ENGINEERING CHARGE PAIR MUTATIONS FOR PAIRING OF HETERO-IgG MOLECULES

Abstract

To provide heterodimers comprising antibody CH3 domains and mutations useful for the facilitation of the formation of heterodimers.SOLUTION: An isolated heteromultimer is provided which comprises a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, the first CH3 domain polypeptide comprising an amino acid modification at position K360, where: (i) the first CH3 domain polypeptide further comprises an amino acid modification at position K370, and (ii) the second CH3 domain polypeptide comprises an amino acid modification at position E357, where the numbering of amino acid residues is according to the EU index as set forth in Kabat. Methods of optimizing purification of the heterodimers at certain pHs are also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of protein engineering. Specifically, the present invention relates to manipulating charged residues in the crystallizable fragment (Fc) of heterodimeric IgG molecules to prevent homodimeric Fc formation and promote heterodimeric Fc formation.

[0002] This application contains an ASCII "txt" Sequence Listing that serves as both a computer-readable form (CRF) and a paper copy as required by 37 CFR Sections 1.821(c) and 1.821(e), and is incorporated herein by reference in its entirety. The "txt" file, created on November 6, 2020, is named A-2389-WO-PCT_SEQ_LIST_11062020_ST25 and is 23.6 kb in size. [Background technology]

[0003] The natural formation of the Fc region of an IgG molecule involves the assembly of two matching Fc chains, independent of the sequence of the Fab arms. Bispecific antibodies have target specificity for two or more different antigens due to the two Fab arms consisting of different sequences. As a result, bispecific molecules with natural Fc regions tend to assemble their heavy chain molecules into three major species: monoclonal antibodies that bind one antigen, monoclonal antibodies that bind a second antigen, and bispecific antibodies that bind both antigens.

[0004] To prevent the formation of monoclonal antibodies during the expression of these recombinant proteins, IgG molecules have been engineered primarily in the CH3 region. Early studies on driving chain pairing between IgG heavy chains began with the use of a knob-in-hole strategy, in which the contours of the interchain surface area are altered by mutating larger and smaller residues. A more recent and widely implemented approach is the use of charge-pair mutations, in which naturally charged residues in one of the two rotationally symmetric regions are reversed by mutating positively charged residues to negatively charged residues and vice versa. Applying this approach to a pair of charged residues creates two repulsive points between matching Fc regions and two attractive points that form salt bridges between different Fc regions. In our study, we screened and optimized three pairs of charged residues in the Fc region and also tested neighboring residues to enhance the drive for pairing specificity and add the benefit of purification at a lower pH. Summary of the Invention [Means for solving the problem]

[0005] The present invention relates to an isolated heteromultimer comprising a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, wherein the first CH3 domain polypeptide comprises an amino acid modification at position K360, wherein (i) the first CH3 domain polypeptide further comprises an amino acid modification at position K370, and (ii) the second CH3 domain polypeptide comprises an amino acid modification at position E357, wherein amino acid residue numbering is according to the EU index as set forth in Kabat for the isolated heteromultimer.

[0006] In another aspect, the invention is directed to a method for stabilizing an isolated heteromultimer at about pH 5.0, wherein the heteromultimer comprises a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, wherein (i) the first CH3 domain polypeptide comprises an amino acid modification at K370 and (ii) the second CH3 domain polypeptide comprises an amino acid modification at position E357, the method comprising introducing an amino acid modification at position K360 of the first CH3 domain, wherein the modification is a substitution of K360 with glutamic acid or aspartic acid, wherein amino acid residue numbering is according to the EU index as set forth in Kabat.

[0007] In one embodiment, the amino acid modification at position K360 is selected from the group consisting of K360E and K360D.

[0008] In one embodiment, the amino acid modification at position K370 is selected from the group consisting of K370E and K370D.

[0009] In one embodiment, the amino acid modification at position E357 is selected from the group consisting of E357K, E357H and E357R.

[0010] In one embodiment, the amino acid modification at position K360 is K360E, the amino acid modification at position K370 is K370D, and the amino acid modification at position E357 is E357K.

