embodiments, provided herein are bispecific antibodies comprising an antibody fragment, the antibody fragment comprising a C-terminal anti-drug antibody epitope and a (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, provided herein are multispecific (e.g., trispecific) antibodies comprising an antibody fragment, the antibody fragment comprising a C-terminal anti-drug antibody epitope and a (His) n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In certain embodiments, the bispecific or multispecific antibody is isolated. In certain embodiments, the multispecific antibody is trispecific.

[0173] In another aspect, provided herein is a protein (e.g., a fusion protein or an antigen-binding protein) comprising an antibody fragment, wherein the antibody fragment has an exposed C-terminus and a terminal (His) at the C-terminus. n (SEQ ID NO: 25), where n is 1, 2, 3, 4, or 5. In some embodiments, (His) n In the sequence, n is 1. In one embodiment, (His) nIn the sequence, n is 2. In some embodiments, (His) n In the sequence, n is 3. In one embodiment, (His) n The n in the sequence is 4 (SEQ ID NO: 27). In some embodiments, (His) n The n in the sequence is 5 (SEQ ID NO: 28). In certain embodiments, the protein is isolated.

[0174] Without being bound by any theory, in certain embodiments, a capping domain (e.g., His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) or (His) capping domain (e.g., His) capping domain (e.g., SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) is added to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein. n The addition of the sequence alters the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope such that the polypeptide or protein no longer interacts with the reference or has reduced interaction with the reference. In some embodiments, the reference is a molecule present in biological material. Thus, in some embodiments, a protein with an exposed C-terminus fused to a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) exhibits reduced interaction with molecules present in biological material. In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In certain embodiments, the biological material comprises serum. In certain embodiments, the biological material comprises plasma. In certain embodiments, the interaction is determined by an agglutination assay described herein (e.g., an assay described in Section 7.9.1) or known to one of skill in the art.

[0175] In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in a reduction in the interaction of the polypeptide or protein with a reference, respectively, compared to the interaction of the same reference with the same polypeptide or protein lacking the cap domain. In some embodiments, the reduction is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the reduction is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In certain embodiments, the interaction is determined by an immunoassay described herein (e.g., an assay described in Section 7.9.1) or known to one of skill in the art.

[0176] In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in a reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 10% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 20% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 30% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine.In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 40% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 50% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 60% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 70% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine.In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about an 80% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 90% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 95% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the single histidine, respectively. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) eliminates the interaction of the polypeptide or protein with a reference. In certain embodiments, the interaction is determined by an immunoassay described herein (e.g., an assay described in Section 7.9.1) or known to one of skill in the art.

[0177] In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in a reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines, respectively. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 10% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines, respectively. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 20% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 30% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines.In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 40% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 50% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 60% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 70% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines.In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about an 80% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 90% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 95% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the two histidines, respectively. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) eliminates the interaction of the polypeptide or protein with a reference. In certain embodiments, the interaction is determined by an immunoassay described herein (e.g., an assay described in Section 7.9.1) or known to one of skill in the art.

[0178] In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in a reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines, respectively. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 10% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines, respectively. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 20% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 30% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines.In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 40% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 50% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 60% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 70% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines.In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about an 80% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 90% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 95% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the three histidines, respectively. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) eliminates the interaction of the polypeptide or protein with a reference. In certain embodiments, the interaction is determined (e.g., by assays described in Section 7.9.1) or known to one of skill in the art.

[0179] In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)), results in a reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines, respectively. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)), results in at least about a 10% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines, respectively. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 20% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 30% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines.In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 40% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 50% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 60% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 70% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines.In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about an 80% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 90% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 95% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the four histidines, respectively. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) eliminates the interaction of the polypeptide or protein with a reference. In certain embodiments, the interaction is determined (e.g., by assays described in Section 7.9.1) or known to one of skill in the art.

[0180] In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in a reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 10% reduction in the interaction of the polypeptide or protein with a reference, compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 20% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 30% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively.In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 40% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 50% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 60% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 70% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively.In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about an 80% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 90% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 95% reduction in the interaction of the polypeptide or protein with a reference compared to the interaction of the same reference with the same polypeptide or protein lacking the five histidines, respectively. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) eliminates the interaction of the polypeptide or protein with a reference. In certain embodiments, the interaction is determined (e.g., by assays described in Section 7.9.1) or known to one of skill in the art.

[0181] In some embodiments, the reference comprises a molecule present in a biological material, where the reference is a molecule present in the biological material. In some embodiments, the biological material comprises serum. In some embodiments, the biological material comprises plasma. In some embodiments, the biological material comprises serum and / or plasma (e.g., human serum and / or human plasma). In some embodiments, the reference comprises an anti-drug antibody. In some embodiments, the anti-drug antibody is pre-existing in the serum and / or plasma of a subject (e.g., a human subject).

[0182] In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in a reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the capping domain. In some embodiments, the reduction is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the reduction is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0183] In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His) n (SEQ ID NO: 25) sequence, resulting in a reduced interaction of the protein with ADA compared to the interaction of the same protein with ADA. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His)n (SEQ ID NO: 25) sequence, resulting in at least about a 10% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal end of an anti-drug antibody epitope can be achieved by adding (His) n (SEQ ID NO: 25) sequence, resulting in at least about a 20% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His) n (SEQ ID NO: 25) sequence, resulting in at least about a 30% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His) n (SEQ ID NO: 25) sequence, resulting in at least about a 40% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n Addition of the sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal end of an anti-drug antibody epitope may be accomplished by adding (His) n (SEQ ID NO: 25) sequence, resulting in at least about a 50% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) nThe addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a therapeutic protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His) n (SEQ ID NO: 25) sequence, resulting in at least about a 60% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His) n (SEQ ID NO: 25) sequence, resulting in at least about a 70% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His) n (SEQ ID NO: 25) sequence, resulting in at least about an 80% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His) n (SEQ ID NO: 25) sequence, resulting in at least about a 90% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. In some embodiments, n is 1, 2, 3, 4, or 5 (His) n The addition of the (SEQ ID NO: 25) sequence to the exposed C-terminus of a protein (e.g., an antigen binding protein), to the non-naturally occurring C-terminus, or to the C-terminal anti-drug antibody epitope may be accomplished by adding (His) n(SEQ ID NO: 25) sequence, resulting in at least about a 95% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the sequence. The interaction of the protein with ADA can be measured and / or detected using a variety of assays known to those of skill in the art or described herein, such as the assays described in Section 7.9.1. In certain embodiments, the interaction between the protein and ADA is determined by ELISA, a surface plasmon resonance assay (e.g., BIACORE), an electrochemiluminescence (ECL)-based assay, or other immunoassay described herein or known to those of skill in the art.

