Bispecific antibodies with cleavable c-terminal charge pair tags

Abstract

To provide novel engineered proteins that are multispecific protein complexes comprising multispecific antibodies, methods of constructing them and methods of producing them.SOLUTION: Provided is a CH3-containing molecule comprising (a) a first polypeptide comprising a CH3 domain and a negatively charged domain comprising consecutive negatively charged amino acid residues; and (b) a second polypeptide comprising a CH3 domain and a positively charged domain comprising consecutive positively charged amino acid residues. Also provided are methods for conjugating synthetic molecules to multispecific antigen-binding molecules.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel engineered proteins that are multispecific protein complexes, including multispecific antibodies, methods for constructing them, and methods for producing them. The present invention also relates to new applications of techniques useful for obtaining multispecific protein complexes.

[0002] This application contains an ASCII "txt" Sequence Listing available in both computer readable form (CRF) and as a paper copy as required by 37 CFR sections 1.821(c) and 1.821(e), and is incorporated herein by reference in its entirety. The "txt" file, created on May 8, 2019, is named A-2238-WO-PCT_SEQ_LIST_050719_ST25 and is 30kb in size. [Background technology]

[0003] The development of multispecific antibodies as therapeutic agents for human diseases holds great clinical potential. Multispecific antibodies can simultaneously recognize two different antigens, neutralize different pathogenic mediators, recruit different types of effector cells, and modulate signaling pathways. However, the generation of multispecific antibodies has been extremely challenging. The widespread application of multispecific antibodies has been hindered by the difficulty of developing a platform for generating multispecific antibodies that exhibits favorable half-lives, high stability, lack of immunogenicity, and feasibility for large-scale production and purification. Promising multispecific antibody formats such as DVD-Ig (dual variable domain Ig) (Nature Biotechnology 25, 1290-1297 (2007)); crossover Ig [Schaefer W et al (2011) PNAS 108(27):11187-11192]; two-in-one Ig (Science 2009, 323, 1610); and BiTE® antibodies [PNAS 92(15):7021-7025; 1995] allow the generation of multispecific antibodies, but they have different kinds of burdens.

[0004] Several innovative techniques have enabled the almost exclusive assembly of Fc heterodimers, providing a framework for engineering multispecific antibodies (e.g., knob-in-hole (Ridgway et al., Protein Eng. 9:617, 1996), electrostatic steering (Gunasekaran et al., J. Biol. Chem. 285:19637, 2010), and strand-exchange engineered domains (SEED) (Davis, Protein Eng. Des. & Sel. 23:195, 2010)). In the dual variable domain (DVD)-Ig approach, the VL and VH of a second antibody are fused via flexible linkers to the N-terminus of the light and heavy chains of a first antibody, respectively, to create two tandem variable domains (VDs), termed the outer VD and inner VD (Wu et al., ibid.). Due to the steric hindrance caused by the proximity of the outer VD to the ligand-binding site of the inner VD, retaining the binding affinity of the inner VD requires extensive optimization, including VD selection from several available monoclonal antibodies, VD orientation, and linker design, most of which must be determined experimentally (DiGiammarino et al., Methods Mol. Biol. 899:145, 2012).

[0005] Another method utilizes species-restricted heavy and light chain pairing in rat / mouse quadromas (Lindhofer et al., J. Immunol. 155:219, 1995). However, the multispecific antibodies generated are rat / mouse antibodies, which have obvious immunogenicity problems as therapeutics.

[0006] Crossmab technology, based on knob-into-hole heterodimerized heavy chains, also uses immunoglobulin domain crossover as a general method for the production of multispecific IgG antibodies (Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11-187, 2011). Nevertheless, correct pairing of the heavy chain heterodimer and the cognate Fv is not exclusive, and unwanted by-products must be removed during purification.

[0007] An extension of the Crossmab approach has been used to generate tetravalent multispecific antibodies by tagging the C-terminus of the Crossmab with an additional set of Fab and Crossmab Fab fragments (Regula et al., US Patent Application Publication No. 2010 / 0322934), but the challenge of obtaining exclusively correct pairing of the H-chain heterodimer and cognate Fv remains.

[0008] A further approach to multispecificity is the use of a single binding site that targets two different antigens, as demonstrated by "two-in-one" antibodies. One such "two-in-one" antibody is a variant of the antibody Herceptin, which interacts with both Her2 and VEGF (Bostrom et al., Science 323:1610, 2009). This approach is attractive for clinical applications because it results in a multispecific antibody with a format identical to that of a normal IgG. However, screening for such variants is very labor-intensive, and there is no guarantee that a single binding site capable of binding both antigens of interest will be obtained.

[0009] Finding a technique for conjugating multispecific antibodies that is useful and adaptable for commercial and therapeutic purposes has been elusive. Many approaches have been attempted, but almost all suffer from significant drawbacks, including, among others, poor solubility; lack of expression in mammalian cells; low yields of heterodimeric structures; technical difficulty in production; immunogenicity; short half-life in vivo; and instability (see, e.g., Hollinger et al., (1993) PNAS 90:6444-6448; U.S. Pat. Nos. 5,932,448; 6,833,441; 5,591,828; 7,129,330; 7,507,796; Fischer et al., (2007) Pathobiology 74:3-14; Booy (2006) Arch. Immunol. Ther. Exp. 54:85-101; Cao et al. (2003) 55:171-197; and Marvin et al. al., (2006) Current Opinion in Drug Discovery & Development 9(2):184-193. Therefore, improved techniques and processes for generating multispecific antibodies are needed. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US Patent Application Publication No. 2010 / 0322934 [Patent Document 2] U.S. Patent No. 5,932,448 [Patent Document 3] U.S. Patent No. 6,833,441 [Patent Document 4] U.S. Patent No. 5,591,828 [Patent Document 5] U.S. Patent No. 7,129,330 [Patent Document 6] U.S. Patent No. 7,507,796 [Non-patent literature]

[0011]

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[0012] In one aspect, the invention relates to a CH3-containing molecule comprising: (a) a first polypeptide comprising a CH3 domain and a negatively charged domain comprising at least four consecutive negatively charged amino acid residues; and (b) a second polypeptide comprising a CH3 domain and a positively charged domain comprising at least four consecutive positively charged amino acid residues.

[0013] In one embodiment, the CH3 domain and negatively charged domain of the first polypeptide are arranged relative to each other in the N-terminal to C-terminal direction, and the CH3 domain and positively charged domain of the second polypeptide are arranged relative to each other in the N-terminal to C-terminal direction. In one embodiment, each of the first and second polypeptides further comprises a CH2 domain. In one embodiment, each of the first and second polypeptides further comprises a hinge domain. In one embodiment, each of the first and second polypeptides comprises a VH, CH1, hinge, CH2, and CH3 domain arranged relative to each other in the N-terminal to C-terminal direction.

[0014] In one embodiment, the molecule further comprises a third and a fourth polypeptide, wherein the third polypeptide comprises a first VL domain and the fourth polypeptide comprises a second VL domain, hi one embodiment, the third polypeptide further comprises a first CL domain, wherein the first VL and CL domains are arranged relative to each other in the N-terminal to C-terminal direction within the third polypeptide, and the fourth polypeptide further comprises a second CL domain, wherein the second VL and CL domains are arranged relative to each other in the N-terminal to C-terminal direction within the fourth polypeptide.

[0015] In one embodiment, the negatively charged domain comprises at least 5, at least 6, or at least 7 consecutive negatively charged amino acid residues, and the positively charged domain comprises at least 5 consecutive positively charged amino acid residues.

[0016] In one embodiment, the negatively charged amino acid residue is an aspartic acid residue and the positively charged amino acid residue is selected from the group consisting of lysine, arginine, and histidine residues, hi one embodiment, the negatively charged amino acid residue is an aspartic acid residue and the positively charged amino acid residue is a lysine residue.

[0017] In one embodiment, the negatively charged amino acid residue is a glutamic acid residue and the positively charged amino acid residue is selected from the group consisting of lysine, arginine, and histidine residues.

[0018] In one embodiment, the first polypeptide further comprises a linker sequence covalently attached to the C-terminus of the CH3 domain, wherein the linker is attached to the N-terminus of the negatively charged domain; and the second polypeptide further comprises a linker sequence covalently attached to the C-terminus of the CH3 domain, wherein the linker is attached to the N-terminus of the positively charged domain.

[0019] In one embodiment, the linker of the first polypeptide is the same as the linker of the second polypeptide.

[0020] In one embodiment, the linker may be cleaved by an enzyme, hi one embodiment, the enzyme is selected from the group consisting of sortase A, sortase B, sortase C, sortase D, sortase E, and sortase F.

[0021] In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO:1 (LPETGGEEST); SEQ ID NO:2 (LPXTG, where X can be any amino acid); SEQ ID NO:3 (LPETG); SEQ ID NO:4 (LPETGG); SEQ ID NO:5 (LPXTA, where X can be any amino acid); SEQ ID NO:6 (NPX[T / S][N / G / S], where X can be any amino acid); SEQ ID NO:7 (IPXTG, where X can be any amino acid); and SEQ ID NO:8 (LAXTG, where X can be any amino acid).

[0022] In one embodiment, either the first polypeptide, the second polypeptide, or both, further comprise a purification tag attached to its C-terminus.

[0023] In one embodiment, the purification tag is selected from the group consisting of a his-tag, a strep-tag, a flag-tag, a T7-tag, a V5-peptide-tag, a GST-tag, a CBP-tag, a MBP-tag, and a c-Myc-tag, hi one embodiment, the purification tag is a his-tag comprising at least five consecutive histidine amino acid residues.

[0024] In one embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 9 (LPETGGEESTDDDDDDD) and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 10 (LPETGGEESTKKKKKKKHHHHHH).

[0025] In one embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11 (LPETGGEESTDDDDDDDHHHHHH) and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12 (LPETGGEESTKKKKKKK).

[0026] In one aspect, the invention relates to a multispecific antibody comprising a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, and a second light chain polypeptide, the first heavy chain polypeptide comprises a CH3 domain and a negatively charged domain comprising at least four consecutive negatively charged amino acid residues, and the first heavy chain polypeptide and the first light chain polypeptide bind to a first antigen; and The second heavy chain polypeptide comprises a CH3 domain and a positively charged domain comprising at least four consecutive positively charged amino acid residues, and the second heavy chain polypeptide and the second light chain polypeptide bind to a second antigen.

[0027] In one embodiment, the multispecific antibody is expressed by a mammalian cell. In one embodiment, the mammalian cell is a HEK cell or a CHO cell.

[0028] In one aspect, the invention relates to a method for producing a multispecific antibody, the method comprising culturing cells comprising a vector encoding a multispecific antibody of the invention in a culture medium.