[0011] In one embodiment, one CH3 domain polypeptide further comprises an amino acid modification at position K409, and the other CH3 domain polypeptide further comprises an amino acid modification at position D399.

[0012] In one embodiment, the first CH3 domain polypeptide comprises an amino acid modification at position K409 and the second CH3 domain polypeptide comprises an amino acid modification at position D399.

[0013] In one embodiment, the first CH3 domain polypeptide comprises an amino acid modification at position D399 and the second CH3 domain polypeptide comprises an amino acid modification at position K409.

[0014] In one embodiment, the amino acid modification at position K409 is selected from the group consisting of K409E and K409D, and the amino acid modification at position D399 is selected from the group consisting of D399K, D399H and D399R.

[0015] In one embodiment, the amino acid modification at position K409 is K409D and the amino acid modification at position D399 is D399K.

[0016] In one embodiment, the CH3 domain polypeptide comprising an amino acid modification at position K409 further comprises an amino acid modification at position K392.

[0017] In one embodiment, the amino acid modification at position K392 is selected from the group consisting of K392E and K392D.

[0018] In one embodiment, one CH3 domain polypeptide further comprises an amino acid modification at position K439, and the other CH3 domain polypeptide further comprises an amino acid modification at position E356.

[0019] In one embodiment, the first CH3 domain polypeptide comprises an amino acid modification at position K439 and the second CH3 domain polypeptide comprises an amino acid modification at position E356.

[0020] In one embodiment, the first CH3 domain polypeptide comprises an amino acid modification at position E356 and the second CH3 domain polypeptide comprises an amino acid modification at position K439.

[0021] In one embodiment, the amino acid modification at position K439 is selected from the group consisting of K439E and K439D, and the amino acid modification at position E356 is selected from the group consisting of E356K, E356H, and E356R.

[0022] In one embodiment, the amino acid modification at position K439 is K439E and the amino acid modification at position E356 is E356K.

[0023] In one embodiment, the first CH3 domain polypeptide comprises K360E, K370D, K409D and K392D mutations, and the second CH3 domain polypeptide comprises E357K and D399K mutations.

[0024] In one embodiment, the first CH3 domain polypeptide further comprises a K439E mutation and the second CH3 domain polypeptide further comprises an E356K mutation.

[0025] In one embodiment, the heterodimeric CH3 domain is comprised in an Fc region based on an IgG Fc region.

[0026] In one embodiment, the IgG Fc region is an IgG1 Fc region.

[0027] In one embodiment, the heteromultimer is a bispecific or multispecific antibody. [Brief explanation of the drawings]

[0028] [Figure 1] Shown are single-chain variable regions (scFv) fused to an Fc chain and an Fc chain lacking a warhead. Molecules assembled into homodimers with two scFv warheads (A) or lacking the two warheads (C) have molecular weights of approximately 100 kDa or 50 kDa, respectively. When assembled into the intended hetero-Fc form (B), the molecular weight is approximately 75 kilodaltons (kDa). [Figure 2] The lead scFv-Fc molecules from the screening are shown. [Figure 3] 1 shows a modified monoclonal antibody, including the addition of charge pair mutations in the CH3 region and the addition of a DEVD cleavage site in the hinge between one of the Fab regions and the Fc region. [Figure 4] Cation exchange of the heterodimer at pH 6.0, 5.6, and 5.0 is shown. [Figure 5] Cation exchange of the heterodimer at pH 6.0, 5.6, and 5.0 compared to the K360 mutant is shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] As used herein, a "multimeric protein" refers to a protein containing two or more distinct polypeptide or protein chains that associate with each other to form a single protein in vitro or in vivo. A multimeric protein can be composed of two or more polypeptides of the same type forming a "homomultimer." Alternatively, a multimeric protein can also be composed of two or more polypeptides of different sequences forming a "heteromultimer." Thus, a "heteromultimer" is a molecule comprising at least a first polypeptide and a second polypeptide, where the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue. Heteromultimers can include "heterodimers" formed by a first and a second polypeptide, or can form higher-order tertiary structures in which three or more polypeptides are present.