[0184] In certain embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in a reduced interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 10% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 20% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 30% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 40% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 50% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine.In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 60% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 70% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about an 80% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 90% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 95% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the single histidine. In certain embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) eliminates the interaction of the protein with ADA.A variety of assays known to those of skill in the art or described herein, such as the assays described in Section 7.9.1, may be used to measure and / or detect the interaction between the protein and ADA. In certain embodiments, the interaction between the protein and ADA is determined by ELISA, a surface plasmon resonance assay (e.g., BIACORE), or other immunoassay described herein or known to those of skill in the art.

[0185] In certain embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in a reduced interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 10% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 20% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 30% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 40% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 50% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines.In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 60% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 70% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about an 80% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 90% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 95% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the two histidines. In certain embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) eliminates the interaction of the protein with ADA.A variety of assays known to those of skill in the art or described herein, such as the assays described in Section 7.9.1, may be used to measure and / or detect the interaction between the protein and ADA. In certain embodiments, the interaction between the protein and ADA is determined by ELISA, a surface plasmon resonance assay (e.g., BIACORE), or other immunoassay described herein or known to those of skill in the art.

[0186] In certain embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in a reduced interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 10% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 20% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 30% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 40% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 50% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines.In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 60% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 70% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about an 80% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 90% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 95% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the three histidines. In certain embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) eliminates the interaction of the protein with ADA.A variety of assays known to those of skill in the art or described herein, such as the assays described in Section 7.9.1, may be used to measure and / or detect the interaction between the protein and ADA. In certain embodiments, the interaction between the protein and ADA is determined by ELISA, a surface plasmon resonance assay (e.g., BIACORE), or other immunoassay described herein or known to those of skill in the art.

[0187] In certain embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in a reduced interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 10% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 20% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 30% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 40% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 50% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines.In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 60% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 70% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about an 80% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 90% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 95% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the four histidines. In certain embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) eliminates the interaction of the protein with ADA.A variety of assays known to those of skill in the art or described herein, such as the assays described in Section 7.9.1, may be used to measure and / or detect the interaction between the protein and ADA. In certain embodiments, the interaction between the protein and ADA is determined by ELISA, a surface plasmon resonance assay (e.g., BIACORE), or other immunoassay described herein or known to those of skill in the art.

[0188] In certain embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in a reduced interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 10% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 20% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 30% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 40% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 50% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines.In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 60% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 70% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about an 80% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 90% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) results in at least about a 95% reduction in the interaction of the protein with ADA compared to the interaction of the same protein with ADA lacking the five histidines. In certain embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antigen binding protein) eliminates the interaction of the protein with ADA.A variety of assays known to those of skill in the art or described herein, such as the assays described in Section 7.9.1, may be used to measure and / or detect the interaction between the protein and ADA. In certain embodiments, the interaction between the protein and ADA is determined by ELISA, a surface plasmon resonance assay (e.g., BIACORE), or other immunoassay described herein or known to those of skill in the art.

[0189] In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in reduced aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 10% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively.In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 20% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 30% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively.In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 40% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 50% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively.In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 60% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 70% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively.In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about an 80% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 90% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37), respectively).In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in at least about a 95% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37), respectively). In certain embodiments, aggregation is determined by an aggregation assay described herein (e.g., an assay described in Section 7.9.7) or known to one of skill in the art.

[0190] In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 30% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine.In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 40% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 50% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 60% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 70% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine.In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about an 80% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 90% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a trispecific antibody)) results in about a 95% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein, respectively, lacking the single histidine. In certain embodiments, aggregation is determined by an agglutination assay described herein (e.g., the assay described in Section 7.9.7) or an agglutination assay known to one of skill in the art.In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein, respectively, lacking the two histidines. In certain embodiments, aggregation is determined by an agglutination assay described herein (e.g., an assay described in Section 7.9.7) or known to one of skill in the art.

[0191] In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 30% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 40% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines.In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 50% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 60% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 70% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about an 80% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines.In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 90% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 95% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the two histidines. In certain embodiments, aggregation is determined by an agglutination assay described herein (eg, an assay described in Section 7.9.7) or known to one of skill in the art.

[0192] In some embodiments, the addition of three histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the three histidines. In some embodiments, the addition of three histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein with the same substance lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 30% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein with the same substance lacking the three histidines.In some embodiments, the addition of three histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 40% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the three histidines. In some embodiments, the addition of three histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 50% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the three histidines. In some embodiments, the addition of three histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 60% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the three histidines. In some embodiments, the addition of three histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 70% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the three histidines.In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about an 80% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 90% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 95% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein, respectively, lacking the three histidines. In certain embodiments, aggregation is determined by an agglutination assay described herein (e.g., the assay described in Section 7.9.7) or an agglutination assay known to one of skill in the art.

[0193] In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced aggregation upon incubation with a biological material, compared to the aggregation exhibited upon incubation of the same polypeptide or protein lacking the four histidines, respectively. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in aggregation upon incubation with a biological material, compared to the aggregation exhibited upon incubation of the same polypeptide or protein lacking the four histidines, respectively. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein with the same substance lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 30% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein with the same substance lacking the four histidines.In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 40% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 50% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 60% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein with the same protein lacking the four histidines, respectively. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 70% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein with the same protein lacking the four histidines, respectively.In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about an 80% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein with the same substance lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 90% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein with the same substance lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 95% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein, respectively, lacking the four histidines. In certain embodiments, aggregation is determined by an agglutination assay described herein (e.g., the assay described in Section 7.9.7) or an agglutination assay known to one of skill in the art.

