Generation of hetromultimeric proteins using mammalian cells

Abstract

To provide methods for efficiently producing antibodies and other multimeric protein complexes capable of specifically binding to plural targets.SOLUTION: Disclosed is a method for preparing a heteromultimeric protein that comprises a first hinge-containing polypeptide comprising a first heterodimerization domain bound to a first light chain, and a second hinge-containing polypeptide comprising a second heterodimerization domain bound to a second light chain, where the second heterodimerization domain interacts with the first heterodimerized domain, the first and second hinge-containing polypeptides are linked via an interchain disulfide bond, where the method comprises the steps of: culturing a first host cell expressing the first hinge-containing polypeptide and the first light chain; culturing a second host cell expressing the second hinge-containing polypeptide and the second heavy chain; and obtaining a mixed culture medium of the first and second host cells without disrupting the cell membranes of the first and second host cells.SELECTED DRAWING: Figure 1B

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 61 / 989,509, filed May 6, 2014, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a method for producing a heteromultimeric protein. [Background technology]

[0003] Monoclonal antibodies of the IgG type contain two identical antigen-binding arms and a constant domain (Fc). Antibodies with different specificities in their binding arms usually do not exist in nature and therefore must be created with the aid of chemical engineering (e.g., chemical cross-linking), recombinant DNA and / or cell fusion techniques.

[0004] Bispecific antibodies can bind to two different antigens simultaneously. This property allows the development of therapeutic strategies that are not possible with conventional monoclonal antibodies. The large panel of imaginable bispecific antibody formats that have been developed reflects the intense interest in such molecules. See Berg J, Lotscher E, Steimer KS, et al., "Bispecific antibodies that mediate killing of cells infected with human immunodeficiency virus of any strain," Proc Natl Acad Sci USA (1991) 88(11): 4723-4727 and Fischer N and Leger O., "Biospecific antibodies: Molecules That Enable Novel Therapeutic Strategies," Pathobiology (2007) 74:3-14.

[0005] Another class of multispecific molecules is recombinant fusion proteins. Recombinant fusion proteins consisting of the extracellular domain of an immunomodulatory protein and the constant (Fc) domain of an immunoglobulin (Ig) are a growing field of human therapeutics. Immunoadhesins combine the binding region of a protein sequence with the effector domain of an antibody with the desired specificity. Immunoadhesins have two important properties that are significant for their therapeutic potential: target specificity and pharmacokinetic stability (half-life in vivo equivalent to that of an antibody). Immunoadhesins can be used as antagonists to inhibit or block deleterious interactions, or as agonists to mimic or enhance physiological responses. See Chamow SM, Zhang DZ, Tan XY, et al., "A humanized, bispecific immunoadhesin-antibody that retargets CD3+ effectors to kill HIV-1-infected cells," J Hematother 1995; 4(5): 439-446.

[0006] Other multispecific molecules have been discussed elsewhere. Examples include, but are not limited to, Fisher et al., Pathobiology (2007) 74:3-14 (review of various bispecific formats); U.S. Patent No. 6,660,843 by Feige et al., issued December 9, 2003 (peptibodies); U.S. Patent Application Publication No. 2002-004587, issued January 10, 2002 (multispecific antibodies); U.S. Patent No. 7,612,181 by Wu et al., issued November 3, 2009 (dual variable domain formats); U.S. Patent No. 6,534,628 by Nord K et al., Prot Eng (1995) 8:601-608, Nord K et al., Nat Biotech (1997) 15:772-777, and Gronwall et al., Biotechnol Appl Biochem. (2008) Jun;50 (Pt 2):97-112 (affibodies); Martens et al., Clin Cancer Res (2006), 12: 6144-6152 and Jin et al., Cancer Res (2008) 68(11):4360-4368 (one-arm antibodies); Bostrom et al., Science (2009) 323:1610-1614 (dual-acting Fab, aka mixed valency antibodies). Other formats are known to those of skill in the art.

[0007] The production of clinical grade material remains challenging for such multispecific molecules. As mentioned above, there are numerous routes to the generation of molecules with mixed binding arms, i.e., binding arms that are not identical to each other. Each of these methods has its drawbacks.

[0008] Chemical cross-linking is laborious because it requires purification of related species from homodimers and other undesired by-products. In addition, the chemical modification process can alter the integrity of the protein, leading to instability. Thus, this method is often inefficient and can lead to loss of antibody activity.

[0009] Cell fusion techniques (e.g., hybrid hybridomas) express two heavy and two light chains that randomly assemble to give rise to 10 antibody combinations. The desired heteromultimeric antibodies represent only a small proportion of the antibodies thus produced. Purification of the desired heteromultimeric proteins dramatically reduces production yields and increases manufacturing costs.

[0010] Recombinant DNA technology has been used to produce various heteromultimeric formats, such as single chain Fvs, diabodies, etc., that do not contain an Fc domain. A major drawback of this type of antibody molecule is the lack of an Fc domain and the resulting lack of the antibody's ability to trigger effector functions (e.g., complement activation, Fc-receptor binding, etc.). Therefore, bispecific antibodies that contain a functional Fc domain are desirable.

[0011] Recombinant DNA technology has also been used to produce "knobs-into-holes" bispecific antibodies. See U.S. Patent Application Publication No. 20030078385 (Arathoon et al. - Applicants). A limitation of this strategy is that the light chains of the two parent antibodies must be identical to prevent mispairing and the formation of unwanted and / or inactive molecules due to their expression in the same cell.

[0012] Thus, there remains a need for alternative methods for producing heteromultimeric proteins. The invention described herein provides such methods. These and other aspects of the invention will become apparent from the description of the invention provided herein. Summary of the Invention

[0013] The present invention provides efficient and novel methods for producing multispecific immunoglobulin complexes (e.g., multispecific antibodies) and other multimeric proteins (collectively referred to herein as heteromultimeric proteins) in mammalian cells that go beyond methods known in the art, see WO 2013 / 055958 and WO 2011 / 133886.

[0014] Thus, in a first aspect, there is provided a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell capable of expressing a first hinge-containing polypeptide and a first light chain; (B) culturing a second host cell capable of expressing a second hinge-containing polypeptide and a second light chain; and (c) obtaining a mixed culture medium of the first host cell and the second host cell; wherein the first host cell and the second host cell are each a mammalian cell. In certain embodiments, the mixed culture medium is obtained without disrupting the cell membranes of the first and second host cells. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0015] Also disclosed is a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, wherein: (a) culturing a first host cell capable of expressing a first hinge-containing polypeptide and a first light chain, wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted from the first host cell; (b) culturing a second host cell capable of expressing the second hinge-containing polypeptide and the second light chain, wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted from the second host cell; (c) obtaining a mixed culture medium of the first host cell and the second host cell without disrupting the cell membranes of the first and second host cells, wherein the mixed culture medium comprises the first homodimer and the second homodimer; (d) incubating the mixed culture medium under reducing conditions sufficient to allow for the formation of heteromultimeric proteins; and (e) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium. In certain embodiments according to (or as applied to) any of the above embodiments, the first hinge-containing polypeptide and the second hinge-containing polypeptide comprise first and second heavy chains. In certain embodiments according to (or as applied to) any of the embodiments described herein, the first hinge-containing polypeptide and the first light chain comprise a first half antibody. In certain embodiments according to (or as applied to) any of the embodiments described herein, the second hinge-containing polypeptide and the second light chain comprise a second half antibody.

[0016] In certain embodiments according to (or as applied to) any of the above embodiments, the step of obtaining the mixed culture medium includes: (1) collecting a first culture medium for the first host cell culture; (2) harvesting a second culture medium for the second host cell culture; and (3) Mixing the first culture medium and the second culture medium to obtain a mixed culture medium. Includes.

[0017] In certain embodiments according to (or as applied to) any of the above embodiments, obtaining the mixed culture medium comprises recovering a culture medium of a mixed cell culture comprising a first host cell and a host cell, In certain embodiments, the mixed culture medium is obtained without disrupting the cell membranes of the first and second host cells.

[0018] The first and second host cells in the methods of the invention can be cultured in any environment that allows for the expression and isolation of the polypeptide of interest. In certain embodiments according to (or as applied to) any of the above embodiments, the first host cell and the second host cell are cultured separately before being combined into a mixed cell culture.

[0019] In certain embodiments according to (or as applied to) any of the above embodiments, the method further comprises culturing the mixed cell culture at a temperature of about 25° C. to about 40° C. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium is incubated for about 24 hours to about 7 days after the mixed culture medium is obtained. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium is incubated at about 4° C. to about 8° C. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium is stirred.

[0020] In certain embodiments according to (or as applied to) any of the above embodiments, the method further comprises isolating the heteromultimeric protein from the mixed culture medium. In certain embodiments according to (or as applied to) any of the above embodiments, the heteromultimeric protein is isolated using a Protein A column. In certain embodiments, the heteromultimeric protein is further purified using methods known in the art.

[0021] In certain embodiments according to (or as applied to) any of the above embodiments, the method further comprises adding a reducing agent to the first cell culture medium and / or the second cell culture medium before or after the first and second cell culture media are harvested. In certain embodiments according to (or as applied to) any of the above embodiments, the method further comprises adding a reducing agent to the culture medium of the mixed cell culture before the culture medium of the mixed cell culture is harvested. In certain embodiments according to (or as applied to) any of the above embodiments, the reducing agent is added about 4 to about 24 hours, about 5 to about 20 hours, about 10 to about 20 hours, about 10 to about 15 hours, or about 15 to about 18 hours before the harvesting step. In certain embodiments according to (or as applied to) any of the above embodiments, the reducing agent is added about 15 hours before the harvesting step.

[0022] In certain embodiments according to (or as applied to) any of the above embodiments, the method further comprises adding a reducing agent to the mixed cell medium. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium containing the reducing agent is further incubated for about 4 hours to about 7 days. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium containing the reducing agent is further incubated for about 15 hours. In certain embodiments according to (or as applied to) any of the above embodiments, the reducing agent is added to the mixed culture medium prior to isolating the heteromultimeric protein from the mixed culture medium. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium containing the reducing agent is incubated for at least about 24 hours prior to isolating the heteromultimeric protein. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium containing the reducing agent is incubated for at least about 48 hours prior to isolating the heteromultimeric protein. In certain embodiments according to (or as applied to) any of the above embodiments, the heteromultimeric protein is isolated using a Protein A column.

[0023] In certain embodiments according to (or as applied to) any of the above embodiments, the reducing agent is selected from the group consisting of glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), cysteine, cysteine, dithiothreitol, cysteine ​​dithiothreitol, dithiolbutylamine, or combinations thereof. In certain embodiments according to (or as applied to) any of the above embodiments, the reducing agent is glutathione, and the glutathione is added at a concentration of about 5 mM to about 20 mM or less. In certain embodiments according to (or as applied to) any of the above embodiments, the reducing agent is glutathione, and the glutathione is added at a concentration of about 2 mM to about 10 mM. In certain embodiments according to (or as applied to) any of the above embodiments, the reducing agent is glutathione, and the glutathione is added at a concentration of about 5 mM to less than about 20 mM. In a particular embodiment according to (or as applied to) any of the above embodiments, the reducing agent is glutathione, and the glutathione is added at a concentration of about 15 mM.

[0024] In certain embodiments according to (or as applied to) any of the above embodiments, the first host cell is a stable cell line. In certain embodiments according to (or as applied to) any of the above embodiments, the second host cell is a stable cell line. In certain embodiments according to (or as applied to) any of the above embodiments, the first host cell is a CHO cell. In certain embodiments according to (or as applied to) any of the above embodiments, the second host cell is a CHO cell.

[0025] In certain embodiments according to (or as applied to) any of the above embodiments, the ratio of the first host cells to the second host cells is adjusted such that when the first host cell culture and the second host cell culture are mixed to form a mixed culture, the molar ratio of the first hinge-containing polypeptide to the second hinge-containing polypeptide is about 1:10 to about 10:1. In certain embodiments according to (or as applied to) any of the above embodiments, when the host cell culture and the second host cell culture are mixed to form a mixed culture, the molar ratio of the first hinge-containing polypeptide expressed by the first host cells to the second hinge-containing polypeptide expressed by the second host cells is 1:1.

[0026] In certain embodiments according to (or as applied to) any of the above embodiments, the hinge-containing polypeptide comprises an Fc region or a variant thereof. In certain embodiments according to (or as applied to) any of the above embodiments, the first and / or second hinge-containing polypeptide comprises an antibody heavy chain.

[0027] In certain embodiments according to (or as applied to) any of the above embodiments, the first heterodimerization domain comprises a knob modification at its contact surface and the second heterodimerization domain comprises a hole modification at its contact surface. In certain embodiments according to (or as applied to) any of the above embodiments, the knob modification comprises replacing an original amino acid residue from the first heterodimerization domain with an amino acid residue having a larger side chain than the original amino acid residue. In certain embodiments according to (or as applied to) any of the above embodiments, the replacing amino acid residue is selected from the group consisting of tryptophan, phenylalanine, tyrosine and arginine. In certain embodiments according to (or as applied to) any of the above embodiments, the hole modification comprises replacing an original amino acid residue from the second heterodimerization domain with an amino acid residue having a smaller side chain than the original amino acid residue. In certain embodiments according to (or as applied to) any of the above embodiments, the replacing amino acid residue is selected from the group consisting of serine, threonine, valine and alanine. In certain embodiments according to (or as applied to) any of the above embodiments, the knob modification comprises a substitution of T366W (EU numbering). In certain embodiments according to (or as applied to) any of the above embodiments, the hole modification comprises a substitution of two or more amino acids selected from the group consisting of T366S, L368A and Y407V (EU numbering).

[0028] In certain embodiments according to (or as applied to) any of the above embodiments, the interchain disulfide bond is between the hinge regions. In certain embodiments according to (or as applied to) any of the above embodiments, the heteromultimeric protein is an antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the heteromultimeric protein is a bispecific antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is a humanized or human antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is a full-length antibody. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is a human C H 2 and / or C H In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is selected from the group consisting of IgG, IgA and IgD. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is an IgG. In certain embodiments according to (or as applied to) any of the above embodiments, the antibody is an IgG1, IgG2 or IgG4. In certain embodiments according to (or as applied to) any of the above embodiments, the first light chain and the second light chain comprise different variable domain sequences.

[0029] In another aspect, a method for preparing a heteromultimeric protein comprising: i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain; and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, comprising: (a) culturing a mixed culture of a first host cell and a second host cell, wherein the first host cell is capable of expressing a first hinge-containing polypeptide and a first light chain, and the second host cell is capable of expressing a second hinge-containing polypeptide and a second light chain, and wherein each of the first host cell and the second host cell is a mammalian cell; (b) adding a reducing agent to the mixed culture; and (c) recovering a mixed culture medium containing the heteromultimeric protein from the mixed culture without disrupting the cell membrane. A method is provided that includes:

[0030] In certain embodiments, the first host cell secretes a first homodimer comprising two first hinge-containing polypeptides and two first light chains, the second host cell secretes a second homodimer comprising two second hinge-containing polypeptides and two second light chains, and the mixed culture comprises the first homodimer and the second homodimer. In certain embodiments, adding a reducing agent to the mixed culture allows for the formation of the heteromultimeric protein.

[0031] In certain embodiments according to (or as applied to) any of the above embodiments, the reducing agent is added after the mixed culture has been cultured for about 18 days or less. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium is harvested 4 hours to 24 hours after the reducing agent is added. In certain embodiments according to (or as applied to) any of the above embodiments, the step of harvesting the mixed culture medium comprises removing the first host cells and the second host cells from the mixed culture medium. In certain embodiments according to (or as applied to) any of the above embodiments, the mixed culture medium is incubated for 4 hours to 7 days.

[0032] In certain embodiments, the method further comprises adjusting the cell:cell ratio of the first host cell and the second host cell in the mixed culture. In certain embodiments, the cell:cell ratio is adjusted to reach a desired molar ratio of the first hinge-containing polypeptide (including an associated light chain) expressed from the first host cell to the second hinge-containing polypeptide (including an associated light chain) expressed from the second host cell in the mixed culture. In certain embodiments, the host cell is a stable cell line. In certain embodiments, the stable cell line is stably transfected with a nucleic acid molecule capable of expressing the hinge-containing polypeptide and the light chain.

[0033] It is understood that the method of the present invention may include other steps that are generally obvious routine steps to initiate and / or complete the process encompassed by the method of the present invention described herein. For example, in one embodiment, the method of the present invention is preceded by a step of introducing a nucleic acid encoding a first hinge-containing polypeptide into a first host cell and a nucleic acid encoding a second hinge-containing polypeptide into a second host cell. In one embodiment, the method of the present invention further includes a step of purifying the heteromultimeric protein having binding specificities for at least two different targets.

[0034] In certain embodiments according to (or as applied to) any of the above embodiments, the first hinge-containing polypeptide and the first light chain (or its associated light chain) comprise a first binding domain for a first target. In certain embodiments according to (or as applied to) any of the above embodiments, the second hinge-containing polypeptide and the second light chain (or its associated light chain) comprise a second binding domain for a second target. The first and second targets can be different epitopes located on a single molecule or on different molecules.

[0035] In another aspect, a heteromultimeric protein produced by any of the above methods is provided. In certain embodiments according to (or as applied to) any of the above embodiments, the heteromultimeric protein is a bispecific antibody. Further provided is a composition comprising a heteromultimeric protein (e.g., a bispecific antibody) produced by any of the above methods and a pharma- ceutically acceptable carrier.

[0036] The heteromultimeric proteins of the invention are capable of binding, typically, preferably specifically, to an antigen. Such antigens include, for example, tumor antigens, cell survival regulators, cell proliferation regulators, molecules associated with (e.g., known or predicted to have a functional contribution to) tissue development or differentiation, cell surface molecules, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis, and molecules associated with (e.g., known or predicted to have a functional contribution to) angiogenesis. The antigens to which the heteromultimeric proteins of the invention can bind are members of one subset of the above categories, with other subsets of the categories including other molecules / antigens with different characteristics (with respect to the antigen of interest). The antigen of interest can also be considered to belong to more than one category. In one embodiment, the invention provides heteromultimeric proteins that bind, preferably specifically, to a tumor antigen that is not a cell surface molecule. In one embodiment, the tumor antigen is a cell surface molecule, such as a receptor polypeptide. In another example of some embodiments, the heteromultimeric proteins of the invention bind, preferably specifically, to a tumor antigen that is not a cluster differentiation factor. In another example, the heteromultimeric proteins of the invention bind, preferably specifically, to a cluster differentiation factor, which in some embodiments is not, for example, CD3 or CD4. In some embodiments, the heteromultimeric proteins of the invention are anti-VEGF antibodies. In some embodiments, the heteromultimeric proteins of the invention bind to a cluster differentiation factor, preferably specifically, which in some embodiments is not, for example, CD3 or CD4. In some embodiments, the heteromultimeric proteins of the invention are anti-VEGF antibodies. In some embodiments, the heteromultimeric proteins of the invention bind to a cluster differentiation factor, such as IL-1 alpha / IL-1 beta, IL-12 / IL-18; IL-13 / IL-9; IL-13 / IL-4; IL-13 / IL-5; IL-5 / IL-4; IL-13 / IL-1 beta; IL-13 / IL-25; IL-13 / TARC; IL-13 / MDC; IL-13 / MEF; IL-13 / TGF-β; IL-13 / LHRα. IL-12 / TWEAK, IL-13 / CL25;IL-13 / SPRR2a;IL-13 / SPRR2b;IL-13 / ADAM8, IL-13 / PED2, IL17A / IL17F, C D3 / CD19, CD138 / CD20;CD138 / CD40;CD19 / CD20;CD20 / CD3;CD38 / CD138;CD38 / CD20;CD38 / CD40;CD40 / CD20;CD-8 / IL-6; CD20 / BR3, TNF alpha / TGF-beta, TNF alpha / IL-1 beta; TNF alpha / IL-2, TNF alpha / IL-3, TNF alpha / IL-4, TNF alpha / IL-5, TNF alpha / IL-6, TNF alpha / IL-8, TNF alpha / IL-9, TNF alpha / IL-10, TNF alpha / IL-11, TNF alpha / IL-12, TNF alpha / IL-13, TNF alpha / IL-14, TNF alpha / IL-15, TNF alpha / IL-16, TNF alpha / IL-17, TNF alpha / IL-18, TNF alpha / IL-19, TNF alpha / IL-20, TNF alpha / IL-23, TNF alpha / IFN alpha, TNF alpha / CD4, TNF alpha / VEGF, TNF alpha / MIF, TNF alpha / ICAM-1, TNF alpha / PGE4, TNF alpha / PEG2, TNF alpha / RANK ligand, TNF alpha / Te38; TNF alpha / BAFF; TNF alpha / CD22; TNF alpha / CTLA-4; TNF alpha / GP130; TNFα / IL-12p40; VEGF / HER2, VEGF-A / HER2, VEGF-A / PDGF, HER1 / HER2, VEGF-A / VEGF-C, VEGF-C / VEGF-D, HER2 / DR5, VEGF / IL-8, VE GF / MET, VEGFR / MET receptor, VEGFR / EGFR, HER2 / CD64, HER2 / CD3, HER2 / CD16, HER2 / HER3;EGFR / HER2, EGFR / HER3, EGFR / HER4 , IL-13 / CD40L, IL4 / CD40L, TNFR1 / IL-1R, TNFR1 / IL-6R, TNFR1 / IL-18R, EpCAM / CD3, MAPG / CD28, EGFR / CD64, CSPGs / RGM A;CTLA-4 / BTNO2;IGF1 / IGF2;IGF1 / 2 / Erb2B;MAG / RGM A;NgR / RGM A;NogoA / RGM A;OMGp / RGM A;PDL-I / CTLA-4;and RGM A / RGM B, IL1β / IL18, NRP1 / VEGFA, VEGFA / NRP2, cMET / EGFR, ALK1 / BMP9, VEGFA / α5β1, HER1 / HER3-BU, and CMV. In some embodiments, the heteromultimeric proteins of the invention are: α5β1, ALK1, BMP9, IL-1 alpha, IL-1 beta, TARC, MDC, MEF, TGF-β, LHR agonist, TWEAK, CL25, SPRR2a, SPRR2b, ADAM8, PED2, CD3, CD4, CD16, CD19, CD20, CD22, CD28, CD40, CD38, CD64, CD138, CD-8, BR3, TNF alpha, TGF-beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9 , IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17A, IL-17F, IL-18, IL-19, IL-20, IL-23, IL-25, IFN alpha, MIF, ICAM-1, PGE4, PE G2, RANK ligand, Te38, BAFF, CTLA-4, GP130, IL-12p40, VEGF, VEGF-A, PDGF, HER1, HER2, HER3, HER3-BU, HER4, VEGF-C, VEGF-D, DR5, cMET, MET, MET receptor, VEGFR, EGFR, CD40L, TNFR1, IL-1R, IL-6R, IL-18R, EpCAM, MAPG, CSPGs, BTNO2, IGF1, IGF2, IGF1 / 2, Erb2B, MAG, NgR, NogoA, NRP1, NRP2, OMGp, PDL-I, RGM A, and RGM B. In some embodiments, the heteromultimeric protein of the present invention binds to CD3 and at least one additional target molecule selected from BLR1, BR3, CD19, CD20, CD22, CD72, CD79A, CD79B, CD180 (RP105), CR2, FcRH1, FcRH2, FcRH5, FCER2, FCRL4, HLA-DOB, and NAG14;

[0037] The heteromultimeric proteins may be modified to enhance and / or add additional desired properties. Such properties include biological functions such as immune effector functions, desirable in vivo half-life / clearance, bioavailability, biodistribution or other pharmacokinetic properties. Such modifications are well known in the art and may be empirically determined, and may include modifications with moieties that may or may not be peptide-based. For example, antibodies are usually glycosylated or non-glycosylated, depending at least in part on the nature of the host cell. Preferably, the antibodies of the invention are non-glycosylated. Non-glycosylated antibodies produced by the methods of the invention can then be glycosylated, for example, by using in vitro glycosylation methods well known in the art. As described above and herein, the heteromultimeric proteins of the invention can be produced in prokaryotic cells, such as, for example, E. coli. Heteromultimeric proteins produced in E. coli are generally non-glycosylated and lack the biological functions normally associated with the glycosylated features found in heteromultimeric proteins produced in mammalian host cells (e.g., CHO).