[0030] As used herein, the term "antigen-binding protein" refers to a protein that specifically binds to one or more target antigens. Antigen-binding proteins can include antibodies and functional fragments thereof. A "functional antibody fragment" is a portion of an antibody that lacks at least some of the amino acids present in a full-length heavy and / or light chain, yet is still capable of specifically binding to an antigen. Functional antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments, and can be derived from any mammalian source, such as human, mouse, rat, rabbit, or camelid. Functional antibody fragments can be comparable to intact antibodies for target antigen binding, and fragments can be produced by modification of intact antibodies (e.g., enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technology or peptide synthesis.

[0031] "Heavy chain" and "light chain" refer to the two polypeptides that make up IgG. The heavy chain can be broken down into the following domains from N- to C-terminus: VH, CH1, CH2, and CH3. The light chain can be broken down into the following domains from N- to C-terminus: VL and CL. The CH1 and CL domains interact to form the functional structure of the VH and VL domains.

[0032] Antigen-binding proteins can also include proteins comprising one or more functional antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, antigen-binding proteins include diabodies (see, e.g., EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, Vol. 90:6444-6448, 1993); intrabodies; domain antibodies (a single VL or VH domain or two or more VH domains joined by a peptide linker; see Ward et al., Nature, Vol. 341:544-546, 1989); maxibodies (two scFvs fused to an Fc region; see Fredericks et al., Protein Engineering, Design & Selection, Vol. 17:95-106, 2004 and Powers et al., Journal of Immunological minibodies (scFv fused to a CH3 domain, see Olafsen et al., Protein Eng Des Sel., Vol. 17:315-23, 2004); peptibodies (one or more peptides attached to an Fc region, see WO 00 / 24782); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with complementary light chain polypeptides, see Zapata et al., Protein Eng Des Sel., Vol. 251:123-135, 2001); triabodies; tetrabodies; Eng., Vol. 8:1057-1062, 1995); small modular immunopharmaceuticals (see U.S. Patent Application Publication No. 2003 / 0133939); and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0033] In certain embodiments, the antigen-binding proteins of the present invention are "bispecific," meaning that they can specifically bind to two different antigens. In another embodiment, the antigen-binding proteins of the present invention are "trispecific," meaning that they can specifically bind to three different antigens. In another embodiment, the antigen-binding proteins of the present invention are "tetraspecific," meaning that they can specifically bind to four different antigens. As used herein, an antigen-binding protein "specifically binds" to a target antigen if, under similar binding assay conditions, it has a significantly higher binding affinity for the target antigen compared to its affinity for other unrelated proteins, thereby being able to distinguish between the antigens. An antigen-binding protein that specifically binds to an antigen has a binding affinity of 1×10 -6 The equilibrium dissociation constant (K D The antigen binding protein may have a K D is 1×10 -8 In one embodiment, an antigen-binding protein of the invention specifically binds an antigen with a binding affinity of 5×10 -7 K below M D In another embodiment, the antigen binding proteins of the invention bind to the target antigen at 1 x 10 -7 K below M D binds to the target antigen.

