Production of heteromultimeric proteins using mammalian cells

Abstract

Described herein are methods for the efficient production of antibodies and other multimeric protein complexes (collectively referred to herein as heteromultimeric proteins) capable of specifically binding to more than one target. The targets may be, for example, different epitopes located on a single molecule or on different molecules.

Description

[0001] This application is a divisional application of Chinese Application No. 201580036549.9, filed on May 6, 2015, with the invention title of "Producing heteromultimeric proteins using mammalian cells".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of priority of U.S. Provisional Application No. 61 / 989,509, filed on May 6, 2014, the disclosure of which is hereby incorporated by reference in its entirety. Technical field

[0004] The present invention relates to methods for producing heteromultimeric proteins. Background art

[0005] IgG-type monoclonal antibodies contain two identical antigen-binding arms and constant domains (Fc). Antibodies with different specificities in their binding arms generally do not exist in nature and thus have to be created by means of chemical engineering (e.g., chemical cross-linking, etc.), recombinant DNA, and / or cell fusion techniques.

[0006] Bispecific antibodies can bind two different antigens simultaneously. This property enables the development of therapeutic strategies that are not possible with conventional monoclonal antibodies. A large class of envisioned bispecific antibody formats has been developed, reflecting the strong 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.

[0007] Another class of multispecific molecules are recombinant fusion proteins. Recombinant fusion proteins consisting of the extracellular domain of an immunomodulatory protein and the constant (Fc) domain of an immunoglobulin (Ig) represent a growing class of human therapeutic agents. Immunoadhesins combine a binding region of a protein sequence with the desired specificity with an effector domain of an antibody. Immunoadhesins have two important properties that are significant for their potential as therapeutic agents: target specificity and pharmacokinetic stability (in vivo half-life comparable to that of an antibody). Immunoadhesins can be used as antagonists to inhibit or block harmful 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.

[0008] 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 to Feige et al., issued December 9, 2003 (peptibodies); U.S. Patent Publication No. 2002-004587, published January 10, 2002 (multispecific antibodies); U.S. Patent No. 7,612,181 to Wu et al., issued November 3, 2009 (dual variable domain format); U.S. Patent No. 6,534,628; Nord K et al., Prot Eng (1995) 8:601-608; Nord K et al., Nat Biotech (1997) 15:772-777 and 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 (single-chain antibodies); Bostrom et al., Science (2009) 323:1610-1614 (dual-action Fab, aka mixed valence antibodies). Other formats are known to those of skill in the art.

[0009] For the multispecific molecules described above, the production of clinical-grade materials remains challenging. As shown above, there are many ways to generate molecules with hybrid binding arms (i.e., binding arms that are different from each other). Each of these methods has its drawbacks.

[0010] The chemical cross-linking method is labor-intensive because it may still be necessary to purify the relevant species from homodimers and other unwanted by-products. In addition, the chemical modification step may alter the integrity of the protein and thus result in poor stability. Therefore, this method is often ineffective and may lead to loss of antibody activity.

[0011] Cell-fusion techniques (e.g., hybridomas) express two randomly assembled heavy chains and two light chains, resulting in the production of 10 antibody combinations. The desired heteropolymeric antibody is only a small fraction of the antibodies thus produced. Purifying the desired heteropolymeric protein significantly reduces the yield and increases the production cost.

[0012] Recombinant DNA technology has been used to generate various heteropolymeric formats that do not contain an Fc domain, e.g., single-chain Fv, diabodies, etc. A major drawback of this type of antibody molecule is the lack of an Fc domain and thus the lack of the ability of the antibody to trigger effector functions (e.g., complement activation, Fc-receptor binding, etc.). Therefore, bispecific antibodies that contain a functional Fc domain are needed.

[0013] Recombinant DNA technology has also been used to generate 'knobs-into-holes' bispecific antibodies. See U.S. Patent Application 20030078385 (Arathoon et al., - Genentech). A limitation of this strategy is that the light chains of the two parental antibodies have to be the same to prevent mispairing and the formation of unintended and / or inactive molecules because they are expressed in the same cell.

[0014] Therefore, alternative methods for producing heteropolymeric proteins are still needed. The inventions described herein provide such methods. These and other aspects and advantages of the present invention will be apparent from the invention description provided herein. BRIEF DESCRIPTION OF THE INVENTION

[0016] 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 heteropolymeric proteins) in mammalian cells that are superior to methods known in the art. See WO 2013 / 055958 and WO 2011 / 133886.

[0017] Accordingly, in a first aspect, there is provided a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0018] (a) culturing a first host cell capable of expressing the first hinge-containing polypeptide and the first light chain;

[0019] (b) culturing a second host cell capable of expressing the second hinge-containing polypeptide and the second light chain; and,

[0020] (c) obtaining a combined culture medium of the first host cell and the second host cell, wherein the combined culture medium contains the heteromultimeric protein, and wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, the combined 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 combined culture.

[0021] There is also provided a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0022] (a) culturing a first host cell capable of expressing the first hinge-containing polypeptide and the first light chain, wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted;

[0023] (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;

[0024] (c) obtaining a combined 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 combined culture medium contains the first homodimer and the second homodimer;

[0025] (d) Incubate the combined culture medium under reducing conditions sufficient to allow the formation of the heteromultimeric protein, and;

[0026] (e) Obtain the 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 combined culture. In certain embodiments according to (or as applicable to) any of the above embodiments, the first hinge-containing polypeptide and the second hinge-containing polypeptide comprise the first and second heavy chains. In certain embodiments according to (or as applicable to) any of the embodiments described herein, the first hinge-containing polypeptide and the first light chain comprise the first half-antibody. In certain embodiments according to (or as applicable to) any of the embodiments described herein, the second hinge-containing polypeptide and the second light chain comprise the second half-antibody.

[0027] In certain embodiments according to (or as applicable to) any of the above embodiments, obtaining the combined culture medium comprises: (1) harvesting a first culture medium of a first host cell culture;

[0028] (2) harvesting a second culture medium of a second host cell culture; and

[0029] (3) combining the first culture medium and the second culture medium to obtain the combined culture medium.

[0030] In certain embodiments according to (or as applicable to) any of the above embodiments, obtaining the combined culture medium comprises harvesting the culture medium of a combined cell culture comprising the first host cell and the host cell. In certain embodiments, the combined culture medium is obtained without disrupting the cell membranes of the first and second host cells.

[0031] The first and second host cells in the method of the present invention can be cultured in any environment that permits the expression and isolation of the polypeptide of interest. In certain embodiments according to (or as applicable to) any of the above embodiments, the first host cell and the second host cell are cultured separately before being combined into the combined cell culture.

[0032] In certain embodiments according to (or as applicable to) any of the above embodiments, the method further comprises the step of culturing the combined cell culture at a temperature of about 25 °C to about 40 °C. In certain embodiments according to (or as applicable to) any of the above embodiments, after obtaining the combined culture medium, the combined culture medium is incubated for about 24 hours to about 7 days. In certain embodiments according to (or as applicable to) any of the above embodiments, the combined culture medium is incubated at about 4 °C to about 8 °C. In certain embodiments according to (or as applicable to) any of the above embodiments, the combined culture medium is stirred.

[0033] In certain embodiments according to (or as applicable to) any of the above embodiments, the method further comprises separating the heteromultimeric protein from the combined culture medium. In certain embodiments according to (or as applicable to) any of the above embodiments, a protein A column is used to separate the heteromultimeric protein. In certain embodiments, the heteromultimeric protein is further purified using methods known in the art.

[0034] In certain embodiments according to (or as applicable to) any of the above embodiments, the method further comprises adding a reducing agent to the first cell culture medium and / or to the second cell culture medium, before or after harvesting the first and second cell culture media. In certain embodiments according to (or as applicable to) any of the above embodiments, the method further comprises adding a reducing agent to the medium of the combined cell culture before harvesting the medium of the combined cell culture. In certain embodiments according to (or as applicable 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 applicable to) any of the above embodiments, the reducing agent is added about 15 hours before the harvesting step.

[0035] In certain embodiments according to (or as applicable to) any of the above embodiments, the method further comprises adding a reducing agent to the combined cell culture medium. In certain embodiments according to (or as applicable to) any of the above embodiments, the combined culture medium containing the reducing agent is further incubated for about 4 hours to about 7 days. In certain embodiments according to (or as applicable to) any of the above embodiments, the combined culture medium containing the reducing agent is further incubated for about 15 hours. In certain embodiments according to (or as applicable to) any of the above embodiments, a reducing agent is added to the combined culture medium before separating the heteromultimeric protein from the combined culture medium. In certain embodiments according to (or as applicable to) any of the above embodiments, the combined culture medium containing the reducing agent is incubated for at least about 24 hours before separating the heteromultimeric protein. In certain embodiments according to (or as applicable to) any of the above embodiments, the combined culture medium containing the reducing agent is incubated for at least about 48 hours before separating the heteromultimeric protein. In certain embodiments according to (or as applicable to) any of the above embodiments, a protein A column is used to separate the heteromultimeric protein.

[0036] In certain embodiments according to (or as applicable to) any of the above embodiments, the reducing agent is selected from glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), cysteine, cysteine, dithiothreitol, cysteine dithiothreitol, dithiobutylamine, or a combination thereof. In certain embodiments according to (or as applicable to) any of the above embodiments, the reducing agent is glutathione, and wherein glutathione is added at a concentration of from about 5 mM to no more than about 20 mM. In certain embodiments according to (or as applicable to) any of the above embodiments, the reducing agent is glutathione, and wherein glutathione is added at a concentration of from about 2 mM to about 10 mM. In certain embodiments according to (or as applicable to) any of the above embodiments, the reducing agent is glutathione, and wherein glutathione is added at a concentration of from about 5 mM to less than about 20 mM. In certain embodiments according to (or as applicable to) any of the above embodiments, the reducing agent is glutathione, and wherein glutathione is added at a concentration of about 15 mM.

[0037] In certain embodiments according to (or as applicable to) any of the above embodiments, the first host cell is a stable cell line. In certain embodiments according to (or as applicable to) any of the above embodiments, the second host cell is a stable cell line. In certain embodiments according to (or as applicable to) any of the above embodiments, the first host cell is a CHO cell. In certain embodiments according to (or as applicable to) any of the above embodiments, the second host cell is a CHO cell.

[0038] In certain embodiments according to (or as applicable to) any of the above embodiments, the ratio of the first host cell to the second host cell is adjusted such that when the first host cell culture and the second host cell culture are combined to form a combined 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. In certain embodiments according to (or as applicable to) any of the above embodiments, when the first host cell culture and the second host cell culture are combined to form a combined 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 1:1.

[0039] In certain embodiments according to (or as applicable 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 applicable to) any of the above embodiments, the first and / or second hinge-containing polypeptide comprises an antibody heavy chain.

[0040] In certain embodiments according to (or as applicable to) any of the above embodiments, the first heterodimerization domain comprises a knot modification at the interface and the second heterodimerization domain comprises a pore modification at the interface. In certain embodiments according to (or as applicable to) any of the above embodiments, the knot modification comprises replacement of 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 applicable to) any of the above embodiments, the replacing amino acid residue is selected from tryptophan, phenylalanine, tyrosine, and arginine. In certain embodiments according to (or as applicable to) any of the above embodiments, the pore modification comprises replacement of 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 applicable to) any of the above embodiments, the replacing amino acid residue is selected from serine, threonine, valine, and alanine. In certain embodiments according to (or as applicable to) any of the above embodiments, the knot modification comprises a T366W substitution (EU numbering). In certain embodiments according to (or as applicable to) any of the above embodiments, the pore modification comprises two or more amino acid substitutions selected from T366S, L368A, and Y407V (EU numbering).

[0041] In certain embodiments according to (or as applicable to) any of the above embodiments, the interchain disulfide bond is between the hinge regions. In certain embodiments according to (or as applicable to) any of the above embodiments, the heteromultimeric protein is an antibody. In certain embodiments according to (or as applicable to) any of the above embodiments, the heteromultimeric protein is a bispecific antibody. In certain embodiments according to (or as applicable to) any of the above embodiments, the antibody is a humanized antibody or a human antibody. In certain embodiments according to (or as applicable to) any of the above embodiments, the antibody is a full-length antibody. In certain embodiments according to (or as applicable to) any of the above embodiments, the antibody is an antibody fragment comprising at least a portion of the human CH2 and / or CH3 domain. In certain embodiments according to (or as applicable to) any of the above embodiments, the antibody is selected from IgG, IgA, and IgD. In certain embodiments according to (or as applicable to) any of the above embodiments, the antibody is IgG. In certain embodiments according to (or as applicable to) any of the above embodiments, the antibody is IgG1, IgG2, or IgG4. In certain embodiments according to (or as applicable to) any of the above embodiments, the first light chain and the second light chain comprise different variable domain sequences.

[0042] In another aspect, there is provided a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0043] (a) Culturing a co-culture of a first host cell and a second host cell, wherein the first host cell is capable of expressing the first hinge-containing polypeptide and the first light chain, wherein the second host cell is capable of expressing the second hinge-containing polypeptide and the second light chain, and wherein the first host cell and the second host cell are each mammalian cells;

[0044] (b) Adding a reducing agent to the co-culture; and

[0045] (c) Harvesting the combined culture medium from the co-culture without disrupting the cell membranes, wherein the combined culture medium contains the heteromultimeric protein.

[0046] In certain embodiments, the first host cell secretes a first homodimer comprising two first hinge-containing polypeptides and two first light chains, wherein the second host cell secretes a second homodimer comprising two second hinge-containing polypeptides and two second light chains, wherein the co-culture contains the first homodimer and the second homodimer. In certain embodiments, adding a reducing agent to the co-culture permits the formation of the heteromultimeric protein.

[0047] In certain embodiments according to (or as applicable to) any of the above embodiments, the reducing agent is added no more than about 18 days after the co-culture has been cultured. In certain embodiments according to (or as applicable to) any of the above embodiments, the combined culture medium is harvested 4 hours to 24 hours after adding the reducing agent. In certain embodiments according to (or as applicable to) any of the above embodiments, the step of harvesting the combined culture medium comprises removing the first host cell and the second host cell from the combined culture medium. In certain embodiments according to (or as applicable to) any of the above embodiments, the combined culture medium is incubated for 4 hours to 7 days.

[0048] In certain embodiments, the method further comprises the step of adjusting the cell:cell ratio of the first host cell and the second host cell in the combined culture. In certain embodiments, the cell:cell ratio is adjusted such that the molar ratio of the first hinge-containing polypeptide (with an associated light chain) expressed from the first host cell to the second hinge-containing polypeptide (with an associated light chain) expressed from the second host cell in the combined culture medium reaches a desired molar ratio. 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 a hinge-containing polypeptide and a light chain.

[0049] It should be understood that the methods of the present invention may include other steps, which are generally routine steps that are obvious for initiating and / or completing the processes covered by the methods of the present invention as described herein. For example, in one embodiment, step (a) of the method of the present invention is preceded by a step in which a nucleic acid encoding a first hinge-containing polypeptide is introduced into a first host cell and a nucleic acid encoding a second hinge-containing polypeptide is introduced into a second host cell. In one embodiment, the method of the present invention further comprises the step of purifying a heteromultimeric protein having binding specificities for at least two different targets.

[0050] In certain embodiments according to (or as applicable 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 applicable 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 may be different epitopes located on a single molecule or on different molecules.

[0051] In another aspect, there is provided a heteromultimeric protein produced by any of the above methods. In certain embodiments according to (or as applicable to) any of the above embodiments, the heteromultimeric protein is a bispecific antibody. There is also provided a composition comprising a heteromultimeric protein (such as a bispecific antibody) produced by any of the above methods and a pharmaceutically acceptable carrier.

[0052] The heteromeric proteins of the present invention are generally capable of binding, preferably specifically binding, to an antigen. Such antigens include, for example, tumor antigens, cell survival regulators, cell proliferation regulators, molecules associated with (e.g., known or suspected to contribute functionally to) tissue development or differentiation, cell surface molecules, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules associated with (e.g., known or suspected to contribute functionally to) vasculogenesis. The antigen capable of binding to the heteromeric protein of the present invention can be a member of a subclass of one of the above classifications, where other subclasses of the classification contain other molecules / antigens with different characteristics (relative to the antigen of interest). The antigen of interest can also be considered to belong to two or more categories. In one embodiment, the present invention provides a heteromeric protein that binds, preferably specifically binds, 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, in some embodiments, the heteromeric protein of the present invention binds, preferably specifically binds, to a tumor antigen that is not a cluster of differentiation factor. In another example, the heteromeric protein of the present invention is capable of binding, preferably specifically binding, to a cluster of differentiation factor that is not, for example, CD3 or CD4 in some embodiments. In some embodiments, the heteromeric protein of the present invention is an anti-VEGF antibody. In some embodiments, the heteromeric protein of the present invention is a bispecific antibody selected from the following: IL-1α / IL-1β, IL-12 / IL-18; IL-13 / IL-9; IL-13 / IL-4; IL-13 / IL-5; IL-5 / IL-4; IL-13 / IL-lβ; IL-13 / IL-25; IL-13 / TARC; IL-13 / MDC; IL-13 / MEF; IL-13 / TGF-β; IL-13 / LHR agonist; IL-12 / TWEAK, IL-13 / CL25; IL-13 / SPRR2a; IL-13 / SPRR2b; IL-13 / ADAM8, IL-13 / PED2, IL17A / IL17F, CD3 / CD19, CD138 / CD20; CD138 / CD40; CD19 / CD20; CD20 / CD3; CD38 / CD138; CD38 / CD20; CD38 / CD40; CD40 / CD20; CD-8 / IL-6; CD20 / BR3, TNFα / TGF-β, TNFα / IL-1β;TNFα / IL-2, TNFα / IL-3, TNFα / IL-4, TNFα / IL-5, TNFα / IL6, TNFα / IL8, TNFα / IL-9, TNFα / IL-10, TNFα / IL-11, TNFα / IL-12, TNFα / IL-13, TNFα / IL-14, TNFα / IL-15, TNFα / IL-16, TNFα / IL-17, TNFα / IL-18, TNFα / IL-19, TNFα / IL-2, TNFα / IL-2, TNFα / IFNα, TNFα / CD4, TNFα / VEGF, TNFα / MIF, TNFα / ICAM-1, TNFα / PGE4, TNFα / PEG2, TNFα / RANK ligand, TNFα / Te38; TNFα / BAFF; TNFα / CD22; TNFα / CTLA-4; TNFα / 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, VEGF / 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 RGMA / RGMB, IL1β / IL18, NRP1 / VEGFA, VEGFA / NRP2, cMET / EGFR, ALK1 / BMP9, VEGFA / α5β1, HER1 / HER3-BU, and CMV. In some embodiments, the heteromultimeric protein of the invention binds to at least two target molecules selected from: α5β1, ALK1, BMP9, IL-1α, IL-1β, 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, TNFα, TGF-β, 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, IFNα, MIF, ICAM-1, PGE4, PEG2, 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, RGMA, and RGMB. In some embodiments, the heteromultimeric protein of the 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.;

[0053] Heteromultimeric proteins can be modified to enhance and / or add additional desired characteristics. Such characteristics include biological functions such as immune effector functions, desirable in vivo half-life / clearance rate, bioavailability, biodistribution, or other pharmacokinetic characteristics. Such modifications are well known in the art and can also be determined empirically, and can include modifications by means of peptide-based or non-peptide-based moieties. For example, antibodies can be glycosylated or non-glycosylated, typically at least in part depending on the nature of the host cell. Preferably, the antibodies of the present invention are non-glycosylated. The non-glycosylated antibodies produced by the methods of the present invention can subsequently 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 present invention can be produced in prokaryotic cells (e.g., Escherichia coli). Heteromultimeric proteins produced by Escherichia coli are generally non-glycosylated and lack biological functions normally associated with glycosylation characteristics present in heteromultimeric proteins produced by mammalian host cells (e.g., CHO).

[0054] The present invention also provides immunoconjugates comprising a heteromultimeric protein of the present invention conjugated to a heterologous moiety. Any heterologous moiety will be suitable as long as its conjugation to the antibody does not substantially reduce the desired functions and / or characteristics of the antibody. For example, in some embodiments, the immunoconjugate comprises a heterologous moiety that is a cytotoxic drug. In some embodiments, the cytotoxic drug is selected from radioactive isotopes, chemotherapeutic agents, and toxins. In some embodiments, the toxin is selected from calicheamicin, maytansine, and trichothecene. In some embodiments, the immunoconjugate comprises a heterologous moiety that is a detectable label. In some embodiments, the detectable label is selected from radioactive isotopes, members of ligand-receptor pairs, members of enzyme-substrate pairs, and members of fluorescence resonance energy transfer pairs.