[0194] In some embodiments, the addition of five histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 30% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the five histidines.In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 40% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 50% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 60% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 70% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines.In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about an 80% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 90% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a trispecific antibody)) results in about a 95% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein, respectively, lacking the five histidines. In certain embodiments, autoaggregation is determined by an agglutination assay described herein (e.g., the assay described in Section 7.9.7) or an agglutination assay known to one of skill in the art.

[0195] In certain embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide, protein (e.g., antibody), bispecific antibody, or multispecific antibody (e.g., triabody) results in reduced self-aggregation of the polypeptide, protein (e.g., antibody), bispecific antibody, or multispecific antibody compared to self-aggregation of the same polypeptide, protein (e.g., antibody), bispecific antibody, or multispecific antibody (e.g., triabody), respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 10% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 20% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines.In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 30% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 40% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 50% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines.In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 60% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 70% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about an 80% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines.In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 90% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines. In some embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) results in at least about a 95% reduction in self-aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) compared to self-aggregation of the same protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody), respectively, lacking the four histidines. In certain embodiments, the addition of four histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a protein (e.g., an antibody), bispecific antibody, or multispecific antibody (e.g., a triabody) eliminates aggregation of the polypeptide, protein (e.g., an antibody), bispecific antibody, or multispecific antibody. In certain embodiments, autoagglutination is determined by an agglutination assay described herein (eg, an assay described in Section 7.9.7) or known to one of skill in the art.

[0196] In some embodiments, the addition of five histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 30% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or the same protein, respectively, lacking the five histidines.In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 40% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 50% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 60% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 70% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines.In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about an 80% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 90% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the same polypeptide or protein, respectively, lacking the five histidines. In some embodiments, the addition of five histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a trispecific antibody)) results in about a 95% reduction in aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of the biological material with the same polypeptide or protein, respectively, lacking the five histidines. In certain embodiments, aggregation is determined by an agglutination assay described herein (e.g., the assay described in Section 7.9.7) or an agglutination assay known to one of skill in the art.

[0197] In some embodiments, the addition of a cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in reduced self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the cap domain (e.g., a His cap domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about a 10% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about a 20% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively.In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about a 30% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about a 40% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about a 50% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively.In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about a 60% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about a 70% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about an 80% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively.In some embodiments, the addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminal end, a non-naturally occurring C-terminal end, or a C-terminal anti-drug antibody epitope end of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)), results in at least about a 90% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In some embodiments, addition of a capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)) to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody or a multispecific antibody (e.g., a triabody)) results in at least about a 95% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the capping domain (e.g., a His capping domain, SLSLSPGK (SEQ ID NO: 36), or TVAPTESS (SEQ ID NO: 37)), respectively. In certain embodiments, autoaggregation is determined by an agglutination assay described herein (e.g., an assay described in Section 7.9.7) or known to one of skill in the art.

[0198] In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 30% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine.In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 40% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 50% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 60% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 70% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine.In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about an 80% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 90% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine. In some embodiments, the addition of a single histidine to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)) results in about a 95% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the single histidine, respectively. In certain embodiments, autoaggregation is determined by an agglutination assay described herein (e.g., the assay described in Section 7.9.7) or known to one of skill in the art.

[0199] In some embodiments, the addition of two histidines to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 30% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines.In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 40% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 50% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 60% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 70% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines.In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about an 80% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 90% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines. In some embodiments, the addition of two histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific antibody, or a multispecific antibody (e.g., a triabody)) results in about a 95% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the two histidines, respectively. In certain embodiments, autoaggregation is determined by an agglutination assay described herein (e.g., the assay described in Section 7.9.7) or known to one of skill in the art.

[0200] In some embodiments, the addition of three histidines to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in reduced self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the three histidines. In some embodiments, the addition of three histidines to an exposed C-terminus, a non-naturally occurring C-terminus, or a C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 10% reduction in self-aggregation compared to the self-aggregation exhibited by the same polypeptide or protein lacking the three histidines. In some embodiments, the addition of three histidines to the exposed C-terminus, non-naturally occurring C-terminus, or C-terminal anti-drug antibody epitope of a polypeptide or protein (e.g., an antibody, e.g., a bispecific or multispecific antibody (e.g., a triabody)) results in about a 20% reduction in self-aggregation compared to the self-aggregation...

Claims

1. 1. An isolated polypeptide comprising one or more target binding regions and an exposed C-terminus fused to a cap domain, wherein the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37); An isolated polypeptide, wherein the isolated polypeptide exhibits reduced interaction with the reference compared to interaction of the isolated polypeptide lacking the cap domain with the reference.

2. The isolated polypeptide of claim 1 , wherein the reference is a molecule present in a biological material.

3. The isolated polypeptide of claim 2 , wherein the biological material comprises serum and / or plasma.

4. 4. The isolated polypeptide of any one of claims 1 to 3, wherein the reference comprises an anti-drug antibody, and optionally the anti-drug antibody is pre-existing in the serum and / or plasma of the subject.

5. The isolated polypeptide of any one of claims 1 to 4, wherein the interaction with the reference is determined by immunoassay.

6. The isolated polypeptide of any one of claims 1 to 4, wherein the interaction with the reference is assessed in vitro or in vivo.

7. 1. An isolated polypeptide comprising one or more target binding regions and an exposed C-terminus fused to a cap domain, wherein the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37); The isolated polypeptide, wherein the isolated polypeptide exhibits reduced self-aggregation compared to self-aggregation exhibited by an isolated polypeptide lacking the cap domain under the same conditions.

8. 8. The isolated polypeptide of claim 7, wherein autoaggregation is reduced in a high concentration liquid formulation (HCLF).

9. 9. The isolated polypeptide of claim 8, wherein the HCLF concentration comprises about 50 mg / mL to about 150 mg / mL of the isolated polypeptide.

10. 10. The isolated polypeptide of any one of claims 7 to 9, wherein self-aggregation is reduced under heat stress.

11. 11. The isolated polypeptide of claim 10, wherein the heat stress comprises incubation at 40°C for about two weeks.