[0029] In one embodiment, the method further comprises recovering the multispecific antibody from the cells or culture medium.

[0030] In one aspect, the present invention provides a method for making an antibody conjugate, comprising: a) providing a multispecific antibody according to any one of claims 40 or 41; and b) treating the antibody with a sortase enzyme in the presence of a synthetic molecule, the molecule comprising a gly-gly-gly sequence. In one embodiment, step b) is performed using a synthetic molecule:antibody molar ratio of about 2 to about 1000.

[0031] Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims. [Brief explanation of the drawings]

[0032] [Figure 1] 1 shows engineering of bispecific antibodies via a C-terminal charge-pair tag. [Figure 2] 1 shows a bispecific antibody based on a C-terminal charge pair tag. [Figure 3] 1 shows a bispecific antibody-based C-terminal charge-pair tag. [Figure 4] 1 shows the cleavage of the C-terminal charge-pair tag from a bispecific antibody by sortase. [Figure 5] 1 shows transpeptidation of a bispecific antibody generated via a C-terminal charge-pair tag. [Figure 6] 1 shows a bispecific antibody drug conjugate generated via a C-terminal charge pair tag. [Figure 7] Transient expression of HEK 293-6e-TFNα / TLA1 is shown. [Figure 8] Transient expression of HEK 293-6e-PAC1 / CGRPR1 is shown. [Figure 9] The expression levels of C-terminal charge-paired dual specific TNFα / TLA1 and PAC1 / CGRPR1 are shown (150-250 mg / L). [Figure 10] Antibody A purified-TNFα / TLA1 is shown. [Figure 11] Elution profile: Protein A affinity purification of PAC1 / CGRPR1. [Figure 12] LCMS: Shows Pro A pool-TNFα / TLA1. [Figure 13] LCMS: Shows Pro A pool-PAC1 / CGRPR1. [Figure 14] SDS-Page: Protein A affinity purification of PAC1 / CGRPR1. [Figure 15] Analysis of TNFα / TLA1 purity by analytical size exclusion and cation exchange chromatography. [Figure 16] Analysis of PAC1 / CGRPR1 purity by analytical size exclusion and cation exchange chromatography. [Figure 17]1 shows the analytical Sec profile of PAC1 / CGRPR1 after UPLC:Pro A purification. [Figure 18] TNFα / TLA1: Preparative cation exchange chromatography. [Figure 19] Preparative cation exchange chromatography is shown. [Figure 20] 1 shows Caliper analysis of PAC1 / CGRPR1 bispecific antibodies generated with a C-terminal charge pair tag. [Figure 21] 1 shows bispecific PAC1 / CGRPR1 antibody binding analysis. [Figure 22] Site-specific cleavage of the C-terminal charge-pair tag is shown. [Figure 23] 1 shows site-specific conjugation of bispecific antibodies via transpeptidation. [Figure 24] Sortase-mediated tag removal is shown. [Figure 25] Extensive transpeptidation is shown. [Figure 26] LCMS removal of charge pair tags from TNFα / TLA1. [Figure 27] LCMS removal of charge pair tags from PAC1 / CGRPR1 is shown. [Figure 28] 1 shows C-terminal conjugation of charge pair-derived bispecific antibodies. [Figure 29] 1 shows site-specific labeling of bispecific antibodies. [Figure 30] Figure 1 shows sortase A-mediated biotinylation of TNFα / TLA1. [Figure 31] 1 shows sortase A-mediated biotinylation of PAC1 / CGRPR1. [Figure 32] 1 shows sortase A-mediated PEGylation of PAC1 / CGRPR1 and TNFα / TLA1. [Figure 33] PEGylation of TNFα / TLA1 with GGG-PEG20 by Sortase A. [Figure 34] PEGylation of PAC1 / CGRPR1 with GGG-PEG20 by Sortase A. [Figure 35] SDS Page: PEGylation of PAC1 / CGRPR1 and TNFα / TLA1 bispecifics with 20kd-PEG using Sortase A-Biab+Sortase A+G3c-PEG20k. [Figure 36] 1 shows analytical Cex of a bispecific antibody PEGylated with PEG20k. [Figure 37] 1 shows site-specific fusion of GGG-Alexa fluorophore with sortase A-TNFα / TLA1. [Figure 38] 1 shows site-specific conjugation of -GGG-Alexa fluorophore by sortase A-PAC1 / CGRPR1. [Figure 39] We show that sortases can also utilize non-standard nucleophiles. [Figure 40] PAC1 / CGRPR1-PEG3-N3+Dibo-Alexa488 click chemistry reaction—24 hour incubation. DETAILED DESCRIPTION OF THE INVENTION

[0033] Without being bound by theory, Applicants believe that the C-terminal charge-pair tag described herein provides an initial trigger that drives the binding of two or more molecules together with a surprisingly high degree of precision and efficiency, even in the presence of the Fc regions of immunoglobulins, which are also naturally attracted to each other under cell culture conditions.

[0034] By reducing heavy chain homodimerization, the use of oppositely charged C-terminal charge-pair tags described herein offers a breakthrough in the ability to generate homogeneous populations of protein complexes containing CH3, CH2-CH3, or hinge-CH2-CH3 components (e.g., multispecific or one-arm antibodies). Multispecific complexes are advantageous for use in therapeutic applications, for example, because they can induce colocalization of a target (e.g., tumor cells) and an agent directed against the target (e.g., T cells), or because they can eliminate the need for combination therapy and the risks associated with administering two or more therapeutic agents to a subject. Furthermore, proteases can be used to facilitate removal of C-terminal charge-pair tags from heterodimers.

[0035] The recombinant polypeptide and nucleic acid methods used herein, including the Examples, are generally those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) or Current Protocols in Molecular Biology (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons 1994), both of which are incorporated herein by reference for any purpose.

[0036] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0037] As used herein, unless otherwise defined, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0038] Generally, the nomenclatures and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present application are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0039] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the disclosure, which is defined solely by the claims.

[0040] Except as otherwise noted in the examples, all numbers indicating quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0041] Unless otherwise specified, as used herein, "a" and "an" are used conventionally to mean "one or more."

[0042] As used herein, the terms "amino acid" and "residue" are used interchangeably and, when used in the context of a peptide or polypeptide, refer to both naturally occurring and synthetic amino acids, as well as amino acid analogs, amino acid mimetics, and non-naturally occurring amino acids that are chemically similar to the naturally occurring amino acids.

[0043] "Naturally occurring amino acids" are those amino acids encoded by the genetic code, as well as those amino acids encoded by the genetic code that are synthesized and later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methylmethionine sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

[0044] "Amino acid mimetics" are chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Examples include methacryloyl or acryloyl derivatives of amides, β-amino acids, γ-amino acids, δ-amino acids (such as piperidine-4-carboxylic acid), and the like.

[0045] A "non-naturally occurring amino acid" is a compound that has the same basic chemical structure as a naturally occurring amino acid, but that is not incorporated into a growing polypeptide chain by the translation complex. "Non-naturally occurring amino acids" also include, but are not limited to, amino acids that result from modification (e.g., post-translational modification) of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids), but that are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. An exemplary list of non-naturally occurring amino acids that can be inserted into or used in place of wild-type residues in a polypeptide sequence includes, but is not limited to, β-amino acids, homoamino acids, cyclic amino acids, and side chain derivatized amino acids. Examples include citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn), Nα-methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Nα-methylarginine (NM eR), Nα-methylleucine (Nα-MeL or NMeL), N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-Tetrahydroisoquinoline (Tic), 2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-amino-Phe), 4-guanidinophenylalanine (Guf), glycyllysine (abbreviated as "K(Nε-glycyl)" or "K(glycyl)" or "K(gly)"), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methylvaline (NMeVal), N-α-methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine; 4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidine acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, Included are N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-amino-O-phthalic acid (4APA), and other similar amino acids, as well as derivatized forms of any of the specifically mentioned amino acids, in either the L- or D-form; abbreviations are given in parentheses.

[0046] The term "isolated nucleic acid molecule" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogs thereof, read from the 5' to the 3' end, from which at least about 50 percent of the polypeptides, peptides, lipids, carbohydrates, polynucleotides, or other materials naturally found with the nucleic acid when the total nucleic acid is isolated from a source cell are removed. Preferably, an isolated nucleic acid molecule is substantially free of any other contaminating nucleic acid molecules or other molecules that are found in the nucleic acid's natural environment and that would interfere with its use in the production of a polypeptide or its therapeutic, diagnostic, prophylactic, or research uses.

[0047] The term "isolated polypeptide" refers to a polypeptide that is separated from at least about 50 percent of the polypeptides, peptides, lipids, carbohydrates, polynucleotides, or other materials with which the polypeptide is naturally found when isolated from the source cell. Preferably, an isolated polypeptide is substantially free of any other contaminating polypeptides or other contaminants that are found in its natural environment and that would interfere with its therapeutic, diagnostic, prophylactic, or research use.

[0048] The term "encoding" refers to a polynucleotide sequence that encodes one or more amino acids. The term does not require a start or stop codon.

[0049] The terms "identical" and percent "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are identical. "Percent identity" refers to the percent residue identity between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. In such calculations, gaps in the alignment, if any, can be addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, A.M., ed.), (1988) New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., (1987) Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., (1988) SIAM J. Applied Math. 48:1073.

[0050] When calculating percent identity, the sequences being compared are aligned in a way that maximizes the match between the sequences. Computer programs used to determine percent identity include the GCG program package, which includes GAP (Devereux et al., (1984) Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. The sequences are aligned so that their respective amino acids or nucleotides are optimally matched (the "match span" determined by the algorithm). A gap opening penalty (calculated as 3 x average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used; "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (usually 1 / 10 of the gap opening penalty), and a comparison matrix such as PAM 250 or BLOSUM 62 are used with the algorithm. In certain embodiments, standard comparison matrices (for the PAM 250 comparison matrix, see Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) are also used by the algorithm.

[0051] Recommended parameters for determining percent identity of polypeptide or nucleotide sequences using the GAP program are as follows: Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., 1992 (supra); Gap penalty: 12 (but no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0

[0052] Using a particular alignment scheme for aligning two amino acid sequences, only short regions of the two sequences may match, and this short aligned region may have very high sequence identity even if there is no significant relatedness between the two full-length sequences. Thus, if desired, the alignment method selected (e.g., the GAP program) can be adjusted to obtain an alignment spanning at least 50 consecutive amino acids of the target polypeptide.

[0053] As used herein, "antigen-binding protein" refers to any protein that specifically binds to a particular target antigen. The term encompasses intact antibodies comprising at least two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutants thereof. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments. Antigen-binding proteins also include domain antibodies, such as nanobodies and scFvs, which are described further below.