[0055] In another aspect, there is provided a host cell comprising a polynucleotide or recombinant vector encoding a first hinge-containing polypeptide of a heteromultimeric protein described above, wherein the host cell does not express a second hinge-containing polypeptide of the heteromultimeric protein. In certain embodiments according to (or as applicable to) any of the above embodiments, the hinge-containing polypeptide is an antibody heavy chain. In certain embodiments according to (or as applicable to) any of the above embodiments, the hinge-containing polypeptide pairs with an antibody light chain. In certain embodiments according to (or as applicable to) any of the above embodiments, the host cell is a stable cell line. In certain embodiments according to (or as applicable to) any of the above embodiments, the host cell is a mammalian cell. In certain embodiments according to (or as applicable to) any of the above embodiments, the host cell is a CHO cell.

[0056] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that although the detailed description and the specific examples, which indicate preferred embodiments of the invention, are given by way of illustration only, various changes and modifications within the spirit of the invention will become apparent to those skilled in the art from this detailed description.

[0057] All references cited herein are incorporated by reference in their entirety. Brief Description of the Drawings

[0059] Figure 1A Shows a fully oxidized half-antibody. The "knobs" or "holes" or other heterodimerization domains are not shown. The half-antibody described in this figure is of the IgG1 isotype. Those skilled in the art will understand that other immunoglobulin isotypes can be envisioned as half-antibodies with corresponding inter-chain and intra-chain bonds. In a full Ab, the hinge cysteines would form inter-chain disulfide bonds.

[0060] Figure 1B Shows a full-length bispecific antibody. The inter-heavy chain disulfide bonds in the hinge region are not described.

[0061] Figure 2 Shows a flow chart of two assays that can be used to determine the % of half-antibody and the % of covalent bispecific antibody.

[0062] Figure 3 Shows the % of bispecific antibody formed when a reducing agent was added to a pooled cell culture 4 hours, 15 hours or 24 hours before harvesting the pooled medium, said pooled cell culture comprising a first mammalian host cell expressing anti-target A (knob) and a second mammalian host cell expressing anti-target B (hole).

[0063] Figure 4 Shows a knob capture pool and a hole capture pool run on a 4%-20% Tris-glycine SDS PAGE.

[0064] Figure 5 A shows a chromatogram of a sample separated based on hydrophobicity for anti-target G, where the homodimer and the half-antibody co-elute into a broad peak. Figure 5 B shows a chromatogram of a sample separated based on hydrophobicity for anti-target H.

[0065] Figure 6 Shows the mass spectrometry results for an anti-target G / anti-target H bispecific antibody.

[0066] Figure 7A Shows the experimental results of electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) performed on untreated and GSH-treated pooled medium, where the anti-target A half-antibody and the anti-target B half-antibody are secreted into the pooled medium.Figure 7B An enlarged view showing the m / z range of the bispecific antibody peak. Figure 7C An enlarged view showing the m / z range of the half-antibody peak.

[0067] Abbreviations

[0068] ADCC = antibody-dependent cell-mediated cytotoxicity

[0069] API = anti-pathogen immunoadhesin

[0070] BP = bactericidal / permeability-increasing protein

[0071] C1q = complement factor 1q

[0072] CD = cluster of differentiation

[0073] CDC = complement-dependent cytotoxicity

[0074] CH1 or C H 1 = the first constant domain of the heavy chain

[0075] CH2 or C H 2 = the second constant domain of the heavy chain

[0076] CH3 or C H 3 = the third constant domain of the heavy chain

[0077] CH4 or C H 4 = the fourth constant domain of the heavy chain

[0078] CL or C L = the constant domain of the light chain

[0079] CT = cytotoxic T lymphocyte-associated molecule

[0080] Fc = crystallizable fragment

[0081] FcR = receptor γ for the Fc portion of IgG

[0082] HIV = human immunodeficiency virus

[0083] ICAM = intercellular adhesion molecule

[0084] BsAb = bispecific antibody

[0085] BsDb = bispecific diabody

[0086] dsFv = disulfide-stabilized Fv

[0087] Fc = the constant fragment of an antibody

[0088] Fd = V of an antibody H +C H 1

[0089] FcR = Fc receptor

[0090] Fv = variable fragment of an antibody

[0091] IgG = immunoglobulin G

[0092] mAb = monoclonal antibody

[0093] PBL = peripheral blood lymphocyte

[0094] scDb = single-chain diabody

[0095] scFv = single-chain Fv

[0096] (scFv)2 = scFv - scFv tandem

[0097] Tandab = tandem diabody

[0098] VH or V H = variable domain of the heavy chain of an antibody

[0099] VL or V L = variable domain of the light chain of an antibody DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention will now be described in detail by way of reference using only the following definitions and examples. All patents and publications mentioned herein, including all sequences disclosed within these patents and publications, are hereby expressly incorporated by reference.

[0102] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994) and Hale & Markham, Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide one of ordinary skill in the art with a general meaning of many of the terms used in the present invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' direction; amino acid sequences are written left to right in the amino to carboxy direction. Regarding definitions and terms in the art, the practitioner is particularly directed to Sambrook et al., 1989, and Ausubel FM et al., 1993. It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described, as they may vary.

[0103] Numerical ranges include the numbers defining the range.

[0104] Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' direction; amino acid sequences are written left to right in the amino to carboxy direction.

[0105] The section headings provided herein are not limitations of the various aspects or embodiments of the invention, and the invention may be obtained by reference to the entire specification as a whole. Accordingly, the terms defined immediately below are more fully described by reference to the entire specification.

[0106] I. Definitions

[0107] "Heteromultimer", "heteromultimer complex", or "heteromultimeric protein" refers to a molecule that comprises a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond. A heteromultimer can comprise a "heterodimer" formed by the first hinge-containing polypeptide, the first light chain, the second hinge-containing polypeptide, and the second light chain. Alternatively, a heteromultimer can form, for example, a bispecific antibody. The polypeptides of the heteromultimer can interact with each other by 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).

[0108] As used herein, "heterodimerization domain" refers to such an alteration or addition to a biomolecule that promotes heteromultimer formation and impedes homomultimer formation. Any heterodimerization domain having a strong preference for forming heterodimers over homodimers is within the scope of the present invention. Illustrative examples include, but are not limited to, for example, U.S. Patent Application 20030078385 (Arathoon et al., - Genentech; describes the knob-into-hole method); WO2007147901( et al., - Novo Nordisk: describes ionic interactions); WO 2009089004 (Kannan et al., - Amgen: describes electrostatic steering effects); WO 2011 / 034605 (Christensen et al., - Genentech; describes coiled coils). See also, for example, Pack, P. and Plueckthun, A., Biochemistry 31, 1579-1584 (1992) describing leucine zippers or Pack et al., Bio / Technology 11, 1271-1277 (1993) describing helix-turn-helix motifs. The phrases "heterodimerization domain" and "heteromultimerization domain" are used interchangeably herein.

[0109] The phrase "hinge-containing polypeptide" as used herein refers to a polypeptide that comprises a region corresponding to an immunoglobulin hinge region as understood in the art (e.g., between the C H 1 domain and the C HA polypeptide between the 2 domains). "Hinge region", "hinge sequence" and variants thereof, as used herein, include the meanings known in the art, such meanings being described, for example, in: Janeway’s Immunobiology, (Garland Science, Taylor&Francis Group, LLC, NY) (7th Edition, 2008); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209:193-202. See also, for example, Burton, Molec. Immunol. 22:161-206 (1985) and Papadea, C. and I.J. 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 appreciate that the number of amino acids available for inter-chain disulfide bond formation and the number of cysteine residues vary between the various classes and isotypes of immunoglobulins. All such hinge regions can be within the polypeptides containing a hinge and within the scope of the present invention. In certain embodiments, the first polypeptide containing a hinge comprises a first antibody heavy chain. In certain embodiments, the first heavy chain associates with a first light chain to form a first half-antibody. The term "antibody" is used herein in the broadest sense and refers to any immunoglobulin (Ig) molecule containing two heavy chains and two light chains and any fragment, mutant variant or derivative thereof, provided they exhibit 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 as described herein. Antibodies can be human, humanized and / or affinity matured.

[0110] As a reference system, as used herein, an antibody will refer to the structure of immunoglobulin G (IgG). However, those skilled in the art will understand / recognize that antibodies of any immunoglobulin class can be used in the methods of the present invention 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 has a variable domain (V L ) and a constant domain (C L ), while each HC has a variable (V H ) and three constant domains (C H 1, C H 2, and CH 3). C H 1 domain and C H 2 domains are connected by a hinge region. This structure is well known in the art. See Figure 1B .

[0111] As used herein, "half antibody" refers to an immunoglobulin heavy chain associated with an immunoglobulin light chain. Figure 1A Exemplary half antibodies are described in. Those skilled in the art will readily understand that a half antibody can also have an antigen-binding domain consisting of a single variable domain.

[0112] The term "maxibody" refers to a fusion protein comprising an scFv fused to an Fc polypeptide. See Figure 8a of WO2009089004. For bispecific maxibodies, see Figure 2 .

[0113] According to the EU numbering system, the "C H 2 domain" of the human IgG / Fc region generally extends from about residue 231 to about residue 340 of IgG. C H The uniqueness of the C H 2 domain is that it does not pair tightly with another domain. Instead, two N-linked branched glycans are interposed between the two C H 2 domains of the intact native IgG molecule. It has been speculated that the sugars can provide an alternative to domain-domain pairing and contribute to the stabilization of the C H "C H 3 domain" comprises a segment of residues at the C-terminus of the C H 2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG according to the EU numbering system).

[0115] The term "Fc region", as used herein, generally refers to a dimeric complex comprising the C-terminal polypeptide sequences of immunoglobulin heavy chains, where the C-terminal polypeptide sequences are those that can be obtained by papain digestion of intact antibodies. The Fc region can comprise native or variant Fc sequences. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc sequence is generally defined by the fragment of the Fc sequence from the amino acid residue at about position Cys226 or from about position Pro230 to its carboxyl terminus. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991, also referred to as the EU index. The Fc sequence of an immunoglobulin generally comprises two constant domains: C H 2 domain and C H 3 domain, and optionally comprises C H 4 domain. "Fc polypeptide" as used herein means one of the polypeptides that constitute the Fc region, e.g., monomeric Fc. Fc polypeptides can be obtained from any suitable immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, or IgM. The Fc region comprises the carboxyl-terminal portions of two H chains linked together by disulfide bonds; the effector functions of an antibody are determined by the sequences in the Fc region; this region is also the part recognized by Fc receptors (FcRs) present on certain types of cells. In some embodiments, the Fc polypeptide comprises part or all of the wild-type hinge sequence (usually at its N-terminus). In some embodiments, the Fc polypeptide does not comprise a functional or wild-type hinge sequence.

[0116] A "functional Fc region" has the "effector functions" 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), etc. Such effector functions generally require the combination of the Fc region with a binding domain (e.g., an antibody variable domain) and can be evaluated using, for example, the various assays disclosed in the definitions herein.

[0117] "Native sequence Fc region" encompasses an amino acid sequence identical to the amino acid sequence of an Fc region that exists in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region (non-A isotype and A allotype); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0118] "Variant Fc region" refers to an amino acid sequence that differs from a native sequence Fc region due to at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, compared to the native sequence Fc region or to the Fc region of a parental polypeptide, the variant Fc region has at least one amino acid substitution in the native sequence Fc region or in the Fc region of the parental polypeptide, e.g., from about 1 to about 10 amino acid substitutions, and preferably from about 1 to about 5 amino acid substitutions. Variant Fc regions herein will preferably have at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parental polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology therewith.

[0119] As used herein, "Fc moiety" refers to the hinge region, C H 2 domain or C H 3 domain of the Fc region.

[0120] In certain embodiments, a polypeptide containing a hinge comprises an IgG / Fc region, preferably derived from a wild-type human IgG / Fc region. For "wild-type" human IgG / Fc, it means the amino acid sequence that occurs naturally within the population. Of course, just as the Fc sequence can vary slightly between individuals, one or more changes can be made to the wild-type sequence and still be within the scope of the present invention. For example, the Fc region can contain additional changes not relevant to the present invention, such as mutations in glycosylation sites or incorporation of non-natural amino acids.

[0121] The terms "variable region" or "variable domain" refer to the domain of an antibody heavy or light chain that participates in binding of the antibody to an antigen. The variable domains of native antibody heavy and light chains (V H and V L ) generally have similar structures, each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single V H domain or V L domain may be sufficient to confer antigen-binding specificity. Additionally, an antibody that binds a particular antigen can utilize the V from an antibody that binds that antigenH or V L domains are separated to separately screen for complementary V L domains or V H domain libraries. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0122] As used herein, the term "Fab" refers to the antigen-binding fragment of an antibody. As shown above, papain can be used to digest intact antibodies. Papain digestion of an antibody produces two identical antigen-binding fragments, namely, "Fab fragments" and the residual "Fc fragment" (i.e., the Fc region above). The Fab fragment consists of the entire L chain together with the variable domain of the H chain (V H ) and the first constant domain of one heavy chain (C H 1).

[0123] As used herein, the phrases "antigen-binding arm", "target molecule-binding arm", "target-binding arm", and variations thereof refer to a component part of the heteromultimeric protein of the invention that has the ability to specifically bind to a target of interest. Generally and preferably, the antigen-binding arm is a complex of immunoglobulin polypeptide sequences (e.g., CDRs and / or variable domain sequences of immunoglobulin light and heavy chains).

[0124] "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 can be an epitope, either 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 will be the cognate binding partner of the receptor. One of ordinary skill 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 can recognize different targets. The target preferably binds to the heteromultimeric protein of the invention with an affinity 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, virus particles (e.g., RSV F protein, CMV, StaphA, influenza virus, hepatitis C virus), microorganisms (e.g., bacterial cell proteins, fungal cells), adhesins, CD proteins and their receptors.

[0125] An example of a "complete" or "full-length" antibody is one that contains an antigen-binding arm as well as C L and at least the heavy chain constant domains, C H 1, C H 2, and C HAn antibody of 3. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof.

[0126] As used herein, the term "coupling" refers to the steps necessary to join a first and a second hinge-containing polypeptide to each other (e.g., to form a covalent bond). Such steps include reducing, renaturing, and / or oxidizing cysteine residues in the first and second hinge-containing polypeptides to form interchain disulfide bonds. Coupling can be achieved by chemical crosslinking or using a redox system. See, e.g., Humphreys et al., J. Immunol. Methods (1998) 217:1-10 and Zhu et al., Cancer Lett., (1994) 86:127-134.

[0127] The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies having multiple epitope specificities. Such multispecific antibodies include, but are not limited to, antibodies comprising heavy chain variable domains (V H ) and light chain variable domains (V L ), wherein the V H V L units have multiple epitope specificities; antibodies having two or more V L domains and V H domains wherein each V H V L unit binds to a different epitope; antibodies having two or more single variable domains wherein each single variable domain binds to a different epitope; full-length antibodies; antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, and triabodies, antibody fragments that have been covalently or noncovalently linked. "Multiple epitope specificity" refers to the ability to specifically bind two or more different epitopes on the same target or different targets. "Monospecificity" refers to the ability to bind only one epitope. According to 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. Figure 1B A schematic diagram of a bispecific antibody is provided in

[0128] "Antibody fragment" includes a portion of a full antibody, preferably the antigen-binding region or variable region of a full 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. Patent No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, and (scFV)4-Fc).

[0129] The term "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (V H or V L ) can confer antigen-binding activity. In other words, a single variable domain does not need to interact with another variable domain to recognize a target antigen. A single domain antibody consists of a single monomeric variable antibody domain (V H or V L ) on each antigen-binding arm. Examples of single domain antibodies include those derived from camelids (alpacas and camels) and cartilaginous fish (e.g., nurse sharks) and those derived from recombinant methods from human and murine 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; WO00 / 29004; WO 02 / 051870). A single variable domain antibody can be present in an antigen-binding arm (e.g., a homomultimer or heteromultimer) having other variable regions or variable domains, in which case it is not a single domain antibody.

[0130] The term "linear antibody" generally refers to the antibodies described in Zapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies contain a pair of tandem Fd segments (V H -C H 1-V H-C H 1), which together with a complementary light chain polypeptide form a pair of antigen-binding regions. The linear antibody can be bispecific or monospecific.

[0131] As used herein, the term "knobs-into-holes" or "KnH" technology refers to a technique for guiding the pairing of two polypeptides together 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 has been introduced at the Fc:Fc binding interface, C L :C H 1 interface or V H / V L interface of antibodies (e.g., US2007 / 0178552, WO 96 / 027011, WO 98 / 050431, and Zhu et al., (1997) Protein Science 6:781-788). This can be particularly useful for driving the pairing of two different heavy chains together during the manufacture of multispecific antibodies. For example, a multispecific antibody having KnH in its Fc region can also contain a single variable domain linked to each Fc region or can also contain different heavy chain variable domains paired with similar or different light chain variable domains. The KnH technology can also be used to pair together two different receptor extracellular domains or any other polypeptide sequences containing different target recognition sequences (e.g., including affibodies, peptibodies, and other Fc fusions).

[0132] "Fv" consists of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly and non-covalently associated. The six hypervariable loops (three loops each from the H and L chains) that contribute the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody are derived from the folding of these two domains. However, even a single variable domain (or half of an Fv containing only 3 antigen-specific CDRs) has the ability to recognize and bind an antigen, although often with an affinity less than that of the complete binding site.

[0133] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment that contains V H and V L antibody domains joined into a single polypeptide chain. Preferably, the sFv polypeptide also contains a linker between the V H domain and the V LA polypeptide linker between domains, said polypeptide linker being capable of enabling the scFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315 (1994); Malmborg et al., J. Immunol. Methods 183:7-13, 1995.

[0134] The term "diabody" refers to small antibody fragments prepared as follows (see the foregoing paragraphs): constructing an sFv fragment with a short linker (about 5-10 residues) between the V H and V L domains, thereby enabling inter-chain rather than intra-chain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossed" sFv fragments, where the V H and V L domains of the two antibodies are on different polypeptide chains. Diabodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0135] The term "monovalent antibody" refers to an antibody that comprises (1) a variable domain linked by a peptide bond to a polypeptide that comprises a C H 2 domain, a C H 3 domain, or a C H 2-C H 3 domain, and (2) a second C H 2, C H 3, or C H 2-C H 3 domain, wherein the variable domain is not linked by a peptide bond to the polypeptide comprising the second C H 2, C H 3, or C H 2-C H 3 domain. In one embodiment, a monovalent antibody comprises three polypeptides: (1) a first polypeptide comprising a variable domain (e.g., V H ), a C H 1, a C H 2, and a C H 3, (2) a second polypeptide comprising a variable domain (e.g., V L ) and a C L domain, and (3) a polypeptide comprising a CH 2 and C H The third polypeptide of the 3 domain. In another embodiment, one-armed antibody has a partial hinge region containing two cysteine residues, and the cysteine residues form a disulfide bond connecting the constant heavy chains. In one embodiment, the variable domain of one-armed antibody forms an antigen-binding region. In another embodiment, the variable domain of one-armed antibody is a single variable domain, and each single variable domain is an antigen-binding region. In one embodiment, the single-armed antibody is a single variable domain antibody.

[0136] The antibodies of the present invention can be "chimeric" antibodies, wherein portions of the heavy and / or light chains are identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portions of the chains are identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. 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 non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0137] A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that contains minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) having the desired specificity, affinity, and capacity, wherein residues in the hypervariable regions of the recipient are replaced by residues in the hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, humanized antibodies can contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will contain substantially all of at least 1, and typically 2, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are those having human immunoglobulin sequences. A humanized antibody optionally will also contain at least a portion of the immunoglobulin constant region (Fc), typically a portion of the human immunoglobulin constant region. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0138] "Peptibody" refers to a fusion of a randomly generated peptide and an Fc domain. See U.S. Patent No. 6,660,843, issued December 9, 2003 to Feige et al. (said document is incorporated herein by reference in its entirety). They include one or more peptides linked to the N-terminus, C-terminus, amino acid side chains, or more than one of these sites. Peptibody technology enables the design of therapeutics incorporating peptides that target one or more ligands or receptors, tumor-homing peptides, membrane transport peptides, etc. Peptibody technology has been shown to be useful for designing numerous such molecules, including linear and disulfide-constrained peptides, "tandem peptide multimers" (i.e., more than one peptide on a single chain of the Fc domain). 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); 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 said documents being hereby incorporated herein by reference in its entirety.