12. The isolated polypeptide of any one of claims 7 to 11, wherein the self-aggregation is assessed by size exclusion chromatography.

13. 1. An isolated polypeptide comprising one or more target binding regions and an exposed C-terminus fused to a cap domain, wherein the cap domain is a His cap domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37); The isolated polypeptide exhibits reduced aggregation upon incubation with a biological material compared to the aggregation exhibited upon incubation of an isolated polypeptide lacking the cap domain with the biological material.

14. 14. The isolated polypeptide of claim 13, wherein the biological material is human serum and / or human plasma.

15. 15. The isolated polypeptide of claim 14, wherein the incubation with the biological material comprises: (a) incubation at about 37°C for about 2 to about 5 days, about 2 to about 7 days, or about 5 to about 14 days; (b) incubation at about 40°C for about 2 to about 5 days, about 2 to about 7 days, or about 7 to about 14 days; (c) incubation at about 4°C for about 2 to about 5 days, about 2 to about 7 days, or about 7 to about 14 days; or (d) incubation at about 25°C for about 7 to about 14 days.

16. 16. The isolated polypeptide of any one of claims 13 to 15, wherein the biological material comprises anti-drug antibodies, and optionally the anti-drug antibodies are pre-present in the serum and / or plasma of the subject.

17. 17. The isolated polypeptide of any one of claims 1 to 16, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

18. 18. The isolated polypeptide of any one of claims 1 to 17, wherein the one or more target binding regions comprise an antibody variable domain or an antigen-binding fragment thereof.

19. 18. The isolated polypeptide of any one of claims 1 to 17, wherein the one or more target binding regions comprise a light chain antibody variable domain or an antigen-binding fragment thereof and / or a heavy chain antibody variable domain or an antigen-binding fragment thereof.

20. 20. The isolated polypeptide of any one of claims 1-19, wherein the His cap domain consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO:27), or five histidine residues (SEQ ID NO:28).

21. the exposed C-terminus fused to the His cap domain, (a) an amino acid sequence selected from the group consisting of VTVSSH (SEQ ID NO:1), VTVSSHH (SEQ ID NO:2), VTVSSHHH (SEQ ID NO:3), VTVSSHHHH (SEQ ID NO:4), and VTVSSHHHHH (SEQ ID NO:5); (b) an amino acid sequence selected from the group consisting of VEIKH (SEQ ID NO:6), VEIKHH (SEQ ID NO:7), VEIKHHH (SEQ ID NO:8), VEIKHHHH (SEQ ID NO:9), and VEIKHHHHH (SEQ ID NO:10); (c) an amino acid sequence selected from the group consisting of VEIKRH (SEQ ID NO:11), VEIKRHH (SEQ ID NO:12), VEIKRHHH (SEQ ID NO:13), VEIKRHHHH (SEQ ID NO:14), and VEIKRHHHHH (SEQ ID NO:15); (d) an amino acid sequence selected from the group consisting of VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20); or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; 19. The isolated polypeptide according to any one of claims 1 to 18, comprising:

22. 1. An isolated protein comprising: (a) a first polypeptide comprising a target binding region and an exposed C-terminus fused to a cap domain, said cap domain being a His cap domain or consisting of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37); (b) a second polypeptide comprising a target binding region; and Including, The isolated protein exhibits a reduced interaction with the reference compared to an interaction of the isolated protein lacking the cap domain with the reference.

23. 23. The isolated protein of claim 22, wherein the target binding regions of the first polypeptide and the second polypeptide are capable of binding to different targets.

24. 24. The isolated protein of claim 22 or 23, wherein the exposed C-terminus of the first polypeptide comprises an anti-drug antibody epitope.

25. 25. The isolated protein of any one of claims 22 to 24, wherein the target binding region of the second polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof.

26. 25. The isolated protein of any one of claims 22 to 24, wherein the target binding region of the second polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof and / or a heavy chain antibody variable domain or an antigen-binding fragment thereof.

27. 27. The isolated protein of any one of claims 22-26, wherein the second polypeptide comprises an exposed C-terminus fused to a capping domain, the capping domain being a His capping domain or consisting of the amino acid sequence of SLSLSPGK (SEQ ID NO:36), TVAPTESS (SEQ ID NO:37), HHHHHH (SEQ ID NO:26), or AS.

28. 28. The isolated protein of claim 27, wherein the exposed C-terminus of the second polypeptide comprises an anti-drug antibody epitope.

29. 29. The isolated protein of claim 27 or 28, wherein the His cap domain of the second polypeptide consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO: 27), or five histidine residues (SEQ ID NO: 28).

30. the exposed C-terminus fused to the His cap domain of the second polypeptide is (a) VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5); (b) VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10); (c) VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15); (d) VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20); or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; 29. The isolated protein of claim 27 or 28, consisting of an amino acid sequence selected from the group consisting of:

31. 31. The isolated protein of any one of claims 22 to 30, wherein the target binding region of the first polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof.

32. 31. The isolated protein of any one of claims 22 to 30, wherein the target binding region of the first polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof and / or a heavy chain antibody variable domain or an antigen-binding fragment thereof.

33. 33. The isolated protein of any one of claims 22-32, wherein the His cap domain of the first polypeptide consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO:27), or five histidine residues (SEQ ID NO:28).

34. the exposed C-terminus fused to the His cap domain of the first polypeptide is (a) VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5); (b) VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10); (c) VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15); (d) VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20); or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; 32. The isolated protein according to any one of claims 22 to 31, consisting of an amino acid sequence selected from the group consisting of:

35. 35. The isolated protein of any one of claims 22 to 34, wherein the isolated protein further comprises a third polypeptide comprising a target binding region.

36. 36. The isolated protein of claim 35, wherein the third polypeptide is capable of binding to a target that is different from the first polypeptide, the second polypeptide, or both.

37. 37. The isolated protein of claim 35 or 36, wherein the target binding region of the third polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof.

38. 37. The isolated protein of claim 35 or 36, wherein the target binding region of the third polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof and / or a heavy chain antibody variable domain or an antigen-binding fragment thereof.