[0054] Generally, an antigen-binding protein "specifically binds" to its target antigen when it exhibits essentially background binding to non-target antigen molecules. However, an antigen-binding protein that specifically binds to its target antigen may cross-react with target antigen polypeptides from different species. Typically, an antigen-binding protein specifically binds to its target antigen when the dissociation constant (KD) measured by surface plasma resonance techniques (e.g., BIACore, GE-Healthcare Uppsala, Sweden) or equilibrium exclusion methods (KinExA, Sapidyne, Boise, Idaho) is ≦10 M. An antigen-binding protein specifically binds to its target antigen with "high affinity" when the KD measured using the described method is ≦5×10 M, and specifically binds to its target antigen with "very high affinity" when the KD measured using the described method is ≦5×10 M.

[0055] "Antigen-binding region" refers to a protein or a portion of a protein that specifically binds to a particular antigen. For example, that portion of an antigen-binding protein containing amino acid residues that interact with an antigen and confer its specificity and affinity for the antigen to the antigen-binding protein is referred to as the "antigen-binding region." An antigen-binding region typically comprises one or more "complementary binding regions" ("CDRs") of an immunoglobulin, single-chain immunoglobulin, or camelid antibody. A particular antigen-binding region also comprises one or more "framework" regions. "CDRs" are amino acid sequences that contribute to antigen-binding specificity and affinity. "Framework" regions can facilitate binding between the antigen-binding region and the antigen by helping to maintain the proper conformation of the CDRs.

[0056] A "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known in the art.

[0057] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and multispecific antibodies. Thus, an "antibody" is a type of antigen-binding protein. An intact antibody generally comprises at least two full-length heavy chains and two full-length light chains. Antibodies may be derived from only a single source, or may be "chimeric," i.e., different portions of the antibody may be derived from two different antibodies, as described further below. Antigen-binding proteins, antibodies, or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.

[0058] The term "light chain" as used with respect to an antibody or a fragment thereof includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain contains a variable region domain (VL) and a constant region domain (CL). The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0059] The term "heavy chain" as used with respect to antibodies or fragments thereof includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain contains a variable region domain (VH) and three constant region domains (CH1, CH2, and CH3). The VH domain is located at the amino terminus of the polypeptide, the CH domain is located at the carboxyl terminus, and CH3 is closest to the carboxy terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0060] As used herein, the term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) is an antigen-binding protein that lacks at least some of the amino acids present in the full-length chain, but that comprises a portion of an antibody (regardless of how that portion is obtained or synthesized) that retains the ability to specifically bind to an antigen. Such a fragment is biologically active in that it specifically binds to a target antigen and can compete with other antigen-binding proteins, including intact antibodies, for specific binding to a given epitope.

[0061] Such biologically active fragments may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of antigen-binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', and F(ab')2 fragments.

[0062] In another embodiment are Fvs, domain antibodies and scFvs, which may be derived from the antibodies of the invention.

[0063] It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, such as, for example, one or more CDRs, can be covalently attached to a second protein or small molecule to create a therapeutic agent that is directed to a specific target in the body, has bifunctional therapeutic properties, or has extended serum half-life.

[0064] A "Fab fragment" consists of one light chain and the CH1 and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0065] The "Fc" region comprises two heavy chain fragments containing the CH2 and CH3 domains of an antibody, held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

[0066] An "Fab' fragment" comprises one light chain and a portion of one heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, such that an F(ab')2 molecule can be formed by interchain disulfide bond formation between the two heavy chains of two Fab' fragments.

[0067] An "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, resulting in the formation of an interchain disulfide bond between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0068] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0069] A "single-chain antibody" or "scFv" is an Fv molecule in which the heavy and light chain variable regions are linked by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. scFvs are discussed in detail in WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0070] A "domain antibody" or "single-chain immunoglobulin" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. Examples of domain antibodies include Nanobodies®. In some cases, two or more VH regions are covalently linked via a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

[0071] A "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding regions. In some cases, the two binding regions have the same antigen specificity. Bivalent antigen-binding proteins and bivalent antibodies may be bispecific, see below.

[0072] A "multispecific antigen-binding protein" or "multispecific antibody" is one that targets more than one antigen or epitope.

[0073] A "bispecific," "dual-specific," or "bifunctional" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody, respectively, having two different antigen-binding sites. Bispecific antigen-binding proteins and antibodies are a type of multispecific antigen-binding protein or multispecific antibody and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes, which may reside on the same or different protein targets.

[0074] The term "compete" when used in the context of antigen-binding proteins (e.g., antibodies) means that competition between antigen-binding proteins is determined by an assay in which the antigen-binding protein (e.g., an antibody or an immunologically functional fragment thereof) under test blocks or inhibits specific binding of a reference antigen-binding protein to a common antigen. Many types of competitive binding assays can be used, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahl et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct label assays, solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 labels (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1986, J. Immunol. 137:3614-3619); al., 1990, Virology 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays use purified antigen bound to a solid surface or cells bearing either such antigen, an unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Further details regarding methods for determining competitive binding are provided in the Examples herein.Typically, when a competing antigen-binding protein is present in excess, it inhibits specific binding of the reference antigen-binding protein to a common antigen by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%. In some cases, binding is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% or more.

[0075] The term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a selective binding agent, such as an antigen-binding protein (including, for example, an antibody), and that can be used in an animal to generate antibodies capable of binding to that antigen. An antigen can have one or more epitopes that are capable of interacting with different antigen-binding proteins, e.g., antibodies.

[0076] The term "epitope" refers to the portion of a molecule that is bound by an antigen-binding protein (e.g., an antibody). The term includes any determinant capable of specifically binding to an antigen-binding protein, such as an antibody. Epitopes can be contiguous or discontinuous (discontinuous) (e.g., in a polypeptide, amino acid residues that are not contiguous with each other in the polypeptide sequence but that share connections within the molecule are subject to binding by an antigen-binding protein). A conformational epitope is an epitope that is present in the conformation of the active protein but not in the denatured protein. In certain embodiments, an epitope can be mimetic in that it contains a three-dimensional structure similar to the epitope used to generate the antigen-binding protein, but does not contain, or contains only some of, the amino acid residues found in the epitope used to generate the antigen-binding protein. Epitopes are most often found on proteins, but can occasionally be found on other types of molecules, such as nucleic acids. Epitopic determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, an antigen-binding protein specific for a particular target antigen will preferentially recognize epitopes present on the target antigen in a complex mixture of proteins and / or macromolecules.

[0077] As used herein, "substantially pure" means that the recited molecular species is the predominant species present, i.e., more abundant on a molar basis than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the reference species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all macromolecular species present in the composition. In other embodiments, the reference species is purified to substantial homogeneity, such that contaminating species cannot be detected in the composition by conventional detection methods, and thus the composition consists of a single detectable macromolecular species.

[0078] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate.

[0079] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotides. In some embodiments, oligonucleotides are 10-60 bases in length. In other embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, for example, for use in constructing mutant genes. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides can contain a label, including a radiolabel, fluorescent label, hapten, or antigenic label for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers, or hybridization probes.

[0080] An "isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or any combination thereof, where the isolated polynucleotide is unaccompanied by all or part of polynucleotides with which it is found in nature or is linked to polynucleotides with which it is not naturally linked. For purposes of this disclosure, a "nucleic acid molecule comprising" a particular nucleotide sequence should be understood to not encompass intact chromosomes. An isolated nucleic acid molecule "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include sequences encoding up to 10 or even up to 20 other proteins or portions thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0081] Unless otherwise stated, the left-hand end of any single-stranded polynucleotide sequence discussed 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 sequence region on the DNA strand that is 5' to the 5' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "upstream sequence"; the sequence region on the DNA strand that is 3' to the 3' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "downstream sequence".

[0082] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences may depend on the host organism. In particular embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes may include a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence, and a transcription termination sequence. "Control sequences" may include leader sequences and / or fusion partner sequences.

[0083] The term "vector" refers to any molecule or entity (eg, nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell.

[0084] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that (in cooperation with the host cell) direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of the coding regions operably linked thereto.

[0085] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under appropriate conditions. For example, a control sequence "operably linked" to a protein-coding sequence in a vector is ligated thereto so that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0086] The term "host cell" refers to a cell that has been transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present.

[0087] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms can also include amino acid polymers that have been modified, for example, by the addition of carbohydrate residues to form glycoproteins or by phosphorylation. Polypeptides and proteins can be naturally occurring and produced by non-recombinant cells; or they can be produced by genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a naturally occurring protein or molecules with one or more amino acid deletions, additions, and / or substitutions from the naturally occurring sequence. The terms "polypeptide" and "protein" specifically encompass antigen-binding proteins, antibodies, or sequences with one or more amino acid deletions, additions, and / or substitutions from an antigen-binding protein. The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length protein. Such fragments can also contain modified amino acids compared to the full-length protein. In certain embodiments, fragments are between about 5 and 500 amino acids in length. For example, fragments can be at least 5, at least 6, at least 8, at least 10, at least 14, at least 20, at least 50, at least 70, at least 100, at least 110, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies that contain the binding domain.

[0088] The term "isolated protein" means that the subject protein (1) is free from at least some other proteins with which it is normally expected to be found; (2) is substantially free from other proteins from the same source, e.g., the same species; (3) is expressed by cells from a different species; (4) has been removed from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials that naturally accompany it; (5) is operably associated (by covalent or noncovalent interactions) with polypeptides that are not naturally associated with it; or (6) is not naturally occurring. Generally, an "isolated protein" will comprise at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof. Preferably, an isolated protein is substantially free of proteins or polypeptides or other contaminants that are found in its natural environment and that would interfere with its therapeutic, diagnostic, preventative, research, or other use.

[0089] "Variants" of a polypeptide (e.g., an antigen-binding protein such as an antibody) include amino acid sequences in which one or more amino acid residues have been inserted, deleted, and / or substituted compared to another polypeptide sequence. Variants include fusion proteins.

[0090] A "derivative" of a polypeptide is a polypeptide (e.g., an antigen-binding protein such as an antibody) that has been chemically modified in some way that differs from the variant by insertion, deletion, or substitution, for example, by conjugation to another chemical moiety.

[0091] The term "naturally occurring" as used throughout this specification in reference to biological material, such as a polypeptide, nucleic acid, host cell, etc., refers to a material that is found in nature.

[0092] As used herein, a "subject" or "patient" can be any mammal. In typical embodiments, the subject or patient is a human.

[0093] A "conservative amino acid substitution" can involve the substitution of a naturally occurring amino acid residue (i.e., a residue found at a given position in a polypeptide sequence) with a non-naturally occurring residue (i.e., a residue not found at the same given position in a polypeptide sequence), resulting in little or no effect on the polarity or charge of the amino acid residue at that position. Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in a biological system. These include peptidomimetics and other forms in which amino acid moieties are reversed or inverted.