[0139] "Affibody" refers to a protein that uses a protein linked to an Fc region by a peptide bond, where the protein is used as a scaffold to provide a binding surface to a target molecule. The protein is often a naturally occurring protein such as staphylococcal protein A or the B domain that binds IgG or the Z protein derived therefrom (see Nilsson et al. (1987), Prot Eng 1, 107-133, and U.S. Patent No. 5,143,844) or fragments or derivatives thereof. For example, an affibody can be generated from a variant of the Z protein with an altered target molecule binding affinity, where a segment of the Z protein has been mutated by random mutagenesis to generate a library of variants capable of binding the target molecule. Examples of affibodies include U.S. Patent 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.

[0140] As used herein, the term "immunoadhesin" refers to a molecule that combines the binding specificity of a heterologous protein ("adhesin protein") with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises an amino acid sequence with the desired binding specificity that is not the antigen recognition and binding site of an antibody (i.e., is "heterologous" compared to the constant region of an antibody) fused to an immunoglobulin constant domain sequence (e.g., the C H 2 and / or C H 3 sequence). Exemplary adhesin protein sequences comprise a contiguous amino acid sequence that comprises the portion of a receptor or ligand that binds to the protein of interest. The adhesin protein sequence can also be a sequence that binds to the protein of interest but is not a receptor sequence or ligand sequence (e.g., the adhesin protein sequence in a peptibody). Such polypeptide sequences can be selected or identified by various methods, including phage display techniques and high-throughput sorting methods. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4 subtypes, IgA (including IgA1 and IgA2), IgE, IgD, or IgM.

[0141] As used herein, "complex" or "complexed" refers to the association of two or more molecules that interact with each other by means of bonds and / or forces that are not peptide bonds (e.g., van der Waals forces, hydrophobic forces, hydrophilic forces). In one embodiment, the complex is a heteromultimer. It should be understood that the term "protein complex" or "polypeptide complex" as used herein includes complexes having non-protein entities conjugated to the proteins in the protein complex (e.g., including, but not limited to, chemical molecules such as toxins or detection agents).

[0142] The inventive heteromultimeric protein that "binds" to the target antigen is a heteromultimeric protein that binds to the target with sufficient affinity such that the heteromultimeric protein can be used as a diagnostic and / or therapeutic agent for targeting the protein or the cell or tissue expressing the target and does not significantly cross-react with other proteins. In such embodiments, the degree of binding of the heteromultimeric protein to a "non-target" protein will be less than about 10% of the binding of the antibody to its specific target protein, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmuno-precipitation (RIA) or ELISA). With respect to the binding of the heteromultimeric protein to the target molecule, the term "specifically binds" or "specifically binds to" a particular polypeptide or an epitope on a particular polypeptide target or is "specific for it" means a binding that is measurably different from non-specific interactions (e.g., non-specific interactions can be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining the binding of a molecule in comparison to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by the excess unlabeled target, specific binding is demonstrated. As used herein, the term "specifically binds" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target can be demonstrated, for example, by a molecule having a Kd of at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM or greater. In one embodiment, the term "specifically binds" refers to a binding in which a heteromultimeric protein binds to a particular polypeptide or an epitope on a particular polypeptide and binds essentially not at all to any other polypeptide or polypeptide epitope.

[0143] "Binding affinity" generally refers to the strength of the sum of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). For example, the Kd can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or stronger. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, while high-affinity antibodies generally bind antigen more rapidly and tend to remain bound for longer. A variety of methods for measuring binding affinity are known in the art, and any one of them can be used for the purposes of the present invention.

[0144] In one embodiment, the "Kd" or "Kd value" of the present invention is measured at 25 °C using a surface plasmon resonance assay, using a BIAcoreTM-2000 or BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) with a CM5 chip immobilized with a target (e.g., antigen) at approximately 10 response units (RU). Briefly, according to the supplier's instructions, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then loaded at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of the coupled protein. After injecting the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, serial two-fold dilutions of Fab (e.g., 0.78 nM to 500 nM) in PBS containing 0.05% Tween 20 (PBST) are injected at a flow rate of approximately 25 μl / min at 25 °C. The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate exceeds 10 6 M -1 S -1, the association rate can be determined using fluorescence quenching techniques, wherein in the presence of increasing concentrations of antigen measured, for example, in a spectrometer such as a spectrophotometer equipped with stop-flow (Aviv Instruments) or an 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette, the fluorescence quenching technique measures the increase or decrease in the fluorescence emission intensity of 20 nM antigen-specific antibody (Fab form) in PBS, pH 7.2 at 25 °C (excitation = 295 nm; emission = 340 nm, 16 nm bandpass).

[0145] Unless otherwise specified, for the heteromultimeric proteins (such as antibodies, fragments, or derivatives thereof) of the present invention, "biologically active" and "biological activity" and "biological characteristics" mean having the ability to bind to a biomolecule.

[0146] When used to describe various heteromultimeric polypeptides, "isolated" means a heteromultimer that has been separated and / or recovered from the cell or cell culture in which it was expressed. The impurity components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the heteromultimer and can 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 greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal residues or an internal amino acid sequence by a spinning cup sequenator, or (3) to a homogeneity determined by Coomassie blue or preferably silver-stained SDS-PAGE under reducing or non-reducing conditions. However, generally, an isolated polypeptide will be prepared by at least one purification step.

[0147] The heteromultimers of the present invention are typically purified to substantially homogeneous. The phrases "substantially homogeneous", "substantially homogeneous form", and "substantial homogeneity" are used to indicate that the product is substantially free of by-products (e.g., homomultimers) from unwanted polypeptide combinations.

[0148] When 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 less than 5%.

[0149] "Biomolecule" refers to nucleic acids, proteins, sugars, lipids, and combinations thereof. In one embodiment, the biomolecule exists in nature.

[0150] "Linked" or "linkage" as used herein means a direct peptide bond linkage between a first and a second amino acid sequence or a bond involving a third amino acid sequence that is a peptide that binds to and is between the first and second amino acid sequences. For example, a linker peptide binds to the C-terminus of one amino acid sequence and to the N-terminus of another amino acid sequence.

[0151] "Linker" as used herein means an amino acid sequence of two or more amino acids in length. The linker can consist of neutral, polar or nonpolar amino acids. The linker can be, for example, from 2 to 100 amino acids in length, such as between 2 and 50 amino acids in length, for example, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length. The linker can be "cleavable", for example, cleavable by a self-cleavage process or an enzymatic cleavage process or a chemical cleavage process. Cleavage sites in amino acid sequences and enzymes and chemicals that cleave at such sites are well known in the art and are also described herein.

[0152] "Tether" as used herein means an amino acid linker that joins two other amino acid sequences. A tether as described herein can join the N-terminus of an immunoglobulin heavy chain variable domain to the C-terminus of an immunoglobulin light chain constant domain. In a particular embodiment, the tether length is between about 15 and 50 amino acids in length, such as between 20 and 26 amino acids in length (e.g., 20, 21, 22, 23, 24, 25 or 26 amino acids in length). The linker tether can be "cleavable, for example, "cleavable by a self-cleavage process or an enzymatic cleavage process or a chemical cleavage process using standard methods and reagents in the art.

[0153] Enzymatic cleavage of a "linker" or "tether" can involve the use of endopeptidases such as, for example, 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), kallikrein, and subtilisin-like proprotein convertases (e.g., furin (PC1), PC2 or PC3) or N-arginine dibasic convertase. Chemical cleavage can include, for example, the use of hydroxylamine, N-chlorosuccinimide, N-bromosuccinimide or cyanogen bromide.

[0154] "Lys-C endopeptidase cleavage site" as used herein is a lysine residue in an amino acid sequence that can be cleaved by Lys-C endopeptidase on the C-terminal side. Lys-C endopeptidase cleaves on the C-terminal side of a lysine residue.

[0155] "Chaotropic agent" means a water-soluble substance that disrupts the three-dimensional structure of a protein (e.g., an antibody) by interfering with stabilizing intramolecular interactions (e.g., hydrogen bonds, van der Waals forces, or hydrophobic effects). Exemplary chaotropic agents include, but are not limited to, urea, guanidine-HCl, lithium perchlorate, histidine, and arginine.

[0156] "Mild detergent" means a water-soluble substance that disrupts the three-dimensional structure of a protein (e.g., an antibody) by interfering with stabilizing intramolecular interactions (e.g., hydrogen bonds, van der Waals forces, or hydrophobic effects), but does not permanently disrupt the protein structure to the extent that biological activity is lost (i.e., does not denature the protein). Exemplary mild detergents include, but are not limited to, Tween (e.g., Tween-20), Triton (e.g., Triton X-100), NP-40 (nonylphenoxypolyethoxyethanol), Nonidet P-40 (octylphenoxypolyethoxyethanol), and sodium dodecyl sulfate (SDS).

[0157] Antibody "effector function" refers to those biological activities attributed to the Fc region of an antibody (natural sequence Fc region or amino acid sequence variant Fc region) and varies with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0158] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig that binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxic agents. The antibody "arms" the cytotoxic cells and is absolutely required for this type of killing. The major cell used to mediate ADCC, the NK cell, expresses only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression 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 test molecule, an in vitro ADCC assay can be performed, such as that described in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a test molecule can be evaluated in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998).

[0159] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are human FcRs. Additionally, preferred FcRs are a receptor that binds an IgG antibody (γ receptor) and include the FcγRI, FcγRII, and FcγRIII subclasses of receptors, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily within their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain (see M Review 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). The term "FcR" as used herein encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0160] "Human effector cells" are white blood cells that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human white blood cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, preferably PBMCs and NK cells. Effector cells can be isolated from natural sources (e.g., from blood).

[0161] "Complement-dependent cytotoxicity" or "ADDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) bound to its 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).

[0162] "Therapeutically effective amount" refers to the amount of an antibody, antibody fragment, or derivative that treats a disease or disorder in a subject. 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 terminate) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow to some extent and preferably terminate) tumor metastasis; inhibit tumor growth to some extent and / or alleviate one or more symptoms associated with the disorder to some extent. To the extent that the antibody or antibody fragment can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, e.g., by assessing survival duration, time to progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0163] "Reduce or inhibit" means the ability to cause an overall reduction, preferably of 20% or more, more preferably of 50% or more, and most preferably of 75%, 85%, 90%, 95% or more. Reduction or inhibition can refer to the symptoms of the condition being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of blood vessels in an angiogenesis disorder.

[0164] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is generally characterized by dysregulated cell growth / proliferation. This definition includes both benign and malignant cancers. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer 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 cancer, salivary gland cancer, kidney cancer (e.g., renal cell carcinoma), liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and various types of head and neck cancers. "Early cancer" means a cancer that has not invaded or metastasized or is classified as stage 0, I, or II cancer. The term "precancerous" refers to a condition or growth that generally precedes or forms cancer. "Non-metastatic" means a cancer that is either benign or remains at the primary site and has not penetrated into the lymphatic or vascular system or into tissues outside the primary site. Generally, non-metastatic cancers are any cancers that are stage 0, I, or II cancer and occasionally stage III cancer.

[0165] "Allergic or inflammatory diseases" herein are diseases or disorders that overactivate the immune system of an individual. 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.

[0166] As used herein, "autoimmune disease" refers to a disease or disorder or manifestation thereof or a condition resulting therefrom that originates from and targets an individual's own tissues or co-segregating. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (such as rheumatoid arthritis like acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, hypertrophic arthritis, psoriatic arthritis, osteoarthritis, and juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, degenerative arthritis, chronic primary polyarthritis, reactive arthritis, and ankylosing spondylitis), inflammatory hyperplastic skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis, 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 urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spinal-optic MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, and ataxic sclerosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal disease, colitis such as ulcerative colitis, ulcerative colitis (colitisulcerosa), microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis and transmural colitis and autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematological diseases, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic rhinitis and atopic rhinitis, encephalitis such as Rasmussen encephalitis and marginal and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens antigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome, such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous (membrano) or membranoproliferative GN (MPGN) (including type I and type II) and rapidly progressive GN, allergic diseases, allergy, eczema, including allergic or idiopathic eczema, asthma, such as bronchial asthma, bronchial asthma and autoimmune asthma, conditions involving T cell infiltration and chronic inflammatory responses, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritis, encephalitis, pediatric, non-renal, extra-renal, discoid, alopecic lupus), juvenile-onset (type I) diabetes, including pediatric insulin-dependent diabetes (IDDM), adult-onset diabetes (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses related to cytokine- and T-lymphocyte-mediated acute and delayed-type hypersensitivity, tuberculosis, sarcoidosis, granulomas, including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis (including large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), microscopic polyangiitis, CNS vasculitis, necrotizing, cutaneous or hypersensitivity vasculitis, systemic necrotizing vasculitis and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond-Blackfan anemia, hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiaperniciosa), Addison's disease, pure red cell anemia or pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, CNS inflammatory diseases, multiple organ injury syndromes such as those secondary to sepsis, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet's disease or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigus such as bullous pemphigus and cutaneous pemphigus, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membranepemphigoid) and pemphigus erythematosus), autoimmune polyendocrinopathy, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathy, chronic neuropathies such as IgM polyneuropathy or IgM-mediated neuropathy, thrombocytopenia (such as formed in patients with myocardial infarction), including thrombotic thrombocytopenic purpura (TTP) and autoimmune or immune-mediated thrombocytopenia, such as idiopathic thrombocytopenic purpura (ITP) (including chronic or acute ITP), autoimmune diseases of the testis and ovary, including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases (including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyendocrine syndromes, such as autoimmune polyendocrine syndrome (or polyendocrine endocrinopathy syndrome), paraneoplastic syndromes, including neurological paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-person or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS) and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia, bronchiolitis obliterans (non-graft) with NSIP, Guillain-Barré syndrome, Berger's disease (IgA neuropathy), idiopathic IgA neuropathy, linear IgA dermatosis, primary biliary cirrhosis, pulmonary cirrhosis, autoimmune enteropathy syndrome, small intestinal malabsorption (Celiac disease), steatorrhea (Coeliac disease), celiac disease (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Louamyotrophic lateral sclerosis (ALS), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis, which includes monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, ion channelopathies such as epilepsy, migraine, arrhythmia, muscular disorder, deafness, blindness, periodic paralysis and CNS ion channelopathies, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, choroidoretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine failure, Schmidt syndrome, adrenalitis, atrophic gastritis, Alzheimer's disease, demyelinating diseases such as autoimmune demyelinating diseases, diabetic neuropathy, Dressler syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis such as hypersensitivity alveolitis and fibrotic alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter syndrome, Caplan syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, fetal erythroblastosis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis such as chronic cyclitis, heterochronic cyclitiscyclitis), iridocyclitis or Fuch's 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, Evan syndrome, autoimmune gonadal failure, Sydenham chorea, post-streptococcal glomerulonephritis, thromboangitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, endocrine ophthalmopathy, chronic allergic pneumonia, sicca keratoconjunctivitis, epidemic keratoconjunctivitis, idiopathic nephrotic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerosis, aspermiogenesis, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylactic endophthalmitis, allergic enteritis, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich disease, sensorineural hearing loss, paroxysmal nocturnal hemoglobinuria, hypogonadism, regional enteritis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, De Quervain's thyroiditis, acquired splenic atrophy, infertility due to anti-sperm antibodies, non-malignant thymic tumors, vitiligo, SCID and Epstein-Barr virus-related diseases, acquired immunodeficiency syndrome (AIDS), parasitic diseases such as leishmaniasis, toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, leukocyte adhesion deficiency, immune responses related to cytokine- and T-lymphocyte-mediated acute and delayed-type hypersensitivity, diseases involving leukocyte extravasation, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, ophthalmia symphatica, rheumatism, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure (polyendocrinefailure), peripheral neuropathy, autoimmune polyendocrine 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 nonsuppurative sinusitis, acute or chronic sinusitis, ethmoiditis, frontal sinusitis, maxillary sinusitis or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary eosinophilic infiltration, eosinophilia-myalgia syndrome, Löffler syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma or eosinophil-containing granuloma, allergic reactions, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, scleral candidiasis, episcleral candidiasis, chronic mucocutaneous candidiasis, Bruton syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune diseases associated with collagen diseases, rheumatism, neurological diseases, ischemic reperfusion diseases, decreased blood pressure response, vascular dysfunction, angiectasis, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia and diseases associated with vascularization, allergic hypersensitivity disorder, glomerulonephritis, reperfusion injury, reperfusion injury of myocardial tissue or other tissues, skin diseases with acute inflammatory components, acute purpuric meningitis or other central nervous system inflammatory diseases, ocular and orbital inflammatory disorders, granulocyte transfusion-related syndrome, cytokine-induced toxicity, acute severe inflammation, chronic intractable inflammation, pyelonephritis, cirrhosis of the lung, diabetic retinopathy, large artery diseases of diabetes, intimal hyperplasia, peptic ulcer, valvulitis and endometriosis.

[0167] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or impedes cell function and / or causes cell destruction. The term is intended to include radioactive isotopes (e.g., radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, Ra223, P32, and Lu); chemotherapeutic agents (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), daunorubicin, melphalan, mitomycin C, chlorambucil, zorubicin, or other intercalating agents); enzymes and fragments thereof such as ribonucleases; antibiotics; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various anti-tumor, anti-cancer, and chemotherapeutic agents disclosed herein. Other cytotoxic agents are described herein. An anti-tumor agent causes the destruction of tumor cells.

[0168] "Chemotherapeutic agent" is a chemical compound used in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); β-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan CPT-11 (irinotecan, ) acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin A; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, nitrogen mustard oxide hydrochloride, melphalan, novembichin, phenyl mustard azide, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1 (see, e.g., Agnew, Chem Intl Ed Engl 33:183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin D, anthramycin, azaserine, bleomycin, actinomycin c, carabicin, carminomycin, carcinomycin, chromomycinis, dactinomycin, zorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin (including morpholino-doxorubicin, cyano-morpholino-doxorubicin, 2-pyrrolino-doxorubicin, and desoxydoxorubicin), epirubicin, isoxorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodomycin, streptothricin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dihydrofolic acid, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioguanine, tioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, thiotriol, methyltestosterone, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as leucovorin; glucuronolactone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; aclarubicin; bestrabucil; bisantrene; edatrexate; defofamine; colchicine amide; diaziquone; elfornithine; elisidepsin; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuzoic acid; triaziquone; 2,2’,2”-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, baccharicin A, and enniatin); urethane; vindesine dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, for example, Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANETM Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL) and docetaxel ( Rorer, Antony, France); chlorambucil; gemcitabine 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine oxaliplatin; leucovovin; vinorelbine mitoxantrone; edatrexate; daunorubicin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid (tretinoin); capecitabine (Xeloda); pharmaceutically acceptable salts, acids or derivatives of any of the above drugs; and combinations of two or more of the above drugs such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisone combination therapy) and FOLFOX (abbreviation for a treatment regimen using oxaliplatin (ELOXATINTM) in combination with 5-FU and leucovovin).

[0169] Also included in this definition are antihormonal agents that act to regulate, reduce, block or inhibit hormonal effects, where the hormones can promote cancer growth and are often in a systemic or overall treatment form. They can be the hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERM), such as including tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene; antiprogesterone; estrogen receptor downregulators (ERD); drugs that act to suppress or shut down the ovaries, e.g., luteinizing hormone-releasing hormone (LHRH) agonists such as and leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other antiandrogens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit aromatase (regulate estrogen production in the adrenal glands), such as 4(5)-imidazoles, aminoglutethimide, Megestrol Acetate, Arimidex exemestane, Formestane, fadrozole, Volclozole, Femara letrozole and anastrozole. In addition, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g., or ), etidronate, NE-58095, zoledronic acid / zoledronate, alendronate, pamidronate@, tiludronate, or risedronate; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit the expression of genes (such as, PK-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R)) involved in signal transduction pathways related to abnormal cell proliferation; vaccines such as vaccines and gene therapy vaccines, e.g., vaccines, vaccines, and vaccines; topoisomerase 1 inhibitors; rmRH; lapatinib ditosylate (a small molecule dual tyrosine kinase inhibitor of ErbB-2 and EGFR also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above drugs.