[0034] Affinity can be determined using a variety of techniques, one example being an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). Using this methodology, the association rate constant (k a , unit:M -1 s -1 ) and dissociation rate constant (k d , unit: s -1 ) can then be measured. D , unit: M) is expressed as the ratio of kinetic rate constants (k d / k aIn some embodiments, affinity can be determined by a kinetic method, such as the equilibrium exclusion binding assay (KExA), as described in Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008. The KinExA assay can be used to determine the equilibrium dissociation constant (K D , unit: M) and association rate constant (k a , unit:M -1 s -1 The dissociation rate constant (k d , unit: s -1 ) are the values of these (K D ×k a ) can be calculated from the affinity. In other embodiments, the affinity is determined by an equilibrium / solution method. In certain embodiments, the affinity is determined by a FACS binding assay. In certain embodiments of the invention, the antigen-binding protein has an affinity of 20 nM (2.0 x 10) for a target antigen expressed by mammalian cells (e.g., CHO, HEK293, Jurkat) as determined by the equilibrium exclusion method performed by the method described in Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008. -8 M) K below D , 10 nM (1.0 × 10 -8 M) K below D , 1 nM (1.0 × 10 -9 M) K below D , 500 pM (5.0 × 10 -10 M) K below D , 200 pM (2.0 × 10 -10 M) K below D , 150 pM (1.50 × 10 -10 M) K below D , 125 pM (1.25 × 10 -10 M) K below D , 105 pM (1.05 × 10 -10 M) K below D , 50 pM (5.0 × 10 -11 M) K below D , or 20 pM (2.0 × 10 -11 M) K belowD In some embodiments, the bispecific antigen-binding proteins described herein specifically bind with k d The binding avidity for the target antigen, as measured by the dissociation rate constant (DCR), is approximately 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 s -1 or less (lower values indicate higher binding avidity), and / or K D The binding affinity for the target antigen, as measured by the equilibrium dissociation constant (Eq.) is approximately 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 M or less (lower values indicate higher binding affinity).

[0035] In certain embodiments of the invention, antigen-binding proteins are multivalent. The valency of a binding protein refers to the number of individual antigen-binding domains within the binding protein. For example, the terms "monovalent," "bivalent," "trivalent," and "tetravalent" with respect to antigen-binding proteins of the invention refer to binding proteins having one, two, three, and four antigen-binding domains, respectively. Thus, a tetravalent antigen-binding protein comprises four or more antigen-binding domains. In other embodiments, bispecific antigen-binding proteins are multivalent. For example, in certain embodiments, a bispecific antigen-binding protein is tetravalent, comprising four antigen-binding domains: two antigen-binding domains that bind to a first target antigen and two antigen-binding domains that bind to a second target antigen. A tetraspecific antigen-binding protein is tetravalent and comprises four antigen-binding domains: one antigen-binding domain that binds to a first target antigen, one antigen-binding domain that binds to a second target antigen, one antigen-binding domain that binds to a third target antigen, and one antigen-binding domain that binds to a fourth target antigen.

[0036] As used herein, the term "antigen-binding domain," used interchangeably with "binding domain," refers to a region of an antigen-binding protein that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antigen-binding protein for that antigen. In some embodiments, the binding domain may be derived from the natural ligand of the target antigen. As used herein, the term "target antigen" refers to the first and / or second target antigen of a bispecific molecule, and to the first, second, third, and / or fourth target antigen of a tetraspecific molecule.

[0037] In certain embodiments of the antigen-binding proteins of the present invention, the binding domain may be derived from an antibody or a functional fragment thereof. For example, the binding domain of the antigen-binding proteins of the present invention may comprise one or more complementarity-determining regions (CDRs) from the light and heavy chain variable regions of an antibody that specifically binds to a target antigen. As used herein, the term "CDR" refers to a complementarity-determining region (also called a "minimal recognition unit" or "hypervariable region") within an antibody variable sequence. There are three heavy chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2, and CDRL3). As used herein, the term "CDR region" refers to a group of three CDRs (i.e., three light chain CDRs or three heavy chain CDRs) present in a single variable region. The CDRs in each of the two chains are typically aligned by framework regions to form a structure that specifically binds to a specific epitope or domain of the target protein. From N-terminus to C-terminus, naturally occurring variable light and heavy chain regions typically have the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0038] Thus, both the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and the AHo numbering scheme (Honegger A. and Plueckthun AJ Mol Biol. 2001 Jun 8;309(3):657-70) can be used in the present invention. The amino acid positions of a given antibody, as well as its complementarity-determining regions (CDRs) and framework regions (FRs), can be identified using either system. For example, EU heavy chain positions 39, 44, 183, 356, 357, 360, 370, 392, 399, and 409 are identical to AHo heavy chain positions 46, 51, 230, 484, 485, 491, 501, 528, 535, and 551, respectively.