[0094] Naturally occurring residues can be grouped into classes based on the following common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) basic: Asn, Gln, His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0095] Additional groups of amino acids can also be delineated using the principles described, for example, in Creighton (1984) PROTEINS: STRUCTURE AND MOLECULAR PROPERTIES (2d Ed. 1993), W.H. Freeman and Company. In some cases, it may be useful to further characterize substitutions based on two or more of such properties (e.g., substitution with a "less polar" residue such as a Thr residue may be a highly conservative substitution in appropriate circumstances).

[0096] Conservative substitutions may involve the exchange of a member of one such class for another member of the same class. Non-conservative substitutions may involve the exchange of a member of one such class for a member of another class.

[0097] Synthetic, rare, or modified amino acid residues known to have similar physicochemical properties to those in the above classes can be used as "conservative" substitutions for particular amino acid residues in a sequence. For example, a D-Arg residue can serve as a substitute for a typical L-Arg residue. In some cases, a particular substitution can be described in terms of more than one of the above classes (e.g., substitution with a small, hydrophobic residue refers to substitution of one amino acid with a residue found in both of the above classes, or with another synthetic, rare, or modified residue known in the art to have similar physicochemical properties as such a residue that meets both definitions).

[0098] To express a nucleic acid sequence, an appropriate coding sequence can be cloned into a suitable vector according to standard cloning and expression techniques, and after introduction into a suitable host, the sequence can be expressed to produce the encoded polypeptide; such techniques are known in the art (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). The present invention also relates to such vectors containing the nucleic acid sequence according to the present invention.

[0099] "Vector" refers to (a) a delivery vehicle that facilitates expression of a nucleic acid sequence encoding a polypeptide; (b) a delivery vehicle that facilitates production of a polypeptide therefrom; (c) a delivery vehicle that facilitates transduction / transformation of a target cell therewith; (d) a delivery vehicle that facilitates replication of the nucleic acid sequence; (e) a delivery vehicle that facilitates stability of the nucleic acid; (f) a delivery vehicle that facilitates detection of the nucleic acid and / or transformed / transfected cells; and / or (g) a delivery vehicle that otherwise confers a beneficial biological and / or physiochemical function to the nucleic acid encoding the polypeptide. The vector may be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors.

[0100] Recombinant expression vectors can be designed to express proteins in prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., insect cells, yeast cells, or mammalian cells using baculovirus expression vectors). In one embodiment, the host cell is a mammalian, non-human host cell. Exemplary host cells include hosts typically used for cloning and expression, such as Escherichia coli strains TOP10F', TOP10, DH10B, DH5a, HB101, W3110, BL21(DE3), and BL21(DE3)pLysS, BLUESCRIPT (Stratagene), mammalian cell lines CHO, CHO-K1, HEK293, 293-EBNA pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509 (1989)); pET vectors (Novagen, Madison, MD). Wis. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and T7 polymerase and an in vitro translation system. Preferably, the vector contains a promoter upstream of the cloning site containing the nucleic acid sequence encoding the polypeptide. Examples of promoters that can be switched on and off include the lac promoter, T7 promoter, trc promoter, tac promoter, and trp promoter.

[0101] The vector can include or be associated with any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer, RSV promoter, SV40 promoter, SL3-3 promoter, MMTV promoter, or HIV LTR promoter, EF1 alpha promoter, or CAG promoter), an efficient poly(A) termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The vector can also include an inducible promoter, as opposed to a constitutive promoter such as CMV IE. In one aspect, a nucleic acid is provided that includes a polypeptide-encoding sequence operably linked to a tissue-specific promoter that promotes expression of the sequence in metabolically relevant tissues, such as liver or pancreatic tissue.

[0102] In another aspect of the present disclosure, host cells are provided comprising the nucleic acids and vectors disclosed herein. In various embodiments, the vector or nucleic acid is integrated into the host cell genome, while in other embodiments, the vector or nucleic acid is present extrachromosomally.

[0103] Recombinant cells, such as yeast cells, bacterial cells (e.g., E. coli) and mammalian cells (e.g., immortalized mammalian cells), containing such nucleic acids, vectors, or a combination of either or both, are provided. In various embodiments, cells are provided that contain a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, that includes a sequence coding for expression of a polypeptide.

[0104] Vectors containing nucleic acid sequences encoding the polypeptides provided herein can be introduced into host cells by transformation or transfection. Methods for transforming cells with expression vectors are well known.

[0105] Nucleic acids can be placed and / or delivered into host cells or host animals by viral vectors. Any suitable viral vector with this capability can be used. Viral vectors can contain any number of viral polynucleotides, alone or in combination with one or more viral proteins that facilitate delivery, replication, and / or expression of the nucleic acids of the invention in desired host cells. Viral vectors can be polynucleotides comprising all or part of the viral genome, viral protein / nucleic acid conjugates, virus-like particles (VLPs), or intact viral particles comprising viral nucleic acids and nucleic acids encoding polypeptides. Viral vectors that are viral particles can comprise wild-type viral particles or modified viral particles. Viral vectors can be vectors that require the presence of another vector or wild-type virus for replication and / or expression, such as adenoviral vector amplicons (e.g., viral vectors can be helper-dependent viruses). Typically, such viral vectors consist of wild-type viral particles or viral particles whose protein and / or nucleic acid content has been modified to increase transgene capacity or to facilitate gene transfer and / or expression of the nucleic acid (examples of such vectors include herpesvirus / AAV amplicons). Typically, viral vectors are similar to and / or derived from viruses that normally infect humans. Suitable viral vector particles in this regard include, for example, adenoviral vector particles (including any virus in the adenoviridae family or any virus derived from a virus in the adenoviridae family), adeno-associated viral vector particles (AAV vector particles) or other parvoviruses and parvoviral vector particles, papillomavirus vector particles, flavivirus vectors, alphavirus vectors, herpesvirus vectors, poxvirus vectors, retroviral vectors (including lentiviral vectors).

[0106] Protein purification methods that can be used to isolate polypeptides, as well as related materials and reagents, are known in the art. Additional purification methods that can be useful for isolating polypeptides can be found in references such as Bootcov MR, 1997, Proc. Natl. Acad. Sci. USA 94:11514-9, Fairlie WD, 2000, Gene 254:67-76.

[0107] The antigen binding proteins provided are polypeptides incorporating and / or linked to one or more complementarity determining regions (CDRs) as described herein. In some antigen binding proteins, the CDRs are incorporated into "framework" regions that orient the CDRs so that their proper antigen binding properties are achieved. Certain antigen binding proteins described herein are antibodies or are derived from antibodies. In other antigen binding proteins, the CDR sequences are incorporated into different types of protein scaffolds. The various structures are further described below.

[0108] In general, the antigen binding proteins provided typically comprise one or more (e.g., one, two, three, four, five, or six) CDRs described herein. In some cases, the antigen binding proteins comprise (a) a polypeptide structure and (b) one or more CDRs inserted into and / or linked to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, the polypeptide structure can be or comprise the framework of a naturally occurring antibody or fragment or variant thereof, or can be entirely synthetic in nature. Examples of various polypeptide structures are described further below.

[0109] In certain embodiments, the polypeptide structure of the antigen-binding protein is or is derived from an antibody. Accordingly, examples of specific antigen-binding proteins provided include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies such as Nanobodies®, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, and portions or fragments thereof. In some cases, the antigen-binding protein is an immunological fragment of a complete antibody (e.g., Fab, Fab', F(ab')2). In other cases, the antigen-binding protein is an scFv that uses CDRs derived from an antibody of the invention.

[0110] In one embodiment, the antigen binding protein has one or more of the following activities: (a) binds to a target antigen such that the KD is ≦200 nM, ≦150 nM, ≦100 nM, ≦50 nM, ≦10 nM, ≦5 nM, ≦2 nM, or ≦1 nM, as measured, for example, via surface plasma resonance or equilibrium exclusion binding techniques. (b) a half-life in human serum of at least 3 days;

[0111] Some of the antigen binding proteins provided have a binding rate (ka) for a target antigen of at least 10, e.g., as measured as described below. 4 / Mx seconds, at least 10 5 / Mx seconds, or at least 10 6 / Mx seconds. Certain antigen-binding proteins provided have slow dissociation or off-rates. Some antigen-binding proteins have dissociation rates of, for example, 1 x 10 -2 seconds -1 , or 1×10 -3 seconds -1 , or 1×10 -4 seconds -1 , or 1×10 -5 seconds -1In certain embodiments, the antigen binding protein has a KD (equilibrium binding affinity) of less than 25 pM, less than 50 pM, less than 100 pM, less than 500 pM, less than 1 nM, less than 5 nM, less than 10 nM, less than 25 nM, or less than 50 nM.

[0112] In another aspect, antigen binding proteins are provided that have an in vitro or in vivo (e.g., when administered to a human subject) half-life of at least 1 day. In one embodiment, the antigen binding protein has a half-life of at least 3 days. In various other embodiments, the antigen binding protein has a half-life of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 days or more. In another embodiment, the antigen binding protein is derivatized or modified to increase its half-life compared to an underivatized or unmodified antibody. In another embodiment, the antigen binding protein comprises point mutations to increase serum half-life. Further details regarding such variants and derivatized forms are provided below.

[0113] Some of the antigen-binding proteins provided typically have a structure associated with naturally occurring antibodies. The structural unit of such antibodies typically comprises one or more tetramers, each composed of two identical couplets of polypeptide chains, although some mammalian species also produce antibodies with only a single heavy chain. In a typical antibody, each pair or couplet comprises one full-length "light" chain (in certain embodiments, approximately 25 kDa) and one full-length "heavy" chain (in certain embodiments, approximately 50-70 kDa). Each individual immunoglobulin chain is composed of several "immunoglobulin domains," each of which consists of approximately 90-110 amino acids and exhibits a characteristic folding pattern. These domains are the basic units that make up antibody polypeptides. The amino-terminal portion of each chain typically contains a variable domain responsible for antigen recognition. The carboxy-terminal portion is more evolutionarily conserved than the other end of the chain and is referred to as the "constant region" or "C region." Human light chains are generally classified as kappa and lambda light chains, each containing one variable domain and one constant domain. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and these chains define the antibody's isotype: IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM and IgM2. IgA subtypes include IgA1 and IgA2. In humans, IgA and IgD isotypes contain four heavy chains and four light chains; IgG and IgE isotypes contain two heavy chains and two light chains; and IgM isotypes contain five heavy chains and five light chains. The C region of the heavy chain typically contains one or more domains that may be responsible for effector function. The number of heavy chain constant region domains depends on the isotype. For example, the heavy chains of IgG each contain three C region domains known as CH1, CH2, and CH3. The provided antibodies can have any of these isotypes and subtypes.In certain embodiments, the antibody is of the IgG1, IgG2, or IgG4 subtype.