[0038] The present invention also provides an immunoconjugate comprising a heteromultimeric protein of the present invention conjugated to a heterologous moiety. Any heterologous moiety is suitable as long as conjugation to the antibody does not diminish the desired function and / or properties of the antibody. For example, in some embodiments, the immunoconjugate comprises a heterologous moiety that is a cytotoxic agent. In some embodiments, the cytotoxic agent is selected from the group consisting of a radioisotope, a chemotherapeutic agent, and a toxin. In some embodiments, the toxin is selected from the group consisting of a calicheamicin, a maytansine, and a trichothene. In some embodiments, the immunoconjugate comprises a heterologous moiety that is a detectable marker. In some embodiments, the detectable marker is selected from the group consisting of a radioisotope, a member of a ligand-receptor pair, a member of an enzyme-substrate pair, and a member of a fluorescence resonance energy transfer pair.

[0039] In another aspect, a host cell is provided comprising a polynucleotide or recombinant vector encoding a first hinge-containing polypeptide of the heteromultimeric protein as described above, which does not express a second hinge-containing polypeptide of the host cell of the heteromultimeric protein. In certain embodiments according to (or as applied to) any of the above embodiments, the hinge-containing polypeptide is an antibody heavy chain. In certain embodiments according to (or as applied to) any of the above embodiments, the hinge-containing polypeptide is paired with an antibody light chain. In certain embodiments according to (or as applied to) any of the above embodiments, the host cell is a stable cell line. In certain embodiments according to (or as applied to) any of the above embodiments, the host cell is a mammalian cell. In certain embodiments according to (or as applied to) any of the above embodiments, the host cell is a CHO cell.

[0040] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to those skilled in the art from the detailed description.

[0041] All references cited herein are incorporated by reference in their entirety. [Brief description of the drawings]

[0042] [Figure 1A] A fully oxidized half antibody is shown. The "knob" or "hole" or other heterodimerization domains are not shown. The half antibody shown in this figure is of IgG1 isotype. One of skill in the art will understand that other immunoglobulin isotypes can be considered as half antibodies with corresponding inter-chain and intra-chain bonds. In an intact Ab, the hinge cysteines will form inter-chain disulfide bonds. [Figure 1B]The full-length bispecific antibody is shown. The inter-heavy chain disulfide bond in the hinge region is not shown. [Diagram 2] FIG. 1 shows a flow diagram of two assays that can be used to determine % half antibodies and % covalent bispecific antibodies. [Diagram 3] 1 shows the % bispecific antibody formed when a reducing agent was added to a mixed cell culture comprising a first mammalian host cell expressing anti-target A (knob) and a second mammalian host cell expressing anti-target B (hole) 4 hours, 15 hours, or 24 hours prior to harvesting the mixed culture medium. [Figure 4] Shown is the knob and hole capture pools run on a 4-20% Tris-glycine SDS PAGE. [Figure 5A] Chromatogram of a sample separated based on the hydrophobicity of anti-target G in which the homodimer and half-antibody co-elute in one broad peak. [Figure 5B] Chromatograms of samples separated based on the hydrophobicity of anti-target H are shown. [Figure 6] 1 shows the results of mass spectrometry of anti-target G / anti-target H bispecific antibodies. [Figure 7A] 1 shows the results of electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) experiments performed on untreated and GSH-treated mixed culture medium into which anti-target A and anti-target B half antibodies have been secreted. [Figure 7B] FIG. 13 is a magnification of the m / z range of the bispecific antibody peak. [Figure 7C] FIG. 1 is a magnified view of the m / z range of the half antibody peak.

[0043] Abbreviation ADCC=antibody-dependent cell-mediated cytotoxicity API = Antipathogenic Immunoadhesin BPI = bactericidal / permeability increasing protein C1q = complement factor 1q CD = Cluster of Differentiation CDC = complement-dependent cytotoxicity CH1 or C H 1 = first constant domain of the heavy chain CH2 or C H 2 = second constant domain of the heavy chain CH3 or C H 3 = third constant domain of the heavy chain CH4 or C H 4 = fourth constant domain of the heavy chain CL or C L = Light chain constant domain CTLA=cytotoxic T lymphocyte binding molecule Fc=crystalline fragment Fc (R = receptor gamma for the Fc portion of IgG HIV = Human Immunodeficiency Virus ICAM=intercellular adhesion molecule BsAb=bispecific antibody BsDb = bispecific diabody dsFv = disulfide stabilized Fv Fc = constant fragment of antibody Fd = V of antibody H +C H 1 FcR = Fc receptor Fv = variable fragment of antibody IgG = immunoglobulin G mAb = monoclonal antibody PBL = peripheral blood lymphocytes scDb = single chain diabody scFv=single chain Fv (scFv)2 = scFv-scFv tandem Tandab = Tandem Diabody VH or V H = variable domain of the antibody heavy chain VL or V L = variable domain of the antibody light chain DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present invention will now be described in detail, for reference purposes only, by way of the following definitions and examples. All patent documents and literature references mentioned herein are expressly incorporated by reference, including all sequences disclosed in such documents.

[0045] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice and testing of this invention, preferred methods and materials are described. Numerical ranges are inclusive of the numbers that define the range. Unless otherwise indicated, nucleic acids are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxy orientation, respectively. Practitioners are directed to particular reference to Sambrook et al., 1989, and Ausubel FM et al., 1993 for definitions and terminology of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0046] Numeric ranges are inclusive of the numbers defining the range.

[0047] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0048] The headings provided herein are not limitations of the various aspects or embodiments of the invention that can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0049] I. Definition A "heteromultimer," "heteromultimeric complex," or "heteromultimeric protein" refers to a molecule comprising a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain and a second hinge-containing polypeptide having a second heterodimerization domain associated with a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond. A heteromultimer can include a "heterodimer" formed by a first hinge-containing polypeptide, a first light chain, a second hinge-containing polypeptide, and a second light chain. Alternatively, a heteromultimer can form, for example, a bispecific antibody. The polypeptides of a heteromultimer can interact with each other through non-peptide covalent bonds (e.g., disulfide bonds) and / or non-covalent interactions (e.g., hydrogen bonds, ionic bonds, van der Waals forces, and / or hydrophobic interactions).

[0050] As used herein, a "heteromultimerization domain" refers to a modification or addition to a biomolecule that promotes heteromultimer formation and discourages homomultimer formation. Any heterodimerization domain that strongly favors the formation of heterodimers compared to homodimers is within the scope of the present invention. Specific examples include, but are not limited to, for example, US Patent Publication No. 20030078385 (Arathoon et al. - Genentech; describing knobs-into-holes); WO 2007147901 (Kjaergaard et al. - Novo Nordisk; describing ionic interactions); WO 2009089004 (Kannan et al. - Amgen; describing electrostatic steering effects); WO 2011 / 034605 (Christensen et al. - Genentech; describing coiled coils). See also, for example, Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584 (1992), which describes leucine zippers, or Pack et al., Bio / Technology 11, 1271-1277 (1993), which describes the helix-turn-helix motif. The terms "heteromultimerization domain" and "heterodimerization domain" are used interchangeably herein.

[0051] As used herein, the term "hinge-containing polypeptide" refers to a hinge region of an immunoglobulin as understood in the art (e.g., the C H 1 and C HThe term "hinge region", "hinge sequence" and variations thereof as used herein include the meanings known in the art, e.g., as described in Janeway's Immunobiology, (Garland Science, Taylor & Francis Group, LLC, NY) (7th ed., 2008); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209:193-202. See also, e.g., Burton, Molec. Immunol.22:161-206 (1985) and Papadea, C. and IJ Check (1989) "Human immunoglobulin G and immunoglobulin G subclasses: biochemical, genetic, and clinical aspects." Crit Rev Clin Lab Sci 27(1): 27-58. Those skilled in the art will understand that the number of amino acids available for interchain disulfide bond formation as well as the number of cysteine ​​residues varies between immunoglobulin classes and isotypes. All such hinge regions may be within the hinge-containing polypeptide and are within the scope of the present invention. In certain embodiments, the first hinge-containing polypeptide comprises a first antibody heavy chain. In certain embodiments, the first heavy chain combines with a first light chain to form a first half antibody. As used herein, the term "antibody" is used in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, as well as any fragment, mutant, variant or derivative thereof, so long as it exhibits the desired biological activity (e.g., epitope binding activity). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments described herein. Antibodies may be human, humanized and / or affinity matured.

[0052] As a frame of reference, antibody as used herein refers to the structure of immunoglobulin G (IgG). However, one of skill in the art will understand / recognize that antibodies of any immunoglobulin class may be utilized in the inventive methods described herein. For clarity, an IgG molecule contains a pair of identical heavy chains (HC) and a pair of identical light chains (LC). Each LC contains one variable domain (V L ) and one constant domain (C L ), and each HC has one variable domain (V H ) and three constant domains (C H 1. C H 2, and C H 3) C H 1 and C H The two domains are connected by a hinge region, the structure of which is well known in the art, see Figure 1B.

[0053] As used herein, a "half antibody" refers to one immunoglobulin heavy chain bound to one immunoglobulin light chain. An exemplary half antibody is shown in Figure 1A. Those skilled in the art will readily appreciate that a half antibody can also have an antigen-binding domain consisting of a single variable domain.

[0054] The term "maxibody" refers to a fusion protein comprising an scFv fused to an Fc polypeptide. See Figure 8a of WO2009089004. See Figure 2 of WO2009089004 for bispecific maxibodies.

[0055] The term "C" for the human IgG Fc region H The "C2 domain" typically extends from about residue 231 to about 340 of IgG, according to the EU numbering system. H The IgG2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched glycans (carbohydrates) are paired with the two C domains of an intact native IgG molecule. H Inserted between the two domains. Sugar (carbohydrate) replaces the domain-domain pairing to form C HIt has been speculated that this may help stabilize the 2 domain. Burton, Molec. Immunol.22:161-206 (1985).

[0056] The term “C H The "3 domain" is a C-terminal C domain of the Fc region. H It contains a stretch of up to two domains (ie, from about amino acid residue 341 to about amino acid residue 447 of IgG according to the EU numbering system).

[0057] The term "Fc region" as used herein generally refers to a dimeric complex comprising a C-terminal polypeptide sequence of an immunoglobulin heavy chain, where the C-terminal polypeptide sequence can be obtained by papain digestion of an intact antibody. The Fc region can comprise a native or mutated Fc sequence. Although the boundaries of an immunoglobulin heavy chain Fc sequence can vary, the human IgG heavy chain Fc sequence is usually defined as extending from an amino acid residue at about Cys226 or about Pro230 to the carboxyl terminus of the Fc sequence. Unless otherwise specified herein, the numbering of amino acid residues within an Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc sequence of an immunoglobulin generally comprises the C-terminal polypeptide sequence of an immunoglobulin heavy chain, which is a C-terminal polypeptide sequence of an immunoglobulin heavy chain. H 2 Domain and C H It contains two constant domains of 3 domains, and optionally C HThe Fc polypeptide comprises four domains. As used herein, "Fc polypeptide" refers to an Fc region, e.g., one of the polypeptides that form a monomeric Fc. An Fc polypeptide can be obtained from any suitable immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4 subtype, IgA, IgE, IgD, or IgM. The Fc region comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences within the Fc region; this region is also the portion recognized by Fc receptors (FcRs) found on certain cell types. In some embodiments, an Fc polypeptide comprises part or all of a wild-type hinge sequence (usually at its N-terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild-type hinge sequence.

[0058] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR); and the like. Such effector functions generally require the presence of a mixture of binding domains in the Fc region (e.g., antibody variable domains) and can be assessed, for example, using the various assays disclosed in the definitions herein.

[0059] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence IgG3 Fc regions; and native sequence human IgG4 Fc regions; and naturally occurring variants thereof.

[0060] A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions, in a native sequence Fc region or in the Fc region of a parent polypeptide, compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology, even more preferably at least about 95% homology, at least about 96% homology, at least about 97%, at least about 98% or at least about 99% homology.

[0061] As used herein, an "Fc component" refers to the hinge region, C H 2 domains or C H Refers to 3 domains.

[0062] In a particular embodiment, the hinge-containing polypeptide comprises an IgG Fc region, preferably an IgG Fc region derived from a wild-type human IgG Fc region. By "wild-type" human IgG Fc is meant a sequence of amino acids that occur naturally in the human population. Of course, since Fc sequences vary slightly between individuals, one or more modifications to the wild-type sequence are possible and are within the scope of the present invention. For example, the Fc region may contain further modifications not relevant to the present invention, such as mutations at glycosylation sites or the inclusion of non-natural amino acids.

[0063] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, each containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6 thed., WH Freeman and Co., page 91 (2007). H Or V L The V domain may be sufficient to confer antigen-binding specificity. Moreover, an antibody that binds to a particular antigen may have a complementary V domain. L Or V H To screen the domain libraries, we used V from an antibody that binds to the antigen. H Or V L Domains can be used to isolate the IL-15 ...

[0064] As used herein, the term "Fab" refers to an antigen-binding fragment of an antibody. As mentioned above, papain is used to digest intact antibodies. Papain digestion of an antibody produces two identical antigen-binding fragments, the "Fab" fragment and the remaining "Fc" fragment (i.e., the Fc region as described above). The Fab fragment contains the entire L chain as well as the variable region domain (V H ), and the first constant domain of one heavy chain (C H 1)

[0065] As used herein, the expressions "antigen-binding arm", "target molecule-binding arm", "target-binding arm" and variations thereof refer to a component of a heteromultimeric protein of the invention having the ability to specifically bind to a target of interest. Typically and preferably, the antigen-binding arm is a complex of immunoglobulin polypeptide sequences, e.g., CDR and / or variable domain sequences of immunoglobulin light and heavy chains.

[0066] "Target" or "target molecule" refers to the moiety recognized by the binding arm of the heteromultimeric protein. For example, if the heteromultimeric protein is an antibody, the target is an epitope on a single molecule or on different molecules, or a pathogen or tumor cell, depending on the context. Similarly, if the heteromultimeric protein is a receptor-Fc fusion protein, the target is the cognate binding partner of the receptor. Those skilled in the art will understand that the target is determined by the binding specificity of the target binding arm, and that different target binding arms recognize different targets. The target preferably binds to the heteromultimeric protein of the present invention with an affinity of greater than 1 uM Kd (by Scatchard analysis). Examples of target molecules include, but are not limited to, serum soluble proteins and / or their receptors, such as cytokines and / or cytokine receptors, adhesins, growth factors and / or their receptors, hormones, viral particles (e.g., RSV F protein, CMV, StaphA, influenza, Hepatitis C virus), microorganisms (e.g., bacterial cell proteins, fungal cells), adhesins, CD proteins and their receptors.

[0067] An example of an "intact" or "full-length" antibody is an antibody that comprises an antigen-binding site and a C L and at least the heavy chain constant domain C H 1. C H 2 and C H 3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.

[0068] The term "coupling" as used herein refers to the steps necessary to link the first and second hinge-containing polypeptides together, e.g., the formation of covalent bonds. Such steps include the formation of interchain disulfide bonds by reduction, annealing, and / or oxidation of cysteine ​​residues in the first and second hinge-containing polypeptides. Coupling is accomplished by chemical crosslinking or the use of acid ring systems. See, e.g., Humphreys et al., J. Immunol. Methods (1998) 217:1-10 and Zhu et al., Cancer Lett., (1994) 86: 127-134.

[0069] The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies with polyepitopic specificity. Such multispecific antibodies include, but are not limited to, V H V L The heavy chain variable domain (V H ) and the light chain variable domain (V L ), antibodies containing each V H V L Two or more V units bind to different epitopes L and V H These include antibodies with domains, antibodies with two or more single variable domains where each single variable domain binds a different epitope, full-length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, and antibody fragments that are covalently or non-covalently linked. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. "Monospecificity" refers to the ability to bind to only one epitope. In one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM. An exemplary diagram of bispecificity is shown in FIG. 1B.

[0070] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies (Db); tandem diabodies (taDb), linear antibodies (e.g., U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); one-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; antibody fragments (e.g., but not limited to, Db-Fc, taDb-Fc, taDb-C, taDb-D ... H 3, and (scFV)4-Fc).

[0071] The term "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" refers to an antibody that contains a single variable domain (SVD). H Or V L ) on each antigen-binding arm. In other words, a single variable domain does not need to interact with another variable domain to recognize the target antigen. Single domain antibodies contain a single monomeric variable antibody domain (V H Or V LExamples of single domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks), as well as those derived recombinantly from human and mouse antibodies (Ward et al., Nature (1989) 341:544-546; Dooley and Flajnik, Dev Comp Immunol (2006) 30:43-56; Muyldermans et al., Trend Biochem Sci (2001) 26:230-235; Holt et al., Trends Biotechnol (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; Davies and Riechmann, Febs Lett (1994) 339:285-290; WO 00 / 29004; WO 02 / 051870). Single variable domain antibodies can also be present in antigen-binding arms with other variable regions or variable domains (e.g., homo- or heteromultimers), in which case the antibody is not a single domain antibody.

[0072] The term "linear antibody" generally refers to the antibodies described in Zapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VFs) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. H -C H 1-V H -C H 1). Linear antibodies can be bispecific or monospecific.

[0073] The term "knob-into-hole" or "KnH" technology as referred to herein refers to a technology that induces pairing of two polypeptides in vitro or in vivo by introducing a protrusion (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface where the two polypeptides interact. For example, KnH is used to induce pairing of two polypeptides at the Fc:Fc binding interface of an antibody, C L:C H 1 Contact surface or V H / V L The KnH technology can also be used to pair two different receptor extracellular domains, or any other polypeptide sequence that contains different target recognition sequences (e.g., affibodies, peptibodies, and other Fc fusions).

[0074] An "Fv" consists of a dimer of one heavy and one light chain variable domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although often with a lower affinity than the entire binding site.

[0075] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a Fv that is composed of VFs linked together in a single polypeptide chain. H and V L An sFv polypeptide is an antibody fragment that contains an antibody domain. H and V LIt further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Malmborg et al., J. Immunol. Methods 183:7-13, 1995.

[0076] The term "diabody" refers to a sFv fragment (see previous paragraph) that is H Domain and V L This refers to small antibody fragments prepared by constructing them with short linkers (approximately 5-10 residues) between the domains and achieving interchain pairing rather than intrachain pairing of the V domains to create bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are fragments that combine the V domains of two antibodies. H Domain and V L Diabodies are heterodimers of two "crossover" Fv fragments whose domains are present on different polypeptide chains. Diabodies are described in further detail in, for example, EP 404097; WO 93 / 01161; and Hudson et al., Nat. Med. 9:129-134 (11161); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0077] The term "one-arm antibody" or "one-arm antibodies" refers to: H 2 domain, C H 3 Domain or C H 2-C H (1) a variable domain linked by a peptide bond to a polypeptide comprising a C3 domain; and (2) a second C H 2. C H 3 or C H 2-C Hrefers to an antibody that comprises three domains, where the variable domain is the second C H 2. C H 3 or C H 2-C H In one embodiment, a one-arm antibody is comprised of three polypeptides: (1) a variable domain (e.g., a V H ), C H 1. C H 2 and C H 3, (2) a first polypeptide comprising a variable domain (e.g., V L ) and C L a second polypeptide comprising a C domain; and H 2 and C H In another embodiment, the one-arm antibody comprises a third polypeptide comprising three domains. In another embodiment, the one-arm antibody has a partial hinge region comprising two cysteine ​​residues that form disulfide bonds linking the constant heavy chain. In one embodiment, the variable domains of the one-arm antibody form the antigen binding region. In another embodiment, the variable domains of the one-arm antibody are a single variable domain, and each single variable domain is an antigen binding region. In one embodiment, the one-arm antibody is a single variable domain antibody.

[0078] The antibodies of the invention include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0079] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FRs being those of human immunoglobulin sequences. Humanized antibodies also optionally comprise at least a portion of a constant region (Fc) of an immunoglobulin, typically a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0080] "Peptibody" (singular and plural) refers to the fusion of randomly generated peptides with an Fc domain. See U.S. Patent No. 6,660,843, issued Dec. 9, 2003 to Feige et al., which is incorporated by reference in its entirety. Peptibodies contain one or more peptides linked to the N-terminus, C-terminus, amino acid side chains, or two or more of these sites. Peptibody technology allows for the design of therapeutic agents that incorporate one or more ligand or receptor targeting peptides, tumor homing peptides, membrane transport peptides, and the like. Peptibody technology has proven useful in the design of many such molecules, including "tandem peptide multimers" (i.e., two or more peptides on a single chain of an Fc domain), which are linear peptides constrained by disulfides. See, for example, U.S. Patent No. 6,660,843; U.S. Patent Application Publication No. 2003 / 0195156, published October 16, 2003 (corresponding to WO 02 / 092620, published November 21, 2002); U.S. Patent Application Publication No. 2003 / 0176352, published September 18, 2003 (corresponding to WO 03 / 031589, published April 17, 2003); U.S. Patent No. 6,835,809 (corresponding to WO 00 / 24770, published May 4, 2000). See U.S. Patent Application Publication No. 2003 / 0229023, published December 11, 2003; WO 03 / 057134, published July 17, 2003; U.S. Patent Application Publication No. 2003 / 0236193, published December 25, 2003 (corresponding to PCT / US04 / 010989, filed April 8, 2004); U.S. Patent No. 6,919,426, filed September 18, 2003 (corresponding to WO 04 / 026329, published April 1, 2004), each of which is incorporated herein by reference in its entirety.