39. 39. The isolated protein of any one of claims 35-38, wherein the third polypeptide comprises an exposed C-terminus fused to a capping domain, the capping domain being a His capping domain or consisting of the amino acid sequence of SLSLSPGK (SEQ ID NO:36), TVAPTESS (SEQ ID NO:37), HHHHHH (SEQ ID NO:26), or AS.

40. 40. The isolated protein of claim 39, wherein the exposed C-terminus of the third polypeptide comprises an anti-drug antibody epitope.

41. 41. The isolated protein of claim 39 or 40, wherein the His cap domain of the third polypeptide consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO: 27), or five histidine residues (SEQ ID NO: 28).

42. the exposed C-terminus of the third polypeptide fused to the His cap domain, (a) VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5); (b) VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10); (c) VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15); (d) VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20); or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; 41. The isolated protein of claim 39 or 40, consisting of an amino acid sequence selected from the group consisting of:

43. 43. The isolated protein of any one of claims 22 to 42, wherein the isolated protein is bispecific, trispecific, or multispecific.

44. The isolated protein of any one of claims 22 to 43, wherein the isolated protein is a heterodimer.

45. An isolated nucleic acid sequence comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 21 or a protein according to any one of claims 22 to 44.

46. 46. ​​An expression vector comprising the nucleic acid sequence of claim 45.

47. An isolated cell expressing a polypeptide according to any one of claims 1 to 21 or a protein according to any one of claims 22 to 44.

48. 47. A host cell comprising a nucleic acid sequence according to claim 45 or a vector according to claim 46.

49. A method for producing a polypeptide according to any one of claims 1 to 21 or a protein according to any one of claims 22 to 44, comprising culturing a cell according to claim 47 or 48.

50. 50. The method of claim 49, further comprising isolating the polypeptide or protein.

1. A bispecific or multispecific antibody comprising one or more target binding regions and an exposed C-terminus fused to a capping domain, said capping domain being a His capping domain or consisting of the amino acid sequence SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37), wherein said antibody exhibits reduced interaction with an anti-drug antibody compared to the interaction of said anti-drug antibody with an antibody lacking said capping domain.

51. The antibody of claim 50, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

52. The antibody of claim 50 or 51, wherein the antibody exhibits reduced interaction with an anti-drug antibody in an immunoassay compared to the interaction of an antibody lacking the His cap domain with the anti-drug antibody.

53. 53. The antibody of any one of claims 50 to 52, wherein the one or more target binding regions comprise at least one antibody variable domain.

54. 54. The antibody of claim 53, wherein the at least one antibody variable domain comprises a heavy chain variable domain or a light chain variable domain.

55. 55. The antibody of any one of claims 50 to 54, wherein the His cap domain consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO:27), or five histidine residues (SEQ ID NO:28).

56. the exposed C-terminus fused to a His cap domain, (a) VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), or VTVSSHHHHH (SEQ ID NO: 5); (b) VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), or VEIKHHHHH (SEQ ID NO: 10); (c) VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), or VEIKRHHHHH (SEQ ID NO: 15); (d) VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), or VEIKRTHHHHH (SEQ ID NO: 20), or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; The antibody according to any one of claims 50 to 54, comprising an amino acid sequence selected from the group consisting of:

57. The antibody of any one of claims 50 to 56, wherein the multispecific antibody is a trispecific antibody.

58. 60. A nucleic acid sequence comprising a nucleotide sequence encoding the antibody of any one of claims 50 or 57.

59. 59. An expression vector comprising the nucleic acid sequence of claim 58.

60. 60. A host cell comprising a nucleic acid sequence according to claim 58 or a vector according to claim 59.

61. An isolated cell expressing an antibody according to any one of claims 50 to 57.

62. A method for producing an antibody according to any one of claims 50 to 57, comprising culturing a cell according to claim 60 or 61.

63. 63. The method of claim 62, further comprising isolating the antibody.

64. 1. A method for reducing an interaction between an isolated polypeptide and a reference, wherein the isolated polypeptide comprises one or more target binding regions and an exposed C-terminus, the method comprising fusing a capping domain to an end of the exposed C-terminus, the capping domain being a His capping domain or consisting of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

65. 65. The method of claim 64, wherein the reference is a molecule present in a biological material.

66. 66. The method of claim 65, wherein the biological material comprises serum and / or plasma.

67. 67. The method of any one of claims 64 to 66, wherein the reference comprises an anti-drug antibody, optionally wherein the anti-drug antibody is pre-present in the serum and / or plasma of the subject.

68. The method of any one of claims 64 to 67, wherein the interaction with the reference is determined by immunoassay.

69. 68. The method of any one of claims 64 to 67, wherein the interaction with the reference is assessed in vitro or in vivo.

70. 1. A method for reducing aggregation of an isolated polypeptide in a biological material, the isolated polypeptide comprising one or more target binding regions and an exposed C-terminus, the method comprising fusing a capping domain to an end of the exposed C-terminus, the capping domain being a His capping domain or consisting of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

71. 71. The method of claim 70, wherein the biological material is human serum and / or plasma.

72. 72. The method of claim 71, wherein the incubation with the biological material comprises: (a) incubation at about 37°C for about 2 to about 5, about 2 to about 7, or about 5 to 14 days; (b) incubation at about 40°C for about 2 to about 5, about 2 to about 7, or about 7 to about 14 days; (c) incubation at about 4°C for about 2 to about 5, about 2 to about 7, or about 7 to about 14 days; or (d) incubation at about 25°C for about 2 to about 5, about 2 to about 7, or about 7 to about 14 days.

73. 73. The method of any one of claims 70 to 72, wherein the biological material comprises anti-drug antibodies, and optionally the anti-drug antibodies are pre-present in the serum and / or plasma of the subject.

74. The method of any one of claims 70 to 73, wherein the interaction with the biological material is assessed by size exclusion chromatography.

75. 1. A method for reducing self-aggregation of an isolated polypeptide, the isolated polypeptide comprising one or more target binding regions and an exposed C-terminus, the method comprising fusing a capping domain to an end of the exposed C-terminus, the capping domain being a His capping domain or consisting of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

76. 76. The method of claim 75, wherein autoaggregation is reduced in a high concentration liquid formulation (HCLF).

77. 77. The method of claim 76, wherein the HCLF concentration comprises about 50 mg / mL to about 150 mg / mL of the isolated polypeptide.