[0114] In full-length light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989, New York: Raven Press (incorporated herein by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form the antigen-binding site.

[0115] In the antibodies provided herein, the variable regions of the immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FRs) connected by three hypervariable regions (more often referred to as "complementarity-determining regions" or CDRs). The CDRs from the two chains of each heavy / light chain pair described above are typically aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target antigen. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically have these elements in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids occupying each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883.

[0116] Thus, both the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and the AHo numbering scheme (Honegger A. and Plueckthun AJ Mol Biol. 2001 Jun 8;309(3):657-70) can be used in the present invention. The amino acid positions of a given antibody, as well as its complementarity-determining regions (CDRs) and framework regions (FRs), can be identified using either system. For example, EU heavy chain positions 39, 44, 183, 356, 357, 370, 392, 399, and 409 are identical to AHo heavy chain positions 46, 51, 230, 484, 485, 501, 528, 535, and 551, respectively. Similarly, EU light chain positions 38, 100, and 176 are identical to AHO light chain positions 46, 141, and 230, respectively. Tables 1, 2, and 3 below demonstrate the equivalence between the numbered positions.

[0117] In one aspect, the invention relates to a CH3-containing molecule comprising: (a) a first polypeptide comprising a CH3 domain and a negatively charged domain comprising at least four consecutive negatively charged amino acid residues; and (b) a second polypeptide comprising a CH3 domain and a positively charged domain comprising at least four consecutive positively charged amino acid residues.

[0118] In one embodiment, the CH3 domain and negatively charged domain of the first polypeptide are arranged relative to each other in the N-terminal to C-terminal direction, and the CH3 domain and positively charged domain of the second polypeptide are arranged relative to each other in the N-terminal to C-terminal direction. In one embodiment, each of the first and second polypeptides further comprises a CH2 domain. In one embodiment, each of the first and second polypeptides further comprises a hinge domain. In one embodiment, each of the first and second polypeptides comprises a VH, CH1, hinge, CH2, and CH3 domain arranged relative to each other in the N-terminal to C-terminal direction.

[0119] In one embodiment, the molecule further comprises a third and a fourth polypeptide, wherein the third polypeptide comprises a first VL domain and the fourth polypeptide comprises a second VL domain, hi one embodiment, the third polypeptide further comprises a first CL domain, wherein the first VL and CL domains are arranged relative to each other in the N-terminal to C-terminal direction within the third polypeptide, and the fourth polypeptide further comprises a second CL domain, wherein the second VL and CL domains are arranged relative to each other in the N-terminal to C-terminal direction within the fourth polypeptide.

[0120] Mutations in the heavy and light chains can be introduced to promote proper pairing of the heavy and light chains. In Tables 1, 2, and 3 below, LC-E will pair with HC-K, while LC-K will pair with HC-E. The common negative charge found on LC-E and HC-E will repel these two chains away from each other. Similarly, the common positive charge found on LC-K and HC-K will repel these two chains away from each other. Therefore, pairing between LC-E and HC-K and pairing between LC-K and HC-E will be favored.

[0121] [Table 1]

[0122] [Table 2]

[0123] [Table 3]

[0124] To promote association of a particular heavy chain with its cognate light chain, both the heavy and light chains may contain complementary amino acid substitutions. As used herein, "complementary amino acid substitution" refers to a substitution of a negatively charged amino acid in one chain paired with a substitution of a positively charged amino acid in the other chain. For example, in some embodiments, a heavy chain contains at least one amino acid substitution to introduce a charged amino acid, and the corresponding light chain contains at least one amino acid substitution to introduce a charged amino acid, the charged amino acid introduced in the heavy chain having the opposite charge of the amino acid introduced in the light chain. In certain embodiments, one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the first light chain (LC1) and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into the heavy chain (HC1) that pairs at the LC1 / HC1 binding interface, while one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into the second light chain (LC2) and one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the heavy chain (HC2) that pairs at the LC2 / HC2 binding interface. Electrostatic interactions will induce LC1 to pair with HC1 and LC2 to pair with HC2 due to the attraction of oppositely charged residues (polarity) at the interface. Heavy / light chain pairs with the same charged residues (polarity) at the interface (eg, LC1 / HC2 and LC2 / HC1) will repel each other, resulting in the suppression of unwanted HC / LC pairing.

[0125] In these and other embodiments, the CH1 domain of the heavy chain or the CL domain of the light chain comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more positively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more negatively charged amino acids. Alternatively, the CH1 domain of the heavy chain or the CL domain of the light chain comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more positively charged amino acids. In some embodiments, one or more amino acids in the CH1 domain of the first and / or second heavy chain in the heterodimeric antibody at EU positions selected from F126, P127, L128, A141, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S183, V185, and K213 are replaced with charged amino acids. In certain embodiments, the heavy chain residue for substitution with a negatively or positively charged amino acid is S183 (EU numbering system). In some embodiments, S183 is substituted with a positively charged amino acid. In alternative embodiments, S183 is substituted with a negatively charged amino acid. For example, in one embodiment, S183 is substituted with a negatively charged amino acid (e.g., S183E) in the first heavy chain, and S183 is substituted with a positively charged amino acid (e.g., S183K) in the second heavy chain.

[0126] In embodiments where the light chain is a kappa light chain, one or more amino acids in the CL domain of the first and / or second light chain in the heterodimeric antibody at a position selected from F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174 and S176 (EU numbering for a kappa light chain) are replaced with a charged amino acid. In embodiments where the light chain is a lambda light chain, one or more amino acids in the CL domain of the first and / or second light chain in the heterodimeric antibody at a position selected from T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176, and Y178 (EU numbering in the lambda chain) are replaced with a charged amino acid. In some embodiments, the residue for substitution with a negatively or positively charged amino acid is S176 (EU numbering system) of the CL domain of either a kappa or lambda light chain. In certain embodiments, S176 of the CL domain is replaced with a positively charged amino acid. In alternative embodiments, S176 of the CL domain is replaced with a negatively charged amino acid. In one embodiment, S176 is substituted with a positively charged amino acid (e.g., S176K) in the first light chain and S176 is substituted with a negatively charged amino acid (e.g., S176E) in the second light chain.

[0127] In addition to or instead of complementary amino acid substitutions in the CH1 and CL domains, the light and heavy chain variable regions of a heterodimeric antibody may contain one or more complementary amino acid substitutions to introduce charged amino acids. For example, in some embodiments, the heavy chain VH region or light chain VL region of a heterodimeric antibody comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more positively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more negatively charged amino acids. Alternatively, the heavy chain VH region or light chain VL region comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more positively charged amino acids.

[0128] V region interface residues in the VH region (i.e., amino acid residues that mediate assembly of the VH and VL regions) include EU positions 1, 3, 35, 37, 39, 43, 44, 45, 46, 47, 50, 59, 89, 91, and 93. One or more of these interface residues in the VH region may be substituted with a charged (positively or negatively charged) amino acid. In certain embodiments, the amino acid at EU position 39 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at EU position 39 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In some embodiments, the amino acid at EU position 39 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G39E) and the amino acid at EU position 39 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G39K). In some embodiments, the amino acid at EU position 44 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at EU position 44 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In certain embodiments, the amino acid at EU position 44 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G44E) and the amino acid at EU position 44 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G44K).

[0129] V region interface residues in the VL region (i.e., amino acid residues that mediate assembly of the VH and VL regions) include EU positions 32, 34, 35, 36, 38, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 85, 87, 89, 90, 91, and 100. One or more interface residues in the VL region may be substituted with a charged amino acid, preferably an amino acid with the opposite charge to that introduced into the VH region of the cognate heavy chain. In some embodiments, the amino acid at EU position 100 in the VL region of the first and / or second light chain is substituted with a positively charged amino acid, e.g., lysine. In an alternative embodiment, the amino acid at EU position 100 in the VL region of the first and / or second light chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In certain embodiments, the amino acid at EU position 100 in the VL region of the first light chain is substituted with a positively charged amino acid (e.g., G100K) and the amino acid at EU position 100 in the VL region of the second light chain is substituted with a negatively charged amino acid (e.g., G100E).

[0130] Additionally or alternatively, correct heavy-light chain pairing can be promoted by swapping the CH1 and CL domains in the carboxyl-terminal Fab-binding domain. As an example, a first polypeptide fused to the carboxyl terminus of a heavy chain may comprise the VL and CH1 domains from a second antibody, and a second polypeptide may comprise the VH and CL domains from a second antibody. In another embodiment, a first polypeptide fused to the carboxyl terminus of a heavy chain may comprise the VH and CL domains from a second antibody, and a second polypeptide may comprise the VL and CH1 domains from a second antibody.

[0131] The heavy chain constant region or Fc region of the bispecific antigen-binding proteins described herein may contain one or more amino acid substitutions that affect the glycosylation and / or effector function of the antigen-binding protein. One of the functions of the Fc region of an immunoglobulin is to signal to the immune system when the immunoglobulin binds to its target. This is commonly referred to as "effector function." Signaling leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through binding of the Fc region to Fc receptors on the surface of cells of the immune system. CDC is mediated through binding of Fc to proteins of the complement system, such as C1q. In some embodiments, the bispecific antigen-binding proteins of the invention contain one or more amino acid substitutions in the constant region that enhance effector function, such as ADCC activity, CDC activity, ADCP activity, and / or the clearance or half-life of the antigen-binding protein. Exemplary amino acid substitutions (EU numbering) that can enhance effector function include E233L, L234I, L234Y, L235S, G236A, S239D, F243L, F243V, P247I, D280H, K290S, K290E, K290N, K290Y, R292P, E294L, Y296W, S298A, S298D, S298V, S298G , S298T, T299A, Y300L, V305I, Q311M, K326A, K326E, K326W, A330S, A330L, A330M, A330F, I332E, D333A, E333S, E333A, K334A, K334V, A339D, A339Q, P396L, or any combination thereof.

[0132] In other embodiments, the bispecific antigen binding proteins of the invention comprise one or more amino acid substitutions in the constant region that reduce effector function. Exemplary amino acid substitutions (EU numbering) that can reduce effector function include, but are not limited to, C220S, C226S, C229S, E233P, L234A, L234V, V234A, L234F, L235A, L235E, G237A, P238S, S267E, H268Q, N297A, N297G, V309L, E318A, L328F, A330S, A331S, P331S, or any combination thereof.