[0081] "Affibody" or "affibodies" refers to the use of proteins linked to an Fc region by a peptide bond, where the protein is used as a scaffold to provide a binding surface for a target molecule. The protein is often a naturally occurring protein such as Staphylococcus protein A or IgG binding B domain, or the Z protein derived therefrom (see Nilsson et al (1987), Prot Eng 1, 107-133, and U.S. Pat. No. 5,143,844), or a fragment or derivative thereof. For example, affibodies can be generated from Z proteins with altered binding affinities for target molecules, where a segment of the Z protein is mutated by random mutagenesis to generate a library of variants capable of binding to the target molecule. Examples of affibodies include U.S. Pat. No. 6,534,628, Nord K et al, Prot Eng 8:601-608 (1995) and Nord K et al, Nat Biotech 15:772-777 (1997). Biotechnol Appl Biochem. 2008 Jun;50(Pt 2):97-112.

[0082] As used herein, the term "immunoadhesin" refers to a molecule which combines the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins consist of an amino acid sequence with the desired binding specificity other than the antigen recognition and binding site of an antibody (i.e., which is "heterologous" compared to the constant region of an antibody), and an immunoglobulin constant domain sequence (e.g., the C H 2 and / or C HExemplary adhesin sequences include fusions with a receptor or ligand sequence (e.g., an adhesin sequence in a peptibody). Exemplary adhesin sequences include contiguous amino acid sequences that include a portion of a receptor or ligand that binds to a protein of interest. An adhesin sequence can also be a sequence that binds to a protein of interest but does not bind to a receptor or ligand sequence (e.g., an adhesin sequence in a peptibody). Such polypeptide sequences can be selected or identified by a variety of methods, including phage display techniques and high-throughput selection methods. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, for example, IgG1, IgG2, IgG3 or IgG4 subtypes, IgA (including IgA1 and IgA2), IgE, IgD, or IgM.

[0083] As used herein, "complex" or "complexed" refers to an association of two or more molecules that interact with each other through bonds and / or forces (e.g., van der Waals, hydrophobic, hydrophilic forces) that are not peptide bonds. In one embodiment, the complex is a heteromultimer. As used herein, the term "protein complex" or "polypeptide complex" includes complexes having a non-protein moiety (e.g., including but not limited to, a chemical molecule such as a toxin or detection agent) conjugated to a protein in the protein complex.

[0084] A heteromultimeric protein of the invention that "binds to an antigen of interest" is one that binds to the target with sufficient affinity and does not significantly cross-react with other proteins so that the heteromultimeric protein is useful as a diagnostic and / or therapeutic agent in targeting proteins or cells or tissues expressing the target. In such an embodiment, the degree of binding of the heteromultimeric protein to a "non-target" protein will be less than about 10% of the binding of an antibody to its particular target protein, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) or ELISA. With respect to the binding of a heteromultimeric protein to a target molecule, the term "specific binding" or "specifically binding" to or "specific for" a particular polypeptide or epitope on a particular polypeptide target refers to binding that is measurably different from a non-specific interaction (e.g., a non-specific interaction can be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as excess unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or "is specific" for a particular polypeptide or epitope on a particular polypeptide target as used herein can be represented by a molecule having a Kd for the target of, for example, at least about 200nM, alternatively at least about 150nM, alternatively at least about 100nM, alternatively at least about 60nM, alternatively at least about 50nM, alternatively at least about 40nM, alternatively at least about 30nM, alternatively at least about 20nM, alternatively at least about 10nM, alternatively at least about 8nM, alternatively at least about 6nM, alternatively at least about 4nM, alternatively at least about 2nM, alternatively at least about 1nM, or more.In one embodiment, the term "specific binding" refers to the binding of a heteromultimeric protein to a particular polypeptide or an epitope on a particular polypeptide without binding to substantially any other polypeptides or polypeptide epitopes.

[0085] Generally, "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). For example, Kd can be about 200nM, 150nM, 100nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 8nM, 6nM, 4nM, 2nM, 1nM, or stronger. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally tend to bind antigens slowly and dissociate quickly, whereas high affinity antibodies generally tend to bind antigens faster and remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0086] In one embodiment, a "Kd" or "Kd value" according to the present invention is measured using a BIAcore™ ELISA at 25° C. with an immobilized target (e.g., antigen) CM5 chip of ∼10 response units (RU). TM -2000 or BIAcore TMThe activity is measured by using a surface plasmon resonance assay with a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (~0.2 μM) in 10 mM sodium acetate (pH 4.8) and injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block non-reactive groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS containing 0.05% Tween 20 (PBST) at a flow rate of approximately 25 ul / min at 25° C. Association rates (k on ) and dissociation rate (k off ) is calculated. The equilibrium dissociation constant (Kd) is k off / k on The ratio is calculated as a ratio of 10 to 10. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 S -1 above, the association rate can be measured using a fluorescence quenching technique measuring the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, bandpass=16 nm) of 20 nM anti-antigen antibody (Fab type) in PBS (pH 7.2) at 25° C. in the presence of increasing concentrations of antigen as measured in a spectrometer, e.g. a spectrophotometer equipped with a flow stop (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0087] "Biologically active" and "biological activity" and "biological properties" with respect to a heteromultimeric protein of the invention, such as an antibody, fragment, or derivative thereof, means, unless otherwise specified, having the ability to bind to a biological molecule.

[0088] When used to describe various heteromultimeric polypeptides, "isolated" refers to a heteromultimer that has been separated and / or recovered from a cell or cell culture that expresses it. Contaminant components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the heteromultimer and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the heteromultimer is purified (1) to greater than 95% by weight of the protein as determined by the Lowry method, and most preferably to greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.

[0089] The heteromultimers of the invention are typically purified to substantial homogeneity. The terms "substantially homogeneous," "substantially homogeneous form," and "substantial homogeneity" are used to indicate that the product is substantially free of by-products resulting from undesired combinations of polypeptides (e.g., homomultimers).

[0090] Expressed in terms of purity, substantial homogeneity means that the amount of by-products does not exceed 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2%, or 1% by weight, or is less than 1% by weight. In one embodiment, the by-products are below 5%.

[0091] "Biomolecule" refers to nucleic acids, proteins, sugars (carbohydrates), lipids, and combinations thereof. In one embodiment, biomolecules are naturally occurring.

[0092] As used herein, "linked" or "linking" refers to a direct peptide bond linkage between a first and a second amino acid sequence or a linkage involving a third amino acid sequence that is a peptide bonded to and between the first and second amino acid sequences, e.g., a linker peptide bonded to the C-terminus of one amino acid sequence and the N-terminus of the other amino acid sequence.

[0093] As used herein, "linker" refers to an amino acid sequence of two or more amino acids in length. Linkers can be composed of naturally occurring polar or non-polar amino acids. Linkers can be, for example, 2 to 100 amino acids in length, for example, 2 to 50 amino acids in length, for example, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. Linkers can be "cleavable", for example, by autocleavage or enzymatic or chemical cleavage. Cleavage sites in amino acid sequences and enzymes and chemicals that cleave at such sites are well known in the art and are described herein.

[0094] As used herein, a "tether" refers to an amino acid linker that connects two other amino acid sequences. A tether as described herein can link the N-terminus of an immunoglobulin heavy chain variable domain and the C-terminus of an immunoglobulin light chain constant domain. In certain embodiments, the tether is about 15 to 50 amino acids in length, e.g., 20 to 26 amino acids in length (e.g., 20, 21, 22, 23, 24, 25, or 26 amino acids in length). A tether can be "cleavable," e.g., by autocleavage or enzymatic or chemical cleavage, using methods and reagents standard in the art.

[0095] Enzymatic cleavage of a "linker" or "tether" involves, for example, the use of endopeptidases such as Lys-C, Asp-N, Arg-C, V8, Glu-C, chymotrypsin, trypsin, pepsin, papain, thrombin, Genenase, Factor Xa, TEV (tobacco etch virus cysteine ​​protease), enterokinase, HRV C3 (human rhinovirus C3 protease), kininogenase, and subtilisin-like proprotein convertases (e.g., furin (PC1), PC2, or PC3) or N-arginine dibasic convertase. Chemical cleavage involves, for example, the use of hydroxylamine, N-chlorosuccinimide, N-bromosuccinimide, or cyanogen bromide.

[0096] As used herein, a "Lys-C endopeptidase cleavage site" is a lysine residue in an amino acid sequence that can be cleaved C-terminally by Lys-C endopeptidase. Lys-C endopeptidase cleaves C-terminally of a lysine residue.

[0097] "Chaotropic agent" means a water-soluble substance that disrupts the three-dimensional structure of a protein (e.g., an antibody) by disrupting stabilizing intramolecular interactions (e.g., hydrogen bonding, van der Waals forces, or hydrophobic effects). Exemplary chaotropic agents include, but are not limited to, urea, guanidine-HCl, lithium perchlorate, histidine, and arginine.

[0098] By "weak detergent" is meant a water-soluble substance that disrupts the three-dimensional structure of a protein (e.g., an antibody) by disrupting stabilizing intramolecular interactions (e.g., hydrogen bonds, van der Waals forces, or hydrophobic effects), but does not permanently destroy the protein structure and causes loss of biological activity (i.e., does not denature the protein). Exemplary weak detergents include, but are not limited to, Tween (e.g., Tween-20), Triton (e.g., Triton X-100), NP-40 (nonyl phenoxyl polyethoxyl ethanol), Nonidet P-40 (octyl phenoxyl polyethoxyl ethanol), and sodium dodecyl sulfate (SDS).

[0099] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0100] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxic agents. Antibodies "enable" the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, e.g., as described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model as disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0101] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibitory receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. This term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0102] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from natural sources, such as blood.

[0103] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the canonical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) bound to their cognate antigen. To assess complement activation, a CDC assay can be performed, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0104] The term "therapeutically effective amount" refers to an amount of an antibody, antibody fragment, or derivative to treat a disease or disorder of interest. In the case of a tumor (e.g., a cancerous tumor), a therapeutically effective amount of an antibody or antibody fragment (e.g., a multispecific antibody or antibody fragment) can reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent that the antibody or antibody fragment can prevent growth and / or kill existing cancer cells, the amount can be cytostatic and / or cytotoxic. In the case of cancer therapy, efficacy in vivo can be measured, for example, by assessing survival time, time to progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0105] By "reduce" or "inhibit" is meant the ability to cause an overall decrease, preferably by 20% or more, more preferably by 50% or more, and most preferably by 75%, 85%, 90%, 95% or more. Reducing or inhibiting can refer to symptoms of the disease being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of blood vessels in angiogenic disorders.

[0106] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. This definition includes benign and malignant cancers. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (e.g., renal cell carcinoma), liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, anal carcinoma, penile cancer, melanoma, and various head and neck cancers. "Early cancer" refers to cancer that is not invasive or metastatic, or is classified as stage 0, I, or II cancer. The term "precancerous" refers to a condition or growth that typically precedes or develops into cancer. "Non-metastatic" refers to a cancer that is benign, or that remains at the primary site and has not spread to the lymphatic or vascular system or tissues outside the primary site. In general, a non-metastatic cancer is any cancer that is a stage 0, I, or II cancer, and sometimes a stage III cancer.

[0107] An "allergic or inflammatory disorder," as used herein, is a disease or disorder resulting from overactivation of an individual's immune system. Exemplary allergic or inflammatory disorders include, but are not limited to, asthma, psoriasis, rheumatoid arthritis, atopic dermatitis, multiple sclerosis, systemic lupus erythematosus, eczema, organ transplantation, age-related macular degeneration, Crohn's disease, ulcerative colitis, eosinophilic esophagitis, and autoimmune diseases associated with inflammation.

[0108] An "autoimmune disease" herein is an immune disorder or condition arising from or directed against an individual's own tissues, or a co-segregation or manifestation thereof, or resulting therefrom. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, osteoarthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis, and juvenile onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis (arthritis chronica progrediente), osteoarthritis (arthritis deformans), early chronic polyarthritis (polyarthritis chronica), and other conditions. psoriasis, such as psoriasis vulgaris, guttate psoriasis, cystic psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria, such as chronic allergic urticaria including chronic autoimmune urticaria and chronic idiopathic urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic dermonecrolysis, scleroderma (including systemic sclerosis), sclerosis, such as systemic sclerosis, multiple sclerosis (MS), such as spinal-ocular MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, multiple sclerosis disseminate), and ataxic sclerosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis, e.g., ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enteritis, and transmural colitis, and autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), adult or acute respiratory distress syndrome Respiratory distress syndrome, including acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematological diseases, rheumatoid spondylitis, sudden deafness, IgE mediated diseases, such as anaphylaxis and allergic and atopic rhinitis, encephalitis, such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis,Granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis or autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome, e.g. chronic or acute glomerulonephritis, e.g. primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous- or membranous proliferative GN (MPGN), including types I and II, and rapidly progressive GN, allergic symptoms, allergic reactions, eczema, including allergic or atopic eczema, asthma, e.g. asthma bronchiale, bronchial asthma asthma, and autoimmune asthma, conditions with T cell infiltration and chronic inflammatory responses, chronic inflammatory lung diseases, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematosus, e.g. cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritis, encephalitis, pediatric, non-renal, extrarenal, alopecia areata), childhood insulin-dependent diabetes mellitus Juvenile-onset (type I) diabetes mellitus (IDDM), adult-onset diabetes mellitus (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, lymphomatoid granulomatosis, granulomatous diseases including Wegener's granulomatosis, agranulocytosis, vasculitis (large vessel vasculitis including polymyalgia rheumatica and giant cell (Takayasu) arteritis), Vasculitis, including medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), microscopic polyarteritis, central nervous system vasculitis, necrotizing, cutaneous, or hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, including Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond-Blackfan anemia, autoimmune hemolytic anemia, hemolytic anemia or immune hemolytic anemia, including anemia of chronic hemolysis (AIHA), pernicious anemia (anemia of malignant fever), Addison's disease, pure red cell anemia or deficiency (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, central nervous system inflammatory diseases, multiple organ injury syndromes, e.g. secondary to sepsis, trauma or hemorrhage, antigen-antibody complex mediated diseases,Antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid, e.g. pemphigoid bullous and cutaneous pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucous membrane pemphigoid, and pemphigus erythematous), autoimmune polyendocrinopathy, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathy, chronic neuropathies, e.g. IgM polyneuropathy or IgM-mediated neuropathy, thrombocytopenic purpura (TTP) and thrombocytopenia including autoimmune or immune-mediated thrombocytopenia (e.g. occurring in patients with myocardial infarction) autoimmune diseases including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis, e.g. autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid diseases, idiopathic hypothyroidism, Graves' disease, polyglandular syndromes such as autoimmune polyglandular syndrome (or polyendocrinopathy syndrome), paraneoplastic syndromes including neurological paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff man or stiff person syndrome, encephalomyelitis, e.g. allergic encephalomyelitis or encephalomyelitis allergica) and experimental allergic encephalomyelitis (EAE), myasthenia gravis, e.g. thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus-ataxia syndrome (OMS), and interval neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia, bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatitis, primary biliary cirrhosis, pulmonary fibrosis, autoimmune enteropathy syndrome, celiac disease, celiac sprue (gluten enteropathy), refractory sprue,Primary causes of lymphoma, including idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear diseases, e.g. autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus-myoclonus ataxia syndrome (OMS), polychondritis, e.g. refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, monoclonal B-cell lymphocytosis (e.g. benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance (MGUS)) Lymphocytosis, peripheral neuropathy, paraneoplastic syndromes, channelopathies such as epilepsy, migraine, cardiac arrhythmias, myopathy, hearing loss, blindness, periodic paralysis, and channelopathies of the central nervous system, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis, retinochoroiditis, autoimmune blood disease, fibromyalgia, polyendocrine glandular deficiency, Schmidt's syndrome, adrenal inflammation, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal hypoperistalsis, severe hypercalcaemia, glaucoma ... dactylism, and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Jagas' disease, rheumatic fever, recurrent abortions, farmer's lung, erythema multiforme, post-cardiotomy syndrome, pituitary basophil adenoma, bird fancier's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport's syndrome, alveolitis, e.g. allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Kaplan's disease syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum, fetal erythroblastosis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, such as chronic cyclitis, metachronous cyclitis, iridocyclitis, or Fuchs' cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection,Epstein-Barr virus infection, mumps, Evans syndrome, autoimmune gonadal dysfunction, Sydenham chorea, poststreptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, spinal fistula, choroiditis, giant cell polymyalgia, endocrine gastro-ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephropathies, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway disease, silicosis, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, beck Chronic leukemia, cryoglobulinemia, Depuytren's contracture, phacosensitivity endophthalmitis, nodular enteritis, leprous erythema nodosum, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hamman-Rich disease, sensorineural hearing loss, hemoglobinuric attacks, hypogonadism, ileitis, leukopenia, mononuclear infections, transverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmic syndromes, anti-carcinoma granuloma, pancreatitis, multiple sclerosis, pyoderma gangrenosum, Carbene thyroiditis, acquired spinal cord atrophy, infertility due to antisperm antibodies, nonmalignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune disorders AIDS, parasitic diseases such as Leishmania, toxic shock syndrome, food poisoning, conditions with T cell infiltration, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases with leukocyte extravasation, multiple organ injury syndrome, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine deficiency, peripheral neuropathy pulmonary disorders, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary infiltrates with eosinophilia, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, Aspergillus species,or eosinophil-containing granulomas, anaphylaxis, seronegative spondyloarthritis, possibly endocrine autoimmune disease, sclerosing cholangitis, scleral, episcleral, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia-telangiectasia, These include autoimmune disorders associated with collagen diseases, rheumatism, neurological diseases, ischemia-reperfusion injury, decreased blood pressure response, vascular insufficiency, antituberculosis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and diseases associated with angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, myocardial or other tissue reperfusion injury, skin diseases with an acute inflammatory component, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, acute severe infections, chronic refractory inflammation, pyelitis, pulmonary fibrosis, diabetic retinopathy, diabetic macroangiopathy, intimal hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0109] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or brings about destruction of cells. This term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , Ra 223 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents), enzymes and fragments thereof, e.g., nucleases, antibiotics, small molecule toxins, or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and the various anti-tumor, anti-cancer and chemotherapeutic agents disclosed herein. Other cytotoxic agents are described herein. Tumoricidal agents cause the destruction of tumor cells.

[0110] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamin. methylamylamines and ethylenimines, including camptothecin; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin). including;bryostatin;callistatin;CC-1065 (including its adrezecin, carzelcin and bizelesin synthetic analogs);podophyllotoxin;podophyllic acid;teniposide;cryptophycins (especially cryptophycin 1 and cryptophycin 8);dolastatins;duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1);eleutherobin;pancratistatin;sarcodictyin;spongistatin;nitrogenma mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Antibiotics, such as enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gamma 1 (see, e.g., Agnew, Chem Intl. Ed. Engl. 33: 183-186 (1994)); the dynemicins, including dynemicin A; the esperamicins; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycins, anthramycins, azaserine, bleomycins, cactinomycins, carabicins, carminomycins, carzinophilins, chromomycinis, dactinomycins, daunorubicins, detorubicins, 6-diaminopyrimidine ... Azo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin , puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine a Naroges, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;Androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;Antiadrenal, such as aminoglutethimide, mitotane, trilostane;Folic acid replacement, such as folinic acid;Aceglatone;Aldophosphamide glycosides;Aminolevulinic acid;Eniluracil;Amsacrine;Bestrabucil; Bisantrene; Edatrexate; Defofamine; Democorsin; Diaziquone; Eflornithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANETM Cremophor free, albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®);Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine (XELODA®); pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP (short for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone), and FOLFOX (short for a treatment regimen using oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™);

[0111] Further included in this definition are anti-hormonal agents that act to regulate, reduce, block or inhibit the action of hormones that may promote cancer growth, and are often systemic or in the form of systemic treatment. These are hormones themselves. Examples of these include anti-estrogens and selective estrogen receptor modulators (SERMs) (e.g., including tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone, and FARESTON® toremifene); anti-progesterones; estrogen receptor downregulators (ERDs); agents that function to suppress or shut down the ovaries, such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin, and tamoxifen. These include luteinizing hormone releasing hormone (LHRH) agonists, such as phosphoacetate and tripterelin; other antiandrogens, such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which block the enzyme aromatase and regulate estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole.Additionally, this definition of chemotherapeutic agent includes bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid / zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that act on signal transduction pathways involved in abnormal cell proliferation. These include those that inhibit the expression of genes involved in the regulation of IL-19, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOOPE® vaccine and gene therapy vaccines, such as the ALLOVECTIN® vaccine, the LEUVECTIN® vaccine, and the VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; lapatinib tosylate (an ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor, also known as GW572016); and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0112] "Growth inhibitor" as used herein refers to a compound or composition that inhibits cell growth in vitro or in vivo. Thus, a growth inhibitor can be one that significantly reduces the proportion of cells in S phase. Examples of growth inhibitors include agents that block cell cycle progression (at a position other than S phase), such as agents that induce G1 arrest or M phase arrest. Classical M phase blockers include vincas (e.g., vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Such agents that arrest G1 also extend to S phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs that are both derived from the yew tree. Docetaxel (TAXOTERE®, Lone Poulenc Rola), derived from the European yew tree, is a semisynthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel stabilize microtubules by promoting the assembly of microtubules from tubulin dimers and preventing depolymerization, which would result in the inhibition of cell mitosis.

[0113] As used herein, "anti-cancer therapy" refers to a therapy that reduces or inhibits cancer in a subject. Examples of anti-cancer therapy include cytotoxic radiation therapy, and administration to a subject of a therapeutically effective amount of a cytotoxic agent, a chemotherapeutic agent, a growth inhibitor, a cancer vaccine, an angiogenesis inhibitor, a prodrug, a cytokine, a cytokine antagonist, a corticosteroid, an immunosuppressant, an antiemetic, an antibody or antibody fragment, or an analgesic.

[0114] The term "prodrug" as used herein refers to a precursor or derivative form of a pharmacologic active substance that is less cytotoxic to tumor cells compared to the parent drug and can be enzymatically activated or converted to a more active parent form.See, for example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted to more active non-cytotoxic drugs. Examples of cytotoxic drugs which can be derivatized into a prodrug form for use in the present invention include, but are not limited to, chemotherapeutic agents such as those listed above.