78. 78. The method of any one of claims 75 to 77, wherein self-aggregation is reduced under thermal stress.

79. 79. The method of claim 78, wherein the heat stress comprises incubation at 40°C for about 2 weeks.

80. The method of any one of claims 75 to 79, wherein the self-aggregation is assessed by size exclusion chromatography.

81. 81. The method of any one of claims 64 to 80, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

82. 82. The method of any one of claims 64 to 81, wherein the one or more target binding regions comprise an antibody variable domain or an antigen-binding fragment thereof.

83. 82. The method of any one of claims 64 to 81, wherein the one or more target binding regions comprise a light chain antibody variable domain or an antigen-binding fragment thereof and / or a heavy chain antibody variable domain or an antigen-binding fragment thereof.

84. 83. The method of any one of claims 64-82, wherein the His cap domain consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO:27), or five histidine residues (SEQ ID NO:28).

85. the exposed C-terminus fused to the His cap domain, (a) VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5); (b) VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10); (c) VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15); (d) VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20); or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; The method of any one of claims 64 to 82, comprising an amino acid sequence selected from the group consisting of:

86. 1. A method for reducing interactions between an isolated protein and a reference, the isolated protein comprising: (a) a first polypeptide comprising a target binding region and an exposed C-terminus; (b) a second polypeptide comprising a target binding region; and Including, The method includes fusing a capping domain to the exposed C-terminus of the first polypeptide, wherein the capping domain is a His capping domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37).

87. 87. The method of claim 86, wherein the target binding regions of the first polypeptide and the second polypeptide are capable of binding to different targets.

88. 88. The method of claim 86 or 87, wherein the exposed C-terminus of the first polypeptide comprises an anti-drug antibody epitope.

89. 89. The method of any one of claims 86 to 88, wherein the target binding region of the second polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof.

90. 89. The method of any one of claims 86 to 88, wherein the target binding region of the second polypeptide comprises a light chain antibody variable domain, or an antigen-binding fragment thereof, and / or a heavy chain antibody variable domain, or an antigen-binding fragment thereof.

91. 91. The method of any one of claims 86-90, wherein the second polypeptide comprises an exposed C-terminus, and the method further comprises fusing a capping domain to the end of the exposed C-terminus of the second polypeptide, wherein the capping domain is a His capping domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36), TVAPTESS (SEQ ID NO: 37), HHHHHH (SEQ ID NO: 26), or AS.

92. 92. The method of claim 91, wherein the exposed C-terminus of the second polypeptide comprises an anti-drug antibody epitope.

93. 93. The method of claim 91 or 92, wherein the His cap domain of the second polypeptide consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO:27), or five histidine residues (SEQ ID NO:28).

94. the exposed C-terminus fused to the His cap domain of the second polypeptide is (a) VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5); (b) VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10); (c) VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15); (d) VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20); or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; 93. The method of claim 91 or 92, comprising an amino acid sequence selected from the group consisting of:

95. 95. The method of any one of claims 86-94, wherein the target binding region of the first polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof.

96. 95. The method of any one of claims 86 to 94, wherein the target binding region of the first polypeptide comprises a light chain antibody variable domain, or an antigen-binding fragment thereof, and / or a heavy chain antibody variable domain, or an antigen-binding fragment thereof.

97. 97. The method of any one of claims 86-96, wherein the His cap domain of the first polypeptide consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO:27), or five histidine residues (SEQ ID NO:28).

98. the exposed C-terminus fused to the His cap domain of the first polypeptide is (a) VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5); (b) VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10); (c) VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15); (d) VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20); or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; The method of any one of claims 86 to 96, comprising an amino acid sequence selected from the group consisting of:

99. 99. The method of any one of claims 86-98, wherein the isolated protein further comprises a third polypeptide comprising a target binding region.

100. 100. The method of claim 99, wherein the third polypeptide is capable of binding to a different target than the first polypeptide, the second polypeptide, or both.

101. 101. The method of claim 99 or 100, wherein the target binding region of the third polypeptide comprises an antibody variable domain or an antigen-binding fragment thereof.

102. 101. The method of claim 99 or 100, wherein the target binding region of the third polypeptide comprises a light chain antibody variable domain or an antigen-binding fragment thereof and / or a heavy chain antibody variable domain or an antigen-binding fragment thereof.

103. 103. The method of any one of claims 99-102, wherein the third polypeptide comprises an exposed C-terminus, and the method further comprises fusing a capping domain to the end of the exposed C-terminus of the third polypeptide, wherein the capping domain is a His capping domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36), TVAPTESS (SEQ ID NO: 37), HHHHHH (SEQ ID NO: 26), or AS.

104. 104. The method of claim 103, wherein the exposed C-terminus of the third polypeptide comprises an anti-drug antibody epitope.

105. 105. The method of claim 103 or 104, wherein the His cap domain of the third polypeptide consists of a single histidine residue, two histidine residues, three histidine residues, four histidine residues (SEQ ID NO:27), or five histidine residues (SEQ ID NO:28).

106. the exposed C-terminus of the third polypeptide fused to the His cap domain, (a) VTVSSH (SEQ ID NO: 1), VTVSSHH (SEQ ID NO: 2), VTVSSHHH (SEQ ID NO: 3), VTVSSHHHH (SEQ ID NO: 4), and VTVSSHHHHH (SEQ ID NO: 5); (b) VEIKH (SEQ ID NO: 6), VEIKHH (SEQ ID NO: 7), VEIKHHH (SEQ ID NO: 8), VEIKHHHH (SEQ ID NO: 9), and VEIKHHHHH (SEQ ID NO: 10); (c) VEIKRH (SEQ ID NO: 11), VEIKRHH (SEQ ID NO: 12), VEIKRHHH (SEQ ID NO: 13), VEIKRHHHH (SEQ ID NO: 14), and VEIKRHHHHH (SEQ ID NO: 15); (d) VEIKRTH (SEQ ID NO: 16), VEIKRTHH (SEQ ID NO: 17), VEIKRTHHH (SEQ ID NO: 18), VEIKRTHHHH (SEQ ID NO: 19), and VEIKRTHHHHH (SEQ ID NO: 20); or (e) the amino acid sequence of TKVTVL(His)n (SEQ ID NO: 46), TKLTVL(His)n (SEQ ID NO: 47), TQLIIL(His)n (SEQ ID NO: 48), TELTVL(His)n (SEQ ID NO: 49), or TQLTVL(His)n (SEQ ID NO: 50), where n is 1, 2, 3, 4, or 5; 105. The method of claim 103 or 104, comprising an amino acid sequence selected from the group consisting of:

107. 107. The method of any one of claims 86 to 106, wherein the isolated protein is bispecific, trispecific, or multispecific.