[0133] Glycosylation can contribute to the effector function of antibodies, particularly IgG1 antibodies. Thus, in some embodiments, bispecific antigen-binding proteins of the invention may contain one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. Glycosylation of polypeptides is typically N-linked or O-linked. N-linkage refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a sugar moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a sugar of N-acetylgalactosamine, galactose, or xylose to a single hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0134] In certain embodiments, glycosylation of the bispecific antigen binding proteins described herein is increased by adding one or more glycosylation sites, for example, to the Fc region of the binding protein. Addition of glycosylation sites to the antigen binding protein can conveniently be achieved by modifying the amino acid sequence to include one or more of the tripeptide sequences described above (in the case of N-linked glycosylation sites). Modifications can also be made by adding to or substituting one or more serine or threonine residues into the starting sequence (in the case of O-linked glycosylation sites). To facilitate this, the antigen binding protein amino acid sequence can be modified by changes at the DNA level, in particular by mutating the DNA encoding the target polypeptide at preselected bases to generate codons that translate into the desired amino acids.

[0135] The present invention also encompasses the production of bispecific antigen-binding protein molecules with modified carbohydrate structures that result in altered effector activity, e.g., antigen-binding proteins with absent or reduced fucosylation that exhibit improved ADCC activity. Various methods for reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by binding of antibody molecules to the FcγRIII receptor, which has been shown to depend on the carbohydrate structure of N-linked glycosylation at residue N297 in the CH2 domain. Nonfucosylated antibodies bind to this receptor with high affinity and elicit FcγRIII-mediated effector function more efficiently than naturally fucosylated antibodies. For example, recombinant production of nonfucosylated antibodies in CHO cells in which the alpha-1,6-fucosyltransferase enzyme has been knocked out results in antibodies with a 100-fold increase in ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. 87(5):614-22, 2004). A similar effect can be achieved by reducing the activity of the alpha-1,6-fucosyltransferase enzyme or other enzymes in the fucosylation pathway, for example, by siRNA or antisense RNA treatment, engineering cell lines to knock out the enzyme, or culturing with selective glycosylation inhibitors (see Rothman et al., Mol Immunol. 26(12):1113-23, 1989). Some host cell lines, such as the Lec13 or rat hybridoma YB2 / 0 cell lines, naturally produce antibodies with lower fucosylation levels (see Shields et al., J Biol Chem. 277(30):26733-40, 2002 and Shinkawa et al., J Biol Chem. 278(5):3466-73, 2003). Increasing the level of bisected carbohydrate chains, for example by recombinantly producing antibodies in cells overexpressing the GnTIII enzyme, has also been shown to increase ADCC activity (see Umana et al., Nat Biotechnol. 17(2):176-80, 1999).

[0136] In other embodiments, glycosylation of the bispecific antigen-binding proteins described herein is reduced or eliminated by removing one or more glycosylation sites, for example, from the Fc region of the binding protein. N-linked glycosylation of an antigen-binding protein can be reduced or eliminated by amino acid substitutions that eliminate or alter N-linked glycosylation sites. In certain embodiments, the bispecific antigen-binding proteins described herein comprise a mutation at position N297 (EU numbering), such as N297Q, N297A, or N297G. In one particular embodiment, the bispecific antigen-binding protein of the invention comprises an Fc region derived from a human IgG1 antibody with an N297G mutation. To improve the stability of molecules containing the N297 mutation, the Fc region of the molecule can be further engineered. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine ​​to promote disulfide bond formation in the dimeric state. Thus, residues corresponding to V259, A287, R292, V302, L306, V323, or I332 (EU numbering) of the IgG1 Fc region may be substituted with cysteine. In one embodiment, specific pairs of residues are substituted with cysteine ​​to preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond scrambling. In certain embodiments, pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In certain embodiments, the bispecific antigen-binding proteins described herein comprise an Fc region derived from a human IgG1 antibody with the R292C and V302C mutations. In such embodiments, the Fc region may also comprise an N297G mutation.

[0137] It may also be desirable to modify the bispecific antigen binding proteins of the invention to increase serum half-life, for example by incorporating or adding a salvage receptor binding epitope (e.g., by mutating the appropriate region, or by incorporating the epitope into a peptide tag which is then fused to the antigen binding protein at either end or in the middle, for example by DNA or peptide synthesis; see, for example, WO 96 / 32478), or by the addition of a molecule such as PEG or other water soluble polymer, e.g., a polysaccharide polymer. The salvage receptor binding epitope preferably constitutes a region in which any one or more amino acid residues from one or two loops of the Fc region are transferred to a similar position in the antigen binding protein. In one embodiment, three or more residues from one or two loops of the Fc region are transferred. In one embodiment, the epitope is taken from the CH2 domain of the Fc region (e.g., an IgG Fc region) and transferred to the CH1, CH3, or VH region, or two or more such regions, of the antigen binding protein. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL or VL region, or both, of the antigen binding protein. See WO 97 / 34631 and WO 96 / 32478 for a description of Fc variants and their interactions with salvage receptors.

[0138] In one embodiment, the negatively charged domain comprises at least two, at least three, at least four, at least five, at least six, or at least seven consecutive negatively charged amino acid residues, and the positively charged domain comprises at least two, at least three, at least four, at least five, at least six, or at least seven consecutive positively charged amino acid residues.

[0139] In one embodiment, the negatively charged amino acid residue is an aspartic acid residue and the positively charged amino acid residue is selected from the group consisting of lysine, arginine, and histidine residues, hi one embodiment, the negatively charged amino acid residue is an aspartic acid residue and the positively charged amino acid residue is a lysine residue.

[0140] In one embodiment, the negatively charged amino acid residue is a glutamic acid residue and the positively charged amino acid residue is selected from the group consisting of lysine, arginine, and histidine residues.

[0141] In one embodiment, the first polypeptide further comprises a linker sequence covalently attached to the C-terminus of the CH3 domain, wherein the linker is attached to the N-terminus of the negatively charged domain; and the second polypeptide further comprises a linker sequence covalently attached to the C-terminus of the CH3 domain, wherein the linker is attached to the N-terminus of the positively charged domain.

[0142] In one embodiment, the linker of the first polypeptide is the same as the linker of the second polypeptide.

[0143] In one embodiment, the linker may be cleaved by an enzyme, hi one embodiment, the enzyme is selected from the group consisting of sortase A, sortase B, sortase C, sortase D, sortase E, and sortase F.

[0144] In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO:1 (LPETGGEEST); SEQ ID NO:2 (LPXTG, where X can be any amino acid); SEQ ID NO:3 (LPETG); SEQ ID NO:4 (LPETGG); SEQ ID NO:5 (LPXTA, where X can be any amino acid); SEQ ID NO:6 (NPX[T / S][N / G / S], where X can be any amino acid); SEQ ID NO:7 (IPXTG, where X can be any amino acid); and SEQ ID NO:8 (LAXTG, where X can be any amino acid).

[0145] In one embodiment, either the first polypeptide, the second polypeptide, or both, further comprise a purification tag attached to its C-terminus.

[0146] In one embodiment, the purification tag is selected from the group consisting of a his-tag, a strep-tag, a flag-tag, a T7-tag, a V5-peptide-tag, a GST-tag, a CBP-tag, a MBP-tag, and a c-Myc-tag, hi one embodiment, the purification tag is a his-tag comprising at least five consecutive histidine amino acid residues.

[0147] In one embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 9 (LPETGGEESTDDDDDDD) and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 10 (LPETGGEESTKKKKKKKHHHHHH).

[0148] In one embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11 (LPETGGEESTDDDDDDDHHHHHH) and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12 (LPETGGEESTKKKKKKK).

[0149] In one aspect, the invention relates to a multispecific antibody comprising a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, and a second light chain polypeptide, the first heavy chain polypeptide comprises a CH3 domain and a negatively charged domain comprising at least four consecutive negatively charged amino acid residues, and the first heavy chain polypeptide and the first light chain polypeptide bind to a first antigen; and The second heavy chain polypeptide comprises a CH3 domain and a positively charged domain comprising at least four consecutive positively charged amino acid residues, and the second heavy chain polypeptide and the second light chain polypeptide bind to a second antigen.

[0150] In one embodiment, the multispecific antibody is expressed by a mammalian cell. In one embodiment, the mammalian cell is a HEK cell or a CHO cell.

[0151] In one aspect, the invention relates to a method for producing a multispecific antibody, the method comprising culturing cells comprising a vector encoding a multispecific antibody of the invention in a culture medium.

[0152] In one embodiment, the method further comprises recovering the multispecific antibody from the cells or culture medium.

[0153] In one aspect, the present invention provides a method for making an antibody conjugate, comprising: a) providing a multispecific antibody according to any one of claims 40 or 41; and b) treating the antibody with a sortase enzyme in the presence of a synthetic molecule, the molecule comprising a gly-gly-gly sequence. In one embodiment, step b) is performed using a synthetic molecule:antibody molar ratio of about 2 to about 1000. [Example]

[0154] Generation of conjugated bispecific antibodies via a sortase-cleavable C-terminal charge-pair tag Construct Design: Heavy chain:heavy chain (HC:HC) pairing: Constructs were designed for two model bispecific antibodies, TNFα / TL1A and PAC1 / CGRP1. Both the TNFα and PAC1 heavy chains were extended to contain a sortase recognition sequence (LPETG (SEQ ID NO: 13)), a short sortase-friendly linker (GEEST (SEQ ID NO: 14)), and seven aspartic acid residues, generating a 17-residue C-terminal extension: LPETGGEESTDDDDDDD (SEQ ID NO: 15).

[0155] Both the TL1A and CGRP1 heavy chains were extended to contain a sortase recognition sequence (LPETG (SEQ ID NO: 13)), generating a 23-residue C-terminal extension, a short sortase-friendly linker (GEEST (SEQ ID NO: 14)), seven lysine residues, and six histidine residues. LPETGGEESTKKKKKKKHHHHHH (SEQ ID NO: 16)

[0156] These terminal extensions were added to existing antibody constructs via site-directed mutagenesis using standard methods (Carrigan et al. 2011).

[0157] Heavy chain:light chain (HC:LC) pairing: The correct HC:LC pairing was designed according to (Liu et al. 2015; Florio et al. 2016).

[0158] Full-length gene sequence for TNFα / TL1A dual specificity: TNFα heavy chain: [ka]

[0159] TNFα light chain: [ka]

[0160] TL1A heavy chain: [ka]

[0161] TL1A light chain: [ka]

[0162] Full-length gene sequence for PAC1 / CGRP1: PAC1 heavy chain: [ka]

[0163] PAC1 light chain: [ka]

[0164] CGRP1 heavy chain: [ka]

[0165] CGRP1 light chain: [ka]

[0166] Cloning Genes for all heavy chains were cloned into the vector pTT5.2 using the Golden Gate method (Engler et al. 2008), whereas genes for TNFα and TL1A light chains were cloned into pTT5.1, and PAC1 and CGRP1 light chains were cloned into pTT5.