[0115] The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on other cells. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone (HGH), N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); epidermal growth factor (EGF); hepatic growth factor; fibroblast growth factor (FGF); prolactin; placental lactogen; tumor necrosis factor alpha and tumor necrosis factor beta; Mullerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGF alpha; platelet growth factors; TGF alpha and TG Included are transforming growth factors (TGFs) such as F beta; insulin-like growth factors I and II; erythropoietin (EPO); bone morphogenetic factors; interferons such as interferon alpha, beta, and gamma; colony stimulating factors (CSFs) such as macrophage CSF (M-CSF); granulocyte macrophage CSF (GM-CSF); and granulocyte CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-18; tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0116] By "cytokine antagonist" is meant a molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of at least one cytokine. For example, a cytokine antagonist can inhibit cytokine activity by inhibiting expression and / or secretion of the cytokine or by binding to the cytokine or cytokine receptor. Cytokine antagonists include antibodies, synthetic or native sequence peptides, immunoadhesins, and small molecule antagonists that bind to the cytokine or cytokine receptor. Cytokine antagonists may be conjugated or fused to a cytotoxic agent. Exemplary TNF antagonists are etanercept (ENBREL®), infliximab (REMICADE®), and adalimumab (HUMIRA). TM ).

[0117] The term "immunosuppressant" as used herein refers to a substance that acts to suppress or block the immune system of the subject being treated. This includes substances that suppress cytokine production, downregulate or suppress the expression of self-antigens, or block MHC antigens. Examples of immunosuppressants include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077); mycophenolate mofetil, e.g., CELLCEPT®; azathioprine (IMURAN®, AZASAN® / 6-mercaptopurine; bromocriptine; danazol; dapsone; glutaraldehyde (to mask MHC antigens as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies against MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids and glucocorticosteroids, e.g., prednisone, prednisolone, e.g., PEDIAPRED® (prednisolone sodium phosphate) or ORAPRED® (prednisolone sodium phosphate oral solution), methylprednisolone, and dexamethasone; methotrexate (oral or subcutaneous) (RHEUMATREX®, TREXALL®, TM); hydroxychloroquine / chloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antagonists (including anti-interferon-gamma, -beta, or -alpha antibodies, anti-tumor necrosis factor-alpha antibodies (infliximab or adalimumab), anti-TNFα immunoadhesins (ENBREL®, etanercept), anti-tumor necrosis factor-beta antibodies, anti-interleukin-2 antibodies, and anti-IL-2 receptor antibodies); anti-LFA-1 antibodies (including anti-CD11a and anti-CD18 antibodies); anti-L 3T4 antibodies; heterologous antilymphocyte globulins; polyclonal or pan-T antibodies, or monoclonal anti-CD3 or anti-CD4 / CD4a antibodies; soluble peptides containing the LFA-3 binding domain (WO 90 / 08187); streptokinase; TGF-β; streptodornase; host-derived RNA or DNA; FK506; RS-61443; deoxyspergualin; rapamycin; T cell receptors (Cohen et al., U.S. Pat. No. 5,114,721); T cell receptor fragments (Offner et al. Science 251: 430-432 (1991); WO 90 / 11294; Ianeway, Nature 341:482 (1989); and WO 91 / 01133; T cell receptor antibodies (EP340109), such as T10B9; cyclophosphamide (CYTOXAN®); dapsone; penicillamine (CUPRIMINE®); plasma exchange; or intravenous immunoglobulin (IVIG). These may be used alone or in combination with each other, particularly in combinations of steroids with another immunosuppressant, or such combinations followed by a maintenance dose of a nonsteroidal drug, to reduce the need for steroids.

[0118] "Analgesic" refers to a drug that acts to inhibit or suppress pain in a subject. Exemplary analgesics include nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen (MOTRIN®), naproxen (NAPROSYN®), acetylsalicylic acid, indomethacin, sulindac, and tolmetin (including salts and derivatives thereof), as well as various other medicines used to reduce possible stinging pain, such as anticonvulsants (gabapentin, phenytoin, carbamazepine) or tricyclic antidepressants. Specific examples include acetaminophen, aspirin, amitriptyline (ELAVIL®), carbamazepine (TEGRETOL®), phenytoin (DILANTIN®), gabapentin (NEURONTIN®), (E)-N-vanillyl-8-methyl-6-noneamid (CAPSAICIN®), or nerve blockers.

[0119] "Corticosteroid" refers to any of a number of synthetic or natural substances that have the general chemical structure of a steroid that mimic or enhance the effects of a naturally occurring corticosteroid. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, triamcinolone, and betamethasone.

[0120] As used herein, a "cancer vaccine" is a composition that stimulates a subject's immune response against cancer. A cancer vaccine typically consists of a source of cancer-related substances or cells (antigens) that are autologous (originating from itself) or allogeneic (originating from elsewhere) to the subject, and other components (e.g., adjuvants) that further stimulate and enhance the immune response against the antigen. A cancer vaccine can ultimately stimulate the subject's immune system to produce antibodies against one or more specific antigens, and / or produce killer T cells that attack cancer cells bearing such antigens.

[0121] As used herein, "cytotoxic radiotherapy" refers to radiotherapy that inhibits or blocks the function of cells and / or causes the destruction of cells. Radiotherapy includes, for example, external beam radiation or therapy with radiolabeled drugs, such as antibodies. The term includes radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , Ra 223 , P 32 , and radioactive isotopes of Lu).

[0122] A "subject" is a vertebrate such as a mammal, e.g., a human. Mammals include, but are not limited to, farm animals (e.g., cows), sport animals, pets (e.g., cats, dogs, and horses), primates, mice, and rats.

[0123] Unless otherwise indicated by context, the terms "first" hinge-containing polypeptide and "second" hinge-containing polypeptide, and variations thereof, are merely generic identifiers and do not identify specific or particular polypeptide or antibody components of the invention.

[0124] Commercially available reagents (if any) referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. In the following examples and throughout the specification, the source of such cells (if any) identified by ATCC deposit numbers is the American Type Culture Collection, Manassas, VA. Unless otherwise indicated, the present invention employs standard procedures of recombinant DNA technology, such as those described above and in the following references. Gait, Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

[0125] Throughout this specification and the claims, the word "comprise" or variations thereof such as "comprising" should be understood to include a stated integer or set of integers, but not to exclude any other integer or set of integers.

[0126] Aspects and embodiments of the invention described herein include those that "consist" and / or "consist essentially of" aspects and embodiments.

[0127] Reference herein to "about" a value or parameter includes (and describes) the variation to that value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0128] As used herein, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Aspects and variations of the invention described herein and in the claims include those "consisting of" and / or "consisting essentially of" aspects and variations.

[0129] II. Methods for preparing heteromultimeric proteins in mammalian host cells The production of heteromultimeric proteins, such as multispecific antibodies, using current technology has drawbacks including, among others, the production of mixtures of products, reduced yields, and reduced / lost effector functions. Therefore, it is desirable to produce heteromultimeric proteins efficiently and at high levels.

[0130] The production of antibody molecules by various means is generally well understood. U.S. Patent No. 6,331,415 (Cabilly et al.) describes, for example, methods for recombinant production of immunoglobulins in which heavy and light chains are expressed simultaneously from a single vector or two separate vectors in a single cell. Wibbenmeyer et al., (1999, Biochim Biophys Acta 1430(2): 191 -202) and Lee and Kwak (2003, J. Biotechnology 101 :189-198) describe the production of monoclonal antibodies from heavy and light chains produced separately using plasmids expressed in separate cultures of E. coli. Various other techniques for the production of antibodies are described, for example, in Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988) and WO2006028936. However, each of these has drawbacks such as low yields and the use of chemicals.

[0131] The method provided herein is based on the surprising discovery that a first hinge-containing polypeptide and a first light chain expressed and secreted from a first mammalian host cell and a second hinge-containing polypeptide and a second light chain expressed and secreted from a second mammalian host cell assemble in a mixed culture medium to form a heteromultimeric protein. As described in detail below, the mixed culture medium can be obtained by culturing a first mammalian host cell in a first cell culture, culturing a second mammalian host cell in a second cell culture, recovering the first and second culture media without disrupting the cell membranes of the first and second host cells, and mixing the recovered first and second culture media to obtain a mixed culture medium containing the heteromultimeric protein. Alternatively, the mixed culture medium can be obtained by culturing a first and a second mammalian host cell in a mixed cell culture and recovering a mixed cell medium containing the heteromultimeric protein from the mixed culture. In certain embodiments, the recovering step comprises removing the first and / or second host cells without disrupting the cell membrane.

[0132] The methods provided herein are surprising because it was generally believed that the protein quality control systems of eukaryotic cells (e.g., mammalian cells) could not efficiently produce truncated antibodies, see, e.g., Spiess et al. (2013) Nature, 31(8): 753-758.

[0133] The inventors have also surprisingly discovered that heteromultimeric proteins can be formed in high yields under reducing conditions in a mixed culture medium comprising a first homodimer comprising two first hinge-containing polypeptides and two first light chains secreted by a first host cell, and a second homodimer comprising two second hinge-containing polypeptides and two second light chains secreted by a second host cell.

[0134] Thus, in a particular embodiment, there is provided a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell capable of expressing and secreting a first hinge-containing polypeptide and a first light chain; (b) culturing a second host cell capable of expressing and secreting the second hinge-containing polypeptide and the second light chain; and (c) obtaining a mixed culture medium of the first host cell and the second host cell without disrupting the cell membranes of the first and second host cells. wherein the mixed culture medium comprises a heteromultimeric protein, and the first host cell and the second host cell are each mammalian cells.

[0135] In certain embodiments, provided is a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell capable of expressing a first hinge-containing polypeptide and a first light chain, wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted from the first host cell; (b) culturing a second host cell capable of expressing the second hinge-containing polypeptide and the second light chain, wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted from the second host cell; (c) obtaining a mixed culture medium of the first host cell and the second host cell, the mixed culture medium comprising the first homodimer and the second homodimer; (d) incubating the mixed culture medium under reducing conditions; and (e) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells.

[0136] In certain embodiments, the mixed culture medium is obtained without disrupting the cell membranes of the first and second host cells. In certain embodiments, the method further comprises adding a reducing agent. In certain embodiments, the reducing conditions are sufficient to allow the formation of heteromultimeric proteins.

[0137] In certain embodiments, the first hinge-containing polypeptide and the first light chain comprise a first half antibody. In certain embodiments, the second hinge-containing polypeptide and the second light chain comprise a second half antibody.

[0138] In certain embodiments, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% (e.g., about 4%, about 3%, about 2%, or about 1%) of the first hinge-containing polypeptide and first light chain (e.g., first half antibody) present in the mixed culture medium are in the form of a first homodimer prior to incubation under reducing conditions or prior to addition of a reducing agent, including any range of these values. In certain embodiments, about 10% to about 75%, about 20% to about 65%, or about 30% to about 55% of the first hinge-containing polypeptide and first light chain (e.g., first half antibody) present in the mixed culture medium are in the first homodimer form prior to incubation under reducing conditions or prior to addition of a reducing agent.

[0139] In certain embodiments, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% (e.g., about 4%, about 3%, about 2%, or about 1%) of the second hinge-containing polypeptide and second light chain (e.g., second half antibody) present in the mixed culture medium are in the form of a second homodimer prior to incubation under reducing conditions or prior to addition of a reducing agent, including any range of these values. In certain embodiments, about 10% to about 75%, about 20% to about 65%, or about 30% to about 55% of the second hinge-containing polypeptide and second light chain (e.g., second half antibody) present in the mixed culture medium are in the second homodimeric form prior to incubation under reducing conditions or prior to addition of a reducing agent.

[0140] In certain embodiments, the mixed culture medium comprises less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%, including any range of these values, of the first homodimer. In certain embodiments, the mixed culture medium comprises from about less than 2% to about less than 20%, from about 5% to about 15%, or from about less than 10% to about 15% of the first homodimer after incubation under reducing conditions or after addition of a reducing agent.

[0141] In certain embodiments, the mixed culture medium contains less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%, including any range of these values, of the second homodimers. In certain embodiments, the mixed culture medium contains from about less than 2% to about less than 20%, from about 5% to about 15%, or from about less than 10% to about 15% of the second homodimer after incubation under reducing conditions or after addition of a reducing agent.

[0142] In certain embodiments, provided is a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain; (b) culturing a second host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain; and (c) obtaining a mixed culture medium of the first host cell and the second host cell; wherein the mixed culture medium comprises the heteromultimeric protein and the first host cell and the second host cell are each mammalian cells. In certain embodiments, the first and second nucleic acids are one nucleic acid molecule, while in other certain embodiments, the first and second nucleic acids are different nucleic acid molecules. In certain embodiments, the third and fourth nucleic acids are one nucleic acid molecule, while in other certain embodiments, the third and fourth nucleic acids are different nucleic acid molecules.

[0143] In a particular embodiment, the present invention provides a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain, wherein the first host cell is capable of expressing the first hinge-containing polypeptide and the first light chain, and wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted; (b) culturing a second host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain, wherein the second host cell is capable of expressing the second hinge-containing polypeptide and the second light chain, and wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted; (c) obtaining a mixed culture medium of the first host cell and the second host cell without disrupting the cell membranes of the first and second host cells, wherein the mixed culture medium comprises the first homodimer and the second homodimer; (d) incubating the mixed culture medium under reducing conditions sufficient to allow for the formation of heteromultimeric proteins; and (e) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, the first and second nucleic acids are one nucleic acid molecule, while in other certain embodiments, the first and second nucleic acids are different nucleic acid molecules. In certain embodiments, the third and fourth nucleic acids are one nucleic acid molecule, while in other certain embodiments, the third and fourth nucleic acids are different nucleic acid molecules. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0144] In certain embodiments, provided is a method for preparing a heteromultimeric protein comprising: i) a first half antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, the first hinge-containing polypeptide attached to a first light chain; and ii) a second half antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, the second hinge-containing polypeptide attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain; (b) culturing a second host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain; and (c) obtaining a mixed culture medium of the first host cell and the second host cell; wherein the mixed culture medium comprises the heteromultimeric protein and the first host cell and the second host cell are each mammalian cells. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0145] In a particular embodiment, the invention provides a method for preparing a heteromultimeric protein comprising: i) a first half-antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, the first hinge-containing polypeptide attached to a first light chain; and ii) a second half-antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, the second hinge-containing polypeptide attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain, wherein the first host cell is capable of expressing the first hinge-containing polypeptide and the first light chain, and wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted; (b) culturing a second host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain, wherein the second host cell is capable of expressing the second hinge-containing polypeptide and the second light chain, and wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted; (c) obtaining a mixed culture medium of the first host cell and the second host cell without disrupting the cell membranes of the first and second host cells, wherein the mixed culture medium comprises the first homodimer and the second homodimer; (d) incubating the mixed culture medium under reducing conditions sufficient to allow for the formation of heteromultimeric proteins; and (e) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0146] In a particular embodiment, the present invention provides a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing in a first cell culture a first mammalian host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain; (b) culturing in a second cell culture a second mammalian host cell that includes a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain; and (c) recovering the first culture medium from the first mammalian host cell; (d) recovering the second culture medium from the second mammalian host cell; (e) mixing the first culture medium and the second culture medium to obtain a mixed culture medium, wherein the mixed culture medium comprises a heteromultimeric protein. In certain embodiments, recovering the first culture medium comprises removing the first host cells from the first cell culture. In certain embodiments, recovering the second culture medium comprises removing the second host cells from the second cell culture. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0147] In certain embodiments, provided is a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing in a first cell culture a first host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain, wherein the first host cell is capable of expressing the first hinge-containing polypeptide and the first light chain, and wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted; (b) culturing in a second cell culture a second host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain, wherein the second host cell is capable of expressing the second hinge-containing polypeptide and the second light chain, and wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted; (c) recovering the first culture medium from the first mammalian host cell, the first culture medium comprising the first homodimer; (d) recovering the second culture medium from the second mammalian host cell, the second culture medium comprising the second homodimer; (e) mixing the first culture medium and the second culture medium to obtain a mixed culture medium containing the first homodimer and the second homodimer; (f) incubating the mixed culture medium under reducing conditions sufficient to allow for the formation of heteromultimeric proteins; and (g) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, recovering the first culture medium comprises removing the first host cell from the first cell culture. In certain embodiments, recovering the second culture medium comprises removing the second host cell from the second cell culture. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0148] In a particular embodiment, the invention provides a method for preparing a heteromultimeric protein comprising: i) a first half-antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, the first hinge-containing polypeptide attached to a first light chain; and ii) a second half-antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, where the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and where the first and second half-antibodies are linked by at least one interchain disulfide bond, the method comprising: (a) culturing in a first cell culture a first mammalian host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain; (b) culturing in a second cell culture a second mammalian host cell that includes a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain; and (c) recovering the first culture medium from the first mammalian host cell; (d) recovering the second culture medium from the second mammalian host cell; (e) mixing the first culture medium and the second culture medium to obtain a mixed culture medium, wherein the mixed culture medium comprises a heteromultimeric protein. In certain embodiments, recovering the first culture medium comprises removing the first host cells from the first cell culture. In certain embodiments, recovering the second culture medium comprises removing the second host cells from the second cell culture.

[0149] In certain embodiments, provided is a method for preparing a heteromultimeric protein comprising: i) a first half-antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, the first hinge-containing polypeptide linked to a first light chain; and ii) a second half-antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, the second hinge-containing polypeptide linked to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing in a first cell culture a first host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain, wherein the first host cell is capable of expressing the first hinge-containing polypeptide and the first light chain, and wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted; (b) culturing in a second cell culture a second host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain, wherein the second host cell is capable of expressing the second hinge-containing polypeptide and the second light chain, and wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted; (c) recovering the first culture medium from the first mammalian host cell, the first culture medium comprising the first homodimer; (d) recovering the second culture medium from the second mammalian host cell, the second culture medium comprising the second homodimer; (e) mixing the first culture medium and the second culture medium to obtain a mixed culture medium containing the first homodimer and the second homodimer; (f) incubating the mixed culture medium under reducing conditions sufficient to allow for the formation of heteromultimeric proteins; and (g) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, recovering the first culture medium comprises removing the first host cell from the first cell culture. In certain embodiments, recovering the second culture medium comprises removing the second host cell from the second cell culture. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0150] In a particular embodiment, the present invention provides a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first mammalian host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain; (b) culturing a second mammalian host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain; and (c) collecting the culture medium of the mixed cell culture comprising the first host cell and the second host cell to obtain a mixed culture medium of the first mammalian host cell and the second mammalian host cell comprising the heteromultimeric protein. Includes.

[0151] In certain embodiments, provided is a method for preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first mammalian host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain, wherein the first host cell is capable of expressing the first hinge-containing polypeptide and the first light chain, and wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted; (b) culturing a second mammalian host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain, wherein the second host cell is capable of expressing the second hinge-containing polypeptide and the second light chain, and wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted; (c) collecting a culture medium of the mixed cell culture comprising the first host cell and the second host cell to obtain a mixed culture medium of the first mammalian host cell and the second mammalian host cell comprising the first homodimer and the second homodimer; (d) incubating the medium under reducing conditions sufficient to allow formation of the heteromultimeric protein; and (e) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0152] In a particular embodiment, the invention provides a method for preparing a heteromultimeric protein comprising: i) a first half antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, the first hinge-containing polypeptide attached to a first light chain; and ii) a second half antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, where the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first mammalian host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain; (b) culturing a second mammalian host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain; and (c) collecting the culture medium of the mixed cell culture comprising the first host cell and the second host cell to obtain a mixed culture medium of the first mammalian host cell and the second mammalian host cell comprising the heteromultimeric protein. Includes.

[0153] In certain embodiments, provided is a method for preparing a heteromultimeric protein comprising: i) a first half-antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, the first hinge-containing polypeptide attached to a first light chain; and ii) a second half-antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, the second hinge-containing polypeptide attached to a second light chain, where the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and where the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first mammalian host cell comprising a first nucleic acid encoding a first hinge-containing polypeptide and a second nucleic acid encoding a first light chain, wherein the first host cell is capable of expressing the first hinge-containing polypeptide and the first light chain, and wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted; (b) culturing a second mammalian host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain, wherein the second host cell is capable of expressing the second hinge-containing polypeptide and the second light chain, and wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted; (c) collecting a culture medium of the mixed cell culture comprising the first host cell and the second host cell to obtain a mixed culture medium of the first mammalian host cell and the second mammalian host cell comprising the first homodimer and the second homodimer; (d) incubating the medium under reducing conditions sufficient to allow formation of the heteromultimeric protein; and (e) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, the method further comprises adding a reducing agent to the mixed culture medium.

[0154] In certain embodiments, culturing the mixed cell culture comprising the first host cell and the second host cell is performed at a temperature between about 25° C. and 40° C. In certain embodiments, culturing the mixed cell culture comprising the first host cell and the second host cell is performed at a temperature between about 30° C. and 37° C. In certain embodiments, culturing the mixed cell culture comprising the first host cell and the second host cell is performed at a pH between about 7.2 and 8.7.

[0155] In certain embodiments, the mixed culture medium is incubated at a temperature of about 4° C. to 40° C. In certain embodiments, the mixed culture medium is incubated at a temperature of about 30° C. to 37° C. In certain embodiments, the mixed culture medium is incubated at a temperature of about 4° C. to 8° C.

[0156] In certain embodiments, the mixed culture medium is stirred. In certain embodiments, the mixed culture medium is stirred for about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or more than 8 days after being obtained. In some embodiments, the mixed culture is stirred intermittently.

[0157] In certain embodiments, the method further comprises isolating the heteromultimeric protein (e.g., bispecific antibody) from the mixed culture medium. In certain embodiments, the heteromultimeric protein (e.g., bispecific antibody) is isolated using a Protein A column.

[0158] In certain embodiments, the method includes adding a reducing agent during the production of the heteromultimeric protein (e.g., bispecific antibody). In certain embodiments of the methods provided herein, the reducing agent used is glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), cysteine, cysteine, dithiothreitol, cysteine ​​dithiothreitol, dithiolbutylamine, or a combination thereof. In certain embodiments, the reducing agent is reduced glutathione. In certain embodiments, the reducing agent is not 2-mercaptoethanol. In certain embodiments, the reducing agent is not dithiothreitol.

[0159] In certain embodiments where the first and second mammalian host cells are cultured separately, i.e., grown in separate cultures, the reducing agent is added to the first cell culture medium and the second cell culture medium before the first and second cell culture media are harvested and mixed to obtain a mixed culture medium. In certain embodiments where the first and second mammalian host cells are grown in the same culture, the reducing agent is added to the culture medium of the mixed cell culture before the mixed cell culture is harvested to obtain a mixed culture medium.

[0160] In certain embodiments, the reducing agent is added about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours (including any ranges between these values) prior to the recovery step.

[0161] In certain embodiments, the reducing agent is added to the mixed culture medium. In certain embodiments, the mixed culture medium containing the reducing agent is incubated for about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days (including any range between these values).

[0162] In certain embodiments, a reducing agent (e.g., glutathione) is added to the mixed cell culture to achieve a final concentration of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM (including any range between these values). In certain embodiments, a reducing agent (e.g., glutathione) is added to the mixed cell culture to achieve a final concentration of less than 20 mM. In certain embodiments, a reducing agent (eg, glutathione) is added to the mixed cell culture to achieve a final concentration of 20 mM or less.