108. The method of any one of claims 86 to 107, wherein the isolated protein is a heterodimer.

109. 1. A capping means for reducing interaction between an isolated polypeptide and a reference, said isolated polypeptide comprising one or more target binding regions and an exposed C-terminus.

110. 110. The capping means of claim 109, wherein the one or more target binding regions comprise an antibody variable domain or an antigen-binding fragment thereof.

111. 111. A capping means according to claim 109 or 110, wherein the reference is a molecule present in a biological material.

112. 112. The capping means of claim 111, wherein said biological material comprises serum.

113. A capping means according to any one of claims 109 to 112, wherein said reference comprises an anti-drug antibody.

114. A capping means according to any one of claims 109 to 113, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

115. The capping means of any one of claims 109 to 114, wherein the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), and TQLTVL (SEQ ID NO:42).

116. 1. A capping means for reducing interactions between an isolated protein and a reference, said isolated protein comprising: (a) a first polypeptide comprising a target binding region and an exposed C-terminus; (b) a second polypeptide comprising a target binding region; and A capping means comprising:

117. 117. The capping means of claim 116, wherein the second polypeptide comprises an exposed C-terminus.

118. 118. A capping means according to claim 116 or 117, wherein the target binding regions of said first polypeptide and said second polypeptide are capable of binding to different targets.

119. A capping means according to any one of claims 116 to 118, wherein the isolated protein further comprises a third polypeptide comprising a target binding region.

120. 120. The capping means of claim 119, wherein the third polypeptide comprises an exposed C-terminus.

121. 121. A capping means according to claim 119 or 120, wherein the third polypeptide is capable of binding to a different target than the first polypeptide, the second polypeptide, or both.

122. A capping means according to any one of claims 116 to 121, wherein the isolated protein is a heterodimer.

123. A capping means according to any one of claims 116 to 122, wherein the isolated protein is bispecific, trispecific or multispecific.

124. A capping means according to any one of claims 116 to 123, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

125. 124. The capping means of any one of claims 116 to 123, wherein the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), and TQLTVL (SEQ ID NO:42).

126. 1. A capping means for reducing interaction between a bispecific or multispecific antibody and a reference, said bispecific or multispecific antibody comprising one or more target binding regions and an exposed C-terminus.

127. 127. A capping means according to claim 126, wherein said one or more target binding regions comprise at least one antibody variable domain.

128. 128. The capping means of claim 127, wherein said at least one antibody variable domain comprises a heavy chain variable domain or a light chain variable domain.

129. A capping means according to any one of claims 126 to 128, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

130. A capping means according to any one of claims 126 to 129, wherein the multispecific antibody is a trispecific antibody.

131. 131. The capping means of any one of claims 109 to 130, wherein the capping means is a histidine capping means.

132. An isolated polypeptide and a capping means for reducing interaction between the isolated polypeptide and a reference, the isolated polypeptide comprising one or more target binding regions and an exposed C-terminus.

133. 133. The isolated polypeptide of claim 132, wherein the one or more target binding regions comprise an antibody variable domain or an antigen-binding fragment thereof.

134. 134. The isolated polypeptide of claim 132 or 133, wherein the reference is a molecule present in a biological material.

135. 135. The isolated polypeptide of claim 134, wherein the biological material comprises serum and / or plasma.

136. 136. The isolated polypeptide of any one of claims 132 to 135, wherein the reference comprises an anti-drug antibody, and optionally the anti-drug antibody is pre-existing in the serum and / or plasma of the subject.

137. 137. The isolated polypeptide of any one of claims 132-136, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

138. 137. The isolated protein of any one of claims 132-136, wherein the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), and TQLTVL (SEQ ID NO:42).

139. 1. An isolated protein and a capping means for reducing interactions between said isolated protein and a reference, said isolated protein comprising: (a) a first polypeptide comprising a target binding region and an exposed C-terminus; (b) a second polypeptide comprising a target binding region; and 1. An isolated protein and a capping means.

140. 140. The isolated protein of claim 139, wherein the second polypeptide comprises an exposed C-terminus.

141. 141. The isolated protein of claim 139 or 140, wherein the target binding regions of the first polypeptide and the second polypeptide are capable of binding to different targets.

142. 142. The isolated protein of any one of claims 139-141, wherein the isolated protein further comprises a third polypeptide comprising a target binding region.

143. 143. The isolated protein of claim 142, wherein the third polypeptide comprises an exposed C-terminus.

144. 144. The isolated protein of claim 142 or 143, wherein the third polypeptide is capable of binding to a target that is different from the first polypeptide, the second polypeptide, or both.

145. 145. The isolated protein of any one of claims 139 to 144, wherein the isolated protein is a heterodimer.

146. 146. The isolated protein of any one of claims 139-145, wherein the isolated protein is bispecific, trispecific, or multispecific.

147. 147. The isolated protein of any one of claims 139-146, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

148. 148. The isolated polypeptide or protein of any one of claims 132-147, wherein said capping means is a histidine capping means.

149. 1. A bispecific or multispecific antibody and a capping means for reducing interaction between said bispecific or multispecific antibody and a reference, said bispecific or multispecific antibody comprising one or more target binding regions and an exposed C-terminus.

150. The antibody of claim 149, wherein the one or more target binding regions comprise at least one antibody variable domain.

151. The antibody of claim 150, wherein the at least one antibody variable domain comprises a heavy chain variable domain or a light chain variable domain.