[0039] Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments (each of which has a single antigen-binding site) and a residual "Fc" fragment (containing the immunoglobulin constant region). The Fab fragment contains the variable domain, as well as all of the constant domains of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is composed of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and one immunoglobulin heavy chain CH1 and variable region (VH). The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The Fc fragment displays carbohydrate and is responsible for many antibody effector functions (such as binding complement and cellular receptors) that distinguish one class of antibody from another. The "Fd fragment" contains the VH and CH1 domains derived from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of a Fab fragment.

[0040] A "Fab' fragment" is a Fab fragment that has one or more cysteine residues from the antibody hinge region at the C-terminus of the CH1 domain.

[0041] A "F(ab')2 fragment" is a bivalent fragment containing two Fab' fragments linked by inter-heavy chain disulfide bridges at the hinge region.

[0042] An "Fv" fragment is the minimum fragment containing a complete antigen-recognition and binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy-chain variable region (VH) and one immunoglobulin light-chain variable region (VL) in tight, non-covalent association. In this configuration, the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single light- or heavy-chain variable region (or half of an Fv fragment containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site comprising both the VH and VL.

[0043] As used herein, the term "variable region," which is used interchangeably with "variable domain" (light chain variable region (VL) and heavy chain variable region (VH)), refers to the region in each of an immunoglobulin light chain and an immunoglobulin heavy chain that is directly involved in binding the antibody to an antigen. As described above, the variable light chain region and the variable heavy chain region have the same general structure, and each region contains four framework (FR) regions, the sequences of which are extensively conserved and connected by three CDRs. The framework regions adopt a beta-sheet structure, and the CDRs may form loops connecting the beta-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form an antigen-binding site together with the CDRs of the other chain.

[0044] An "immunoglobulin domain" refers to a peptide comprising approximately 100 amino acid residues that contains an amino acid sequence similar to that of an immunoglobulin and that includes at least two cysteine residues. Examples of immunoglobulin domains include VH, CH1, CH2, and CH3 of an immunoglobulin heavy chain, and VL and CL of an immunoglobulin light chain. Immunoglobulin domains are also present in proteins other than immunoglobulins. Examples of immunoglobulin domains in proteins other than immunoglobulins include immunoglobulin domains contained in proteins belonging to the immunoglobulin superfamily, such as major histocompatibility complex (MHC), CD1, B7, and T cell receptor (TCR). Any of the immunoglobulin domains can be used as the immunoglobulin domain of the multivalent antibody of the present invention.

[0045] In human antibodies, CH1 refers to the region having the amino acid sequence at positions 118 to 215 of the EU index. A highly flexible amino acid region called the "hinge region" is located between CH1 and CH2. CH2 represents the region having the amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents the region having the amino acid sequence at positions 341 to 446 of the EU index.

[0046] "CL" represents the light chain constant region. In the case of a human antibody κ chain, CL represents the region having the amino acid sequence at positions 108 to 214 of the EU index. In the case of a λ chain, CL represents the region having the amino acid sequence at positions 108 to 215 of the EU index.

[0047] Binding domains that specifically bind to target antigens can be derived from a) known antibodies against these antigens, or b) new antibodies or antibody fragments obtained by novel immunization methods using antigen proteins or fragments thereof, by phage display, or by other conventional methods. The antibodies from which the binding domains of the antigen-binding proteins are derived can be monoclonal, polyclonal, recombinant, human, or humanized. In certain embodiments, the antibodies from which the binding domains are derived are monoclonal. In these and other embodiments, the antibodies are human or humanized and can be of the IgG1, IgG2, IgG3, or IgG4 type.

[0048] As used herein, the terms "stability" and "stabilization" are defined as the maintenance of the chemical or physical integrity and / or biological activity of an antigen-binding polypeptide or protein over a period of time. Stabilization of an antigen-binding polypeptide or protein includes preventing or slowing the degradation or degradation of the antigen-binding polypeptide or protein from its biologically and / or therapeutically active form to an inactive form. Instability can result from events such as aggregation, denaturation, fragmentation, or chemical modification, e.g., oxidation, cross-linking, deamidation, and reaction with other components present in a composition comprising the antigen-binding polypeptide or protein.