[0163] In certain embodiments, the reducing agent is added to the mixed culture medium prior to isolating the heteromultimeric protein (e.g., bispecific antibody) from the mixed culture medium. In certain embodiments, the mixed culture medium containing the reducing agent is incubated for about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days (including any range between these values) prior to isolating the heteromultimeric protein (e.g., bispecific antibody) from the mixed culture medium containing the reducing agent. In some embodiments, the heteromultimeric protein is isolated using a Protein A column.

[0164] In certain embodiments, a reducing agent (e.g., glutathione) is added to the mixed culture medium to achieve a final concentration of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM. In certain embodiments, a reducing agent (e.g., glutathione) is added to the mixed culture medium to achieve a final concentration of less than 20 mM. In certain embodiments, a reducing agent (e.g., glutathione) is added to the mixed culture medium to achieve a final concentration of 20 mM or less.

[0165] In certain embodiments of the method, the first host cell is a stable cell line. In certain embodiments, the second host cell is a stable cell line. In certain embodiments, the first host cell is a CHO cell. In certain embodiments, the second host cell is a CHO cell. In certain embodiments, the first host cell and the second host cell are grown in the same culture, the ratio of the first host cell to the second host cell at the start of the mixed culture is about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1 or about 10:1.

[0166] As used herein, "molar ratio" refers to the ratio of the first hinge-containing polypeptide bound to the first light chain (e.g., the first half antibody) expressed and / or secreted to the second hinge-containing polypeptide bound to the second light chain (e.g., the second half antibody). In some embodiments, the molar ratio of the first hinge-containing polypeptide bound to the first light chain to the second hinge-containing polypeptide bound to the second light chain is about 1.5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1 (including any range between these values). In some embodiments, the molar ratio of the first hinge-containing polypeptide bound to the first light chain to the second hinge-containing polypeptide bound to the second light chain is about 1:1. In some embodiments, the molar ratio of the first half antibody to the second half antibody is about 1.5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, including any ranges between these values. In some embodiments, the molar ratio of the first half antibody to the second half antibody is about 1:1.

[0167] III. Heteromultimeric proteins Further provided by the present invention is a heteromultimeric protein produced by any one of the methods described herein. In certain embodiments, the heteromultimeric protein comprises an Fc region of an antibody or a variant thereof (e.g., a variant having altered ADCC function). In certain embodiments, the heteromultimeric protein comprises a majority of an Fc region of an antibody or a variant thereof (e.g., a variant having altered ADCC function). In certain embodiments, the heteromultimeric protein comprises a C H 1. C H 2, and / or C H In certain embodiments, the heteromultimeric protein comprises a heavy chain that comprises only a portion of the C3 domain. H 1. C H 2, and / or C HIn certain embodiments, the heteromultimeric protein is an antibody fragment that contains only a portion of the three domains. In certain embodiments, the heteromultimeric protein is an antibody. In certain embodiments, the heteromultimeric protein is a bispecific antibody. In certain embodiments, the heteromultimeric protein is a humanized antibody. In certain embodiments, the heteromultimeric protein is a human antibody. In certain embodiments, the antibody is an IgG (e.g., IgG1, IgG2, or IgG4), IgA, or IgD. In certain embodiments, the first light chain and the second light chain of the heteromultimeric protein contain different variable domain sequences. In certain embodiments, the first and second hinge-containing polypeptides of the heteromultimeric protein produced by the methods provided herein comprise an Fc region or a variant thereof. In certain embodiments, the first and second hinge-containing polypeptides of the heteromultimeric protein comprise an antibody heavy chain.

[0168] Heteromultimerization domain The heteromultimeric protein comprises a heteromultimerization domain. To generate a population of substantially homogeneous heterodimeric molecules, the heterodimerization domain must have a strong preference for forming heterodimers over homodimers. The heteromultimeric proteins exemplified herein are conducive to heteromultimerization using knobs-into-holes technology, but other heteromultimerization domains useful in the present invention will be apparent to those skilled in the art.

[0169] Knob-into-Hole The use of knobs-into-holes as a method for generating multispecific antibodies is well known in the art. See U.S. Patent No. 5,731,168, granted to the present applicant on March 24, 1998, International Publication No. WO 2009089004, issued on July 16, 2009, and assigned to Amgen, and U.S. Patent Application Publication No. 20090182127, published on July 16, 2009, and assigned to Novo Nordisk A / S. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol. Sin., 26:1-9. A brief description is provided herein.

[0170] A "bulge" refers to at least one amino acid side chain that protrudes from the interface of a first polypeptide and can therefore be positioned in a complementary cavity in an adjacent interface (i.e., the interface of a second polypeptide). Such positioning stabilizes heteromultimers and thereby promotes heteromultimer formation over homomultimer formation, for example. The bulge may be present in the original interface or may be synthetically introduced (e.g., by modifying the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the first polypeptide is modified to encode the bulge. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with a nucleic acid encoding at least one "import" amino acid residue having a larger side chain volume than the original amino acid residue. It will be understood that there may be multiple original and corresponding imported residues. The upper limit for the number of original residues that can be replaced is the total number of residues in the interface of the first polypeptide. The side chain volumes of various amino acid residues are shown in Table 1 below.

[0171] TIFF2025063070000002.tif255170

[0172] Preferred import residues for the formation of the bulge are commonly naturally occurring amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, the original residue for the formation of the bulge has a small side chain, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

[0173] A "cavity" refers to at least one amino acid side chain that is recessed from the interface of a second polypeptide and thus accommodates a corresponding protuberance on the interface of an adjacent first polypeptide. The cavity may be present in the original interface or may be synthetically introduced (e.g., by modifying the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the second polypeptide is modified to encode the cavity. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one "import" amino acid residue having a smaller side chain volume than the original amino acid residue. It will be understood that there may be multiple original and corresponding imported residues. The upper limit for the number of original residues that can be replaced is the total number of residues in the interface of the second polypeptide. The side chain volumes of various amino acid residues are shown in Table 1 above. Preferred imported residues for the formation of the cavity are usually naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T) and valine (V). Most preferred are serine, alanine or threonine.In one embodiment, the original residue for formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan.

[0174] The "original" amino acid residue is the one that is replaced by the "import" residue, which may have a smaller or larger side chain volume than the original residue. The imported amino acid residue may be a naturally occurring or non-naturally occurring amino acid residue, preferably the former. A "naturally occurring" amino acid residue is a residue that is encoded by the genetic code and is listed in Table 1 above. A "non-naturally occurring" amino acid residue refers to a residue that is not encoded by the genetic code but can be covalently linked to adjacent amino acid residue(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244: 182 (1989) and Ellman et al. (supra) can be used. Briefly, this involves chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA. The method of the invention involves replacing at least one original amino acid residue, although more than one original residue can be replaced. Usually, no more than the total number of residues in the interface of the first or second polypeptide will include the original amino acid residue that is replaced. Typically, the original residue for replacement is "buried". By "buried" we mean that the residue is substantially inaccessible to solvent. Generally, the critical residue is not a cysteine, to prevent oxidation or possible mispairing of disulfide bonds.

[0175] By "positionable" the ridge within the cavity, we mean that the spatial location of the ridge and cavity, respectively, on the interface of the first and second polypeptides, and the size of the ridge and cavity, are such that the ridge can be positioned within the cavity without significantly disrupting the normal association of the first and second polypeptides at the interface. Because ridges such as Tyr, Phe and Trp do not typically have a preferred conformation extending perpendicularly from the axis of the interface, the alignment of the ridge with the corresponding cavity relies on modeling the ridge / cavity pair based on the three-dimensional structure, such as obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be accomplished using techniques that are widely accepted in the art. "Original or template nucleic acid" refers to a nucleic acid that encodes a polypeptide of interest that can be "modified" (i.e., generally engineered or mutated) to encode a ridge or cavity. The original or starting nucleic acid can be a naturally occurring nucleic acid or can include a previously modified nucleic acid (e.g., a humanized antibody fragment). By "modifying" a nucleic acid is meant mutating the original nucleic acid by inserting, deleting, or replacing at least one codon that codes for an amino acid residue of interest. Usually, the codon that codes for the original residue is replaced by a codon that codes for the imported residue. Techniques for genetically modifying DNA in this manner are reviewed in Mutagenesis: a Practical Approach, MJ McPherson, Ed., (IRL Press, Oxford, UK. (1991) and include, for example, site-directed mutagenesis, cassette mutagenesis, and polymerase chain reaction (PCR) mutagenesis. Mutagenizing the original nucleic acid / template nucleic acid results in a corresponding modification of the original polypeptide / template polypeptide encoded by the original nucleic acid / template nucleic acid.

[0176] A protuberance or cavity can be "introduced" into the interface of a first or second polypeptide by synthetic means, such as recombinant techniques, in vitro peptide synthesis, the techniques described above for introducing non-naturally occurring amino acid residues, enzymatic or chemical coupling of peptides, or any combination of these techniques. The protuberance or cavity that is "introduced" is thus "non-naturally occurring" or "non-native", meaning that such protuberance or cavity is not present in the natural or original polypeptide (e.g., a humanized monoclonal antibody).

[0177] In general, amino acid residues important for forming the ridge have a relatively small number of "rotamers" (e.g., about 3-6). A "rotamer" is an energetically favorable conformation of an amino acid side chain. The number of rotamers for various amino acid residues is reviewed in Ponders and Richards, J. Mol. Biol. 193: 775-791 (1987).

[0178] In some embodiments of the methods provided herein, the first heterodimerization domain of the heteromultimeric protein comprises a knob modification at the interface and the second heterodimerization domain comprises a hole modification. In certain embodiments, the knob modification comprises replacing an original amino acid residue from the first heterodimerization domain with an amino acid residue having a larger side chain than the original amino acid residue. In certain embodiments, the amino acid residue replacing the larger side chain is tryptophan, phenylalanine, tyrosine, or arginine. In certain embodiments, the knob modification comprises a T366W substitution (EU numbering). In certain embodiments, the hole modification comprises replacing an amino acid residue from the second heteromultimerization domain with an amino acid residue having a smaller side chain. In certain embodiments, the amino acid replacing the smaller side chain is serine, threonine, valine, or alanine. In some embodiments, the hole modification comprises two or more amino acid substitutions, including T366S, L368A, and / or Y407V (EU numbering).

[0179] IV. Vectors, Host Cells, and Recombinant Methods For recombinant production of the heteromultimeric protein (e.g., antibody) of the present invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. The DNA encoding the antibody is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends, in part, on the host cell to be used. Generally, the host cell is of mammalian origin. It is understood that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

[0180] a. Production of heteromultimeric proteins using mammalian host cells: Generally, vector components include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0181] i. Signal sequence component Vectors for use in mammalian host cells may also include a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. Preferably, the heterologous signal sequence selected is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences are available as well as viral secretory leaders, such as the herpes simplex gD signal. The DNA of such a precursor region is ligated in reading frame to DNA encoding the desired heteromultimeric protein(s) (e.g., an antibody).

[0182] ii. Replication starting point Generally, the origin of replication component is not needed for mammalian expression vectors For example, the SV40 origin is typically used only because it contains the early promoter.

[0183] iii. Selection gene component Expression and cloning vectors can contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where appropriate, or (c) supply critical nutrients not available from complex media.

[0184] One example of a selection scheme utilizes drugs to arrest the growth of host cells. Cells that have been successfully transformed with a heterologous gene will produce a protein that confers drug resistance and therefore survive the selection regime. Examples of such domain selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0185] Further examples of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably a primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc.

[0186] For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in medium containing methotrexate (Mtx), a competitive antagonist of DHFR. A preferred host cell when wild-type DHFR is used is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0187] Alternatively, host cells transformed or co-transformed with an antibody encoding DNA sequence, a wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) (particularly wild-type hosts containing endogenous DHFR) can be selected by growing the cells in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See, e.g., U.S. Patent No. 4,965,199.

[0188] iv. Promoter Component Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the desired hinge-containing polypeptide nucleic acid. Promoter sequences for mammalian cells are known. Virtually all mammalian genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription begins. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most mammalian genes is an AATAAA sequence that may be a signal for addition of a poly A tail to the 3' end of the coding sequence. All of these sequences are appropriately inserted into mammalian expression vectors.

[0189] The desired hinge-containing polypeptide and light chain transcription from the vector in a mammalian host cell is controlled by promoters derived from the genomes of viruses such as, for example, polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, simian virus 40 (SV40), or from heterologous mammalian promoters such as the actin promoter or immunoglobulin promoters, or from heat shock promoters, provided that the promoters are compatible with the host cell system.

[0190] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), for expression of human β-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0191] v. enhancer element component Transcription of DNA encoding the desired hinge-containing polypeptide(s) and light chain(s) by mammalian host cells can be increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, and insulin genes). Enhancers from mammalian cell viruses may also be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) for a description of enhancing elements for eukaryotic promoter activation. The enhancer may be spliced ​​into the vector at a position 5' or 3' from the antibody polypeptide coding sequence, as long as it is functional, but is generally located at a position 5' from the promoter.

[0192] vi. Transcription termination components Expression vectors used in mammalian host cells will typically also contain sequences necessary for transcription termination and for mRNA stabilization. Such sequences can be obtained from the 5', and occasionally the 3', untranslated regions of mammalian or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94 / 11026 and the expression vectors disclosed therein.

[0193] vii. Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein include the mammalian cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture as described in Graham et al., J. Gen Virol. 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y Acad. Sci. 383: 44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0194] Host cells are transformed with the above-described expression or cloning vectors for the desired hinge-containing polypeptide and light chain production and cultured in common nutrient media appropriately modified to contain promoters and to select for transformants or amplify the genes encoding the desired sequences. In some embodiments, the host cells are stably transfected host cells. In certain embodiments, the host cells are stable cell lines.

[0195] viii. Cultivation of host cells The host cells used to produce the desired hinge-containing polypeptides and light chains of the present invention may be cultured in a variety of culture media. Commercially available culture media, such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, the methods described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem.102:255 Any of the culture media described in (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Reissue Patent No. 30985 can be used as a culture medium for the host cells. Any of these culture media can contain additional ingredients such as hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), and the like. TM The culture conditions may include, as needed, supplementation with nutrients (such as drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions such as temperature, pH, etc. will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0196] ix. Purification of heteromultimeric proteins When using recombinant techniques, the hinge-containing polypeptide linked to the light chain polypeptide can be produced intracellularly or can be directly secreted into the medium. If the hinge-containing polypeptide and the light chain polypeptide are produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. If the hinge-containing polypeptide linked to the light chain polypeptide is secreted into the medium, the supernatant of such an expression system is usually first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0197] Heteromultimeric compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification method. The suitability of Protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. The antibody is C H If it contains 3 domains, use Bakerbond ABX™ resin for purification. TMResin (JT Baker, Phillipsburg, NJ) is useful. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, heparin SEPHAROSE TM Chromatography on polyaspartic acid columns, chromatography on anion or cation exchange resins (eg, polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0198] Following any preparatory purification step(s), the mixture containing the antibody of interest and contaminants is subjected to low pH hydrophobic interaction chromatography, preferably performed at a low salt concentration (e.g., about 0-0.25 M salt), with an elution buffer at a pH of about 2.5-4.5. Alternatively or additionally (to any of the specific methods above), production of the heteromultimeric protein can include dialyzing the solution containing the mixture of polypeptides.

[0199] V. Heteromultimeric Protein Formation / Assembly The formation of the complete heteromultimeric protein involves the reassembly of the first hinge-containing polypeptide, the first light chain, the second hinge-containing polypeptide, and the second light chain by disulfide bond formation, which is referred to as refolding in the present invention. Refolding includes the binding of the first hinge-containing polypeptide to the second hinge-containing polypeptide and the formation of interchain disulfide bonds, for example, to form a bispecific antibody. Thus, in some embodiments of the methods provided herein, the interchain disulfide bonds of the heteromultimeric protein are between the hinge regions of the first and second hinge-containing polypeptides. Refolding, also referred to as renaturation in the present invention, is performed in vitro.

[0200] After the hinge-containing polypeptide and the associated light chain are secreted from the cell, the heteromultimerization domain will promote the association of the heteromultimeric protein. Interchain disulfide formation of the associated hinge-containing polypeptide proceeds. The resulting disulfide-linked heteromultimeric protein is then purified. Optionally, such proteins may be formulated for research, diagnostic, therapeutic or other purposes.

[0201] VI.Target molecule Examples of molecules that can be targeted by the heteromultimeric proteins of the present invention include, but are not limited to, soluble serum proteins and their receptors, as well as other membrane-bound proteins (eg, adhesins).

[0202] In another embodiment, the heteromultimeric protein of the present invention comprises one or more cytokines, cytokine-related proteins, and / or ligands selected from the group consisting of BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, CSFl (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNAl, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNBl, IFNG, IFNWl, FELLl, FELLl(EPSELON), FELLl(ZETA), IL 1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL 25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA(TNF-b), LTB, TNF(TNF-a), TNFSF4(OX40 ligand), TNFSF5(CD40 TNFSF6(FasL), TNFSF7(CD27 ligand), TNFSF8(CD30 ligand), TNFSF9(4-1BB ligand), TNFSFl0(TRAIL), TNFSF1l(TRANCE), TNFSF12(APO3L), TNFSF13( April), TNFSF13B, TNFSF14(HVEM-L), TNFSF15(VEGI), TNFSF18, HGF(VEGFD), VEGF, VEGFB, VEGFC, ILlR1, IL1R2, IL1RL1, LL1RL2, IL2RA, IL2RB, IL2RG , IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, ILl0RA, ​​ILl0RB, IL1lRA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA,It can bind to a cytokine receptor selected from the group consisting of IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIFl, HGF, LEP (leptin), PTN, and THPO.

[0203] In another embodiment, the target molecule is a chemokine, a chemokine receptor, or a chemokine-specific agonist such as CCL1 (I-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP-Ia), CCL4 (MIP-Ib), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCLH (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (TARC), CCL21 (TARC), CCL22 (TARC), CCL23 (TARC), CCL24 (TARC), CCL25 (TARC), CCL26 (TARC), CCL27 (TARC), CCL28 (TARC), CCL29 (TARC), CCL30 (TARC), CCL31 (TARC), CCL32 (TARC), CCL33 (TARC), CCL34 (TARC), CCL35 (TARC), CCL36 (TARC), CCL37 (TARC), CCL38 (TARC), CCL39 (TARC), CCL40 (TARC), CCL41 (TARC), CCL42 (TARC), CCL43 (TARC), CCL44 (TARC), CCL45 (TARC), CCL46 (TARC), CCL47 (TARC), CCL48 (TARC), CCL49 (TARC), CCL50 (TARC), CCL51 (TARC), CCL52 (TARC), CCL53 (TARC), CCL54 (TARC), CCL55 (TARC), CCL56 (TARC), CCL57 (TARC), CCL58 (TARC), CCL59 (TARC), CCL60 (TARC), CCL61 (TARC), CCL62 (TARC 20 (MIP-3a), CCL21 (SLC / Exodus-2), CCL22 (MDC / STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2 / Eotaxin-2), CCL25 (TECK), CCL26 (Eotaxin-3), CCL27 (CTACK / ILC), CCL28, CXCLl (GROl), CXCL2 (GRO2), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP10), CXCL11 (I-TAC), CXCL12 (SDFl), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYDl), SCYEl, / RA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR 7 / EBIl), CCR8(CMKBR8 / TERl / CKR-Ll), CCR9(GPR-9-6), CCRLl(VSHKl), CCRL2(L-CCR), XCRl(GPR5 / CCXCRl) , CMKLRl, CMKORl(RDCl), CX3CR1(V28), CXCR4, GPR2(CCRlO), GPR31, GPR81(FKSG80), CXCR3(GPR9 / CKR-L2) , CXCR6(TYMSTR / TRL33 / Bonzo), HM74, IL8RA(IL8Ra), IL8RB(IL8Rb), LTB4R(GPR16), TCPlO, CKLFSF2, CKLFS and a chemokine-related protein selected from the group consisting of F3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCClO(ClO), EPO, FY(DARC), GDF5, HDFlA, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREMl, TREM2, and VHL.