[0170] As used herein, "growth inhibitor" refers to a compound or composition that inhibits cell growth, either in vitro or in vivo. Thus, a growth inhibitor can be a drug that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitors include drugs that block the cell cycle progression (at a position other than the S phase), such as drugs that induce G1 arrest and M phase arrest. Classic M phase blockers include vinca alkaloids (e.g., vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, zorubicin, etoposide, and bleomycin. Drugs that cause G1 arrest also unexpectedly cause S phase arrest, such as DNA alkylating agents like tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and cytarabine (ara-C). Additional information can be found in The Molecular Basis of Cancer, edited by Mendelsohn and Israel, Murakami et al., Chapter 1, titled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WBSaunders: Philadelphia, 1995), particularly on page 13. Taxanes (paclitaxel and docetaxel) are anti-cancer drugs that are both derived from yew. Docetaxel ( Rhone-Poulenc Rorer), which is derived from the European yew, is a semi-synthetic analogue of paclitaxel (Taxol, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing disassembly, which results in mitotic arrest in cells.

[0171] As used herein, "anti-cancer therapy" refers to a treatment that reduces or inhibits cancer in a subject. Examples of anti-cancer therapies include cytotoxic radiotherapy and administration of a therapeutically effective amount of a cytotoxic agent, chemotherapeutic agent, growth inhibitor, cancer vaccine, angiogenesis inhibitor, prodrug, cytokine, cytokine antagonist, corticosteroid, immunosuppressant, antiemetic, antibody or antibody fragment, or analgesic to the subject.

[0172] As used herein, the term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance, wherein the precursor or derivative has lower cytotoxicity to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into a more active parent form. See, e.g., 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. (eds.), pp. 247-267, Human Press (1985). Prodrugs include, but are not limited to, prodrugs containing phosphate esters, prodrugs containing thiophosphate esters, prodrugs containing sulfate esters, prodrugs containing peptides, D-amino acid-modified prodrugs, glycosylated prodrugs, prodrugs containing β-lactams, prodrugs optionally containing substituted phenoxyacetamides or optionally substituted phenylacetamides, 5-fluorocytosine, and other 5-fluorouridine prodrugs, which can be converted into more cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present invention include, but are not limited to, those chemotherapeutic drugs described above.

[0173] The term "cytokine" is a generic term for proteins released by a population of cells that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones. Included among the cytokines are 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); hepatocyte growth factor; fibroblast growth factor (FGF); prolactin; placental lactogen; tumor necrosis factor-α and -β; Müllerian-inhibiting substance; murine gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-α; platelet growth factor; transforming growth factors (Tg) such as Tg-α and TGF-β; insulin-like growth factors-I and -II; erythropoietin (EPO); osteogenic factor; interferons such as interferon-α, -β, and -γ; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs such as IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-18a; tumor necrosis factors such as TNF-α or TNF-β; 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 cultures and biologically active equivalents of native sequence cytokines.

[0174] "Cytokine antagonist" means 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 cytokine expression and / or secretion, or by binding to the cytokine or to the cytokine receptor. Cytokine antagonists include antibodies, synthetic peptides or native sequence peptides, immunoadhesins, and small molecule antagonists that bind to a cytokine or a cytokine receptor. The cytokine antagonist is optionally conjugated or fused to a cytotoxic agent. Exemplary TNF antagonists are etanercept infliximab and adalimumab (HUMIRATM).

[0175] As used herein, the term "immunosuppressant" refers to a substance that acts to suppress or mask the immune system of a subject being treated. This includes substances that inhibit cytokine production, down-regulate or inhibit autoantigen expression, or mask MHC antigens. Examples of immunosuppressants include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077); mycophenolate esters such as azathioprine ( / 6-mercaptopurine; bromocryptine; danazol; dapsone; glutaraldehyde (which masks MHC antigens, as described in U.S. Patent No. 4,120,649); anti-idiotypic antibodies directed against MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids and glucocorticoids, for example, prednisone, prednisolone such as (prednisolone sodium phosphate) or (prednisolone sodium phosphate oral solution), methylprednisolone, and dexamethasone; methotrexate (oral or subcutaneous) ( TREXALLTM); hydroxychloroquine / chloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antagonists, including anti-interferon-γ, -β, or -α antibodies, anti-tumor necrosis factor-α antibodies (infliximab or adalimumab), anti-TNFα immunoadhesin ( etanercept), anti-tumor necrosis factor-β antibodies, anti-interleukin-2 antibodies, and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CD11a antibodies and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; 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; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptors (Cohen et al., U.S. Patent 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 98 / 07409); T-cell receptor antibodies (EP 340,109) such as T10B9; cyclophosphamide dapsone; penicillamine plasmapheresis; or intravenous immunoglobulin (IVIG). These immunosuppressants can be used alone or in combination with each other, particularly in combination of a steroid with another immunosuppressant or such combinations followed by maintenance administration of a non-steroid drug to reduce the need for steroids.

[0176] "Analgesic" refers to a drug that acts to inhibit or suppress pain in a subject. Exemplary analgesics include non-steroidal anti-inflammatory drugs (NSAIDs), including ibuprofen naproxen acetylsalicylic acid, indomethacin, sulindac, and tolmetin, including salts and their derivatives, and various other drugs used to reduce possible stinging, including anticonvulsants (gabapentin, phenytoin, carbamazepine) or tricyclic antidepressants. Specific examples include acetaminophen, aspirin, amitriptyline carbamazepine phenytoin gabapentin (E)-N-vanillyl-8-methyl-6-nonenamide or neuroleptics.

[0177] "Corticosteroid" refers to any of several synthetic or naturally occurring substances having the overall chemical structure of a steroid that mimic or enhance the action of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, triamcinolone, and betamethasone.

[0178] As used herein, a "cancer vaccine" is a composition that stimulates an immune response against cancer in a subject. Cancer vaccines generally consist of a source of cancer-related material or cells (antigen) together with other components (e.g., adjuvants) that further stimulate and enhance the immune response against that antigen, and the source can be autologous (from the subject itself) or allogeneic (from others) relative to the subject. A cancer vaccine can result in stimulation of the subject's immune system to produce antibodies against one or several specific antigens, and / or to produce cytotoxic T cells to attack cancer cells bearing those antigens.

[0179] "Cytotoxic radiotherapy" as used herein refers to radiotherapy that inhibits or disrupts the function of cells and / or causes cell destruction. Radiotherapy can include, for example, external beam irradiation or therapy using radiolabeled drugs (such as antibodies). The term is intended to include the use of radioisotopes (e.g., radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, Ra223, P32, and Lu).

[0180] A "subject" is a vertebrate, such as a mammal, e.g., a human. Mammals include, but are not limited to, livestock (such as cattle), racing animals, pets (such as cats, dogs, and horses), primates, mice, and rats.

[0181] Unless otherwise indicated by the context, the terms "first" hinge-containing polypeptide and "second" hinge-containing polypeptide and variants thereof are merely generic identifiers and should not be construed as defining specific or particular polypeptides or components of the antibodies of the invention.

[0182] Unless otherwise noted, commercially available reagents (if present) referred to in the examples were used according to the manufacturers' instructions. The source of those cells identified by ATCC accession number (if present) in the following examples and throughout this specification is the American Type Culture Collection, Manassas, VA. Unless otherwise indicated, standard methods of recombinant DNA technology were used in the present invention, such as those described above and in the following textbooks: Sambrook et al., supra; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, NY, 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc., NY, 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

[0183] Throughout this specification, the word "comprising" or variations thereof such as "comprises" or "comprising" shall be understood to imply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0184] It is understood that aspects and embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0185] References herein to "about" a particular value or parameter include (and describe) variations that relate to that value or parameter per se. For example, a description of "about X" includes a description of "X".

[0186] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0187] II. Method for Preparing Heteromultimeric Proteins in Mammalian Host Cells

[0188] Generating heteromultimeric proteins (e.g., bispecific antibodies) using prior art has several drawbacks, including generating a mixture of products, reduced yields, and diminished / abolished effector function among others. Accordingly, there is a need to generate heteromultimeric proteins efficiently and at high levels.

[0189] Generating antibody molecules by various means is generally well understood. For example, U.S. Patent 6331415 (Cabilly et al.) describes a method of recombinantly generating immunoglobulins wherein the heavy and light chains are co-expressed from a single vector or from 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 generating monoclonal antibodies from separately produced heavy and light chains using plasmids expressed in separate E. coli cultures. A variety of other techniques related to generating antibodies are described in the following documents, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988) and WO2006028936. However, each of these techniques has drawbacks such as low yields, use of chemicals.

[0190] The methods provided herein are based on the following surprising results: 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 to form a heteromultimeric protein in the combined culture medium. As discussed further in detail below, the combined culture medium can be obtained by culturing the first mammalian host cell in a first cell culture, culturing the second mammalian host cell in a second cell culture, harvesting the first and second culture media without disrupting the cell membranes of the first and second host cells, and combining the harvested first and second culture media to obtain a combined culture medium containing the heteromultimeric protein. Alternatively, the combined culture medium can be obtained by culturing the first and second mammalian host cells in a combined cell culture and harvesting the combined cell culture medium containing the heteromultimeric protein from the combined culture. In certain embodiments, the harvesting step includes removing the first and / or second host cells without disrupting the cell membranes.

[0191] The methods provided herein are surprising because it is generally believed that the protein quality control systems of eukaryotic cells, such as mammalian cells, may not be able to efficiently produce incomplete antibodies. See, e.g., Spiess et al. (2013) Nature, 31(8):753-758.

[0192] The Applicant has also surprisingly found that the heteromultimeric protein can be formed in high yield in the combined culture medium under reducing conditions, the combined culture medium containing a first homodimer comprising two first hinge-containing polypeptides and two first light chains that have been secreted by the first host cell and a second homodimer comprising two second hinge-containing polypeptides and two second light chains that have been secreted by the second host cell.

[0193] Accordingly, in certain embodiments, there is provided a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide associates with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide associates with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0194] (a) culturing a first host cell capable of expressing and secreting a first hinge-containing polypeptide and a first light chain,

[0195] (b) culturing a second host cell capable of expressing and secreting a second hinge-containing polypeptide and a second light chain; and

[0196] (c) Obtaining a combined culture medium of a first host cell and a second host cell without disrupting the cell membranes of the first and second host cells, wherein the combined culture medium contains a heteromultimeric protein, and wherein the first host cell and the second host cell are each mammalian cells.

[0197] In certain embodiments, there is provided a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide associates with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide associates with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0198] (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;

[0199] (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;

[0200] (c) Obtaining a combined culture medium of the first host cell and the second host cell, wherein the combined culture medium contains the first homodimer and the second homodimer;

[0201] (d) Incubating the combined culture medium under reducing conditions, and;

[0202] (e) Obtaining the heteromultimeric protein, wherein the first host cell and the second host cell are each mammalian cells.

[0203] In certain embodiments, the combined 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 formation of the heteromultimeric protein.

[0204] 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.

[0205] In certain embodiments, prior to incubation under reducing conditions or prior to addition of a reducing agent, 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%) (including any range between these values) of the first hinge-containing polypeptide and the first light chain (e.g., the first half-antibody) present in the combined medium are in the first homodimeric form. In certain embodiments, prior to incubation under reducing conditions or prior to addition of a reducing agent, about 10% to about 75%, about 20% to about 65%, or about 30% to about 55% of the first hinge-containing polypeptide and the first light chain (e.g., the first half-antibody) present in the combined medium are in the first homodimeric form.

[0206] In certain embodiments, prior to incubation under reducing conditions or prior to addition of a reducing agent, 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%) (including any range between these values) of the second hinge-containing polypeptide and the second light chain (e.g., the second half-antibody) present in the combined medium are in the second homodimeric form. In certain embodiments, prior to incubation under reducing conditions or prior to addition of a reducing agent, about 10% to about 75%, about 20% to about 65%, or about 30% to about 55% of the second hinge-containing polypeptide and the second light chain (e.g., the second half-antibody) present in the combined medium are in the second homodimeric form.

[0207] In certain embodiments, after incubation under reducing conditions or after addition of a reducing agent, the pooled 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 between these values) of a first homodimer. In certain embodiments, after incubation under reducing conditions or after addition of a reducing agent, the pooled medium contains from less than about 2% to about 20%, from less than about 5% to about 15%, or from less than about 10% to about 15% of the first homodimer.

[0208] In certain embodiments, after incubation under reducing conditions or after addition of a reducing agent, the pooled 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 between these values) of a second homodimer. In certain embodiments, after incubation under reducing conditions or after addition of a reducing agent, the pooled medium contains from less than about 2% to about 20%, from less than about 5% to about 15%, or from less than about 10% to about 15% of the second homodimer.

[0209] In certain embodiments, provided is a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide associates with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide associates with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0210] (a) culturing a first host cell, the first host cell comprising a first nucleic acid encoding the first hinge-containing polypeptide and a second nucleic acid encoding the first light chain;

[0211] (b) Culturing a second host cell that comprises a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain; and

[0212] (c) Obtaining a combined culture medium of the first host cell and the second host cell, wherein the combined culture medium comprises a heteromultimeric protein, and 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; and in certain other 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; and in certain other embodiments, the third and fourth nucleic acids are different nucleic acid molecules.

[0213] In certain embodiments, the present invention provides a method for preparing a heteromultimeric protein that comprises i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide associates with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide associates with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0214] (a) Culturing a first host cell that comprises 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;

[0215] (b) Culturing a second host cell that comprises 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;

[0216] (c) Obtaining a combined 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 combined culture medium comprises the first homodimer and the second homodimer;

[0217] (d) Incubating the combined culture medium under reducing conditions sufficient to permit the heteromultimeric protein; and

[0218] (e) Obtain 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; and in certain other 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; and in certain other 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 combined culture medium.

[0219] In certain embodiments, provided is a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first half-antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second half-antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0220] (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;

[0221] (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

[0222] (c) Obtaining the combined culture medium of the first host cell and the second host cell, wherein the combined culture medium comprises the heteromultimeric protein, and 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 combined culture.

[0223] In certain embodiments, the present invention provides a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first half-antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second half-antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0224] (a) Cultivate a first host cell, the 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;

[0225] (b) Cultivate a second host cell, the 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;

[0226] (c) Obtain a combined 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 combined culture medium comprises the first homodimer and the second homodimer;

[0227] (d) Incubate the combined culture medium under reducing conditions sufficient to permit the formation of a heteromultimeric protein, and;

[0228] (e) Obtain 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 combined culture medium.

[0229] In certain embodiments, the present invention provides a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide associates with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide associates with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0230] (a) Cultivate a first mammalian host cell in a first cell culture, the 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;

[0231] (b) Cultivate a second mammalian host cell in a second cell culture, the second host cell comprising a third nucleic acid encoding a second hinge-containing polypeptide and a fourth nucleic acid encoding a second light chain;

[0232] (c) Harvest a first culture medium from the first mammalian host cell;

[0233] (d) Harvest a second culture medium from the second mammalian host cell;

[0234] (e) Combine the first culture medium and the second culture medium to obtain a combined culture medium, wherein the combined culture medium contains the heteromultimeric protein. In certain embodiments, harvesting the first culture medium includes removing the first host cells from the first cell culture. In certain embodiments, harvesting the second culture medium includes removing the second host cells from the second cell culture. In certain embodiments, the method further includes adding a reducing agent to the combined culture medium.

[0235] In certain embodiments, there is provided a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0236] (a) Culturing a first host cell in a first cell culture, the first host cell comprising a first nucleic acid encoding the first hinge-containing polypeptide and a second nucleic acid encoding the 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;

[0237] (b) Culturing a second host cell in a second cell culture, the second host cell comprising a third nucleic acid encoding the second hinge-containing polypeptide and a fourth nucleic acid encoding the 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;

[0238] (c) Harvesting a first culture medium from the first mammalian host cell, wherein the first culture medium contains the first homodimer;

[0239] (d) Harvesting a second culture medium from the second mammalian host cell, wherein the second culture medium contains the second homodimer;

[0240] (e) Combining the first culture medium and the second culture medium to obtain a combined culture medium, wherein the combined culture medium contains the first homodimer and the second homodimer;

[0241] (f) Incubating the combined culture medium under reducing conditions sufficient to allow formation of the heteromultimeric protein, and;

[0242] (g) Obtain a heteromultimeric protein, wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, harvesting the first medium comprises removing the first host cells from the first cell culture. In certain embodiments, harvesting the second 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 combined medium.

[0243] In certain embodiments, the present invention provides a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first half - antibody comprising a first hinge - containing polypeptide having a first heterodimerization domain, wherein the first hinge - containing polypeptide is associated with a first light chain, and ii) a second half - antibody comprising a second hinge - containing polypeptide having a second heterodimerization domain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second half - antibodies are linked by at least one inter - chain disulfide bond, the method comprising the steps of:

[0244] (a) Culturing a first mammalian host cell in a first cell culture, the first mammalian host cell comprising a first nucleic acid encoding the first hinge - containing polypeptide and a second nucleic acid encoding the first light chain;

[0245] (b) Culturing a second mammalian host cell in a second cell culture, the second host cell comprising a third nucleic acid encoding the second hinge - containing polypeptide and a fourth nucleic acid encoding the second light chain;

[0246] (c) Harvesting a first medium from the first mammalian host cell;

[0247] (d) Harvesting a second medium from the second mammalian host cell;

[0248] (e) Combining the first medium and the second medium to obtain a combined medium, wherein the combined medium comprises the heteromultimeric protein. In certain embodiments, harvesting the first medium comprises removing the first host cells from the first cell culture. In certain embodiments, harvesting the second medium comprises removing the second host cells from the second cell culture.

[0249] In certain embodiments, a method for preparing a heteromultimeric protein is provided, the heteromultimeric protein comprising i) a first half-antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second half-antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps:

[0250] (a) Culturing a first host cell in a first cell culture, the 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;

[0251] (b) Culturing a second host cell in a second cell culture, the 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;

[0252] (c) Harvesting a first culture medium from the first mammalian host cell, wherein the first culture medium comprises the first homodimer;

[0253] (d) Harvesting a second culture medium from the second mammalian host cell, wherein the second culture medium comprises the second homodimer;

[0254] (e) Combining the first culture medium and the second culture medium to obtain a combined culture medium, wherein the combined culture medium comprises the first homodimer and the second homodimer;

[0255] (f) Incubating the combined culture medium under reducing conditions sufficient to permit formation of the heteromultimeric protein, and;

[0256] (g) Obtaining the heteromultimeric protein, wherein the first host cell and the second host cell are each mammalian cells. In certain embodiments, harvesting the first culture medium comprises removing the first host cell from the first cell culture. In certain embodiments, harvesting 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 combined culture medium.

[0257] In certain embodiments, the present invention provides a method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps:

[0258] (a) Culturing a first mammalian host cell comprising a first nucleic acid encoding the first hinge-containing polypeptide and a second nucleic acid encoding the first light chain;

[0259] (b) Culturing a second mammalian host cell comprising a third nucleic acid encoding the second hinge-containing polypeptide and a fourth nucleic acid encoding the second light chain; and

[0260] (c) Harvesting the medium of a combined cell culture comprising the first host cell and the second host cell to obtain a combined medium of the first mammalian host cell and the second mammalian host cell, wherein the combined medium comprises the heteromultimeric protein.

[0261] In certain embodiments, a method for preparing a heteromultimeric protein is provided, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps:

[0262] (a) Culturing a first mammalian host cell comprising a first nucleic acid encoding the first hinge-containing polypeptide and a second nucleic acid encoding the 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;

[0263] (b) Culturing a second mammalian host cell comprising a third nucleic acid encoding the second hinge-containing polypeptide and a fourth nucleic acid encoding the 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;

[0264] (c) Harvesting the medium of a combined cell culture comprising a first host cell and a second host cell to obtain a combined medium of the first mammalian host cell and the second mammalian host cell, wherein the medium of the combined cell culture comprises a first homodimer and a second homodimer;

[0265] (d) Incubating the medium under reducing conditions sufficient to permit the formation of the heteropolymeric protein, and;

[0266] (e) Obtaining the heteropolymeric 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 combined medium.

[0267] In certain embodiments, the present invention provides a method for preparing a heteropolymeric protein, the heteropolymeric protein comprising i) a first half-antibody comprising a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second half-antibody comprising a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0268] (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;

[0269] (b) Culturing a second mammalian host cell, the 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

[0270] (c) Harvesting the medium of a combined cell culture comprising the first host cell and the second host cell to obtain a combined medium of the first mammalian host cell and the second mammalian host cell, wherein the combined medium comprises the heteropolymeric protein.