[0167] Transient HEK 293-6e cell expression HEK 293-6e cells were grown to a VCD of 1.5e6 in FreeStyle F-17 medium (10 mL / L of 10%), Kolliphor P188, 500 μL / L G418, and 6 mM (30 mL / L) L-glutamine at 36°C and 5% CO2. After shaking in flasks at 150 rpm, transfection complexes consisting of FreeStyle F-17 medium (10% of the final culture volume), 0.5 mg / L transfection DNA (1.25 g / L per strand), and 2.0 mg / L PEI Max (pH 7.0) were added (Longo et al. 2013). Three hours after the initial transfection, the cultures were fed with yeastolate to a final concentration of 0.5% w / v and 2.5 g / L glucose.

[0168] Transiently transfected cells were incubated at 37°C, 5% CO2, and shaken in flasks at 150 rpm for 7 days before being harvested by centrifugation at 4000 RPM for 10 minutes. Conditioned medium from each harvest was then collected and vacuum filtered through a 0.22 μm bottle-top filter.

[0169] purification Antibodies were purified from conditioned medium by ProA capture on HiTrap ProA HP resin (GE) and eluted in 100 mM sodium acetate, pH 3.6. 10–20 ml of ProA column eluate was adjusted to pH 5.0 by adding 100–600 μl of 1 M TRIS-HCl, pH 9. The elution fractions were pooled and diluted two-fold to lower the conductivity in preparation for loading onto a cation exchange column. The diluted ProA eluate was loaded onto a HiTrap SP column (GE) pre-equilibrated in 20 mM sodium acetate, pH 5.0, and eluted with a linear gradient of 0–1 M NaCl over 20 column volumes. The highly charged C-terminal extension allowed baseline separation between the desired bispecific heterodimeric antibody (75–85% of the generated material) and monospecific homodimeric antibody contaminants. The CEX eluate was buffer exchanged into 10 mM sodium acetate + 9% sucrose pH 5 through stepwise concentration and dilution in 15 mL 30,000 MWCO centrifugal filters (Millipore) until a dilution factor of >50,000 was reached.

[0170] Sortase cleavage reaction The sortase cleavage reaction, which removes the charge-pair tag via SrtA-mediated hydrolysis (Jacobitz et al. 2017; Ton-That, H., Mazmanian, S.K., Alksne, L., and Schneewind 2000; Clancy et al. 2010), was carried out at 37°C for 24 h using 10 μM bispecific antibody and 20 μM SrtA2.0 (123 Bio) in 50 mM TRIS-HCl, pH 5 (where the low pH was specifically chosen to reduce unwanted SrtA-catalyzed isopeptide bond formation to Lys side chains in the poly-K tail (Dasgupta et al. 2011)). To our knowledge, removal of C-terminal tags from proteins by SrtA has not been reported elsewhere. Furthermore, ours is the first report demonstrating efficient SrtA cleavage of tags at pH 5.0. Typically, SrtA-mediated cleavage is performed at pH 7.0–8.5. The progress of the reaction was monitored by analytical CEX, and the reaction products were verified by LCMS. After 24 h, the reaction typically reached 85-90% completion. The fully detagged bispecific antibody could then be purified from residual tag molecules and sortase by cation exchange chromatography (CEX) as described above.

[0171] Conjugation of bispecific antibodies with molecular entities Sortase transpeptidation reaction Sortase-mediated transpeptidation (Jacobitz et al. 2017; Levary et al. 2011; Mao et al. 2004; Ranganath Parthasarathy et al. 2007; Swee et al. 2013; Popp et al. 2007; Proft 2010; Spirig et al. 2011; Clancy et al. 2010; Antos et al. 2016) was used to remove the charge-pair tags and replace them with various synthetic molecules containing an internal Gly3 sequence for sortase recognition, including Gly3, Gly3-biotin, Gly3-PEG20, Gly3-PEG20kD, and Gly3-Alexa650. Reactions were carried out for 24 hours at 37°C in 50 mM TRIS HCl, pH 6.5, using 10-100 μM bispecific antibody with 0.1-10% w / w SrtA2.0 (123 Bio) in a 10-100-fold molar excess of synthetic substrate:bispecific antibody. Reaction progress was monitored by analytical CEX, and products were analyzed by LCMS. Covalent attachment of a fluorescent Gly3-Alexa650 probe via sortase-mediated transpeptidation was further verified by the appearance of a fluorescent band at the expected molecular weight via SDS-PAGE. After 24 hours, reactions typically reached 85-99% yield. Transpeptidation products were purified from unreacted material and sortase through cation exchange chromatography as described above.

[0172] Generation of "clickable" bispecific antibodies via sortase-mediated transpeptidation with non-standard nucleophiles and subsequent covalent attachment of fluorescent tags by click chemistry An additional sortase-mediated transpeptidation reaction was performed to demonstrate the ability of sortase to covalently attach non-standard nucleophiles containing click chemistry handles (Ranganath Parthasarathy et al. 2007; Ton-That, H., Mazmanian, S.K., Alksne, L., and Schneewind 2000) to the C-terminus of the bispecific. This clickable bispecific could then be used to covalently attach a synthetic fluorophore via azide-DIBO click chemistry. 50 μM of PAC1 / CGRP1 bispecific antibody was incubated with 5 mM NH2-PEG3-N3 and 5 μM SrtA2.0 (123 Bio) in 25 mM TRIS HCl pH 6.5 at 37°C for 24 hours. The reaction progress was monitored by CEX, with a final yield of 87% after 24 hours. The formation of the expected product species was verified by LCMS. The reaction product was removed from unreacted starting materials by CEX using the method detailed above. After incubating the resulting azide-functionalized bispecific with a 10-fold molar excess of DIBO-Alexa 488 at 1 μM for 24 h at room temperature, covalent crosslinking between the fluorophore and the newly formed bispecific was confirmed by the appearance of a fluorescent band at the correct molecular weight on an SDS-PAGE gel and further verified by LCMS.

[0173] All patents, patent applications, published patent applications, and other publications cited or referenced in this specification are herein incorporated by reference to the same extent as if each individual patent, patent application, published patent application, or publication was specifically and individually indicated to be incorporated by reference.

[0174] References Antos,J.M.,Truttmann,M.C.& Ploegh,H.L.,2016.Recent advances in sortase-catalyzed ligation methodology.Current Opinion in Structural Biology,38,pp.111-118.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 27318815[Accessed October 4,2017]. Carrigan,P.E.,Ballar,P.& Tuzmen,S.,2011.Site-Directed Mutagenesis.In J.K.DiStefano,ed.Disease Gene Identification:Methods and Protocols.Totowa,NJ:Humana Press,Totowa,NJ,pp.107-124.Available at:http: / / link.springer.com / 10.1007 / 978-1-61737-954-3_8[Accessed October 4,2017]. Clancy,K.W.,Melvin,J.A.& McCafferty,D.G.,2010.Sortase transpeptidases:insights into mechanism,substrate specificity,and inhibition.Biopolymers,94(4),pp.385-396.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 20593474. Dasgupta,S.et al.,2011.Isopeptide ligation catalyzed by quintessential sortase A:mechanistic cues from cyclic and branched oligomers of indolicidin.The Journal of biological chemistry,286(27),pp.23996-4006.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 21566128[Accessed October 4,2017]. Engler,C.et al.,2008.A One Pot,One Step,Precision Cloning Method with High Throughput Capability H.A.El-Shemy,ed.PLoS ONE,3(11),p.e3647.Available at:http: / / dx.plos.org / 10.1371 / journal.pone.0003647[Accessed July 12,2017]. Florio,M.et al.,2016.A bispecific antibody targeting sclerostin and DKK-1 promotes bone mass accrual and fracture repair.Nature communications,7,p.11505.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 27230681[Accessed October 4,2017]. Jacobitz,A.W.et al.,2017.Sortase Transpeptidases:Structural Biology and Catalytic Mechanism.In Advances in Protein Chemistry and Structural Biology.Available at:http: / / www.sciencedirect.com / science / article / pii / S1876162317300391[Accessed June 13,2017]. Levary,D.A.et al.,2011.Protein-Protein Fusion Catalyzed by Sortase A J.Najbauer,ed.PLoS ONE,6(4),p.e18342.Available at:http: / / dx.plos.org / 10.1371 / journal.pone.0018342[Accessed December 16,2016]. Liu,Z.et al.,2015.A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.The Journal of biological chemistry,290(12),pp.7535-62.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 25583986[Accessed October 4,2017]. Longo,P.A.et al.,2013.Transient mammalian cell transfection with polyethylenimine(PEI).Methods in enzymology,529,pp.227-40.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 24011049[Accessed January 3,2017]. Mao,H.et al.,2004.Sortase-mediated protein ligation:a new method for protein engineering.Journal of the American Chemical Society,126(9),pp.2670-1.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 14995162[Accessed July 1,2015]. Popp,M.W.et al.,2007.Sortagging:a versatile method for protein labeling.Nature Chemical Biology,3(11),pp.707-708.Available at:http: / / www.nature.com / doifinder / 10.1038 / nchembio.2007.31[Accessed May 23,2017]. Proft,T.,2010.Sortase-mediated protein ligation:an emerging biotechnology tool for protein modification and immobilisation.Biotechnology Letters,32(1),pp.1-10.Available at:http: / / link.springer.com / article / 10.1007 / s10529-009-0116-0[Accessed May 23,2013]. Ranganath Parthasarathy,†,Shyamsundar Subramanian,† and & Eric T.Boder*,†,‡,2007.Sortase A as a Novel Molecular “Stapler” for Sequence-Specific Protein Conjugation. Spirig,T.,Weiner,E.M.& Clubb,R.T.,2011.Sortase enzymes in Gram-positive bacteria.Molecular microbiology,82(5),pp.1044-59.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 22026821[Accessed December 20,2012]. Swee,L.K.et al.,2013.Sortase-mediated modification of αDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes.Proceedings of the National Academy of Sciences of the United States of America,110(4),pp.1428-33.Available at:http: / / www.ncbi.nlm.nih.gov / pubmed / 23297227[Accessed December 16,2016]. Ton-That,H.,Mazmanian,S.K.,Alksne,L.,and Schneewind,O.,2000.Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus.SORTASE CATALYZED IN VITRO TRANSPEPTIDATION REACTION USING LPXTG PEPTIDE AND NH2-GLY3 SUBSTRATES.Journal of Biological Chemistry,275(13),pp.9876-9881.Available at:http: / / www.jbc.org / content / 275 / 13 / 9876.full[Accessed June 9,2015].