[0204] In another embodiment, the heteromultimeric protein of the invention is any of the following: ABCFl;ACVRl;ACVRlB;ACVR2;ACVR2B;ACVRLl;AD0RA2A;Aggrecan;AGR2;AICDA;AIFl;AIGl;AKAPl;AKAP2;AMH;AMHR2;ANGPTl;ANGPT2;ANGPTL3;ANGPTL4;ANPEP;APC;APOCl;AR;AZGPl (zinc-a-glycoprotein);B7.1;B7.2;BAD;BAFF(BLys);BAGl;BAIl;BCL2;BCL6;BDNF;BLNK;B LRl(MDR15);BMPl;BMP2;BMP3B(GDFlO);BMP4;BMP6;BMP8;BMPRlA;BMPRlB;BMPR2;BPAGl(plectin);BRCAl;C19orflO(IL27w);C3;C4A;C5;C5R1;CANTl; CASP1;CASP4;CAVl;CCBP2(D6 / JAB61);CCLl(1-309);CCLIl(eotaxin);CCL13(MCP-4);CCL15(MIP-Id);CCL16(HCC-4);CCL17(TARC);CCL18(PARC);CC L19(MIP-3b);CCL2(MCP-1);MCAF;CCL20(MIP-3a);CCL21(MTP-2);SLC;Exodus-2;CCL22(MDC / STC-I);CCL23(MPIF-1);CCL24(MPIF-2 / eotaxin-2);CC L25(TECK);CCL26(eotaxin-3);CCL27(CTACK / ILC);CCL28;CCL3(MTP-Ia);CCL4(MDP-Ib);CCL5(RANTES);CCL7(MCP-3);CCL8(mcp-2);CCNAl;CCNA2;C CNDl;CCNEl;CCNE2;CCRl(CKRl / HM145);CCR2(mcp-lRB / RA);CCR3(CKR3 / CMKBR3);CCR4;CCR5(CMKBR5 / ChemR13);CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6 );CCR7(CKR7 / EBIl);CCR8(CMKBR8 / TERl / CKR-Ll);CCR9(GPR-9-6);CCRLl(VSHKl);CCRL2(L-CCR);CD164;CD19;CDlC;CD20;CD200;CD22;CD24;CD28;CD3;CD37;CD38;CD3E;CD3G;CD3Z;CD4;CD40;CD40L;CD44;CD45RB;CD52;CD69;CD72;CD74;CD79A;CD79B;CD8;CD80;CD81;CD83;CD86;CDHl(E-カドヘリン);CDH10;CDH12;CDH13;CDH18;CDH19;CDH20;CDH5;CDH7;CDH8;CDH9;CDK2;CDK3;CDK4;CDK5;CDK6;CDK7;CDK9;CDKNlA(p21Wapl / Cipl);CDKNlB(p27Kipl);CDKNlC;CDKN2A(P16INK4a);CDKN2B;CDKN2C;CDKN3;CEBPB;CERl;CHGA;CHGB;Chitinase;CHST10;CKLFSF2;CKLFSF3;CKLFSF4;CKLFSF5;CKLFSF6;CKLFSF7;CKLFSF8;CLDN3;CLDN7(クローディン-7);CLN3;CLU(クラステリン);CMKLRl;CMKORl(RDCl);CNRl;COL18A1;COLlAl;COL4A3;COL6A1;CR2;CRP;CSFl(M-CSF);CSF2(GM-CSF);CSF3(GCSF);CTLA4;CTNNBl(b-カテニン);CTSB(カテプシンB);CX3CL1(SCYDl);CX3CR1(V28);CXCLl(GROl);CXCL10(IP-10);CXCLIl(I-TAC / IP-9);CXCL12(SDFl);CXCL13;CXCL14;CXCL16;CXCL2(GRO2);CXCL3(GRO3);CXCL5(ENA-78 / LIX);CXCL6(GCP-2);CXCL9(MIG);CXCR3(GPR9 / CKR-L2);CXCR4;CXCR6(TYMSTR / STRL33 / Bonzo);CYB5;CYCl;CYSLTRl;DAB2IP;DES;DKFZp451J0118;DNCLl;DPP4;E2F1;ECGFl;EDGl;EFNAl;EFNA3;EFNB2;EGF;EGFR;ELAC2;ENG;ENO1;ENO2;ENO3;EPHB4;EPO;ERBB2(Her-2);EREG;ERK8;ESRl;ESR2;F3(TF);FADD;FasL;FASN;FCERlA;FCER2;FCGR3A;FGF;FGFl(aFGF);FGF10;FGF11;FGF12;FGF12B;FGF13;FGF14;FGF16;FGF17;FGF18;FGF19;FGF2(bFGF);FGF20;FGF21;FGF22;FGF23;FGF3(int-2);FGF4(HST); ;FGF5;FGF6(HST-2);FGF7(KGF);FGF8;FGF9;FGFR3;FIGF(VEGFD);FELl(シン);FILl(ZETA);FLJ12584;FLJ25530;FLRTl(floxacin);FLTl;FOS;FOSLl( FRA-I);FY(DARC);GABRP(GABA);GAGEBl;GAGECl;GALNAC4S-6ST;GATA3;GDF5;GFI1;GGT1;GM-CSF;GNASl;GNRHl;GPR2(CCRlO);GPR31;GPR44;GPR81(FK). SG80);GRCClO(ClO);GRP;GSN(Gelsolin);GSTPl;HAVCR2;HDAC4;HDAC5;HDA C7A;HDAC9;HGF;HIFlA;HDPl;Frenchylexin-French Activator;HLA-A;HLA-DRA;HM74;HMOXl ;HUMCYT2A;ICEBERG;ICOSL;ID2;IFN-a;IFNAl;IFNA2;IFNA4;IFNA5;IFNA6;IFNA7;IFNB1;IFNgamma;DFNWl;IGBPl;IGFl;IGFlR;IGF2;IGFBP2;I GFBP3;IGFBP6;IL-I;IL10;IL10RA;IL10RB;IL11;IL11RA;IL-12;IL12A;IL12B;IL12RB1;IL12RB2;IL13;IL13RA1;IL13RA2;IL14;IL15;IL15RA; IL16;IL17;IL17B;IL17C;IL17R;IL18;IL18BP;IL18R1;IL18RAP;IL19;IL1A;IL1B;ILlF10;IL1F5;IL1F6;IL1F7;IL1F8;IL1F9;IL1HYL;IL1Rl;I L1R2;IL1RAP;IL1RAPL1;IL1RAPL2;IL1RL1;IL1RL2、ILlRN;IL2;IL20;IL20RA;IL21R;IL22;IL22R;IL22RA2;IL23;IL24;IL25;IL26;IL27;IL28A;IL28B;IL29;IL2RA;IL2RB;IL2RG;IL3;IL30;IL3RA;IL4;IL4R;IL5;IL5RA;IL6;IL6R;IL6ST(protein130);EL7;EL7R;EL8;IL8RA;DL 8RB;IL8RB;DL9;DL9R;DLK;INHA;INHBA;INSL3;INSL4;IRAQ;ERAK2;ITGAl;ITGA2;ITGA3;ITGA6(a6イテリン);ITGAV;ITGB3;ITGB4(b 4THROACH);JAGl;JAKl;JAK3;JUN;K6HF;KAIl;KDR;KITLG;KLF5(GC Box BP);KLF6;KLKlO;KLK12;KLK13;KLK14;KLK15;KLK3;KLK4;KLK5;KLK6;KLK9;KRT1;KRT19(Keratin 19);KRT2A;KHTHB6(Knight-Transforming H Link);Lep;LEP(Lingo);Lingo-p75;Lingo-Troy; LPS;LTA(TNF-b);LTB;LTB4R(GPR16);LTB4R2;LTBR;MACMARCKS; ;MAP2K7(c-Jun);MDK;MIBl;ツ;MEF;MIP-2;MKI67;(Ki-67);MMP2;MMP9 ;MS4A1;MSMB;MT3(mucosa-III);MTSSl;MUCl(mucosa);MYC;MYD88;NCK2;MYCーカン;NFKBl;NFKB2;NGFB(NGF);NGFR;NgR-Lingo;NgR-Nogo66(Nogo);NgR-p75;NgR-Troy;NMEl(NM23A);N0X5;NPPB;NROBl;NR0B2;NRlDl;NR1D2;NR1H2 ;NR1H3;NR1H4;NR1I2;NR1I3;NR2C1;NR2C2;NR2E1;NR2E3;NR2F1;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRPl;NRP2;NT 5E;NTN4;ODZl;OPRDl;P2RX7;PAP;PARTl;PATE;PAWR;PCA3;PCNA;PDGFA;PDGFB;PECAMl;PF4(CXCL4);PGF;PLXDC1;PPBP(CXCL7);PPID;PRl;PRKCQ;PRKDl;PRL;PROC;PROK2;PSAP;PSCA;PTAFR;PTEN;PTGS2(COX-2);PTN;RAC2(p21Rac2);RARB;RGSl;RGS13;RGS3;RNFIlO(ZNF144);ROBO2;S100A2;SCGB1D2(lipophilin B);SCGB2A1(mammaglobin 2);SCGB2A2(mammaglobin 1);SCYEl(endothelial monocyte-activating cytokine);SDF2;SERPINAl;SERPINA3;SERP1NB5(maspin);SERPINEl(PAI-I);SERPDMF1;SHBG;SLA2;SLC2A2;SLC33A1;SLC43A1;SLIT2;SPPl;SPRRlB(Sprl);ST6GA L1;STABl;STAT6;STEAP;STEAP2;TB4R2;TBX21;TCPlO;TDGFl;TEK;TGFA;TGFBl;TGFBlIl;TGFB2;TGFB3;TG FBI;TGFBRl;TGFBR2;TGFBR3;THlL;THBSl(Thrombospondin-1);THBS2;THBS4;THPO;TIE(Tie-1);TMP3;Tissue factor;TL RlO;TLR2;TLR3;TLR4;TLR5;TLR6;TLR7;TLR8;TLR9;TNF;TNF-a;TNFAEP2(B94);TNFAIP3;TNFRSFIlA;TNFR SFlA;TNFRSFlB;TNFRSF21;TNFRSF5;TNFRSF6(Fas);TNFRSF7;TNFRSF8;TNFRSF9;TNFSFlO(TRAIL);TNFSFl 1(TRANCE);TNFSF12(APO3L);TNFSF13(April);TNFSF13B;TNFSF14(HVEM-L);TNFSF15(VEGI);TNFSF18;TNFSF4(OX40 ligand);TNFSF5(CD40 ligand);TNFSF6(FasL);TNFSF7(CD27 ligand);TNFSF8(CD30 ligand);TNFSF9(4-1BB ligand);TOLLIP;Toll-like receptor;TOP2A(Topoisomerase Ea);TP53;TPMl;TPM2;TRADD;TRAFl;TRAF2;TRAF3;TRAF4;TRAF5;TRAF6;TREMl;Selected from the group consisting of TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCLl (lymphotactin); XCL2 (SCM-Ib); XCRl (GPR5 / CCXCRl); YYl; and ZFPM2; It may bind to one or more targets.

[0205] Preferred molecular targets of antibodies encompassed by the invention include CD proteins such as CD3, CD4, CD8, CD16, CD19, CD20, CD34; CD64, CD200 members of the ErbB receptor family, such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules, such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, alpha4 / beta7 integrin, and alphav / beta3 integrin (including its alpha or beta subunits) (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF-A, VEGF-C; tissue factor (TF); alpha interferon (alphaIFN); TNF alpha, interleukins such as IL-1 beta, IL-3, IL-4, IL-5, IL-8, IL-9, IL-13, IL17A / F, IL-18, IL-13R alpha 1, IL13R alpha 2, IL-4R, IL-5R, IL-9R, IgE; blood group antigens; flk2 / flt3 receptors; obesity (OB) receptors; mpl receptors; CTLA-4; RANKL, RANK, RSV F protein, protein C, and the like.

[0206] In one embodiment, the heteromultimeric protein of the present invention binds to low density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor, and at least one target selected from the group consisting of 1) beta-secretase (BACE1 or BACE2), 2) alpha-secretase, 3) gamma-secretase, 4) tau-secretase, 5) amyloid precursor protein (APP), 6) death receptor 6 (DR6), 7) amyloid beta peptide, 8) alpha-synuclein, 9) parkin, 10) huntingtin, 11) p75 NTR, and 12) caspase-6.

[0207] In one embodiment, the heteromultimeric protein of the present invention is selected from the group consisting of IL-1 alpha and IL-1 beta, IL-12 and IL-18; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-1 beta; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-12 and TWEAK, IL-13 and CL25; IL-13 and SP RR2a;IL-13 and SPRR2b;IL-13 and ADAM8,IL-13 and PED2,IL17A and IL17F,CD3 and CD19,CD138 and CD20;CD138 and CD40;CD19 and CD20;CD20 and CD3;CD38 and CD138;CD38 and CD20;CD38 and CD40;CD40 and CD20;CD-8 and IL-6;CD20 and BR3,TNF alpha and TGF-beta,TNF alpha and IL-1 beta;TNF alpha and IL-2,TNF alpha and IL-3, TNF alpha and IL-4, TNF alpha and IL-5, TNF alpha and IL6, TNF alpha and IL-8, TNF alpha and IL-9, TNF alpha and IL-10, TNF alpha and IL-11, TNF alpha and IL-12, TNF alpha and IL-13, TNF alpha and IL-14, TNF alpha and IL-15, TNF alpha and IL-16, TNF alpha and IL-17, TNF alpha and IL-18, TNF alpha and IL-19, TNF a IL-20; TNF alpha and IL-23; TNF alpha and IFN alpha; TNF alpha and CD4; TNF alpha and VEGF; TNF alpha and MIF; TNF alpha and ICAM-1; TNF alpha and PGE4; TNF alpha and PEG2; TNF alpha and RANK ligand; TNF alpha and Te38; TNF alpha and BAFF; TNF alpha and CD22; TNF alpha and CTLA-4; TNF alpha and GP130; TNF alpha and IL-12p40;VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, VEGFR and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR (HER1) and HER2, EGFR and HER3, EGFR and HER4, IL-13 and CD40L, IL4 and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTNO2; IGF1 and IGF2; IGF1 / 2 and Erb2B; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; PDL-I and CTLA-4; and RGM A and RGM B.

[0208] Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens to generate antibodies. For transmembrane molecules such as receptors, fragments of these (e.g., extracellular domains of receptors) can be used as immunogens. Alternatively, cells expressing the transmembrane molecule can be used as immunogens. Such cells can be from natural sources (e.g., cancer cell lines) or can be cells that have been recombinantly transformed to express the transmembrane molecule. Other antigens and forms thereof useful for the preparation of antibodies will be apparent to those skilled in the art.

[0209] VII. Activity Assays The heteromultimeric proteins of the present invention can be characterized with respect to their physical / chemical properties and biological functions by various assays known in the art.

[0210] The purified heteromultimeric proteins can be further characterized by a range of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size-exclusion high performance liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0211] In certain embodiments of the present invention, the immunoglobulins produced herein are analyzed for their biological activity. In some embodiments, the immunoglobulins of the present invention are tested for their antigen-binding activity. Antigen-binding assays known in the art and can be used herein include, but are not limited to, any direct or competitive binding assay using techniques such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescent immunoassay and protein A immunoassay. Specific antigen-binding assays are provided in the Examples section below.

[0212] In one embodiment, the present invention contemplates engineered antibodies that have some, but not all, effector functions, making them desirable candidates for many applications where in vivo antibody half-life is important, but where certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In certain embodiments, the Fc activity of the generated heteromultimeric protein is measured to ensure that only the desired properties are maintained. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the heteromultimeric protein lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express only Fc(RIII, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII). Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). One example of an in vitro assay to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest can be assessed using immunohistochemistry, e.g., as described in Clynes et al., PNAS (USA) 95:652-656. (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind to C1q and thus lacks CDC activity. To assess complement activation, a CDC assay can be performed, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art.

[0213] VIII. Conjugated Proteins The invention also provides conjugated proteins, such as conjugated antibodies or immunoconjugates (e.g., "antibody-drug conjugates" or "ADCs"), including heteromultimeric proteins described herein (e.g., antibodies made according to the methods described herein) in which one of the constant regions of the light or heavy chains is conjugated to a chemical molecule, such as a dye, or a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin), or a radioisotope (i.e., a radioconjugate). In particular, the use of heteromultimerization domains, as described herein, allows for the construction of antibodies that include two different heavy chains (HC1 and HC2) and two different light chains (LC1 and LC2). Immunoconjugates constructed using the methods described herein may include a cytotoxic agent conjugated to only one of the constant regions of the heavy chain (HC1 or HC2) or only one of the constant regions of the light chain (LC1 or LC2). Also, because an immunoconjugate can have a cytotoxic agent attached to only one of the heavy or light chains, the amount of cytotoxic agent administered to a subject is reduced compared to administration of an antibody having a cytotoxic agent attached to both heavy or light chains. By reducing the amount of cytotoxic agent administered to a subject, adverse side effects associated with the cytotoxic agent are limited.

[0214] The use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, i.e., agents for killing or inhibiting tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Research 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278), allows for targeted delivery of the drug moiety to, and intracellular accumulation in, tumors, where administration of these unconjugated agents can result in unacceptable levels of toxicity not only to the tumor cells that are being eliminated, but also to normal cells (Baldwin et al., Lancet (Mar. 15, 1986):603-605 (1986); Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). Thus, maximum efficacy with minimal toxicity is sought. Both polyclonal and monoclonal antibodies have been reported to be useful in such strategies (Rowland et al., Cancer Immunol. Immunother. 21:183-187 (1986)). Drugs that have been used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, geldanamycin (Mandler et al., Jour. of the Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)). Toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic agents tend to become inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0215] Chemotherapeutic agents useful for generating immunoconjugates are described herein (e.g., above). Enzymatically active toxins and fragments thereof which may be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Abrasion formosum protein, dianthin protein, pokeweed protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the trichothecenes. See, for example, WO 93 / 21232 published Oct. 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include:212 Bi, 131 I, 131 In, 90 Y and 186 Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionuclides to antibodies. See, for example, WO 94 / 11026.

[0216] Conjugates of an antibody and one or more small molecule toxins (e.g., calicheamicin, maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, and derivatives of these toxins that have toxin activity) are also contemplated in the present invention. Details regarding such small molecule toxins are provided in WO 2008 / 021290.

[0217] i. Maytansine and maytansinoids In some embodiments, the immunoconjugate comprises an antibody of the invention (full length or fragments) conjugated to one or more maytansinoid molecules.

[0218] Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinoids and their derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,308,270; 4,308,271; 4,308,272; 4,308,273; 4,308,274; 4,308,275; 4,308,276; 4,308,277; 4,308,278; 4,308,279 ... 9428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663; and 4371533.

[0219] Maytansinoid drug moieties are attractive drug moieties for antibody-drug conjugates because they are (i) relatively easy to prepare by fermentation or chemical modification or derivatization of the fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[0220] Immunoconjugates containing maytansinoids, methods for their preparation and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, and EP 0 425 235, the disclosures of which are incorporated herein by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describe an immunoconjugate containing a maytansinoid designated DM1 that binds to the monoclonal antibody C242 against human colorectal cancer. The conjugate was found to be highly cytotoxic against cultured colon cancer cells and exhibited antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) describe immunoconjugates in which maytansinoids are conjugated to the murine antibody A7, which binds via a disulfide linker to an antigen on a human colon cancer cell line, or to another murine monoclonal antibody, TA.1, which binds to the HER-2 / neu oncogene. The cytotoxicity of the TA.1-maytansinoid conjugates was tested in vitro on the human breast cancer cell line SK-BR-3, with a cytotoxicity of 3x10 per cell. 5 The A7-maytansinoid conjugates showed low systemic cytotoxicity in mice.

[0221] Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly reducing the biological activity of either the antibody or the maytansinoid molecule. See, for example, U.S. Pat. No. 5,208,020, the disclosure of which is expressly incorporated herein by reference. Although one molecule of toxin / antibody would be expected to increase cytotoxicity over the use of naked antibodies, conjugations of an average of 3-4 maytansinoid molecules per antibody molecule have shown efficacy in improving cytotoxicity of target cells without adversely affecting the function or solubility of the antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in the aforementioned U.S. Pat. No. 5,208,020 and other patents, as well as in non-patent literature. Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or other positions of the maytansinol molecule, such as various maytansinol esters.

[0222] There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Pat. No. 0425235B1; Chari et al., Cancer Research 52:127-131 (1992); and U.S. Patent Application Publication No. 2005 / 0169933, the disclosures of which are expressly incorporated herein by reference. Antibody-maytansinoid conjugates containing the linker moiety SMCC can be prepared as disclosed in U.S. Patent Application Publication No. 2005 / 0169933. Such linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups as disclosed in the above-listed patents, with disulfide and thioether groups being preferred. Additional linking groups are described and exemplified herein.

[0223] Conjugates of antibodies and maytansinoids can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Particularly suitable coupling agents for providing disulfide bonds include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP).

[0224] The linker can be attached to the maytansinoid molecule at various positions, depending on the type of linkage. For example, an ester bond is formed by reaction with a hydroxyl group using conventional coupling techniques. Reaction can occur at the C-3 position, which bears a hydroxyl group, the C-14 position, which is modified with a hydroxyl group, the C-15 position, which is modified with a hydroxyl group, and the C-20 position, which bears a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analogue.

[0225] ii. Auristatins and dolastatins In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated to auristatins (U.S. Pat. Nos. 5,635,483 and 5,780,588), which are dolastatins or peptide analogs and derivatives of dolostatins. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al., Antimicrob. Agents and Chemother. 45(12):3580-3584 (2001)), and have anticancer activity (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965 (1998)). The dolastatin or auristatin drug moiety can be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02 / 088172).

[0226] Exemplary auristatin embodiments include N-terminally linked monomethylauristatin drug moieties DE and DF, as disclosed in "Monomethylvaline Compounds Capable of Conjugation to Ligands," U.S. Patent Application Publication No. 2005 / 0238649, the entire disclosure of which is incorporated herein by reference.

[0227] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments, for example, according to liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schroder and K. Lubke, "The Peptides," volume 1, pp. 76-136, 1965, Academic Press). Auristatin / dolastatin drug moieties may be prepared according to the methods described in U.S. Pat. Nos. 5,635,483 and 5,780,588; Pettit et al., J. Nat. Prod. 44:482-485 (1981); Pettit et al., Anti-Cancer Drug Design 13:47-66 (1998); Poncet, Curr. Pharm. Des. 5:139-162 (1999); and Pettit, Fortschr. Chem. Org. Naturst. 70:1-79 (1997). See also, Doronina, Nat. Biotechnol. 21(7):778-784 (2003); and "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Patent Application Publication No. 2005 / 0238649, the entire disclosure of which is incorporated herein by reference (disclosing, e.g., linkers and methods for preparing monomethylvaline compounds, e.g., MMAE and MMAF conjugated to linkers).

[0228] iii. Calicheamicin In another embodiment, the immunoconjugate comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of inducing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that may be used include, but are not limited to, γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG and θ I 1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the U.S. patent to American Cyanamid cited above). Another antitumor drug to which antibodies can be conjugated is the antifolate drug QFA. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, uptake of these drugs into cells via antibody-mediated internalization greatly enhances the cytotoxic effects of the drugs.

[0229] iv. Other cytotoxic agents Other anti-tumor agents that can be conjugated to the antibodies of the present invention or made according to the methods described herein include BCNU, streptozocin, vincristine and 5-fluorouracil, the family of agents described in U.S. Pat. Nos. 5,053,394; 5,770,710 and collectively known as LL-E33288 conjugates, and esperamicin (U.S. Pat. No. 5,877,296).

[0230] Enzymatically active toxins and fragments thereof which may be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes (see, e.g., WO 93 / 21232, published October 28, 1993).

[0231] The present invention further contemplates immunoconjugates formed between an antibody and a compound having nucleolytic activity (eg, a ribonuclease, or a DNA endonuclease such as a deoxyribonuclease (ie, DNase)).

[0232] For selective destruction of tumors, the antibody may contain a highly radioactive atom. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu. When used for detection, the conjugates contain radioactive atoms for scintigraphic examinations, e.g., tc 99m Or I 123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0233] Radiolabels or other labels can be incorporated into the conjugates in known manner. For example, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using appropriate amino acid precursors, e.g., with fluorine-19 instead of hydrogen. Labels, e.g., tc 99m or I 123 , Re 186 , Re 188 and In 111 can be attached through a cysteine ​​residue in the peptide. Yttrium-90 can be attached through a lysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun. 80:49-57 (1978)) can be used to incorporate iodine-123. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0234] Conjugates of the antibody and cytotoxic agent can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionuclides to antibodies. See, for example, WO 94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic agent in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) can be used.

[0235] The compounds of the present invention specifically contemplate ADCs prepared with, but not limited to, the following crosslinkers, which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA): BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate. See pages 467-498 of the 2003-2004 Applications Handbook and Catalog.

[0236] v. Preparation of conjugated antibodies In the conjugated antibodies of the present invention, the antibody is conjugated to one or more moieties (e.g., drug moieties), for example, from about 1 to about 20 moieties per antibody, optionally via a linker. The conjugated antibodies can be prepared by several routes utilizing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including (1) reacting a nucleophilic group of the antibody with a bivalent linker reagent via a covalent bond, followed by reaction with the moiety of interest; and (2) reacting a nucleophilic group of the moiety with a bivalent linker reagent via a covalent bond, followed by reaction with the nucleophilic group of the antibody. Additional methods for preparing conjugated antibodies are described herein.