[0271] In certain embodiments, a method for preparing a heteromultimeric protein is provided, the heteromultimeric protein comprising i) a first half - antibody comprising a first hinge - containing polypeptide having a first heterodimerization domain, wherein the first hinge - containing polypeptide is associated with a first light chain, and ii) a second half - antibody comprising a second hinge - containing polypeptide having a second heterodimerization domain, wherein the second hinge - containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge - containing polypeptides are linked by at least one inter - chain disulfide bond, the method comprising the steps:

[0272] (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;

[0273] (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;

[0274] (c) Harvesting the medium of a combined cell culture comprising the first host cell and the second host cell to obtain a combined medium of the first mammalian host cell and the second mammalian host cell, wherein the medium of the combined cell culture comprises the first homodimer and the second homodimer;

[0275] (d) Incubating the medium under reducing conditions sufficient to allow the formation of the heteromultimeric protein, and;

[0276] (e) Obtaining the 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 combined medium.

[0277] In certain embodiments, culturing the combined cell culture comprising the first host cell and the second host cell is carried out at a temperature between about 25 °C and 40 °C. In certain embodiments, culturing the combined cell culture comprising the first host cell and the second host cell is carried out at a temperature between about 30 °C and 37 °C. In certain embodiments, culturing the combined cell culture comprising the first host cell and the second host cell is carried out at a pH between about 7.2 and 8.7.

[0278] In certain embodiments, the combined medium is incubated at a temperature between about 4°C and 40°C. In certain embodiments, the combined medium is incubated at a temperature between about 30°C and 37°C. In certain embodiments, the combined medium is incubated at a temperature between about 4°C and 8°C.

[0279] In certain embodiments, the combined medium is stirred. In certain embodiments, after obtaining the combined medium, the combined 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. In some embodiments, the combined medium is stirred intermittently.

[0280] In certain embodiments, the method further comprises separating the heteromultimeric protein (such as a bispecific antibody) from the combined medium. In certain embodiments, a Protein A column is used to separate the heteromultimeric protein (such as a bispecific antibody).

[0281] In certain embodiments, the method comprises adding a reducing agent during the production of the heteromultimeric protein (such as a 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, dithiobutylamine, 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.

[0282] In certain embodiments in which the first and second mammalian host cells are cultured separately (i.e., in separate cultures), the reducing agent is added to the first cell culture medium and to the second cell culture medium before harvesting the first and second cell culture media and combining them to obtain the combined medium. In certain embodiments in which the first and second mammalian host cells are cultured in the same culture, the reducing agent is added to the medium of the combined cell culture before harvesting the combined cell culture to obtain the combined medium.

[0283] 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 range between these values) before the harvesting step.

[0284] In certain embodiments, a reducing agent is added to the combined medium. In certain embodiments, the combined medium containing the reducing agent is incubated for about 4 hours, about 5 hours, about 6 hours, 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).

[0285] In certain embodiments, a reducing agent (such as glutathione) is added to the combined 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 (such as glutathione) is added to the combined cell culture to achieve a final concentration of less than 20 mM. In certain embodiments, a reducing agent (such as glutathione) is added to the combined cell culture to achieve a final concentration of no more than 20 mM.

[0286] In certain embodiments, a reducing agent is added to the combined medium before separating the heteromultimeric protein (such as a bispecific antibody) from the combined medium. In certain embodiments, the combined medium containing the reducing agent is incubated for about 4 hours, about 5 hours, about 6 hours, 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) before separating the heteromultimeric protein (such as a bispecific antibody) from the combined medium containing the reducing agent. In some embodiments, the heteromultimeric protein is separated using a protein A column.

[0287] In certain embodiments, a reducing agent (such as glutathione) is added to the combined 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 (such as glutathione) is added to the combined medium to achieve a final concentration of less than 20 mM. In certain embodiments, a reducing agent (such as glutathione) is added to the combined medium to achieve a final concentration of no more than 20 mM.

[0288] 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 where the first host cell and the second host cell are cultured in the same culture, the ratio of the first host cell to the second host cell at the start time of the combined 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.

[0289] As used herein, "molar ratio" refers to the ratio of the first hinge-containing polypeptide (such as the first half-antibody) associated with the first light chain that has been expressed and / or secreted to the second hinge-containing polypeptide (such as the second half-antibody) associated with the second light chain that has been expressed and / or secreted. In some embodiments, the molar ratio of the first hinge-containing polypeptide associated with the first light chain to the second hinge-containing polypeptide associated with the second light chain is between 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 associated with the first light chain to the second hinge-containing polypeptide associated with 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 between 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 half-antibody to the second half-antibody is about 1:1.

[0290] III. Heteromultimeric Proteins

[0291] The present invention also provides heteromultimeric proteins produced by any of the methods described herein. In certain embodiments, the heteromultimeric protein comprises an antibody Fc region or a variant thereof (such as a variant with altered ADCC function). In certain embodiments, the heteromultimeric protein comprises a distinct portion of an antibody Fc region or a variant thereof (e.g., a variant with altered ADCC function). In certain embodiments, the heteromultimeric protein comprises a heavy chain that comprises only the C H 1 domain, C H 2 domain, and / or a portion of the C H 3 domain. In certain embodiments, the heteromultimeric protein is an antibody fragment that comprises only the C H 1 domain, C H 2 domain, and / or a portion of the C H 3 domain. 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 IgG (such as IgG1, IgG2, or IgG4), IgA, or IgD. In certain embodiments, the first and second light chains of the heteromultimeric protein comprise 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 antibody heavy chains.

[0292] Heteromultimerization domain

[0293] The heteromultimeric protein comprises a heteromultimerization domain. In order to produce a substantially homogeneous population of heterodimeric molecules, the heterodimerization domain must have a strong preference for forming heterodimers relative to homodimers. Although the heteromultimeric proteins exemplified herein use the knobs-into-holes technique to facilitate heteromultimerization, those skilled in the art will appreciate that other heteromultimerization domains can be used in the present invention.

[0294] Knobs-into-holes

[0295] The use of incorporation holes as a method for generating multispecific antibodies is well known in the art. See U.S. Patent No. 5,731,168, issued to Genentech on March 24, 1998, PCT Publication No. WO2009089004, published and assigned to Amgen on July 16, 2009, and U.S. Patent Publication No. 20090182127, published and assigned to Novo Nordisk A / S on July 16, 2009. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649 - 658 and Kontermann (2005) Acta Pharacol. Sin., 26:1 - 9. A brief discussion is provided herein.

[0296] "Protrusion" refers to at least one amino acid side chain that extends from the interface of the first polypeptide and can thus be disposed in a complementary cavity adjacent to the interface (i.e., the interface of the second polypeptide), thereby stabilizing the heteromultimer and thus, for example, favoring heteromultimer formation over homomultimer formation. Protrusions can be present in the native interface or can be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). Normally, the nucleic acid encoding the interface of the first polypeptide is altered to encode a protrusion. To achieve this, the nucleic acid encoding at least one "native amino acid residue" in the interface of the first polypeptide is replaced with the nucleic acid encoding at least one "input" amino acid residue having a larger side chain volume than the native amino acid residue. It will be understood that there may be more than one native residue and corresponding input residue. The upper limit of the number of native residues to be replaced is the total number of residues in the interface of the first polypeptide. The side chain volumes of various amino residues are shown in Table 1 below:

[0297] Table 1

[0298] Properties of Amino Acid Residues

[0299]

[0300]

[0301] a Molecular weight of amino acid minus molecular weight of water. Values are from Handbook of Chemistry and Physics , 43rd Edition, Cleveland, Chemical Rubber Publishing Co., 1961.

[0302] b Values are from A.A. Zamyatnin, Prog. Biophys. Mol. Biol. 24:107 - 123, 1972.

[0303] The c value is from C. Chothia, J. Mol. Biol. 105: 1-14, 1975. The accessible surface area is defined in Figure 6-2 0 of that reference.

[0304] Preferred input residues for forming the protrusion are typically naturally occurring amino acid residues and are 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 forming the protrusion has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0305] "Hole" refers to at least one amino acid side chain that is recessed from the interface of the second polypeptide and thus accommodates a corresponding protrusion on the adjacent interface of the first polypeptide. The hole can be present in the original interface or can be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). Normally, the nucleic acid encoding the interface of the second polypeptide is altered to encode a hole. To achieve this, the DNA of at least one "original amino acid residue" in the interface of the second polypeptide is replaced with a nucleic acid encoding at least one "input" amino acid residue having a smaller side chain volume than the original amino acid residue. It will be understood that there may be more than one original residue and corresponding input residue. The upper limit of the number of original residues to be replaced is the total number of residues in the interface of the second polypeptide. Table 1 above shows the side chain volumes of various amino residues: Preferred input residues for forming the hole are typically naturally occurring amino acid residues and are 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 forming the hole has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0306] A "native" amino acid residue is an amino acid residue that is replaced by an "input" residue that can have a smaller or larger side chain volume than the native residue. The input amino acid residue can be a naturally occurring or non-naturally occurring amino acid residue, but preferably the former. "Naturally occurring" amino acid residues are those residues encoded by the genetic code and listed in Table 1 above. "Non-naturally occurring" amino acid residues mean residues that are not encoded by the genetic code but are capable of covalently binding to adjacent amino acid residues 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 methods of Noren et al., Science 244:182 (1989) and Ellman et al. above 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 present invention involves replacing at least one native amino acid residue, but more than one native residue can be replaced. Normally, no more than the total residues in the interface of the first or second polypeptide will constitute the replaced native amino acid residues. Generally, the native residues to be replaced are "buried". "Buried" means that the residue is substantially inaccessible to the solvent. Usually, the input residue No is cysteine to prevent possible oxidation or mispairing of disulfide bonds.

[0307] The protrusion is “disposably” in the cavity, which means that the spatial positions of the protrusion and the cavity on the first polypeptide interface and the second polypeptide interface respectively, and the sizes of the protrusion and the cavity are such that the protrusion can be in the cavity without significantly disturbing the normal association of the first polypeptide and the second polypeptide at the interface. Since protrusions such as Tyr; Phe and Trp generally do not extend perpendicularly from the axis of the interface and have preferred conformations, in some cases, the alignment of the protrusion with the corresponding cavity depends on modeling of the protrusion / cavity pair according to a three-dimensional structure (such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR)). This can be achieved using techniques widely accepted in the art. “Original nucleic acid or template nucleic acid” means a nucleic acid encoding a polypeptide of interest, which can be “altered” (i.e., genetically engineered or mutated) to encode a protrusion or a cavity. The original nucleic acid or starting nucleic acid can be a naturally occurring nucleic acid or can comprise a nucleic acid that has been subjected to prior alteration (e.g., a humanized antibody fragment). “Altering” a nucleic acid means mutating the original nucleic acid by inserting, deleting or substituting at least one codon encoding the amino acid residue of interest. Normally, the codon encoding the original residue is replaced by a codon encoding the input residue. Techniques for genetically modifying DNA in this way have been reviewed in Mutagenesis: a Practical Approach, edited by M.J. McPherson (IRL Press, Oxford, UK. (1991), and include, for example, site-directed mutagenesis, cassette mutagenesis and polymerase chain reaction (PCR) mutagenesis. By mutating the original / template nucleic acid, the original / template polypeptide encoded by the original / template nucleic acid is accordingly altered.

[0308] The protrusion or the cavity can be introduced into the interface of the first or second polypeptide by synthetic means, such as by recombinant techniques, in vitro peptide synthesis methods, those techniques for introducing the aforementioned non-naturally occurring amino acid residues, by enzymatic or chemical coupling of peptides or some combination of these techniques. Thus, the “introduced” protrusion or cavity is “non-naturally occurring” or “non-native”, which means that it does not exist in nature or in the original polypeptide (e.g., a humanized monoclonal antibody).

[0309] Generally, the input amino acid residues used to form the protrusion have a relatively small number (e.g., about 3-6) of “rotamers”. A “rotamer” is an energetically favorable conformation of an amino acid side chain. The number of rotamers of various amino acid residues is reviewed in Ponders and Richards, J. Mol. Biol. 193:775-791 (1987).

[0310] In some embodiments of the methods provided herein, the first heterodimerization domain of the heteromultimeric protein comprises a knot modification at the interface and the second heterodimerization domain comprises a pore modification. In certain embodiments, the knot modification comprises replacement of 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 replacement amino acid residue with the larger side chain is tryptophan, phenylalanine, tyrosine, or arginine. In certain embodiments, the knot modification comprises a T366W replacement (EU numbering). In certain embodiments, the pore modification comprises replacement of an amino acid residue from the second heterodimerization domain with an amino acid residue having fewer side chains. In certain embodiments, the replacement amino acid residue with the smaller side chain is serine, threonine, valine, or alanine. In some embodiments, the pore modification comprises two or more amino acid replacements, the amino acid replacements including T366S, L368A, and / or Y407V (EU numbering).

[0311] IV. Vectors, Host Cells and Recombinant Methods

[0312] To recombinantly produce the heteromultimeric proteins (e.g., antibodies) of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. The DNA encoding the antibody is readily isolated using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to the 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 will be understood that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species.

[0313] a. Production of heteromultimeric proteins using mammalian host cells:

[0314] Vector components generally include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoter, and transcription termination sequences.

[0315] i. Signal sequence component

[0316] Vectors for mammalian host cells can also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected is preferably a heterologous signal sequence recognized and processed by the host cell (i.e., cleaved by signal peptidase). In mammalian cell expression, mammalian signal sequences as well as viral secretory leader sequences, such as the herpes simplex gD signal, are available. The DNA of such a precursor region is ligated in-frame to the DNA encoding the desired heteromultimeric protein (e.g., an antibody).

[0317] ii. Origin of replication

[0318] Typically, mammalian expression vectors do not require an origin of replication component. For example, the SV40 origin of replication can generally be used, but only because it contains an early promoter.

[0319] iii. Selectable gene component

[0320] Expression and cloning vectors can contain a selectable gene, also called a selectable marker. Common selectable genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media.

[0321] An example of a selection scheme utilizes a drug that arrests the growth of host cells. Those cells successfully transformed with the heterologous gene produce a protein conferring drug resistance and thus achieve the selection scheme. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0322] Another example of a suitable selectable marker for mammalian cells is selectable markers that can identify cells capable of taking up antibodies, such as DHF, thymidine kinase, metallothionein-I and -II, preferably the primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc.

[0323] For example, cells transformed with the DHF selectable gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx) (a competitive antagonist of DHF). When using wild-type DHFR, a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0324] Alternatively, host cells (especially wild-type hosts containing endogenous DHF) transformed or co-transformed with a DNA sequence encoding an antibody, wild-type DHF protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by growing the cells in a 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.

[0325] iv. Promoter assembly

[0326] Expression vectors and cloning vectors typically contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the desired polypeptide containing a hinge. Promoter sequences for mammalian cells are known. In fact, all mammalian genes have an AT-rich region located approximately 25 to 30 bases upstream of the site where transcription is initiated. Another sequence located 70 to 80 bases upstream of the transcription start point of many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most mammalian genes is the AATAAA sequence, which can be a signal for adding a polyadenylate tail to the 3' end of the coding sequence. All of these sequences are appropriately inserted into mammalian expression vectors.

[0327] Transcription of the desired polypeptide containing a hinge and light chain transcription from the vector in mammalian host cells are controlled, for example, by promoters obtained from the genomes of viruses (e.g., polyomavirus, fowlpox virus), adenoviruses (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40)), from heterologous mammalian promoters such as the actin promoter or immunoglobulin promoter, or from heat shock promoters, provided that such promoters are compatible with the host cell system.

[0328] The early and late promoters of the SV40 virus are conveniently obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is disclosed in U.S. Patent No. 4,419,446. A modified form of this system is described in U.S. Patent No. 4,601,978. For the expression of human interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus, also see Reyes et al., Nature 297:598-601 (1982). Alternatively, the long terminal repeat sequence of Rous sarcoma virus can be used as a promoter.

[0329] v. Enhancer element assembly

[0330] The DNA encoding the hinge-containing polypeptide and light chain required for transcription in mammalian host cells can be increased by inserting enhancer sequences into the vector. Many enhancer sequences from mammalian genes (e.g., globin genes, elastase genes, albumin genes, α-fetoprotein genes, and insulin genes) are now known. Additionally, enhancers from mammalian cell viruses can be used. Examples include the SV40 enhancer at the late side of the origin of replication (bp 100-270), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer on the late side of the origin of replication, and the adenovirus enhancer. For a description of elements that enhance eukaryotic promoter activation, also see Yaniv, Nature 297:17-18 (1982). The enhancer can be spliced into the vector at a position 5' or 3' of the sequence encoding the antibody polypeptide, provided that enhancement is achieved, but is usually present at a site 5' to the promoter.

[0331] vi. Transcription termination component

[0332] Expression vectors used in mammalian host cells generally will also contain sequences necessary for terminating mRNA transcription and stabilizing the mRNA. Such sequences are often obtainable from the 5' untranslated region of mammalian DNA or cDNA or viral DNA or cDNA and occasionally from its 3' untranslated region. These regions contain nucleotide segments that are transcribed into the polyadenylation segment in the untranslated portion of the mRNA encoding the antibody. One available transcription termination component is the bovine growth hormone polyadenylation region. See WO 94 / 11026 and expression vectors are disclosed herein.

[0333] vii. Selection and transformation of host cells

[0334] Suitable host cells for cloning or expressing the DNA in the vectors herein include the mammalian cells described herein, including vertebrate host cells. The propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of available mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHF (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); dog kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, HB8065); mouse mammary tumor (MM060562, ATCC CCL 51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982))); MRC5 cells; FS4 cells; and human hepatoma cell line (HepG2).

[0335] Transfect or transform the host cells with the above-described expression or cloning vector containing the desired polypeptide and light chain with a hinge and culture in a conventional nutrient medium, adjusting the conventional nutrient medium, if appropriate, to induce the promoter, and select the transformants or amplify the gene encoding the desired sequence. In some embodiments, the host cells are stably transfected host cells. In certain embodiments, the host cells are stable cell lines.

[0336] viii. Culture the host cells

[0337] Host cells for producing the hinge-containing desired polypeptides and light chains of the present invention can be cultured in a variety of media. Commercially available media such as Ham F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any medium described in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Biochem. 102:255 (1980); U.S. Patent 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. Patent Reissue 30,985 can be used as the medium for host cells. Any of these media can be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamicin TM drug), trace elements (defined as inorganic compounds that are typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used with the host cells selected for expression and will be apparent to the ordinary skilled person.

[0338] ix. Purification of heteromultimeric proteins

[0339] When recombinant techniques are used, the hinge-containing polypeptide and the associated light chain polypeptide can be produced intracellularly or secreted directly into the medium. If the hinge-containing polypeptide and the light chain polypeptide are produced intracellularly, then as a first step, particulate debris (host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. In the case where the hinge-containing polypeptide associated with the light chain polypeptide is secreted into the medium, the supernatant from such an expression system is first typically concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration devices. Protease inhibitors such as PMSF can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0340] For example, using hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, heteromultimeric compositions prepared from cells can be purified, and affinity chromatography is the preferred purification technique. The suitability of protein A as an affinity ligand depends on the class and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth., 62:1-13 (1983)). Protein G is recommended for all murine isotypes and for human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene-divinyl)benzene allow faster flow rates and shorter processing times than can be achieved with agarose. In the case where the antibody contains a CH3 domain, Bakerbond ABX TM resin (J.T. Baker; Phillipsburg, NJ) can be used for purification. Depending on the antibody to be recovered, other techniques for protein purification such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica gel, chromatography on heparin SEPHAROSE TM chromatography, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatography focused, SDS-PAGE, and ammonium sulfate precipitation methods are also available.

[0341] After any preliminary purification step, the mixture containing the antibody of interest and impurities can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5 - 4.5, preferably at a low salt concentration (e.g., starting from about 0 - 0.25 M salt). Production of the heteromultimeric protein can alternatively or additionally (in addition to any of the foregoing specific methods) include dialysis of a solution containing the polypeptide mixture.

[0342] V. Heteromultimeric Protein Formation / Assembly

[0343] Formation of the intact heteromultimeric protein involves assembly of a first hinge-containing polypeptide, a first light chain, a second hinge-containing polypeptide, and a second light chain through the formation of disulfide bonds, which is referred to as refolding in the present invention. Refolding includes association of the first hinge-containing polypeptide with the second hinge-containing polypeptide and 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 is performed in vitro in the present invention, also referred to as renaturation.

[0344] Once the hinge-containing polypeptide and the associated light chain are secreted from the cell, the heteropolimerization domain will drive the association of the heteromeric protein. The formation of interchain disulfide bonds in the associated hinge-containing polypeptide continues. The resulting disulfide-linked heteromeric protein is then purified. Optionally, it can be formulated for research purposes, diagnostic purposes, therapeutic purposes, or other purposes.

[0345] VI. Target Molecules

[0346] Examples of molecules that can be targeted by the heteromeric proteins of the invention include, but are not limited to, soluble serum proteins and their receptors and other membrane-bound proteins (e.g., adhesins).