Claims

1. A CH3-containing molecule, (a) a first polypeptide comprising a CH3 domain and a negatively charged domain comprising at least four consecutive negatively charged amino acid residues; and (b) a second polypeptide comprising a CH3 domain and a positively charged domain comprising at least four consecutive positively charged amino acid residues; A CH3-containing molecule comprising:

2. 2. The CH3-containing molecule of claim 1, wherein the CH3 domain and the negatively charged domain of the first polypeptide are arranged relative to each other in an N-terminal to C-terminal direction, and the CH3 domain and the positively charged domain of the second polypeptide are arranged relative to each other in an N-terminal to C-terminal direction.

3. The CH3-containing molecule of claim 1 or 2, wherein each of the first and second polypeptides further comprises a CH2 domain.

4. The CH3-containing molecule of any one of claims 1 to 3, wherein each of the first and second polypeptides further comprises a hinge domain.

5. The CH3-containing molecule of any one of claims 1 to 4, wherein each of the first and second polypeptides comprises a VH, CH1, hinge, CH2, and CH3 domains arranged relative to each other in the N-terminal to C-terminal direction.

6. 6. The CH3-containing molecule of any one of claims 1 to 5, wherein the molecule further comprises a third and a fourth polypeptide, the third polypeptide comprising a first VL domain, and the fourth polypeptide comprising a second VL domain.

7. 7. The CH3-containing molecule of claim 6, wherein the third polypeptide further comprises a first CL domain, and the first VL and CL domains are arranged relative to each other in the N-terminal to C-terminal direction within the third polypeptide, and the fourth polypeptide further comprises a second CL domain, and the second VL and CL domains are arranged relative to each other in the N-terminal to C-terminal direction within the fourth polypeptide.

8. 8. The CH3-containing molecule of claim 1, wherein the negatively charged domain comprises at least five consecutive negatively charged amino acid residues and the positively charged domain comprises at least five consecutive positively charged amino acid residues.

9. 9. The CH3-containing molecule of claim 1, wherein the negatively charged domain comprises at least six consecutive negatively charged amino acid residues and the positively charged domain comprises at least six consecutive positively charged amino acid residues.

10. 10. The CH3-containing molecule of any one of claims 1 to 9, wherein the negatively charged domain comprises at least seven consecutive negatively charged amino acid residues and the positively charged domain comprises at least seven consecutive positively charged amino acid residues.

11. 11. The CH3-containing molecule of any one of claims 1 to 10, wherein the negatively charged amino acid residue is an aspartic acid residue and the positively charged amino acid residue is selected from the group consisting of lysine, arginine, and histidine residues.

12. 12. The CH3-containing molecule of claim 1, wherein the negatively charged amino acid residue is an aspartic acid residue and the positively charged amino acid residue is a lysine residue.

13. 13. The CH3-containing molecule of any one of claims 1 to 12, wherein the negatively charged amino acid residue is a glutamic acid residue and the positively charged amino acid residue is selected from the group consisting of lysine, arginine, and histidine residues.

14. the first polypeptide further comprises a linker sequence covalently attached to the C-terminus of the CH3 domain, and the linker is attached to the N-terminus of the negatively charged domain; and 14. The CH3-containing molecule of any one of claims 1 to 13, wherein the second polypeptide further comprises a linker sequence covalently attached to the C-terminus of the CH3 domain, and the linker is attached to the N-terminus of the positively charged domain.

15. The CH3-containing molecule of claim 14, wherein the linker of the first polypeptide is the same as the linker of the second polypeptide.

16. The CH3-containing molecule of claim 15, wherein the linker can be cleaved by an enzyme.

17. 17. The CH3-containing molecule of claim 16, wherein the enzyme is selected from the group consisting of sortase A, sortase B, sortase C, sortase D, sortase E, and sortase F.

18. 18. The CH3-containing molecule of claim 17, wherein the enzyme is sortase A.

19. 19. The CH3-containing molecule of any of claims 14-18, wherein the linker comprises the amino acid sequence of SEQ ID NO:1 (LPETGGEEST); SEQ ID NO:2 (LPXTG, where X can be any amino acid); SEQ ID NO:3 (LPETG); SEQ ID NO:4 (LPETGG); SEQ ID NO:5 (LPXTA, where X can be any amino acid); SEQ ID NO:6 (NPX[T / S][N / G / S], where X can be any amino acid); SEQ ID NO:7 (IPXTG, where X can be any amino acid); and SEQ ID NO:8 (LAXTG, where X can be any amino acid).

20. The CH3-containing molecule of any one of claims 1 to 19, wherein either the first polypeptide, the second polypeptide, or both further comprise a purification tag attached to its C-terminus.

21. 21. The CH3-containing molecule of claim 20, wherein the purification tag is selected from the group consisting of a his-tag, a strep-tag, a flag-tag, a T7-tag, a V5-peptide-tag, a GST-tag, a CBP-tag, an MBP-tag, and a c-Myc-tag.

22. The CH3-containing molecule of claim 21, wherein the purification tag is a his-tag containing at least five consecutive histidine amino acid residues.

23. 23. The CH3-containing molecule of any one of claims 1 to 22, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 9 (LPETGGEESTDDDDDDDD), and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 10 (LPETGGEESTKKKKKKKHHHHHH).

23. 23. The CH3-containing molecule of any one of claims 1 to 22, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 11 (LPETGGEESTDDDDDDDHHHHHH), and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 12 (LPETGGEESTKKKKKKKK).

24. 1. A multispecific antibody comprising a first heavy chain polypeptide, a first light chain polypeptide, a second heavy chain polypeptide, and a second light chain polypeptide, the first heavy chain polypeptide comprises a CH3 domain and a negatively charged domain comprising at least four consecutive negatively charged amino acid residues, and the first heavy chain polypeptide and the first light chain polypeptide bind to a first antigen; and a second heavy chain polypeptide comprising a CH3 domain and a positively charged domain comprising at least four consecutive positively charged amino acid residues, and wherein the second heavy chain polypeptide and the second light chain polypeptide bind to a second antigen.

25. 3. The multispecific antibody of claim 2, wherein the CH3 domain and the negatively charged domain of the first heavy chain polypeptide are arranged relative to each other in an N-terminal to C-terminal direction, and the CH3 domain and the positively charged domain of the second heavy chain polypeptide are arranged relative to each other in an N-terminal to C-terminal direction.

26. 26. The multispecific antibody of claim 24 or 25, wherein the negatively charged domain comprises at least 5 consecutive negatively charged amino acid residues and the positively charged domain comprises at least 5 consecutive positively charged amino acid residues.

27. 27. The multispecific antibody of claim 26, wherein the negatively charged domain comprises at least 6 consecutive negatively charged amino acid residues and the positively charged domain comprises at least 6 consecutive positively charged amino acid residues.

28. 28. The multispecific antibody of claim 27, wherein the negatively charged domain comprises at least 7 consecutive negatively charged amino acid residues and the positively charged domain comprises at least 7 consecutive positively charged amino acid residues.

29. 29. The multispecific antibody of any of claims 24 to 28, wherein the negatively charged amino acid residue is an aspartic acid residue and the positively charged amino acid residue is selected from the group consisting of lysine, arginine, and histidine residues.

30. 30. The multispecific antibody of any of claims 24 to 29, wherein the negatively charged amino acid residue is an aspartic acid residue and the positively charged amino acid residue is a lysine residue.

31. 29. The multispecific antibody of any of claims 24 to 28, wherein the negatively charged amino acid residue is a glutamic acid residue and the positively charged amino acid residue is selected from the group consisting of lysine, arginine, and histidine residues.

32. the first heavy chain polypeptide further comprises a linker sequence covalently attached to the C-terminus of the CH3 domain, and the linker is attached to the N-terminus of the negatively charged domain; and 32. The multispecific antibody of any of claims 24 to 31, wherein the second heavy chain polypeptide further comprises a linker sequence covalently attached to the C-terminus of the CH3 domain, and wherein the linker is attached to the N-terminus of the positively charged domain.

33. 33. The multispecific antibody of claim 32, wherein the linker of the first heavy chain polypeptide is the same as the linker of the second heavy chain polypeptide.

34. 34. The multispecific antibody of claim 33, wherein the linker is enzymatically cleavable.

35. 35. The multispecific antibody of claim 34, wherein the enzyme is selected from the group consisting of sortase A, sortase B, sortase C, sortase D, sortase E, and sortase F.

36. 36. The multispecific antibody of claim 35, wherein the enzyme is sortase A.

37. 37. The multispecific antibody of any of claims 32 to 36, wherein the linker comprises the amino acid sequence of SEQ ID NO: 1 (LPETGGEEST); SEQ ID NO: 2 (LPXTG, X can be any amino acid); SEQ ID NO: 3 (LPETG); SEQ ID NO: 4 (LPETGG); SEQ ID NO: 5 (LPXTA, X can be any amino acid); SEQ ID NO: 6 (NPX[T / S][N / G / S], X can be any amino acid); SEQ ID NO: 7 (IPXTG, X can be any amino acid); and SEQ ID NO: 8 (LAXTG, X can be any amino acid).

38. The multispecific antibody of any of claims 32 to 37, wherein either the first heavy chain polypeptide, the second heavy chain polypeptide, or both, further comprise a purification tag attached to its C-terminus.

39. 39. The multispecific antibody of claim 38, wherein the purification tag is selected from the group consisting of his-tag, strep-tag, flag-tag, T7-tag, V5-peptide-tag, GST-tag, CBP-tag, MBP-tag and c-Myc-tag.

40. The multispecific antibody of claim 39, wherein the purification tag is a his-tag comprising at least five consecutive histidine amino acid residues.

41. 41. The multispecific antibody of any of claims 32 to 40, wherein the first heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 9 (LPETGGEESTDDDDDDDD), and the second heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 10 (LPETGGEESTKKKKKKKHHHHHH).

41. 41. The multispecific antibody of any of claims 32 to 40, wherein the first heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 11 (LPETGGEESTDDDDDDDHHHHHH), and the second heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 12 (LPETGGEESTKKKKKKKK).

42. The multispecific antibody of any of claims 32 to 41, wherein the multispecific antibody is expressed by a mammalian cell.

43. 43. The multispecific antibody of claim 42, wherein the mammalian cell is a HEK or CHO cell.

44. 42. A method for producing a multispecific antibody, comprising culturing a cell comprising a vector encoding the multispecific antibody of any of claims 32 to 41 in a culture medium.

45. 45. The method of claim 44, wherein the method further comprises recovering the multispecific antibody from the cells or the culture medium.

46. 1. A method of making an antibody conjugate, comprising: a) providing a multispecific antibody according to any one of claims 40 or 41; and b) treating said antibody with a sortase enzyme in the presence of a synthetic molecule, said molecule comprising a gly-gly-gly sequence.

47. 47. The method of claim 46, wherein step b) is carried out using a synthetic molecule:antibody molar ratio of about 2 to about 1000.

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