[0237] The linker reagent may consist of one or more linker moieties. Exemplary linker moieties include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). Additional linker moieties are known in the art, some of which are described herein. See also “Monomethylvaline Compounds Capable of Conjugation to Ligands” (U.S. Patent Application Publication No. 2005 / 0238649), the entire disclosure of which is incorporated herein by reference.

[0238] In some embodiments, the linker may comprise an amino acid residue. Exemplary amino acid linker moieties include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues that comprise the amino acid linker moiety include naturally occurring as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker moieties can be designed and optimized for selectivity of enzymatic cleavage by a particular enzyme, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0239] Nucleophilic groups on an antibody include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, such as lysine, (iii) side chain thiol groups, such as cysteine, and (iv) hydroxyl or amino groups of the sugar on which the antibody is glycosylated. Amine, thiol and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, including (i) active esters, such as NHS esters, HOBt esters, haloformates and acid halides, (ii) alkyl and benzyl halides, such as haloacetamides; (ii) aldehydes, ketones, carboxyls and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine ​​bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with reducing agents, such as DTT (dithiothreitol). Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody by reaction of lysine with 2-iminothiolane (Traut's reagent), resulting in the conversion of an amine to a thiol. Reactive thiol groups can be introduced into the antibody (or fragment thereof) by introducing one, two, three, four or more cysteine ​​residues (e.g., preparing a mutant antibody containing one or more non-native cysteine ​​amino acid residues).

[0240] Conjugated antibodies of the invention can also be generated by modifying an antibody to introduce electrophilic moieties that can react with nucleophilic substituents on a linker reagent or a drug or other moiety. The sugars of a glycosylated antibody can be oxidized, for example with periodate oxidizing agents, to form ketone or aldehyde groups that can react with amine groups on a linker reagent or a drug or other moiety. The resulting imine Schiff base groups can form stable bonds or can be reduced, for example with borohydride reagents, to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with glactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on a drug or other moiety (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate resulting in the generation of an aldehyde in place of the first amino acid (Geoghegan and Stroh, Bioconjugate Chem. 3:138-146 (1992); U.S. Pat. No. 5,362,852). Such aldehydes can react with drug moieties or linker nucleophiles.

[0241] Similarly, nucleophilic groups on a moiety (e.g., drug moiety) include, but are not limited to, (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide groups, which can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including aldehyde, ketone, carboxyl, and maleimide groups.

[0242] Alternatively, a fusion protein comprising the antibody and the cytotoxic agent can be produced, for example by recombinant techniques or peptide synthesis. The length of DNA can include regions encoding the two parts of the conjugate adjacent to each other, or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate. In another embodiment, an antibody is conjugated to a "receptor" (e.g., streptavidin) for use in tumor pretargeting (the antibody-receptor conjugate is administered to an individual), followed by the use of a detergent to remove unbound conjugate from the circulation, and then a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide) is administered.

[0243] IX.Usefulness The method of the present invention provided herein finds industrial applicability in the production of heteromultimeric proteins. The method reduces the amount of work involved in two separate fermentations and isolation, a technical difficulty inherent in two separate fermentations. Furthermore, the elimination of the annealing and redox steps of the prior art process steps may increase yields and reduce processing complexity and costs.

[0244] The heteromultimeric proteins described herein find use in, for example, in vitro, ex vivo and in vivo therapeutic methods. The invention provides various methods based on the use of one or more of such molecules. In certain pathological conditions, it is necessary and / or desirable to utilize heteromultimeric proteins, such as multispecific antibodies. The invention provides such heteromultimeric proteins, which can be used for various purposes, such as therapeutic, preventive and diagnostic purposes. For example, the invention provides a method of treating a disease, comprising administering the heteromultimeric proteins of the invention to a subject in need of treatment, thereby treating the disease. Any of the heteromultimeric proteins of the invention described herein can be used in the therapeutic (or preventive or diagnostic) methods described herein.

[0245] For example, when a heteromultimeric protein is multivalent, a valuable advantage is the increased avidity it provides to its corresponding antigen. In addition to having an inherent high affinity for the antigen on a per-binding unit (e.g., Fab) basis, normal IgG antibodies utilize the avidity effect to increase binding to the antigen as a result of bivalent binding to the target.

[0246] Heteromultimeric proteins directed against two distinct epitopes on the same antigen molecule can not only offer the advantage of enhanced avidity (due to bivalent binding), but can also acquire novel properties not associated with any of the parent antibodies. Thus, the heteromultimeric proteins of the invention find use, for example, in blocking receptor-ligand interactions.

[0247] The heteromultimeric proteins described herein also find use in applications where one molecule is used to simultaneously block the signaling pathways of two targets.

[0248] X. Therapeutic Use The antibodies and antibody fragments described herein (e.g., antibodies and / or fragments thereof generated according to the methods described herein) can be used for therapeutic applications. For example, such heteromultimeric proteins can be used for the treatment of tumors, including precancerous, non-metastatic, metastatic and cancerous tumors (e.g., early stage cancers), for the treatment of allergic or inflammatory disorders, or for the treatment of autoimmune diseases, or for the treatment of subjects at risk of developing cancer (e.g., breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma, or ovarian cancer), allergic or inflammatory disorders, or autoimmune diseases.

[0249] The term cancer encompasses a collection of proliferative disorders, including, but not limited to, precancerous growths, benign tumors, and malignant tumors. Benign tumors remain localized at the site of origin and do not have the ability to invade, invade, or metastasize to distant sites. Malignant tumors invade and damage other surrounding tissues. Malignant tumors can also acquire the ability to break away from where they originate and spread (metastasize) to other parts of the body, usually through the bloodstream or lymphatic system where lymph nodes are located. Primary tumors are classified by the type of tissue they arise from; metastatic tumors are classified by the tissue type from which the cancer cells originate. Over time, the cells of a malignant tumor become more and more abnormal and do not look like normal cells. This change in the appearance of the cancer cells is called the tumor grade, and the cancer cells are described as well-differentiated, moderately differentiated, poorly differentiated, or undifferentiated. Well-differentiated cells have a very normal appearance and resemble the normal cells from which they originated. Undifferentiated cells are cells that have become abnormal and the origin of the cells can no longer be determined.

[0250] The tumor may be a solid tumor or a non-solid or soft tissue tumor. Examples of soft tissue tumors include leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic leukemia, or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include any cancer of body tissues other than blood, bone marrow, or lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, lips, nasopharynx, skin, uterus, male reproductive organs, urinary tract, bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors and bone tumors.

[0251] Epithelial cancers usually progress from benign tumors to a preinvasive stage (eg, carcinoma in situ) to malignant cancer (invading the basement membrane and invading the stroma beneath the epithelium).

[0252] Multispecific protein complexes may also be used in such therapeutic applications, in particular antibodies that bind to HER2 may be used to treat breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma, or ovarian cancer.

[0253] Other subjects who are candidates to receive the compositions of the present invention include those suffering from conditions including fibrovascular hyperplasia, rosacea, acquired immune deficiency syndrome, arterial occlusion, atopic keratitis, bacterial ulcers, Behcet's disease, blood borne tumors, carotid artery occlusive disease, choroidal neovascularization, chronic inflammation, chronic retinal detachment, chronic uveitis, chronic vitritis, contact lens overuse, corneal transplant rejection, corneal neovascularization, corneal graft neovascularization, Crohn's disease, Eales' disease, epidemic keratitis, and the like. Conjunctivitis, fungal ulcers, herpes simplex infections, herpes zoster infections, hyperadhesive syndrome, Kaposi's sarcoma, leukemia, fatty degeneration, Lyme disease, marginal epidermolysis, Mooren's ulcer, non-leprosy mycobacterial infections, myopia, ocular neovascular disease, enophthalmos, Osler-Weber syndrome (Osler-Weber-Rendu), osteoarthritis, Paget's disease, pars planitis, pemphigoid, glomerulitis, polyarteritis nodosa, post-laser complications, protozoal infections, ballistic missiles Pseudoxanthoma, Dry pterygium keratitis, Radial keratotomy, Retinal neovascularization, Retinopathy of prematurity, Retrolental fibroplasia, Sarcoid, Scleritis, Sickle cell anemia, Sjogren's syndrome, Solid tumors, Stargardt's disease, Stevens-Johnson disease, Superior limbal keratitis, Syphilis, Systemic lupus, Therrien's peripheral corneal degeneration, Toxoplasmosis, Ewing's sarcoma tumors, Neuroblastoma tumors, Osteosarcoma tumors, Omental tumors Having or being at risk of developing: mesoblastoma tumors, rhabdomyosarcoma tumors, ulcerative colitis, venous obstruction, vitamin A deficiency, Wegener's sarcoidosis, undesirable angiogenesis associated with diabetes, parasitic diseases, abnormal wound healing, post-surgical enlargement, injury or trauma (e.g., acute lung injury / ARDS), inhibition of hair growth, inhibition of ovulation and corpus luteum formation, inhibition of implantation, and inhibition of intrauterine embryo development.

[0254] Examples of allergic or inflammatory disorders or autoimmune diseases or disorders that can be treated using the antibodies generated according to the methods described herein include, but are not limited to, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, osteoarthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis, and juvenile onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis (arthritis chronica progrediente), arthritis deformans, early chronic polyarthritis (polyarthritis chronica), and / or other chronic conditions. psoriasis, such as psoriasis vulgaris, guttate psoriasis, cystic psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria, such as chronic allergic urticaria including chronic autoimmune urticaria and chronic idiopathic urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic dermonecrolysis, scleroderma (including systemic sclerosis), sclerosis, such as systemic sclerosis, multiple sclerosis (MS), such as spinal-ocular MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, multiple sclerosis disseminate), and ataxic sclerosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal disease, colitis (e.g., ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enteritis, and transmural colitis, and autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), adult or acute respiratory distress syndrome (ARDS). respiratory distress syndrome, including meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematological diseases, rheumatoid spondylitis, sudden deafness, IgE-mediated diseases, such as anaphylaxis and allergic and atopic rhinitis, encephalitis, such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis,phaco-allergic uveitis, posterior uveitis or autoimmune uveitis; glomerulonephritis (GN) with or without nephrotic syndrome, e.g. chronic or acute glomerulonephritis, e.g. primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous- or membranous proliferative GN (MPGN), including types I and II, and rapidly progressive GN; allergic symptoms, allergic reactions, eczema, including allergic or atopic eczema; asthma, e.g. asthma bronchiale, bronchial asthma asthma), and autoimmune asthma, conditions with T cell infiltration and chronic inflammatory responses, chronic inflammatory lung disease, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematosus, e.g. cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritis, encephalitis, childhood, non-renal, extrarenal, alopecia areata), childhood insulin-dependent diabetes mellitus (IDDM) Juvenile-onset (Type I) diabetes mellitus, adult-onset (Type II) diabetes mellitus, autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, lymphomatoid granulomatosis, granulomatous diseases including Wegener's granulomatosis, agranulocytosis, vasculitis (large vessel vasculitis including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vessel vasculitis including Kawasaki disease and Vasculitis, including vasculitis of the central nervous system, necrotizing, cutaneous, or hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, e.g., Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, hemolytic anemia, including aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond-Blackfan anemia, and autoimmune hemolytic anemia (AIHA) or immune hemolytic anemia, pernicious anemia (anemia malignant fever), Addison's disease, pure red cell anemia or deficiency (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases with leukocyte extravasation, central nervous system inflammatory diseases, multiple organ injury syndromes, e.g. secondary to sepsis, trauma or hemorrhage, antigen-antibody complex mediated diseases, antiglomerular basement membrane disease, antiphospholipid antibody syndrome,allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid, e.g. pemphigoid bullous and cutaneous pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucous membrane pemphigoid, and pemphigus erythematous), autoimmune polyendocrinopathy, Reiter's disease or syndrome, immune complex nephritis, antibody mediated nephritis, neuromyelitis optica, polyneuropathy, chronic neuropathy, e.g. IgM polyneuropathy or IgM mediated neuropathy, thrombotic thrombocytopenic purpura (TTP) and thrombocytopenia including autoimmune or immune mediated thrombocytopenia (e.g. occurring in myocardial infarction patients), e.g. chronic or Idiopathic thrombocytopenic purpura (ITP), including acute ITP; autoimmune diseases of the testes and ovaries, including autoimmune orchitis and oophoritis; primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, including thyroiditis, e.g. autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis; autoimmune thyroid diseases, idiopathic hypothyroidism, Graves' disease; polyglandular syndromes, e.g. autoimmune polyglandular syndrome (or polyendocrinopathy syndrome); paraneoplastic syndromes, including neurological paraneoplastic syndromes, e.g. Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome; encephalomyelitis, e.g. allergic encephalomyelitis or encephalomyelitis allergica) and experimental allergic encephalomyelitis (EAE), myasthenia gravis, e.g. thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus-ataxia syndrome (OMS), and interval neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia, bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatitis, primary biliary cirrhosis, pulmonary fibrosis, autoimmune enteropathy syndrome, celiac disease, celiac disease, sprue) (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia,Amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus-myoclonus-ataxia syndrome (OMS), polychondritis, such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g. benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance (MGUS)), peripheral neuropathy, tumors Concomitant syndromes, channelopathies such as epilepsy, migraine, cardiac arrhythmias, myopathy, hearing loss, blindness, periodic paralysis, and channelopathies of the central nervous system, autism, inflammatory myopathies, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis, retinochoroiditis, autoimmune blood disorders, fibromyalgia, polyendocrine deficiency, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal hypoperistalsis, sclerodactyly, and telangiectasia), males and females Autoimmune infertility, mixed connective tissue disease, Jagas' disease, rheumatic fever, recurrent abortions, farmer's lung, erythema multiforme, post-cardiotomy syndrome, pituitary basophil adenoma, bird fancier's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport's syndrome, alveolitis e.g. allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Kaplan's syndrome, dengue fever, endocarditis, endomyocarditis fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, e.g. chronic cyclitis, metachronous cyclitis, iridocyclitis, or Fuchs' cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection,Mumps, Evans syndrome, autoimmune gonadotropic dysfunction, Sydenham's chorea, poststreptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, spinal fistula, choroiditis, giant cell polymyalgia, endocrine gastro-ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephrotic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway disease, silicosis, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Beck's disease, cryoglobulinemia, DePuy's Tran's contracture, phacosensitivity endophthalmitis, nodular enteritis, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hamman-Rich disease, sensorineural hearing loss, hemoglobinuric attacks, hypogonadism, ileitis, leukopenia, mononuclear infections, transverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmic syndromes, anti-carcinoma granuloma, pancreatitis, multiple sclerosis, pyoderma gangrenosum, Carbene thyroiditis, acquired spinal cord atrophy, infertility due to antisperm antibodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-related diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases, e.g. Leishmania, toxic shock syndrome, food poisoning, conditions with T cell infiltration, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases with leukocyte extravasation, multiple organ injury syndrome, antigen-antibody complex-mediated disease, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine deficiency, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary infiltrates with eosinophilia, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, Aspergillus species, or eosinophil-containing granulomas, anaphylaxis,Seronegative spondyloarthritis, possibly endocrine autoimmune disease, sclerosing cholangitis, scleral, episcleral, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of early childhood, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen diseases, rheumatism, These include neurological diseases, ischemic reperfusion injury, decreased blood pressure response, vascular insufficiency, antituberculosis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and diseases associated with angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, reperfusion injury of the myocardium or other tissues, skin diseases with an acute inflammatory component, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion associated syndrome, cytokine induced toxicity, acute severe infections, chronic refractory inflammation, pyelitis, pulmonary fibrosis, diabetic retinopathy, diabetic macroangiopathy, intimal hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0255] In addition to therapeutic uses, the antibodies of the present invention can be used for other purposes, including diagnostic methods, such as for the diseases and disorders described herein.

[0256] XI. Dosage, Formulation and Duration The proteins of the invention are formulated, dispensed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual subject, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the protein to be administered will depend on such considerations and is the minimum amount necessary to prevent, ameliorate, or treat the particular disorder (e.g., cancer, an allergic or inflammatory disorder, or an autoimmune disorder). The protein is optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat a disease. The effective amount of such other agents will depend on the amount of protein present in the formulation, the type of disease or treatment, and other factors not mentioned above. Such other agents are typically used in the same dosages and routes of administration as used above, or at about 1 to 99% of the conventional dosage. In general, mitigating or treating cancer involves alleviating one or more symptoms or medical problems associated with cancer. A therapeutically effective amount of a drug can accomplish one or a combination of the following: reduce the number of cancer cells (by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more); reduce or inhibit tumor size or tumor burden; inhibit (i.e., reduce and / or stop to some extent) the invasion of cancer cells into peripheral organs; reduce hormone secretion in the case of adenomas; reduce vascular density; inhibit tumor metastasis; reduce or inhibit tumor growth; and / or alleviate to some extent one or more symptoms associated with cancer. In some embodiments, the protein is used to prevent the onset or recurrence of cancer or an autoimmune disorder in a subject.

[0257] In one embodiment, the present invention can be used to extend the survival of a human subject susceptible to or diagnosed with cancer or an autoimmune disorder. Survival is defined as the time from the first administration of the drug to death. Survival can also be measured by the stratified hazard ratio (HR) of the treatment group versus the control group, which represents the risk of a subject dying during treatment.

[0258] In yet another embodiment, the treatment of the invention significantly increases the response rate in a group of human subjects susceptible to or diagnosed with cancer treated with various anti-cancer therapies. Response rate is defined as the percentage of treated subjects who respond to the treatment. In one aspect, the combination treatment of the invention using a protein of the invention and surgery, radiation therapy, or one or more chemotherapeutic agents significantly increases the response rate of the treated group compared to a group treated with surgery, radiation therapy, or chemotherapy alone (the increase has a chi-squared p-value of less than 0.005). Further measures of therapeutic efficacy of the treatment of cancer are described in US Patent Application Publication No. 20050186208.

[0259] In certain embodiments, compositions are provided that include a heteromultimeric protein produced according to any of the methods described herein and a pharma- ceutically acceptable carrier. Therapeutic formulations are prepared using standard methods known in the art by mixing the active ingredient having the desired purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (2013)). th (Eds. 1999, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Acceptable carriers include saline or buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN. TM , PLURONICS TM Or PEG.

[0260] Optionally but preferably, the formulation contains a pharma- ceutically acceptable salt (preferably sodium chloride) at approximately physiological concentrations. Optionally, the formulation of the present invention may contain a pharma- ceutically acceptable preservative. In some embodiments, the preservative concentration is typically in the range of 0.1 to 2.0% v / v. Suitable preservatives include those known in the pharmaceutical art. Benzyl alcohol, phenol, m-cresol, methylparaben and propylparaben are preferred preservatives. Optionally, the formulation of the present invention may contain a pharma- ceutically acceptable surfactant at a concentration of 0.005 to 0.02%.

[0261] The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0262] The active ingredient may also be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, e.g., in microcapsules prepared by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0263] Sustained release formulations may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the heteromultimeric protein, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT TM These include degradable lactic acid-glycolic acid copolymers such as lactic acid-glycolic acid copolymers (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow release of molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time. If the encapsulated heteromultimeric protein(s) remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, losing biological activity and possibly altering immunogenicity. Depending on the mechanism involved, rational strategies for stabilization can be devised. For example, if the aggregation mechanism is found to be intermolecular SS bond formation by thio-disulfide exchange, stabilization can be achieved by modification of sulfhydryl residues, lyophilization from acidic solution, control of water content, use of appropriate additives, and development of specific polymer matrix compositions.

[0264] The proteins described herein (e.g., heteromultimeric proteins such as multispecific antibodies produced according to the methods described herein) are administered to human subjects according to known methods, such as intravenous administration by bolus or continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, buccal, topical, or inhalation routes. Local administration may be particularly desirable when extensive side effects or toxicity are associated with antagonism to the target molecule recognized by the protein. Ex vivo strategies may also be used for therapeutic applications. Ex vivo strategies involve transfecting or transducing cells obtained from a subject with a polynucleotide encoding a protein of the invention. The transfected or transduced cells are then returned to the subject. The cells may be any of a wide range of types, including, but not limited to, hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

[0265] In one example, the protein complex (e.g., a heteromultimeric protein such as a multispecific antibody made according to the methods described herein) is administered locally, e.g., by direct injection if the location of the lesion or tumor permits, and the injections can be repeated periodically. The protein complex can be delivered systemically to a subject or directly to tumor cells, e.g., to a tumor or to a tumor bed following surgical resection of a tumor, to prevent or reduce local recurrence or metastasis.

[0266] XII. Manufactured products Another embodiment of the invention is an article of manufacture comprising one or more heteromultimeric proteins described herein and a substance useful for treating or diagnosing a disorder (e.g., an autoimmune disease or cancer). The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating a condition and may have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a heteromultimeric protein of the invention (e.g., an antibody or antibody fragment). The label or package insert indicates that the composition is used to treat a particular condition. The label or package insert would further include instructions for administering the heteromultimeric protein composition to a subject. Articles of manufacture and kits comprising the combinatorial therapies described herein are also contemplated.

[0267] Package insert refers to instructions typically included with commercial packaging of a therapeutic product that contain information about the directions, usage, dosage, administration, contraindications and / or precautions regarding the use of such therapeutic product. In certain embodiments, the package insert indicates that the composition is used to treat breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma, or ovarian cancer.

[0268] In addition, the article of manufacture may further comprise a second container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials considered from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0269] Kits are also provided that are useful for various purposes, such as purification or immunoprecipitation of antigens from cells. For antigen isolation and purification, the kits can include heteromultimeric proteins bound to beads (e.g., sepharose beads). Kits can be provided that include heteromultimeric protein(s) for in vitro antigen detection and quantification in ELISA or Western blot. As with articles of manufacture, kits include a container and a label or package insert on or associated with the container. The container holds a composition that includes at least one heteromultimeric protein of the present invention (e.g., a multispecific antibody or antibody fragment). Additional containers may be included that contain, for example, diluents and buffers or control antibodies. The label or package insert may provide a description of the composition and instructions for the intended in vitro or diagnostic use.

[0270] The foregoing description is believed to be sufficient to enable one skilled in the art to practice the invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any manner. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description, and such modifications are within the scope of the appended claims.