[0347] In another embodiment, the heteromeric protein of the invention is capable of binding one, two or more cytokines, cytokine-related proteins and cytokine receptors selected from the following: BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, CSF1 (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, FELl, FELl (EPSELON), FELl (ZETA), IL1A, 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, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, LL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R,IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.

[0348] In another embodiment, the target molecule is a chemokine, chemokine receptor or chemokine protein selected from the following: 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 (MIP-3a), CCL21 (SLC / secondary lymphoid tissue chemokine-2), CCL22 (MDC / STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECT), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CXCL1 (GRO1), CXCL2 (GRO2), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP10), CXCLIl (I-TAC), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (lymphotactin), XCL2 (SCM-Ib), BLR1 (MDR15), CCBP2 (D6 / JAB61), CCR1 (CKR1 / HM145), CCR2 (mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EBI1), CCR8 (CMKBR8 / TER1 / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHKl), CCRL2 (L-CCR), XCR1 (GPR5 / CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCRlO), GPR31, GPR81 (FKSG80), CXCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R (GPR16), TCPlO, CKLFSF2, CKLFSF3,CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCClO(ClO), EPO, FY (DARC), GDF5, HDFlA, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2 and VHL.

[0349] In another embodiment, the heteromeric protein of the invention is capable of binding to one or more targets selected from the following: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; AD0RA2A; aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF (BLys); BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BMP1; BMP2; BMP3B (GDF1O); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (netrin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (1-309); CCLI1 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-Id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MTP-2); SLC; secondary lymphoid tissue chemokine-2; CCL22 (MDC / STC-I); CCL23 (MPIF-1); CCL24 (MPIF-2 / eotaxin-2); CCL25 (TECT); CCL26 (eotaxin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MTP-Ia); CCL4 (MDP-Ib); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1 / HM145); CCR2 (mcp-1RB / RA); CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKR7 / EBI1); CCR8 (CMKBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; 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; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1 / Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A (P16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (Claudin-7); CLN3; CLU (Clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (β-catenin); CTSB (Cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCLI1 (I-TAC / IP-9); CXCL12 (SDF1); 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; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2;FCGR3A; FGF; FGF1 (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); FEL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-I); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR1O); GPR31; GPR44; GPR81 (FKSG80); GRCC1O (C1O); GRP; GSN (gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HDP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNγ; DFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; 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; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2,IL1RN; 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 (glycoprotein 130); EL7; EL7R; EL8; IL8RA; DL8RB; IL8RB; DL9; DL9R; DLK; INHA; INHBA; INSL3; INSL4; IRAKI; ERAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GCBoxBP); KLF6; KLK1O; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KHTHB6 (hair-specific type H keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MEF; MIP-2; MKI67; (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; neuroglycan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); N0X5; NPPB; NROB1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1;PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; Proc; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNFI1O (ZNF144); ROBO2; S100A2; SCGB1D2 (Lipocalin B); SCGB2A1 (Lactoglobulin 2); SCGB2A2 (Lactoglobulin 1); SCYE1 (Endothelial monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERP1NB5 (Mammary serpin); SERPINE1 (PAI-I); SERPDMF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (Thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; Tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAEP2 (B94); TNFAIP3; TNFRSFI1A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (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; TOP Topoisomerase Ea); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC;Pluripotent glycan; VHLC5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-Ib); XCR1 (GPR5 / CCXCR1); YY1; and ZFPM2.;

[0350] Preferred molecular target molecules of the antibodies covered by the present invention include CD proteins such as CD3, CD4, CD8, CD16, CD19, CD20, CD34; members of the ErbB receptor family such as CD64, CD200, e.g., EGF receptor, HER2, HER3 or HER4 receptors; cell adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α / β7 integrin and α / β3 integrin (including their α or β components) (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF-A, VEGF-C; tissue factor (TF); alpha interferon (αIFN);; TNFα, interleukins such as IL-1β, IL-3, IL-4, IL-5, IL-8, IL-9, IL-13, IL17A / F, IL-18, IL-13Rα1, IL13Rα2, IL-4R, IL-5R, IL-9R, IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; RANKL, RANK, RSV F protein, C protein, etc.

[0351] In one embodiment, the heteromeric 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 following: 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) p75NTR, and 12) caspase-6.

[0352] In one embodiment, the heteromeric protein of the invention binds to at least two target molecules selected from the following: IL-1α and IL-1β, 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-1β; IL-13 and IL-2; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and Tg-β; IL-13 and LHR agonist; IL-12 and TWEAK, IL-13 and CL25; IL-13 and SPRR2a; 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, TNFα and TGF-β, TNFα and IL-1β; TNFα and IL-2, TNFα and IL-3, TNFα and IL-4, TNFα and IL-5, TNFα and IL6, TNFα and IL8, TNFα and IL-9, TNFα and IL-10, TNFα and IL-11, TNFα and IL-12, TNFα and IL-13, TNFα and IL-14, TNFα and IL-15, TNFα and IL-16, TNFα and IL-17, TNFα and IL-18, TNFα and IL-19, TNFα and IL-2, TNFα and IL-2, TNFα and IFNα, TNFα and CD4, TNFα and VEGF, TNFα and MIF, TNFα and ICAM-1, TNFα and PGE4, TNFα and PEG2, TNFα and RANK ligand, TNFα and Te38; TNFα and BAFF; TNFα and CD22; TNFα and CTLA-4; TNFα and GP130; TNFα and IL-12p40; VEGF and HER2, VEGF-A and HER2, VEGF-A and PD, 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 receptors, 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-6 and TNFR1 and IL-18R, EPCAM and CD3, MAP and CD28, EGFR and CD64, CSPG and RGMA; CTLA-4 and BTNO2; IGF1 and IGF2; IGF1 / 2 and Erb2B; MAG and RGMA; ng and RGMA; NogoA and RGMA; OMGp and RGMA; PDL-I and CTLA-4; and RGMA and RGMB.

[0353] Its soluble antigen or fragment, optionally conjugated to other molecules, can be used as an immunogen for generating antibodies. For transmembrane molecules such as receptors, fragments of these molecules (e.g., the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing the transmembrane molecule can be used as immunogens. Such cells can be derived from natural sources (e.g., cancer cell lines) or can be cells that have been transformed by recombinant techniques to express the transmembrane molecule. Other antigens and their forms for use in preparing antibodies will be apparent to those skilled in the art.

[0354] VII. Activity Assays

[0355] The heteromultimeric proteins of the present invention can be characterized for their physical / chemical properties and biological functions by various assays known in the art.

[0356] The purified heteromultimeric proteins can be further characterized by a series 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.

[0357] In certain embodiments of the present invention, the immunoglobulins generated 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 that can be used herein include but are not limited to any direct binding assay or competitive binding assay using a variety of techniques such as western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescence immunoassay, and protein A immunoassay. Schematic antigen binding assays are provided in the Examples section below.

[0358] In one embodiment, the present invention contemplates antibodies having altered effector functions, some but not all, which renders the antibodies favorable candidates for many applications where the in vivo half-life of the antibody is important while certain effector functions, such as complement and ADCC, are unnecessary or harmful. In certain embodiments, the Fc activity of the resulting 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 reduced / depleted CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the heteromultimeric protein lacks FcR binding (and thus likely lacks ADCC activity), but retains FcRn binding ability. Primary cells - NK cells that mediate ADCC express only FcRIII, while monocytes express FcRI, FcRII, and FcRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a target molecule can be evaluated in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. To evaluate 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 can also be determined using methods known in the art.

[0359] VIII. Conjugated Proteins

[0360] The present invention also provides conjugated proteins such as conjugated antibodies or immunoconjugates (e.g., "antibody-drug conjugates" or "ADCs"), including any of the heteromultimeric proteins described herein (e.g., antibodies produced according to the methods described herein), wherein one of the constant regions of the light or heavy chain is conjugated to a chemical molecule such as a dye or a cytotoxic drug such as a chemotherapeutic agent, a drug, a growth inhibitor, 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). Specifically, as described herein, the use of heteromultimerization domains enables the construction of antibodies containing two different heavy chains (HC1 and HC2) and two different light chains (LC1 and LC2). Immunoconjugates constructed using the methods described herein can contain a cytotoxic drug conjugated to the constant region of only one heavy chain (HC1 or HC2) or only one light chain (LC1 or LC2). Additionally, because an immunoconjugate can have a cytotoxic drug linked to only one heavy or light chain, the amount of cytotoxic drug being administered to a subject is reduced relative to administering an antibody having a cytotoxic drug linked to two heavy or light chains. Reducing the amount of cytotoxic drug being administered to a subject limits the adverse side effects associated with the cytotoxic drug.

[0361] Local delivery of cytotoxic or cytostatic drugs using antibody-drug conjugates (i.e., drugs that kill or inhibit tumor cells when treating cancer (Syrigos and Epenetos, Anticancer Research 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Patent No. 4,975,278)) allows for the directed delivery of the drug moiety to the tumor and its intracellular accumulation therein, where systemic administration of these unconjugated drugs can lead to unacceptable levels of toxicity for both normal cells and the tumor cells sought to be eliminated (Baldwin et al., Lancet (Mar. 15, 1986): 603-605 (1986); Thorpe, (1985) “Antibody Carriers OfCytotoxic 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 is sought with minimal toxicity. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21:183-187 (1986)). Drugs used in these methods include daunorubicin, 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, small molecule toxins such as 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 (EP 1391213; 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 can exert their cytotoxic and cytostatic effects through multiple mechanisms, including tubulin binding, DNA binding, or topoisomerase inhibition. When conjugated to large antibodies or protein receptor ligands, some cytotoxic drugs tend to be inactive or less active.

[0362] Chemotherapeutic agents useful for generating immunoconjugates are described herein (e.g., supra). Enzymatically active toxins or fragments thereof that can be used include diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, Dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor protein, curcin, crotin, sapaonaria officinalis inhibitor protein, gelonin, mitogellin, enomycin, phenomycin, and trichothecenes. See, e.g., WO 93 / 21232, published Oct. 28, 1993. A variety of radionuclides can be used to generate radio-conjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. Conjugates of antibodies and cytotoxic drugs are produced using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidates (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azides (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium salt derivatives (such as bis-(p-diazonium salt benzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyl-diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See, e.g., WO 94 / 11026.

[0363] Also contemplated herein are conjugates of antibodies and one or more small molecule toxins such as calicheamicin, maytansine, dolastatin, aurostatin, trichothecene, and CC1065, and toxin-active derivatives of these toxins. Details of such small molecule toxins are provided in WO 2008 / 021290.

[0364] i. Maytansine and maytansinoids

[0365] In some embodiments, the immunoconjugate comprises an antibody (full-length or fragment) of the invention conjugated to one or more maytansinoid molecules.

[0366] Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was found that certain microorganisms also produce maytansinoids, such as maytanvaline and C-3 maytanvaline ester (U.S. Patent No. 4,151,042). Synthetic maytanvaline and its derivatives and analogs are disclosed, for example, in U.S. Patent 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; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0367] Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they: (i) are relatively easy to prepare by fermentation or chemical modification of, and derivatization of, fermentation products, (ii) are suitable for derivatization with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) are stable in plasma, and (iv) are effective against multiple tumor cell lines.

[0368] Immunoconjugates containing maytansinoids, methods for producing the former, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent EP 0 425 235 B1, the disclosures of which are hereby expressly incorporated by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described an immunoconjugate containing a maytansinoid named DM1, which was linked to the monoclonal antibody C242 against human colorectal cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and showed anti-tumor activity in in vivo tumor growth assays. Chari et al., Cancer Research 52:127-131 (1992) described immunoconjugates in which maytansinoids were conjugated to the murine antibody A7 that binds to an antigen on a human colon cancer cell line, or conjugated to another murine monoclonal antibody TA.1 that binds to the HER-2 / neu oncogene. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in the human breast cancer cell line SK-BR-3 that expresses 3 x 105 HER-2 surface antigens per cell in vitro. The drug conjugate achieved a cytotoxicity level similar to that of the free maytansinoid drug, and the cytotoxicity level could be increased by increasing the number of maytansinoid molecules / antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

[0369] Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly impairing the biological activities of the antibody or the maytansinoid molecule. See, for example, U.S. Patent No. 5,208,020 (the disclosure of which is hereby expressly incorporated by reference). It has been shown that 3-4 maytansinoid molecules conjugated per antibody molecule on average effectively enhance the cytotoxicity to target cells without adversely affecting the function or solubility of the antibody, although even one mole of toxin / antibody was expected to enhance cytotoxicity relative to the use of the naked 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 U.S. Patent No. 5,208,020 and in the other patents and non-patent publications mentioned above. Preferred maytansinoids are maytanol and maytanol analogs modified in the aromatic ring or at other positions in the maytanol molecule, such as various maytanol esters.

[0370] There are a variety of linking groups known in the art for generating antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Patent No. 5,208,020 or EP Patent 0,425,235B1; Chari et al., Cancer Research 52:127-131 (1992) and U.S. Patent Application Publication No. 2005 / 0169933, the disclosures of which are hereby expressly incorporated by reference. Antibody-maytansinoid conjugates containing the linker component SMCC can be prepared as disclosed in U.S. Patent Application Publication No. 2005 / 0169933. Linking groups include disulfide, thioether, acid-labile, photo-labile, peptidase-labile or esterase-labile groups as disclosed in the patents identified above, preferably disulfide and thioether groups. Additional linking groups are described and exemplified herein.

[0371] A variety of bifunctional protein coupling agents can be used to generate conjugates of antibodies and maytansinoids, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), iminothiolane (IT), bifunctional derivatives of imidates (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-diazobenzoyl)hexanediamine), bis-diazonium salt derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)valerate (SPP) which provide a disulfide bond.

[0372] Depending on the type of linkage, the linker can be attached to the maytansine molecule at multiple positions. For example, an ester linkage can be formed by reacting with a hydroxyl group using conventional coupling techniques. This reaction can occur at the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analog.

[0373] ii. Auristatins and dolostatins

[0374] In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated to dolastatin or a dolastatin peptide analogue and derivative, auristatin (U.S. Patent Nos. 5,635,483 and 5,780,588). Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and cytokinesis (Woyke et al., Antimicrob. Agents and Chemother. 45(12):3580 - 3584 (2001)) and have anticancer activity (U.S. Patent 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 linked to the antibody via the N (amino) - terminus or C (carboxyl) - terminus of the peptidic drug moiety (WO 02 / 088172).

[0375] Exemplary auristatin embodiments include the N - terminal - linked monomethyl auristatin drug moieties DE and DF of U.S. Application Publication No. 2005 / 0238649, “Monomethylvaline Compounds Capable of Conjugating to Ligands”, the disclosure of which is hereby incorporated by reference in its entirety.

[0376] Generally, peptide - based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid - phase synthesis methods well - known in the field of peptide chemistry (see E. and K. Lübke, “The Peptides”, Vol. 1, pp. 76 - 136, 1965, Academic Press). The auristatin / dolastatin drug moieties can be prepared according to the methods of U.S. Patent 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 U.S. Application Publication No. 2005 / 0238649, “Monomethylvaline Compounds Capable of Conjugating to Ligands” (which, for example, discloses linkers and methods for preparing monomethylvaline compounds conjugated to linkers such as MMA and MMA), hereby incorporated by reference in its entirety.

[0377] iii. Calicheamicin

[0378] In other embodiments, the immunoconjugate comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of causing DNA breaks at subpicomolar concentrations. For the preparation of conjugates of the calicheamicin family, 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 assigned to American Cyanamid Company). Structurally similar analogs of calicheamicin that can be used include, but are not limited to, γ1I, α2I, α3I, N-acetyl-γ1I, PSAG, and θ1I (Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998); and the aforementioned U.S. applications assigned to American Cyanamid). Another anti-tumor drug that can be conjugated to an antibody is QFA, an antifolate. Calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, the uptake of these drugs by cells via antibody-mediated internalization greatly enhances their cytotoxic effects.

[0379] iv. Other cytotoxic drugs

[0380] Other anti-tumor drugs that can be conjugated to the antibodies of the invention or antibodies produced by the methods of the invention include BCNU, streptozocin, vincristine, and 5-fluorouracil, the family of drugs collectively referred to as the LL-E33288 complex described in U.S. Patent Nos. 5,053,394 and 5,770,710, and esperamicin (U.S. Patent No. 5,877,296).

[0381] Enzymatically active toxins or fragments thereof that can be used include diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, restrictocin, trichosanthin, pokeweed antiviral protein (PAPI, PAPII, and PAP-S), momordin, curcin, crotin, saporin, gelonin, enomycin, and trichothecenes (see, e.g., WO93 / 21232, published October 28, 1993).

[0382] The present invention further contemplates forming a single immunoconjugate between a polypeptide and a compound having nucleolytic activity (e.g., ribonuclease or DNA endonuclease such as deoxyribonuclease; DNase).

[0383] For selective destruction of tumors, the antibody can comprise highly radioactive atoms. A variety of radioisotopes can be used to produce radiolabeled antibodies. Examples include radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu. When the conjugate is used for detection, it can comprise radioactive atoms for scintigraphic studies, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also referred to as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0384] The radiolabel or other label can be incorporated into the conjugate in a known manner. For example, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors (including, for example, fluorine-19 which replaces hydrogen). Labels such as tc99m or I123, Re186, Re188, and In111 can be attached via cysteine residues in the peptide. Yttrium-90 can be attached via lysine residues. 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).

[0385] A variety of bifunctional protein conjugating agents can be used to produce conjugates of antibodies and cytotoxic drugs, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-diazobenzoyl)hexanediamine), bis-diazotized derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyl diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides with antibodies. See, e.g., WO 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic agent in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers, or linkers containing disulfide bonds can be used (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0386] The compounds of the present invention are explicitly contemplated, but not limited to, ADCs prepared with the following crosslinking agent reagents: 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 commercially available SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, Illinois, USA). See the 2003-2004 Handbook and Catalog, pages 467-498.

[0387] v. Preparation of Conjugated Antibodies

[0388] In the conjugated antibodies of the present invention, the antibody is conjugated to one or more moieties (e.g., drug moieties) (e.g., from about 1 to about 20 moieties / antibody), optionally via a linker. Conjugated antibodies can be prepared by several routes using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of 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) reaction of a nucleophilic group of a moiety with a bivalent linker reagent via a covalent bond, followed by reaction with a nucleophilic group of the antibody. Additional methods for preparing conjugated antibodies are described herein.

[0389] The linker reagent can consist of one or more linker components. Exemplary linker components include 6-maleimidohexanoyl (“MC”), maleimidopropionyl (“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 components are known in the art and some are described herein. See also the N-terminal-linked monomethyl auristatin drug moieties DE and DF of U.S. Application Publication No. 2005 / 0238649 “Monomethyl valine compounds capable of conjugating to ligands”, the contents of which are hereby incorporated by reference in their entirety.

[0390] In some embodiments, the linker can comprise amino acid residues. Exemplary amino acid linker components 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). The amino acid residues that make up the amino acid linker components include those that are naturally occurring as well as rare amino acids and non-naturally occurring amino acid analogs such as citrulline. Amino acid linker components can be designed and optimized in terms of their selectivity for enzymatic cleavage by specific enzymes (e.g., tumor-associated proteases, cathepsin B, C, and D, or plasmin proteases).

[0391] Nucleophilic groups on the antibody include, but are not limited to: (i) the N-terminal amino group, (ii) side-chain amino groups, such as lysine, (iii) side-chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups in the case where the antibody is glycosylated. Amines, thiols, and hydroxyl groups are nucleophilic and, through sulfide exchange, are capable of reacting with electrophilic groups on the linker moiety and linker reagent to form covalent bonds. The electrophilic groups include: (i) active esters such as NHS esters, HOBt esters, haloformates, and acyl halides; (ii) alkyl halides and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl groups, and maleimide groups. Certain antibodies have reducible interchain disulfide bonds, i.e., cysteine bridges. The antibody can be made reactive towards conjugation to the linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody by reacting lysine with 2-iminothiolane (Traut reagent), resulting in the conversion of the 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-natural cysteine amino acid residues).