152. The antibody of any one of claims 149 to 151, wherein the bispecific or multispecific antibody is a heterodimer.

153. The antibody of any one of claims 149 to 152, wherein the multispecific antibody is a trispecific antibody.

154. The antibody of any one of claims 149 to 153, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

155. The antibody of any one of claims 149 to 154, wherein the capping means is a histidine capping means.

156. 1. An isolated protein for use in a diagnostic assay comprising a target binding region and an exposed C-terminus fused to a cap domain, wherein the cap domain consists of a His cap domain, or the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

157. The protein for use according to claim 156, wherein the protein is an antibody.

158. The antibody is directed against glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), B cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut 158. The protein for use according to claim 157, which binds to hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin.

159. 158. The protein for use according to claim 157, wherein the antibody binds to an antigen of a pathogen.

160. 160. The protein for use according to claim 159, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.

161. 161. The protein for use according to any one of claims 156 to 160, wherein said protein is used in a diagnostic assay for detecting the presence of a molecule in a biological material.

162. The protein for use according to claim 161, wherein the molecule is an antigen.

163. 163. The protein for use according to claim 161 or 162, wherein the biological material is serum and / or plasma.

164. The protein for use according to any one of claims 156 to 163, wherein the diagnostic assay is performed in vitro or in vivo.

165. 165. The protein for use according to any one of claims 156 to 164, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

166. 165. The protein for use according to any one of claims 156 to 164, wherein the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), and TQLTVL (SEQ ID NO:42).

167. 1. An isolated therapeutic protein for use in therapy comprising a target binding region and an exposed C-terminus fused to a capping domain, wherein said capping domain consists of a His capping domain, or the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

168. The protein for use according to claim 167, wherein the protein is a cytokine or an antibody.

169. 169. The protein for use according to claim 168, wherein the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17.

170. The antibody is directed against glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), B cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut 169. The protein for use according to claim 168, which binds to hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin.

171. 169. The protein for use according to claim 168, wherein the antibody binds to an antigen of a pathogen.

172. 172. The protein for use according to claim 171, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.

173. 173. The protein for use according to any one of claims 167 to 172, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

174. 173. The protein for use according to any one of claims 167 to 172, wherein the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), and TQLTVL (SEQ ID NO:42).

175. 1. An isolated protein comprising a target binding region and an exposed C-terminus fused to a cap domain, wherein the exposed C-terminus consists of the amino acid sequence VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), or TQLTVL (SEQ ID NO:42), and wherein the cap domain consists of a His cap domain, or the amino acid sequence of SLSLSPGK (SEQ ID NO:36) or TVAPTESS (SEQ ID NO:37).

176. The protein of claim 175, wherein the protein is an antibody.

177. The antibody is directed against glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), B cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut 177. The protein of claim 176, which binds to hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin.

178. The protein of claim 176, wherein the antibody binds to an antigen of a pathogen.

179. 179. The protein for use according to claim 178, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.

180. 1. A therapeutic bispecific or multispecific antibody for use in therapy comprising two or more target binding regions and an exposed C-terminus fused to a capping domain, said capping domain being a His capping domain or the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

181. 181. A bispecific or multispecific antibody for use according to claim 180, wherein said two or more target binding regions bind to two or more antigens.

182. At least one antigen is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut 182. A bispecific or multispecific antibody for use according to claim 181, selected from the group consisting of hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

183. 181. The bispecific or multispecific antibody of claim 180, wherein the antibody binds to an antigen of a pathogen.

184. 184. The protein for use according to claim 183, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.

185. 185. A bispecific or multispecific antibody for use according to any one of claims 180 to 184, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

186. 186. A bispecific or multispecific antibody for use according to any one of claims 180 to 185, wherein the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), or TQLTVL (SEQ ID NO:42).

187. 180. A pharmaceutical composition for use in therapy comprising a protein according to any one of claims 175 to 179 and a pharma- ceutically acceptable excipient.

188. 180. A pharmaceutical composition for use in a diagnostic assay comprising a protein according to any one of claims 175 to 179 and a pharma- ceutically acceptable excipient.

189. 1. Use of a cap domain to reduce interaction between an anti-drug antibody and a protein, said protein comprising one or more target binding regions and an exposed C-terminus, said cap domain being a His cap domain or consisting of the amino acid sequence SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

190. 1. Use of a cap domain to reduce aggregation in a biological material, wherein the protein comprises one or more target binding regions and an exposed C-terminus, and the cap domain is a His cap domain or consists of the amino acid sequence SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

191. 1. Use of a capping domain to reduce self-aggregation, wherein the protein comprises one or more target binding regions and an exposed C-terminus, and the capping domain consists of a His capping domain or consists of the amino acid sequence of SLSLSPGK (SEQ ID NO: 36) or TVAPTESS (SEQ ID NO: 37).

192. 191. The use of claim 190, wherein the biological material comprises serum and / or plasma.

193. 192. The use according to any one of claims 189 to 191, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

194. 192. The use of any one of claims 189 to 191, wherein the cap domain is a His cap domain consisting of 1, 2, 3, 4, or 5 histidines.

195. 1. An isolated protein comprising a target binding region and an exposed C-terminus fused to a cap domain, wherein the cap domain consists of a His cap domain or the amino acid sequence of HHHHHH (SEQ ID NO:26), SLSLSPGK (SEQ ID NO:36), or AS, TVAPTESS (SEQ ID NO:37).

196. 196. The isolated protein of claim 195, which exhibits reduced interaction with anti-drug antibodies in biological materials.

197. 200. The isolated protein of claim 196, wherein the biological material comprises serum and / or plasma.

198. 202. The isolated protein of any one of claims 195-197, wherein the exposed C-terminus comprises an anti-drug antibody epitope.

199. 200. The isolated protein of any one of claims 195-198, wherein the exposed C-terminus consists of an amino acid sequence selected from the group consisting of VTVSS (SEQ ID NO:21), VEIK (SEQ ID NO:22), VEIKR (SEQ ID NO:23), VEIKRT (SEQ ID NO:24), TKVTVL (SEQ ID NO:38), TKLTVL (SEQ ID NO:39), TQLIIL (SEQ ID NO:40), TELTVL (SEQ ID NO:41), or TQLTVL (SEQ ID NO:42).

200. 200. The isolated protein of any one of claims 195 to 199 for use in a therapeutic or diagnostic assay.

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