[0271] In the experimental disclosure which follows, the following abbreviations are used: eq (equivalent); M (mole); μM (micromolar); N (normal); mol (mole); mmol (millimolar); μmol (micromole); nmol (nanomole); g (gram); mg (milligram); kg (kilogram); μg (microgram); L (liter); ml (millimeter); μl (microliter); cm (centimeter); mm (millimeter); μm (micrometer); nm (nanometer); °C (degrees Celsius); h (hour); min (minute); sec (second); msec (millisecond); ADCC (antibody-dependent cellular cytotoxicity); BsAb (bispecific antibody); C L (light chain constant domain); C H(constant domain of the heavy chain); CMC (complement-mediated cytotoxicity); Fab (antibody binding fragment); Fc (fragment crystallization); Fv (fragment variable (V L +V H )); EGFR (epidermal growth factor receptor); HC (heavy chain); IGFR (insulin-like growth factor receptor); LC (light chain); scFv (single-chain variable fragment (V L and V H );VEGF (vascular endothelial growth factor);VEGFR2 (vascular endothelial growth factor receptor 2);V H (variable heavy domain);V L (variable light domain). EXAMPLES

[0272] The present invention will be described in more detail in the following examples, which are not intended to limit the scope of the invention as claimed in any way. The accompanying drawings are intended to be considered as an integral part of the specification and description of the invention. All references cited are specifically incorporated herein by reference for all that is described therein. The following examples are provided for illustrative purposes, and are not provided to limit the scope of the invention as claimed.

[0273] Example 1: The molar ratio of monomeric components of bispecific antibodies in mixed cell cultures can be controlled by adjusting the cell:cell ratio The following example shows the molar ratio of knob half antibody to whole half antibody when two mammalian cell lines (CHO cells), each expressing either knob half antibody or whole half antibody, were grown in the same culture.

[0274] For each cell line, a production study was performed to determine the production titer (i.e., the total amount of antibody produced by the cells, including heterodimers, homodimers, and monomeric half antibodies). Individual cell lines were subcultured every 3 or 4 days in seed culture medium until production titer data for individual cell lines were available.

[0275] The two cell lines were then grown and induced to express knob half antibody or whole half antibody in separate cultures. Individual knob and whole cell line cultures were subcultured in 40 mL shake flasks in cell culture medium every 3 or 4 days. On the day of mixing the cultures, cell numbers were determined by Vicell (Beckman Coulter).

[0276] The separate cultures were then mixed at a specific knob:whole host cell ratio, which was calculated based on the known production titers for each individual cell line. For each small scale production run, approximately 40x10 6 cells were required. Using the Vicell count (cells / mL), it was possible to determine the volume of each cell line that needed to be added to the mixed culture to achieve the desired knob:whole host cell ratio. The appropriate volumes of each of the knob and whole cell lines were mixed in a new shake flask in a final volume of 40 mL in production medium.

[0277] 15 hours prior to harvest, glutathione (GSH) stock solution was prepared by dissolving GSH in 1 M arginine in 400 mM succinate (pH=9.0) for a final GSH stock concentration of 250 mM. GSH stock solution was added to the production cultures for a final GSH concentration of 15 mM. Mixed culture medium was then harvested and the % knob half antibody and % hole half antibody were determined by reverse phase under reducing conditions for each mixed culture. The % covalent bispecific antibody formed for each ratio tested was determined as described in Figure 2.

[0278] Such experiments were carried out using the following knob half antibody / hole half antibody pairs: Anti-target A (knob) / Anti-target B (hole) Anti-Target C (Knob) / Anti-Target D (Hole) Anti-Target D (knob) / Anti-Target C (hole) Anti-Target E (Knob) / Anti-Target F (Hole)

[0279] The results of these experiments are shown in Table 2 below:

[0280] TIFF2025063070000003.tif127170

[0281] As shown in Table 2, % covalent bispecific antibody yields were improved when the molar ratio of knob half antibody:hole half antibody was about or close to 1:1. The optimal molar ratio for bispecific formation will be determined for each particular bispecific antibody. The knob host cell:hole host cell ratio that produces a 1:1 knob half antibody:hole half antibody molar ratio will vary by cell line and will be determined empirically.

[0282] Example 2: Timing of addition of reducing agent and concentration of reducing agent added during production of bispecific antibodies in mixed cell cultures The following example shows the addition of reducing agents during different steps of the production of bispecific antibodies containing anti-target A (knob) and anti-target B (hole). First, two mammalian cell lines expressing anti-target A (knob) or anti-target B (hole) were grown and induced to express knob or hole half antibodies in separate cultures, which were then mixed in separate production cultures to achieve a 1:1 molar ratio of anti-target A (knob):anti-target B (hole) as described above. GSH stock solution was added to the production cultures to a final concentration of 2 mM, 4 mM, or 10 mM 24 hours, 15 hours, or 4 hours before harvesting, or the cultures were left untreated. The mixed culture medium from each of the production cultures was then harvested, and the % knob half antibody and % hole half antibody were determined for each mixed culture by reverse phase under reducing conditions. The % covalent bispecific antibody formed for each ratio tested was determined as described in Figure 2.

[0283] As shown in Figure 3, the yield of covalent bispecific antibody in production cultures with a final GSH concentration of 10 mM was improved compared to the yield of bispecific antibody in production cultures with final GSH concentrations of 2 mM or 4 mM or the yield in the untreated control group. The production step of adding GSH was not shown to affect the yield of covalent bispecific antibody.

[0284] In further experiments, cell lines expressing anti-target A (knob) or anti-target B (hole) were first grown and induced to express knob or hole half antibodies in separate cultures, which were then mixed in separate production cultures to achieve a 0.82:1 or 1:1 molar ratio of anti-target A (knob):anti-target B (hole) as described above. GSH stock solution was added to the production cultures 15 hours prior to harvest to a final concentration of 5 mM, 10 mM, or left untreated ("0 mM"). Cell viability of each mixed cell culture was determined at harvest, and then the % bispecific antibody formed under each condition tested was determined by the ion exchange assay shown in Figure 2. The results of the experiments are shown in Table 3 below.

[0285] TIFF2025063070000004.tif105170

[0286] As shown in Table 3, the yield of covalent bispecific antibodies in production cultures with a final GSH concentration of 10 mM was improved compared to the yield of bispecific antibodies in production cultures with a final GSH concentration of 5 mM or the untreated yield. Addition of GSH to mixed cell cultures up to a final concentration of 10 mM had no effect on cell viability or total antibody production. No undesirable mixed disulfide formation or protein scrambling was observed at 10 mM GSH (data not shown).

[0287] Similar experiments were performed with two other mammalian cell lines, each expressing anti-target D (knob) or anti-target C (hole). The two cell lines were first grown and induced to express anti-target D (knob) or anti-target C (hole) in separate cultures, and then mixed in separate production cultures to achieve a molar ratio of 0.82:1 for anti-target D (knob):anti-target C (hole), as described above. GSH stock solution was added to the production cultures 15 hours prior to harvest to final concentrations of 10 mM, 15 mM, or 20 mM, or left untreated ("0 mM"). Cell viability of each mixed cell culture was determined at harvest, and then the % bispecific antibody formed under each condition tested was determined as shown in FIG. 2. The results of these experiments are shown in Table 4 below:

[0288] TIFF2025063070000005.tif97170

[0289] As shown in Table 4, the yield of covalent bispecific antibodies in production cultures with a final GSH concentration of 20 mM was improved compared to the yield of bispecific antibodies in production cultures with a final GSH concentration of 10 mM, 15 mM, or untreated. No effect on titer was observed when GSH was added to the mixed cell cultures to a final concentration of 20 mM. However, covalent modification of half antibodies with GSH was observed when GSH was added to the production cultures to a final concentration of 20 mM (data not shown).

[0290] Example 3: Yield of bispecific antibodies formed in mixed culture medium compared to in vitro assembly Additional experiments were performed using anti-target E (knob) and anti-target F (hole) to compare the yield of bispecific antibodies obtained by mixed culture of mammalian host cells expressing anti-target E (e.g., CHO cells) and mammalian cells expressing anti-target F (e.g., CHO cells) with the yield of bispecific antibodies obtained by mixing purified anti-target E (knob) and purified anti-target F (hole) using a Protein A column in vitro. Briefly, two mammalian cell lines, each expressing anti-target E (knob) or anti-target F (hole), were first grown and induced to express knob or hole half antibodies in separate cultures. The separate cultures were then mixed and grown for an additional period of time. The mixed culture medium was harvested and the bispecific antibody (%) formed was then determined by a cation exchange assay as shown in FIG. 2. In parallel, bispecific antibodies were formed in vitro by mixing purified anti-target E (knob) and anti-target F (hole) (see, e.g., WO 2013 / 055958). The final yields of bispecific antibodies formed under both conditions were comparable (data not shown).

[0291] Example 4: Bispecific Generation Additional experiments were performed to test whether bispecific anti-target G / anti-target H antibodies could be formed from purified anti-target G half antibody preparations containing anti-target G / anti-target G homodimers and purified anti-target H half antibody preparations containing anti-target H / anti-target H homodimers using anti-target G(knob) and anti-target H(hole). Separate cultures of mammalian host cells (e.g., CHO cells) transiently expressing the half antibody anti-target G(knob) and mammalian host cells (e.g., CHO cells) transiently expressing the half antibody anti-target H(hole) were grown and harvested as described above.

[0292] Each half antibody was captured on a 5 mL MabSURE SELECT Protein A column. The column was then washed with 10 column volumes (CV) of the following buffer: equilibration buffer consisting of 50 mM TRIS (pH 8.0), 150 mM NaCl, 0.05% Triton X-100, 0.05% Triton X-114, wash buffer consisting of 25 mM sodium citrate (pH 6.0). Each group was eluted in 0.15 M sodium acetate (pH 2.7). The identity of each half antibody was confirmed by MS.

[0293] For bispecifics, each half antibody was titrated 1:10 with 1M TRIS arginine, pH 9.0 to pH 5.0, then combined at a 1:1 ratio. The mixture was then titrated 1:10 with 1M TRIS arginine, pH 9.0 to pH 8.5 and 0.5M extra freshly prepared reduced L-glutathione (Sigma Aldrich) was added at a molar ratio of 1:200 before being left at room temperature for 3 days. The reactions were checked for bispecific ID by MS.

[0294] Knob and hole capture pools were run on a 4-20% Tris-glycine SDS PAGE revealing the presence of bands corresponding to the molecular weights of the half antibody and homodimer in each capture pool. See Figure 4. In addition to the MabSURE SELECT capture below, 0.5 mg of each was loaded onto a hydrophobic column (2.1 x 100 mm). The running buffer was 25 mM potassium phosphate, 1 M ammonium sulfate (pH 6.5) and the elution buffer was 25 mM potassium phosphate (pH 6.5), 25% isopropanol. The chromatograms reflect the heterogeneity seen in the gel and also reveal differences in the retention times of the main peaks. Figures 5A and 5B show the chromatograms for anti-target G (knob) and anti-target H (hole), respectively. The amount of homodimer and half antibody in the capture pool may vary depending on the specific antibody.

[0295] Following glutathione treatment, the bispecific was further loaded onto a hydrophobic column. The chromatogram revealed a single main peak (>90%) whose duration was located between that of each half antibody. See FIG. 6. MS confirmed that the main peak was the bispecific. Such results suggest that covalent homodimers exist in the captured pool of half antibodies and that the homodimers can be disrupted and available to form the bispecific using the assembly conditions described above.

[0296] The assembly conditions of the bispecifics were further tested by comparing the assembly of anti-target A (knob) and anti-target B (hole) in mixed culture medium in the absence versus the presence of 10 mM GSH.

[0297] CHO cells were transfected using Lipofectamine 2000 according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA). Transfected cells were selected in various concentrations of MSX (methionine sulfoximine, 25 and 50 uM). Selected colonies were assessed for antibody production by sampling supernatants from the colonies and analyzing by ELISA. The top 48 colonies based on ELISA titers were mixed and expanded to generate stable pools. Individual clones were also expanded and expanded for evaluation of antibody productivity to determine top clones.

[0298] Next, using the 40 L culture, the cells were diluted to 1.2x10 6 Cells were seeded at 1000 cells / mL in 2.8 L of culture medium. Cultures were split regularly, stirred at 90 rpm, and maintained at a pH set point of 7.0±0.03 and 30% dissolved oxygen (dO2). 15 mM GSH was added on day 11, followed by cell harvest on day 12.

[0299] Untreated and GSH-treated cell-free media were each processed with MabSURE SELECT and the captured pool was analyzed using ESI-TOF, see Figure 7A. Figure 7B is a zoom in on the m / z range of the bispecific antibody peak (right peak in Figure 7A). Figure 7C is a zoom in on the m / z range of the half antibody peak (left peak in 7A).

[0300] 7A-C show that the abundance of the homodimer and half antibody peaks is visibly reduced when 10 mM GSH is added to the cultures, demonstrating that bispecific formation is promoted by the content of both half antibodies and homodimers present in the co-culture. The extent to which each homodimer participates in bispecific antibody formation depends on the particular half antibody.

[0301] Those skilled in the art will appreciate that multiple embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, are not intended to limit its scope in any way.

Claims

1. 1. A method for preparing a heteromultimeric protein comprising: i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain; and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell capable of expressing a first hinge-containing polypeptide and a first light chain; (b) culturing a second host cell capable of expressing a second hinge-containing polypeptide and a second light chain; and (c) obtaining a mixed culture medium of the first host cell and the second host cell without disrupting the cell membranes of the first and second host cells. wherein the mixed culture medium comprises a heteromultimeric protein and the first host cell and the second host cell are each mammalian cells.

2. 1. A method for preparing a heteromultimeric protein comprising: i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain; and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell capable of expressing a first hinge-containing polypeptide and a first light chain, wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted from the first host cell; (b) culturing a second host cell capable of expressing a second hinge-containing polypeptide and a second light chain, wherein a second homodimer comprising two second hinge-containing polypeptides and two second light chains is secreted from the second host cell; (c) obtaining a mixed culture medium of the first host cell and the second host cell without disrupting the cell membranes of the first and second host cells, wherein the mixed culture medium comprises the first homodimer and the second homodimer; (d) incubating the mixed culture medium under reducing conditions sufficient to allow for the formation of heteromultimeric proteins; and (e) Obtaining a heteromultimeric protein wherein the first host cell and the second host cell are each mammalian cells.

3. Obtaining a mixed culture medium comprises: (1) harvesting a first culture medium for a first host cell; (2) harvesting a second culture medium for the second host cell; and (3) Mixing the first culture medium and the second culture medium to obtain a mixed culture medium. The method of claim 1 or 2, comprising:

4. 3. The method of claim 1 or 2, wherein the step of obtaining a mixed culture medium comprises harvesting a culture medium of a mixed cell culture comprising the first host cell and the second host cell.

5. 5. The method of claim 4, wherein the first host cell and the second host cell are cultured separately before being combined into a mixed cell culture.

6. 6. The method of claim 4 or 5, further comprising culturing the mixed cell culture at a temperature of about 25° C. to about 40° C.

7. 7. The method of claim 1, further comprising agitating the mixed culture medium.

8. 8. The method of claim 1, further comprising isolating the heteromultimeric protein from the mixed culture medium.

9. The method of claim 8, wherein the heteromultimeric protein is isolated using a Protein A column.

10. 4. The method of claim 3, comprising adding a reducing agent to the first cell culture medium and / or the second cell culture medium before or after the first and second cell culture media are harvested.

11. 11. The method of any one of claims 4 to 10, comprising adding a reducing agent to the culture medium of the mixed cell culture before the culture medium of the mixed cell culture is harvested.

12. 12. The method of claim 10 or 11, wherein the reducing agent is added from about 4 to about 24 hours prior to the recovering step.

13. 13. The method of claim 12, wherein the reducing agent is added about 15 hours prior to the recovering step.

14. 14. The method of any one of claims 1 to 13, further comprising adding a reducing agent to the mixed cell culture medium.

15. 15. The method of claim 14, wherein the mixed culture medium containing the reducing agent is further incubated for about 4 hours to about 7 days.

16. 16. The method of claim 15, wherein the mixed culture medium containing the reducing agent is further incubated for about 15 hours.

17. 17. The method of claim 16, wherein a reducing agent is added to the mixed culture medium prior to isolating the heteromultimeric protein from the mixed culture medium.

18. 20. The method of claim 17, wherein the mixed culture medium containing the reducing agent is incubated for at least about 24 hours prior to isolation of the heteromultimeric protein.

19. 20. The method of claim 18, wherein the heteromultimeric protein is isolated using a Protein A column.

20. 1. A method for preparing a heteromultimeric protein comprising: i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain; and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a first host cell capable of expressing a first homodimer comprising two first hinge-containing polypeptides and their associated light chains; (b) culturing a second host cell capable of expressing a second homodimer comprising two second hinge-containing polypeptides and their associated light chains; (c) obtaining a mixed culture medium of the first host cell and the second host cell; (c) incubating the mixed culture medium under reducing conditions sufficient to allow for the formation of heteromultimeric proteins; and (d) Obtaining a heteromultimeric protein The method includes:

21. 21. The method of any one of claims 14 to 20, wherein the reducing agent is selected from the group consisting of glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), cysteine, cysteine, dithiothreitol, cysteine ​​dithiothreitol, dithiolbutylamine, or a combination thereof.

22. 22. The method of claim 21, wherein the reducing agent is glutathione, and the glutathione is added at a concentration of about 5 mM to about 20 mM or less.

23. 23. The method of claim 22, wherein the reducing agent is glutathione, and the glutathione is added at a concentration of about 2 mM to about 10 mM.

24. 23. The method of claim 22, wherein the reducing agent is glutathione, and the glutathione is added at a concentration of about 5 mM to less than about 20 mM.

25. 25. The method of claim 24, wherein the reducing agent is glutathione, and the glutathione is added at a concentration of about 15 mM.

26. 26. The method of any one of claims 1 to 25, wherein the first host cell is a stable cell line.

27. 27. The method of any one of claims 1 to 26, wherein the second host cell is a stable cell line.

28. 28. The method of any one of claims 1 to 27, wherein the first host cell is a CHO cell.

29. 29. The method of any one of claims 1 to 28, wherein the second host cell is a CHO cell.

30. 30. The method of any one of claims 1 to 29, wherein the ratio of the first host cells to the second host cells is adjusted such that when the first host cell culture and the second host cell culture are mixed to form a mixed culture, the molar ratio of the first hinge-containing polypeptide to the second hinge-containing polypeptide is from about 1:10 to about 10:

1.

31. 31. The method of claim 30, wherein when the host cell culture and the second host cell culture are mixed to form a mixed culture, the molar ratio of the first hinge-containing polypeptide expressed by the first host cell to the second hinge-containing polypeptide expressed by the second host cell is about 1:

1.

32. 32. The method of any one of claims 1 to 31, wherein the hinge-containing polypeptide comprises an Fc region or a variant thereof.

33. 33. The method of any one of claims 1 to 32, wherein the first and / or second hinge-containing polypeptide comprises an antibody heavy chain.

34. 34. The method of any one of claims 1 to 33, wherein the first heterodimerization domain comprises a knob modification at its contact surface and the second heterodimerization domain comprises a hole modification at its contact surface.

35. 35. The method of claim 34, wherein the knob modification comprises replacing an original amino acid residue from the first heterodimerization domain with an amino acid residue having a larger side chain than the original amino acid residue.

36. 36. The method of claim 35, wherein the substituting amino acid residue is selected from the group consisting of tryptophan, phenylalanine, tyrosine and arginine.

37. 37. The method of claim 36, wherein the hole modification comprises replacing an original amino acid residue from the second heterodimerization domain with an amino acid residue having a smaller side chain than the original amino acid residue.

38. 38. The method of claim 37, wherein the substituting amino acid residue is selected from the group consisting of serine, threonine, valine, and alanine.

39. 39. The method of any one of claims 34 to 38, wherein the knob modification comprises a T366W substitution (EU numbering).

40. 40. The method of any one of claims 34 to 39, wherein the hole modification comprises a substitution of two or more amino acids selected from the group consisting of T366S, L368A and Y407V (EU numbering).

41. 41. The method of any one of claims 1 to 40, wherein the interchain disulfide bond is between the hinge regions.

42. 42. The method of any one of claims 1 to 41, wherein the heteromultimeric protein is an antibody.

43. 43. The method of any one of claims 1 to 42, wherein the heteromultimeric protein is a bispecific antibody.

44. 44. The method of claim 43, wherein the antibody is a humanized or human antibody.

45. 45. The method of claim 44, wherein the antibody is a full-length antibody.

46. The antibody is human C Ｈ 2 and / or C Ｈ The method of claim 45, which is an antibody fragment comprising at least a portion of the three domains.

47. 47. The method of any one of claims 42 to 46, wherein the antibody is selected from the group consisting of IgG, IgA and IgD.

48. 48. The method of claim 47, wherein the antibody is an IgG.

49. 49. The method of claim 48, wherein the antibody is IgG1, IgG2 or IgG4.

50. 50. The method of any one of claims 1 to 49, wherein the first light chain and the second light chain comprise different variable domain sequences.

51. 1. A method for preparing a heteromultimeric protein comprising: i) a first hinge-containing polypeptide having a first heterodimerization domain attached to a first light chain; and ii) a second hinge-containing polypeptide having a second heterodimerization domain attached to a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at a contact interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising: (a) culturing a mixed culture of a first host cell and a second host cell, wherein the first host cell is capable of expressing a first hinge-containing polypeptide and a first light chain, and the second host cell is capable of expressing a second hinge-containing polypeptide and a second light chain, and wherein each of the first host cell and the second host cell is a mammalian cell; (b) adding a reducing agent to the mixed culture; and (c) recovering a mixed culture medium containing the heteromultimeric protein from the mixed culture without disrupting the cell membrane. The method includes:

52. 52. The method of claim 51 , wherein the first host cell secretes a first homodimer comprising two first hinge-containing polypeptides and two first light chains, and the second host cell secretes a second homodimer comprising two second hinge-containing polypeptides and two second light chains.

53. 53. The method of claim 51 or 52, wherein the mixed culture medium is harvested 4 to 24 hours after the reducing agent is added.

54. 54. The method of any one of claims 51 to 53, wherein the step of recovering the mixed culture medium comprises removing the first host cells and the second host cells from the mixed culture medium.

55. 55. The method of any one of claims 51 to 54, wherein the mixed culture medium is incubated for between 4 hours and 7 days.

56. 56. A heteromultimeric protein produced by the method of any one of claims 1 to 55.

57. The heteromultimeric protein of claim 56, which is a bispecific antibody.

58. 58. A composition comprising the heteromultimeric protein of claim 56 or 57 and a pharma- ceutically acceptable carrier.

59. A host cell comprising a polynucleotide or recombinant vector encoding a first hinge-containing polypeptide of a heteromultimeric protein according to claim 56 or 57, wherein the host cell does not express a second hinge-containing polypeptide of the heteromultimeric protein.

60. 60. The host cell of claim 59, wherein the hinge-containing polypeptide is an antibody heavy chain.

61. 61. The host cell of claim 59 or 60, wherein the hinge-containing polypeptide is paired with an antibody light chain.

62. 62. A host cell according to any one of claims 59 to 61, which is a stable cell line.

63. 63. A host cell according to any one of claims 59 to 62, which is a mammalian cell.

64. 64. The host cell of any one of claims 59 to 63, which is a CHO cell.

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