[0392] The conjugated antibodies of the present invention can also be produced by modifying the antibody to introduce an electrophilic moiety that can react with a nucleophilic substituent on the linker reagent, drug, or other moiety. The sugar of the glycosylated antibody can be oxidized, e.g., with a periodate oxidizing reagent, to form an aldehyde or ketone group that can react with the amino group of the linker reagent, drug, or other moiety. The resulting imine Schiff base group can form a stable bond or can be reduced, e.g., by a borohydride reagent, to form a stable amine bond. In one embodiment, the reaction of the sugar moiety of the glycosylated antibody with galactose oxidase or sodium metaperiodate can generate carbonyl groups (aldehyde and ketone groups) in the protein that can react with appropriate groups on the drug or other moiety (Hermanson, Bioconjugate Techniques). In another embodiment, a protein containing an N-terminal serine or threonine residue can react with sodium metaperiodate, resulting in the generation of an aldehyde, replacing the first amino acid (Geoghegan and Stroh, Bioconjugate Chem. 3:138-146 (1992); U.S. Patent No. 5,362,852). Such an aldehyde can react with the drug moiety or linker nucleophile.

[0393] Similarly, the nucleophilic groups on the moiety (such as the drug moiety) include, but are not limited to: amino group, mercapto group, hydroxyl group, hydrazide group, oxime group, hydrazine group, thiosemicarbazone group, carboxylated hydrazide group and aromatic hydrazide group, and the aforementioned groups can react to form covalent bonds with the electrophilic groups on the linker moiety and the linker reagent, and the electrophilic groups include: (i) active esters such as NHS ester, HOBt ester, haloformate and acyl halide; (ii) alkyl and benzyl halides such as haloacetamide; and (iii) aldehyde, ketone, carboxyl and maleimide groups.

[0394] Optionally, for example, by recombinant techniques or peptide synthesis, a fusion protein comprising an antibody and a cytotoxic agent can be produced. The length of the DNA can comprise respective regions encoding the two moieties of the conjugate, and the two moieties are adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate. In yet another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for tumor pretargeting, wherein the antibody-receptor conjugate is administered to an individual, and then the unbound conjugate is removed from the circulation using a scavenger and then a "ligand" (such as avidin) conjugated to a cytotoxic drug (such as a radioactive nucleotide) is administered.

[0395] IX. Utility

[0396] The method of the present invention provided herein has industrial applicability for producing heteromultimeric proteins. The method of the present invention reduces the workload involved in two independent fermentations and separations, and also reduces the technical problems inherent in two independent fermentations. Additionally, eliminating the renaturation step and the redox step of the previous method procedures can increase the yield and reduce the processing complexity and cost.

[0397] The heteromultimeric proteins described herein are, for example, used in in vitro, ex vivo and in vivo therapeutic methods. The present invention provides a variety of methods based on the use of one or more of these molecules. In certain pathological conditions, it is desirable and / or necessary to utilize heteromultimeric proteins, for example, multispecific antibodies. The present invention provides these heteromultimeric proteins, and the heteromultimeric proteins can be used for various purposes, such as therapeutic agents, prophylactic agents and diagnostic agents. For example, the present invention provides a method for treating a disease, the method comprising administering a heteromultimeric protein of the present invention to a subject in need of treatment, thereby treating the disease. Any heteromultimeric protein of the present invention described herein can be used in the therapeutic (or prophylactic or diagnostic) methods described herein.

[0398] For example, when the heteromultimeric protein is multivalent, a valuable benefit is the enhanced affinity they confer for their antigens. In addition to having an intrinsically high affinity for the antigen on the binding unit (i.e., Fab), normal IgG antibodies also utilize the avidity effect to increase their association with the antigen because they bind bivalently to the target.

[0399] Heteromeric proteins directed against two independent epitopes on the same antigen molecule can not only provide the benefit of enhanced binding affinity (due to bivalent binding), but also acquire new properties that do not associate with either of the parental antibodies. Thus, the heteromeric proteins of the invention are useful, for example, for blocking receptor-ligand interactions.

[0400] The heteromeric proteins described herein are also useful in applications where the signaling pathways of two targets are blocked simultaneously by one molecule.

[0401] X. Therapeutic uses

[0402] The heteromeric proteins described herein, such as antibodies and antibody fragments (e.g., antibodies and / or their fragments produced according to the methods of the invention), can be used in therapeutic applications. For example, such heteromeric proteins can be used to treat tumors, including pre-cancerous, non-metastatic, metastatic, and cancerous tumors (e.g., early stage cancers), to treat allergic or inflammatory disorders, or to treat autoimmune diseases, or to treat subjects at risk of developing cancers (e.g., breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma, or ovarian cancer), allergic or inflammatory disorders, or autoimmune diseases.

[0403] The term cancer encompasses the general category of proliferative disorders, including but not limited to pre-cancerous growths, benign tumors, and malignant tumors. Benign tumors remain localized at the site of origin and do not have the ability to invade, infiltrate, or metastasize to distant sites. Malignant tumors will invade and damage other tissues around them. They also acquire the ability to break through their site of origin and spread (metastasize) to other parts of the body, which typically occurs via the bloodstream or via the lymphatic system in which lymph nodes are present. Primary tumors are classified according to the type of tissue from which the primary tumor originated; metastatic tumors are classified according to the type of tissue from which the cancer cells are derived. Over time, malignant tumor cells become more abnormal and appear less like normal cells. This change in the appearance of cancer cells is called tumor grading, and cancer cells are described as well-differentiated, moderately-differentiated, poorly-differentiated, or undifferentiated. Well-differentiated cells appear fairly normal and resemble the normal cells from which they originated. Undifferentiated cells are cells that have become so abnormal that it is no longer possible to determine the origin of the cells.

[0404] Tumors can be solid tumors or non-solid or soft tissue tumors. Examples of soft tissue tumors include leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, prolymphocytic leukemia, or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin lymphoma, cutaneous T-cell lymphoma or Hodgkin disease). Solid tumors include any cancer of body tissues other than the blood, bone marrow or lymphatic system. Solid tumors can be further divided into those solid tumors of epithelial cell origin and those solid tumors 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, lip, nasopharynx, skin, uterus, male reproductive organs, urinary organs, bladder and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors and bone tumors.

[0405] Epithelial cancers typically evolve from a benign tumor to a pre-invasive stage (e.g., carcinoma in situ), to a malignant cancer that has penetrated the basement membrane and invaded the subepithelial stroma.

[0406] Multispecific protein complexes can also be used in these therapeutic applications, and antibodies that bind HER2 can in particular be used to treat breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma or ovarian cancer.

[0407] Other subjects who are candidates for receiving the compositions of the present invention suffer from the following conditions or are at risk of developing the following conditions: abnormal proliferation of fibrovascular tissue, rosacea acne, acquired immunodeficiency syndrome, arterial occlusion, keratitis ectropica, bacterial ulcer, Bechet's disease, hematogenous tumors, carotid obstructive disease, choroidal neovascularization, chronic inflammation, chronic retinal detachment, chronic uveitis, chronic vitritis, overwear of contact lenses, corneal transplant rejection, corneal neovascularization, corneal graft neovascularization, Crohn's disease, Eale's disease, epidemic keratoconjunctivitis, fungal ulcer, herpes simplex infection, herpes zoster infection, hyperviscosity syndrome, Kaposi's sarcoma, leukemia, lipid degeneration, Lyme disease, marginal keratolysis, Mooren's ulcer, mycobacterial infections other than leprosy, myopia, ocular neovascular diseases, optic pit, Osler-Weber syndrome (Osler-Weber-Rendu), osteoarthritis, Paget's disease, pars planitis, pemphigoid, vesicular diseases, polyarteritis, post-laser complications, protozoal infections, pseudoxanthoma elasticum, pterygium, xerophthalmia, radial keratotomy, retinal neovascularization, retinopathy of prematurity, retrolental fibroplasia, sarcoid, scleritis, sickle cell anemia, Sjögren's syndrome, solid tumors, Stargardt's disease, Steven-Johnson disease, superior limbic keratitis, syphilis, systemic lupus, Terrien's marginal degeneration of the cornea, toxoplasmosis, tumors of Ewing's sarcoma, tumors of neuroblastoma, tumors of osteosarcoma, tumors of retinoblastoma, tumors of rhabdomyosarcoma, ulcerative colitis, venous occlusion, vitamin A deficiency, Wegener's granulomatosis, angiogenesis adverse to diabetes, parasitic diseases, abnormal wound healing, postoperative hypertrophy, 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 embryogenesis.

[0408] 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 such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, hypertrophic arthritis, psoriatic arthritis, osteoarthritis, and juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, degenerative arthritis, chronic primary polyarthritis, reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis, dermatitis, including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, X-linked IgM excess syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spinal-optic MS, primary progressive MS (PPMS) and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis and ataxic sclerosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal disease, colitis such as ulcerative colitis, ulcerative colitis (colitisulcerosa), microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis, and transmural colitis associated with autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematologic diseases, rheumatoid spondylitis, sudden sensorineural hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen encephalitis and marginal and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens antigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome, such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous (membrano) or membranoproliferative GN (MPGN), including type I and type II, and rapidly progressive GN, atopy, allergy, eczema, including allergic or idiopathic eczema, asthma, such as bronchial asthma, bronchial asthma and autoimmune asthma, conditions involving T cell infiltration and chronic inflammatory responses, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupuserythematodes) such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritis, encephalitis, pediatric, non-renal, extra-renal, discoid, alopecic lupus), juvenile-onset (type I) diabetes, including pediatric insulin-dependent diabetes mellitus (IDDM), adult-onset diabetes (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses related to cytokine- and T-lymphocyte-mediated acute and delayed-type hypersensitivity reactions, tuberculosis, sarcoidosis, granulomatosis, including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis (including large-vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous or hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, congenital aplastic anemia, hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or dysplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, CNS inflammatory disorders, multiple organ injury syndromes, such as those secondary to sepsis, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet or Behcet's disease, Castleman syndrome, Goodpasture syndrome, Reynaud syndrome, Sjogren syndrome, Stevens-Johnson syndrome, pemphigoid, such as bullous pemphigoid and cutaneous pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membranepemphigoid) and pemphigus erythematosus), autoimmune polyendocrinopathy, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathy, chronic neuropathies such as IgM polyneuropathy or IgM-mediated neuropathy, thrombocytopenia (such as developed in patients with myocardial infarction), including thrombotic thrombocytopenic purpura (TTP), and autoimmune or immune-mediated thrombocytopenia, such as idiopathic thrombocytopenic purpura (ITP) (including chronic or acute ITP), autoimmune diseases of the testis and ovary, including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases (including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid diseases, idiopathic hypothyroidism, Grave's disease, polyendocrine syndrome, such as autoimmune polyendocrine syndrome (or polyendocrine endocrinopathy syndrome), paraneoplastic syndromes, including neurological paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, such as allergic encephalomyelitis or experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), and sensory 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-graft) vs NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac disease, celiac disease, gluten enteropathy, refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; LouAmyotrophic lateral sclerosis (ALS), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis, which includes monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndromes, ion channel diseases such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and ion channel diseases of the central nervous system (CNS), autism, inflammatory myopathies, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, choroidoretinitis, autoimmune hepatological disorders, fibromyalgia, multiple endocrine failure, Schmidt syndrome, adrenalitis, gastric atrophy, Alzheimer's disease, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport syndrome, alveolitis such as hypersensitivity alveolitis and fibrotic alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, Chagas disease, schistosomiasis, ascariasis, aspergillosis, Sampter syndrome, Caplan syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, fetal erythroblastosis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis such as chronic cyclitis, heterochronic cyclitiscyclitis), iridocyclitis or Fuch's cyclitis, Henoch-Schönlein 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, Evan syndrome, autoimmune gonadal failure, Sydenham chorea, post-streptococcal glomerulonephritis, thromboangiitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, endocrine 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, aphthae, aphthous stomatitis, atherosclerotic disease, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, allergic enteritis, lepra nodosa, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich disease, sensorineural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, regional enteritis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxoedema, nephrosis, ophthalmia symphatica, granulomatous orchitis, pancreatitis, acute polyneuritis, pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy (acquired spenicatrophy), infertility due to anti-sperm antibodies, non-malignant thymic tumors, vitiligo, SCID and Epstein-Barr virus-related diseases, acquired immunodeficiency syndrome (AIDS), parasitic diseases such as Leishmania, toxic shock syndrome, food poisoning, conditions involving T cell infiltration, leukocyte adhesion deficiency, immune responses related to cytokine- and T-lymphocyte-mediated acute and delayed-type hypersensitivity, diseases involving 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, multiple endocrine failure, peripheral neuropathy, autoimmune polyendocrine syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, acquired epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoiditis, frontal sinusitis, maxillary sinusitis or sphenoid sinusitis, eosinophil-related conditions such as eosinophilia, eosinophilic pulmonary infiltration, eosinophilia-myalgia syndrome, Löffler syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma, or aspergilloma containing eosinophils, allergic reactions, seronegative spondyloarthritis, polyendocrine autoimmunity, sclerosing cholangitis, scleral candidiasis, episcleral candidiasis, chronic mucocutaneous candidiasis, Bruton syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune dysfunction associated with collagen diseases, rheumatism, neurological diseases, ischemic reperfusion disease, hypotensive responses, vascular dysfunction, angiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and diseases associated with vascularization, allergic hypersensitivity disorder, glomerulonephritides, reperfusion injury, myocardial or other tissue reperfusion injury, skin diseases with an acute inflammatory component, acute purpuric meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-related syndromes, cytokine-induced toxicity, acute severe inflammation, chronic refractory inflammation, pyelonephritis, cirrhosis, diabetic retinopathy, diabetic macroangiopathy, intimal hyperplasia, peptic ulcer, valvulitis and endometriosis.

[0409] In addition to therapeutic uses, the antibodies of the invention may be used for other purposes, including diagnostic methods, such as for the diseases and conditions described herein.

[0410] XI. Medication, preparation, and duration

[0411] The proteins of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual subject, the cause of the condition, 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 be governed by such considerations and will be the minimum amount necessary to prevent, ameliorate, or treat a particular condition (cancer, allergic or inflammatory disease, or autoimmune disease). The protein is not necessarily, but optionally, formulated with one or more drugs currently used to prevent or treat the condition, the effective amount of such other drugs depending on the amount of protein present in the formulation, the type of disease or therapy, and the other factors discussed above. These drugs are generally used in the same dosage and by the same route of administration as previously used or at about 1% to 99% of the dosage previously used. Generally, alleviating or treating cancer includes attenuating one or more symptoms associated with the cancer. Symptoms or medical problems. The therapeutically effective amount of the drug can achieve one or a combination of the following: reduce the number of cancer cells (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 to a certain extent and / or stop) cancer cells from infiltrating peripheral organs; reduce hormone secretion in the case of adenoma; reduce blood vessel density; inhibit tumor metastasis; reduce or inhibit tumor growth; and / or alleviate one or more symptoms associated with cancer to a certain extent. In some embodiments, the protein is used to prevent the appearance or recurrence of cancer or autoimmune disease in a subject.

[0412] In one embodiment, the present invention can be used to increase the survival duration of human subjects susceptible to or diagnosed with cancer or autoimmune diseases. The survival duration is defined as the time from the first administration of the drug to death. The survival duration can also be measured by the stratified hazard ratio (HR) of the treatment group relative to the control group, and the stratified hazard ratio represents the risk of death of the subject during treatment.

[0413] In yet another embodiment, the treatment of the invention significantly increases the remission rate in a population of human subjects predisposed to or diagnosed with cancer that has been treated with multiple anti-cancer therapies. The remission rate is defined as the percentage of subjects who receive treatment and respond to the therapy. In one aspect, compared to a population treated with single surgery, radiotherapy, or chemotherapy, the combination treatment of the invention using the proteins of the invention and surgery, radiotherapy, or one or more chemotherapeutic agents significantly increases the remission rate in the group of treated subjects, with a chi-square p-value of less than 0.005. Additional measures of the therapeutic efficacy of treating cancer are described in U.S. Patent Application Publication No. 20050186208.

[0414] In certain embodiments, a composition is provided that comprises a heteromultimeric protein produced by any of the methods described herein and a pharmaceutically acceptable carrier. Therapeutic formulations are prepared by mixing an active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers using standard methods known in the art (Remington’s Pharmaceutical Sciences (20th 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 sugars, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN, PLURONICS, or PEG.

[0415] Optionally, but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably at about physiological concentration. Optionally, the formulations of the invention may contain a pharmaceutically acceptable preservative. In some embodiments, the preservative concentration is from 0.1 to 2.0%, generally v / v. Suitable preservatives include those known in the pharmaceutical art. Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben are preferred preservatives. Optionally, the formulations of the invention may contain a pharmaceutically acceptable surfactant at a concentration of from 0.005% to 0.02%.

[0416] The formulations herein may also contain more than one active compound, preferably those having complementary activities that do not adversely interact, depending on the particular indication being treated. Such molecules are present in amounts effective for the intended purpose.

[0417] The active ingredient can also be entrapped in, for example, microcapsules prepared by coacervation techniques or interfacial polymerization respectively (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methylmethacrylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences supra.

[0418] Sustained release articles can be prepared. Suitable examples of sustained release articles include solid hydrophobic polymer semipermeable matrices containing heterologous proteins, said matrices being in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), poly(lactic acid) (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for a shorter period of time. When encapsulated heterologous proteins remain in the body for a long time, they may denature or aggregate due to exposure to moisture at 37 °C, resulting in loss of biological activity and possible alteration of immunogenicity. Rational strategies for stabilization can be conceived according to the mechanisms involved. For example, if the aggregation mechanism is due to intermolecular S-S bonds resulting from thiol-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling the moisture content, using suitable additives and developing specific polymer matrix compositions.

[0419] According to known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, the proteins described herein (e.g., heteromultimeric proteins such as multispecific antibodies produced according to the methods described herein) are administered to a subject, e.g., a human subject, by intramuscular, intraperitoneal, intrathecal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Local administration may be particularly desirable if extensive side effects or toxicity are associated with antagonizing the target molecule recognized by the protein. Ex vivo strategies can also be used for therapeutic applications. Ex vivo strategies include 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 can be any of a wide variety of cell 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.

[0420] In one example, when the condition or tumor location permits, the protein complex (e.g., heteromultimeric proteins such as multispecific antibodies produced according to the methods described herein) is administered locally, e.g., by direct injection, and the injection can be repeated periodically. The protein complex can be delivered systemically to the subject or directly to the tumor cells, e.g., to the tumor or tumor bed after surgical removal of the tumor, with the aim of preventing or reducing local recurrence or metastasis.

[0421] XII. Manufacture

[0422] Another embodiment of the invention is a manufacture, which contains one or more heteromultimeric proteins described herein and materials useful for treating or diagnosing a condition (e.g., an autoimmune disease or cancer). The manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition effective for treating the condition and can have a sterile access port (e.g., the container can be an intravenous infusion bag or a vial with a stopper penetrable by a subcutaneous injection needle). At least one active substance 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 for treating a specific condition. The label or package insert will further contain instructions for administering the heteromultimeric protein to a subject. Manufactures and kits containing the combinatorial therapeutics described herein are also contemplated.

[0423] A package insert refers to the instructions habitually included in the commercial packaging of a therapeutic product, which contains information on indications, usage, dosage, administration, contraindications, and / or warnings related to the use of such a therapeutic product. In certain embodiments, the package insert states that the composition is for the treatment of breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma, or ovarian cancer.

[0424] Additionally, the manufactured article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials recognized from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0425] Kits are also provided that can be used for a variety of purposes (e.g., purifying cells or immunoprecipitating antigens). For separating and purifying an antigen, the kit may contain a heteromultimeric protein conjugated to beads (e.g., agarose beads). Kits containing heteromultimeric proteins for detecting and quantifying an antigen in vitro (e.g., in ELISA or Western blotting) may be provided. Like the manufactured article, the kit includes a container and a label or package insert on or associated with the container. The container houses a composition comprising at least one heteromultimeric protein of the invention (e.g., a multispecific antibody fragment or an antibody fragment). Additional containers containing, for example, diluents and buffers or control antibodies may be included. The label or package insert may provide a description of the composition and instructions for the intended in vitro or diagnostic use.

[0426] It is believed that the foregoing written description is sufficient to enable one of ordinary skill 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 way. Indeed, various modifications of the invention will be apparent to those of ordinary skill in the art from the foregoing description and fall within the scope of the appended claims.

[0427] In the following experimental disclosures, the following abbreviations apply: eq (equivalent); M (mole); μM (micromole); N (normal); mol (mole); mmol (millimole); μmol (micromole); nmol (nanomole); g (gram); mg (milligram); kg (kilogram); μg (microgram); L (liter); ml (milliliter); μl (microliter); cm (centimeter); mm (millimeter); μm (micrometer); nm (nanometer); °C (degree Celsius); hr (hour); min (minute); sec (second); msec (millisecond); ADCC (antibody-dependent cell cytotoxicity); BsAb (bispecific antibody); CL (constant light chain domain); CH (constant heavy chain domain); CMC (complement-mediated cell cytotoxicity); Fab (antigen-binding fragment); Fc (crystallizable fragment); Fv (variable fragment (VL + VH)); EGFR (epidermal growth factor receptor); HC (heavy chain); IGFR (insulin-like growth factor receptor); LC (light chain); scFv (single-chain variable fragment (VL and VH tethered by an amino acid linker)); VEGF (vascular endothelial growth factor); VEGFR2 (vascular endothelial growth factor receptor 2); V H (variable heavy chain domain); V L (variable light chain domain). Specific embodiments

[0428] 1. A method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:

[0429] (a) culturing a first host cell capable of expressing the first hinge-containing polypeptide and the first light chain;

[0430] (b) culturing a second host cell capable of expressing the second hinge-containing polypeptide and the second light chain; and,

[0431] (c) obtaining a combined 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 combined culture medium contains the heteromultimeric protein, and wherein the first host cell and the second host cell are each mammalian cells.

[0432] 2. A method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps:

[0433] (a) Culturing a first host cell capable of expressing the first hinge-containing polypeptide and the first light chain, wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted;

[0434] (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;

[0435] (c) Obtaining a combined 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 combined culture medium comprises the first homodimer and the second homodimer;

[0436] (d) Incubating the combined culture medium under reducing conditions sufficient to allow the formation of the heteromultimeric protein, and;

[0437] (e) Obtaining the heteromultimeric protein, wherein the first host cell and the second host cell are each mammalian cells.

[0438] 3. The method of embodiment 1 or embodiment 2, wherein obtaining the combined culture medium comprises:

[0439] (1) Harvesting a first culture medium of the first host cell;

[0440] (2) Harvesting a second culture medium of the second host cell; and,

[0441] (3) Combining the first culture medium and the second culture medium to obtain the combined culture medium.

[0442] 4. The method of embodiment 1 or embodiment 2, wherein obtaining the combined culture medium comprises harvesting the culture medium of a combined cell culture comprising the first host cell and the second host cell.

[0443] 5. The method of embodiment 4, wherein the first host cell and the second host cell are cultured separately before being combined into the combined cell culture.

[0444] 6. The method of any one of embodiments 4-5 further comprises the step of culturing the combined cell culture at a temperature of from about 25°C to about 40°C.

[0445] 7. The method of any one of embodiments 1-6 further comprises agitating the combined culture medium.

[0446] 8. The method of any one of embodiments 1-7 further comprises separating the heteromultimeric protein from the combined culture medium.

[0447] 9. The method of embodiment 8, wherein the heteromultimeric protein is separated using a protein A column.

[0448] 10. The method of embodiment 3, comprising adding a reducing agent to the first cell culture medium and / or to the second cell culture medium before or after harvesting the first and second cell culture media.

[0449] 11. The method of any one of embodiments 4-10, comprising adding a reducing agent to the medium of the combined cell culture before harvesting the medium of the combined cell culture.

[0450] 12. The method of embodiment 10 or embodiment 11, wherein the reducing agent is added about 4 to about 24 hours before the harvesting step.

[0451] 13. The method of embodiment 12, wherein the reducing agent is added about 15 hours before the harvesting step.

[0452] 14. The method of any one of embodiments 1-13 further comprises adding a reducing agent to the combined cell culture medium.

[0453] 15. The method of embodiment 14, wherein the combined culture medium containing the reducing agent is further incubated for about 4 hours to about 7 days.

[0454] 16. The method of embodiment 15, wherein the combined culture medium containing the reducing agent is further incubated for about 15 hours.

[0455] 17. The method of embodiment 16, wherein a reducing agent is added to the combined culture medium before separating the heteromultimeric protein from the combined culture medium.

[0456] 18. The method of embodiment 17, wherein the combined culture medium containing the reducing agent is incubated for at least about 24 hours before separating the heteromultimeric protein.

[0457] 19. The method of embodiment 18, wherein the heteromultimeric protein is separated using a protein A column.

[0458] 20. A method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps:

[0459] (a) Culturing a first host cell capable of expressing a first homodimer, wherein the first homodimer comprises two first hinge-containing polypeptides and their associated light chains;

[0460] (b) Culturing a second host cell capable of expressing a second homodimer, wherein the second homodimer comprises two second hinge-containing polypeptides and their associated light chains;

[0461] (c) Obtaining a combined culture medium of the first host cell and the second host cell;

[0462] (c) Incubating the combined culture medium under reducing conditions sufficient to allow the formation of the heteromultimeric protein, and;

[0463] (d) Obtaining the heteromultimeric protein.

[0464] 21. The method according to any one of embodiments 14-20, wherein the reducing agent is selected from glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), cysteine, cysteine, dithiothreitol, cysteine dithiothreitol, dithiobutylamine or a combination thereof

[0465] 22. The method according to embodiment 21, wherein the reducing agent is glutathione, and wherein glutathione is added at a concentration of about 5 mM to no more than about 20 mM.

[0466] 23. The method according to embodiment 22, wherein the reducing agent is glutathione, and wherein glutathione is added at a concentration of about 2 mM to about 10 mM.

[0467] 24. The method according to embodiment 22, wherein the reducing agent is glutathione, and wherein glutathione is added at a concentration of at least about 5 mM to about 20 mM.

[0468] 25. The method according to embodiment 24, wherein the reducing agent is glutathione, and wherein glutathione is added at a concentration of about 15 mM.

[0469] 26. The method according to any one of embodiments 1-25, wherein the first host cell is a stable cell line.

[0470] 27. The method of any one of embodiments 1-26, wherein the second host cell is a stable cell line.

[0471] 28. The method of any one of embodiments 1-27, wherein the first host cell is a CHO cell.

[0472] 29. The method of any one of embodiments 1-28, wherein the second host cell is a CHO cell.

[0473] 30. The method of any one of embodiments 1-29, wherein the ratio of the first host cell to the second host cell is adjusted such that when the first host cell culture and the second host cell culture are combined to form a combined 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.

[0474] 31. The method of embodiment 30, wherein when the first host cell culture and the second host cell culture are combined to form a combined 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.

[0475] 32. The method of any one of embodiments 1-31, wherein the hinge-containing polypeptide comprises an Fc region or a variant thereof.

[0476] 33. The method of any one of embodiments 1-32, wherein the first and / or the second hinge-containing polypeptide comprises an antibody heavy chain.

[0477] 34. The method of any one of embodiments 1-33, wherein the first heterodimerization domain comprises a knot modification at the interface and the second heterodimerization domain comprises a pore modification at the interface.

[0478] 35. The method of embodiment 34, wherein the knot 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.

[0479] 36. The method of embodiment 35, wherein the replacing amino acid residue is selected from tryptophan, phenylalanine, tyrosine, and arginine.

[0480] 37. The method of embodiment 36, wherein the pore 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.

[0481] 38. The method of embodiment 37, wherein the replacing amino acid residue is selected from serine, threonine, valine, and alanine.

[0482] The method of any one of embodiments 34-38, wherein the knot modification comprises a T366W substitution (EU numbering).

[0483] The method of any one of embodiments 34-39, wherein the pore modification comprises two or more amino acid substitutions selected from T366S, L368A, and Y407V (EU numbering).

[0484] The method of any one of embodiments 1-40, wherein the inter-chain disulfide bond is between hinge regions.

[0485] The method of any one of embodiments 1-41, wherein the heteromultimeric protein is an antibody.

[0486] The method of any one of embodiments 1-42, wherein the heteromultimeric protein is a bispecific antibody.

[0487] The method of embodiment 43, wherein the antibody is a humanized antibody or a human antibody.

[0488] The method of embodiment 44, wherein the antibody is a full-length antibody.

[0489] The method of embodiment 45, wherein the antibody is an antibody fragment comprising at least a portion of the human C H 2 and / or C H 3 domain.

[0490] The method of any one of embodiments 42-46, wherein the antibody is selected from IgG, IgA, and IgD.

[0491] The method of embodiment 47, wherein the antibody is IgG.

[0492] The method of embodiment 48, wherein the antibody is IgG1, IgG2, or IgG4.

[0493] The method of any one of embodiments 1-49, wherein the first light chain and the second light chain comprise different variable domain sequences.

[0494] A method of preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one inter-chain disulfide bond, the method comprising the steps of:

[0495] (a) Cultivate a combined 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, wherein the second host cell is capable of expressing a second hinge-containing polypeptide and a second light chain, and wherein the first host cell and the second host cell are each mammalian cells;

[0496] (b) Add a reducing agent to the combined culture; and

[0497] (c) Harvest the combined culture medium from the combined culture without disrupting the cell membranes, wherein the combined culture medium contains a heteromultimeric protein.

[0498] 52. The method of embodiment 51, wherein the first host cell secretes a first homodimer comprising two first hinge-containing polypeptides and two first light chains, and wherein the second host cell secretes a second homodimer comprising two second hinge-containing polypeptides and two second light chains.

[0499] 53. The method of embodiment 51 or embodiment 52, wherein the combined culture medium is harvested 4 hours to 24 hours after adding the reducing agent.

[0500] 54. The method of any one of embodiments 51-53, wherein the step of harvesting the combined culture medium comprises removing the first host cell and the second host cell from the combined culture medium.

[0501] 55. The method of any one of embodiments 51-54, wherein the combined culture medium is incubated for 4 hours to 7 days.

[0502] 56. A heteromultimeric protein produced by the method of any one of embodiments 1-55.

[0503] 57. The heteromultimeric protein of embodiment 56, wherein the heteromultimeric protein is a bispecific antibody.

[0504] 58. A composition comprising the heteromultimeric protein of embodiment 56 or embodiment 57 and a pharmaceutically acceptable carrier.

[0505] 59. A host cell comprising a polynucleotide or a recombinant vector encoding a first hinge-containing polypeptide of the heteromultimeric protein of embodiment 56 or 57, wherein the host cell does not express the second hinge-containing polypeptide of the heteromultimeric protein.

[0506] 60. The host cell of embodiment 59, wherein the hinge-containing polypeptide is an antibody heavy chain.

[0507] 61. The host cell of embodiment 59 or 60, wherein the hinge-containing polypeptide pairs with an antibody light chain.

[0508] The host cell of any one of embodiments 59 - 61, wherein the host cell is a stable cell line.

[0509] The host cell of any one of embodiments 59 - 62, wherein the host cell is a mammalian cell.

[0510] The host cell of embodiments 59 - 63, wherein the host cell is a CHO cell.

[0511] Example

[0512] The present invention is described in further detail in the following examples, which are not intended to limit the scope of the present invention as claimed in any way. The accompanying drawings are intended to be regarded as an integral part of the specification and description of the present invention. All references cited are incorporated herein by reference in their entirety. The following examples are provided for illustration but not to limit the claimed invention.

[0513] Example 1: The molar ratio of the monomeric components of the bispecific antibody in the combined cell culture can be controlled by adjusting the cell:cell ratio

[0514] The following example shows the molar ratio of the knot half - antibody and the hole half - antibody when culturing two mammalian cell lines (CHO cells) that each express the knot half - antibody or the hole half - antibody in the same culture.

[0515] Production studies were conducted to determine the production titer of each cell line (i.e., the total amount of antibody produced by the cells, which includes heterodimers, homodimers, and monomeric half - antibodies). Each cell line was passaged in the seed train medium every 3 or 4 days until the production titer data for each cell line became available.

[0516] Subsequently, the two cell lines were cultured and induced to express the knot half - antibody or the hole half - antibody in separate cultures. The separate knot cell line cultures and hole cell line cultures were passaged every 3 or 4 days in a 40 mL volume in shake flasks in the seed train medium. On the day the cultures were to be combined, the cell count was measured using Vicell (Beckman Coulter).

[0517] The separate cultures were then combined at a specific knot host cell:hole host cell ratio. Based on the known production titers for each cell line, the knot host cell:hole host cell ratio was calculated. For each small - scale production, approximately 40x10 6 cells were required. Using the Vicell count (cells / mL), the volume of each cell line needed to be added to the combined culture to achieve the desired knot host cell:hole host cell ratio could be determined. The appropriate volumes of each knot cell line and hole cell line were combined in the production medium in a new shake flask at a final volume of 40 mL.

[0518] Fifteen hours before harvest, a glutathione (GSH) stock solution was prepared by dissolving GSH in 1 M arginine in 400 mM succinic acid (pH = 9.0) to a final concentration of 250 mM GSH stock solution. The GSH stock solution was added to the production culture such that the final concentration of GSH was 15 mM. The combined media was then harvested and the percent knot half-antibody and percent well half-antibody of each combined culture were measured by reverse phase under reducing conditions. The percent covalent bispecific antibody formed for each tested ratio was determined as Figure 2 described in.

[0519] These experiments were conducted with the following knot half-antibody / well half-antibody pairs:

[0520] Anti-target A (knot) / anti-target B (well)

[0521] Anti-target C (knot) / anti-target D (well)

[0522] Anti-target D (knot) / anti-target C (well)

[0523] Anti-target E (knot) / anti-target F (well)

[0524] The results of these experiments are provided in Table 2 below:

[0525]

[0526]

[0527] *N / D = Not determined

[0528] As shown in Table 2, the percent covalent bispecific antibody yield improved when the knot half-antibody:well half-antibody molar ratio was approximately or close to 1:1. The optimal molar ratio for bispecific formation may be determined for each specific bispecific antibody. The knot host cell:well host cell ratio that produces a 1:1 knot half-antibody:well half-antibody molar ratio varies with the cell line and is determined experimentally.

[0529] Example 2: Varying the time of addition of a reducing agent and the concentration of the reducing agent added in a combined cell culture during the production of a bispecific antibody

[0530] The following examples show the addition of a reducing agent during different stages of the production of a bispecific antibody comprising anti-target A (knob) and anti-target B (hole). Two mammalian cell lines expressing anti-target A (knob) or anti-target B (hole) are initially cultured and induced to express the knob or hole half-antibodies in separate cultures, which are then combined in multiple independent production cultures to achieve a 1:1 molar ratio of anti-target A (knob):anti-target B (hole), as described above. The GSH stock solution is added to the production cultures 24 hours, 15 hours, or 4 hours before harvest to a final concentration of 2 mM, 4 mM, or 10 mM, or the cultures are left untreated. The combined media are then harvested from each production culture and the % knob half-antibody and % hole half-antibody of each combined culture are measured by reverse phase under reducing conditions. The % covalent bispecific antibody formed for each tested ratio is determined as described in Figure 2 as described in.

[0531] As Figure 3 shown, the yield of covalent bispecific antibody was increased in production cultures with a final GSH concentration of 10 mM compared to the yield of bispecific antibody in production cultures with a final GSH concentration of 2 mM or 4 mM or in the untreated control group. The production stage at which GSH was added did not show an effect on the yield of covalent bispecific antibody.

[0532] In a further experiment, cell lines expressing anti-target A (knob) or anti-target B (hole) are initially cultured and induced to express the knob or hole half-antibodies in separate cultures, which are then combined in multiple independent 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. The GSH stock solution is added to the production cultures 15 hours before harvest to a final concentration of 5 mM or 10 mM, or left untreated (“0 mM”). The cell viability of each combined cell culture is measured at harvest and then the % bispecific antibody formed in each tested case is determined by ion exchange as shown in Figure 2 as shown in. The experimental results are shown in Table 3 below.

[0533] Table 3

[0534]

[0535] *Titer refers to the total amount of antibody produced by the two cell lines in the combined culture, e.g., including homodimers, heterodimers, and monomeric half-antibodies.

[0536] As shown in Chart 3, the yield of the covalent bispecific antibody was increased in the production culture with a final GSH concentration of 10 mM as compared to the production culture or untreated control with a final GSH concentration of 5 mM. It was found that adding GSH to the combined cell culture up to a final concentration of 10 mM did not affect cell viability or the total antibody production. No unwanted mixed disulfide bond formation or protein scrambling was observed at 10 mM GSH (data not shown).

[0537] Similar experiments were performed with two other mammalian cell lines each expressing anti-target D (nodule) or anti-target C (hole). These two cell lines were initially cultured and induced to express anti-target D (nodule) or anti-target C (hole) in separate cultures, which were then combined in multiple independent production cultures to achieve a molar ratio of anti-target D (nodule):anti-target C (hole) of 0.82:1 as described above. The GSH stock solution was added to the production culture 15 hours before harvest to a final concentration of 10 mM, 15 mM or 20 mM, or left untreated ("0 mM"). Cell viability of each combined cell culture was determined at harvest, and then the percentage of bispecific antibody formed in each tested case was determined as shown in Figure 2 The results of these experiments are shown in Table 4 below.

[0538] Table 4

[0539]

[0540] *Titer refers to the total amount of antibody produced by the two cell lines in the combined culture, e.g., including homodimers, heterodimers and monomeric half-antibodies captured by the protein A column.

[0541] As shown in Chart 4, the yield of the covalent bispecific antibody was increased in the production culture with a final GSH concentration of 20 mM as compared to the production culture or untreated control with a final GSH concentration of 10 mM or 15 mM. It was found that adding GSH to the combined cell culture up to a final concentration of 20 mM did not affect the titer. However, when GSH was added to the production culture to a final concentration of 20 mM, covalent modification of the half-antibody by GSH was observed (data not shown).

[0542] Example 3: Yield of bispecific antibody formed in combined medium compared to in vitro assembly

[0543] Additional experiments were performed using anti-target E (hinge) and anti-target F (aperture) to compare the yield of bispecific antibodies obtained from co-cultures 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 in vitro co-incubation of purified anti-target E (hinge) and purified anti-target F (aperture) using a protein A column. Briefly, two mammalian cell lines each expressing anti-target E (hinge) or anti-target F (aperture) were initially cultured and induced to express the hinge or aperture half-antibodies in separate cultures. The separate cultures were then combined and cultured for an additional length of time. The combined culture medium was harvested and the percent bispecific antibody formed was then determined by cation exchange assay as shown in Figure 2 . Meanwhile, specific antibodies were formed by in vitro co-incubation of purified anti-target E (hinge) and anti-target F (aperture) (see, e.g., WO 2013 / 055958). The final yields of bispecific antibodies formed under both conditions were comparable (data not shown).

[0544] Example 4: Bispecific Generation

[0545] Additional experiments were performed using anti-target G ...

Claims

1. A method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps: (a) Culturing a first host cell capable of expressing the first hinge-containing polypeptide and the first light chain; (b) Culturing a second host cell capable of expressing the second hinge-containing polypeptide and the second light chain; and, (c) Obtaining a combined 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 combined culture medium contains the heteromultimeric protein, and wherein the first host cell and the second host cell are each mammalian cells.

2. A method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps: (a) Culturing a first host cell capable of expressing the first hinge-containing polypeptide and the first light chain, wherein a first homodimer comprising two first hinge-containing polypeptides and two first light chains is secreted; (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; (c) Obtaining a combined 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 combined culture medium contains the first homodimer and the second homodimer; (d) Incubating the combined culture medium under reducing conditions sufficient to allow the formation of the heteromultimeric protein, and; (e) Obtaining the heteromultimeric protein, wherein the first host cell and the second host cell are each mammalian cells.

3. A method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps: (a) Culturing a first host cell capable of expressing a first homodimer, wherein the first homodimer comprises two first hinge-containing polypeptides and their associated light chains; (b) Culturing a second host cell capable of expressing a second homodimer, wherein the second homodimer comprises two second hinge-containing polypeptides and their associated light chains; (c) Obtaining a combined culture medium of the first host cell and the second host cell; (c) Incubating the combined culture medium under reducing conditions sufficient to allow the formation of the heteromultimeric protein, and; (d) Obtaining the heteromultimeric protein.

4. A method for preparing a heteromultimeric protein, the heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps: (a) Culturing a combined 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, wherein the second host cell is capable of expressing a second hinge-containing polypeptide and a second light chain, and wherein the first host cell and the second host cell are each mammalian cells; (b) Adding a reducing agent to the combined culture; and (c) Harvesting the combined culture medium from the combined culture without disrupting the cell membrane, wherein the combined culture medium contains the heteromultimeric protein.

5. A heteromultimeric protein produced by the method of any one of claims 1-4.

6. A composition comprising the heteromultimeric protein of claim 56 or claim 57 and a pharmaceutically acceptable carrier.

7. A host cell comprising a polynucleotide or a recombinant vector, wherein the polynucleotide or the recombinant vector encodes a first hinge-containing polypeptide of the heteromultimeric protein of claim 5, and wherein the host cell does not express a second hinge-containing polypeptide of the heteromultimeric protein.

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