[0049] The stability of an antigen-binding protein or polypeptide in a composition can be characterized using methods known in the art, including, but not limited to, measuring biological activity such as antigen-binding activity by immunoassay techniques such as ELISA, or other techniques that determine purity or physical / chemical changes to the antigen-binding protein or polypeptide, such as size exclusion chromatography, capillary gel electrophoresis, circular dichroism, or mass spectrometry. Stability is determined by comparing measurements obtained by these types of characterization methods at an initial time point, such as upon formulation or preparation of the composition (i.e., a suspension or dispersion, as the case may be), with measurements obtained at a later time point, i.e., after storage in a given environment or condition.

[0050] The term "monoclonal antibody" (or "mAb"), as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor natural mutations. Monoclonal antibodies are highly specific and are directed against a distinct antigenic site or epitope, as opposed to polyclonal antibody preparations, which typically contain a variety of antibodies directed against different epitopes. Monoclonal antibodies can be produced using any technique known in the art, for example, by immortalizing spleen cells harvested from transgenic animals after completion of the immunization schedule. Spleen cells can be immortalized using any technique known in the art, for example, by fusing the spleen cells with myeloma cells to produce hybridomas. The myeloma cells used in the fusion procedure to produce hybridomas are non-antibody-producing cells, have high fusion efficiency, and possess enzyme deficiencies that prevent them from growing in specific selective media that support the growth of only the desired fused cells (hybridomas). Examples of cell lines suitable for use in mouse fusions include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag41, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7, and S194 / 5XXO Bul, while examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0051] In some examples, hybridoma cell lines are made by immunizing an animal (e.g., a transgenic animal having human immunoglobulin sequences) with a target antigen, harvesting spleen cells from the immunized animal, fusing the harvested spleen cells with a myeloma cell line, thereby producing hybridoma cells, establishing hybridoma cell lines from the hybridoma cells, and identifying hybridoma cell lines that produce antibodies that bind to the target antigen.

[0052] Monoclonal antibodies secreted by hybridoma cell lines can be purified using any technique known in the art, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Hybridomas or mAbs can be further screened to identify mAbs with particular properties, such as, for example, the ability to bind to cells expressing the target antigen, the ability to block or interfere with the binding of the target antigen to its respective receptor or ligand, or the ability to functionally block either of the target antigens.

[0053] In some embodiments, the binding domain of the antigen-binding protein of the present invention may be derived from a humanized antibody against a target antigen. A "humanized antibody" refers to an antibody in which regions (e.g., framework regions) have been modified to include regions derived from a corresponding human immunoglobulin. Generally, humanized antibodies can be generated from monoclonal antibodies initially generated in non-human animals. Certain amino acid residues of this monoclonal antibody, generally from the non-antigen-recognizing portion of the antibody, are modified to be homologous to corresponding residues in a human antibody of the corresponding isotype. Humanization can be achieved, for example, by substituting at least a portion of a rodent variable region with the corresponding region of a human antibody using various methods (see, for example, U.S. Pat. Nos. 5,585,089 and 5,693,762; Jones et al., Nature, Vol. 321:522-525, 1986; Riechmann et al., Nature, Vol. 332:323-27, 1988; Verhoeyen et al., Science, Vol. 239:1534-1536, 1988). The CDRs of the light and heavy chain variable regions of an antibody generated in another species can be grafted onto consensus human FRs. To create consensus human FRs, FRs derived from multiple human heavy chain or light chain amino acid sequences can be aligned to identify a consensus amino acid sequence.

[0054] Novel antibodies generated against target antigens from which the binding domains of the antigen-binding proteins of the present invention can be derived can be fully human antibodies. A "fully human antibody" is an antibody comprising variable and constant regions derived from human germline immunoglobulin sequences. One specific means provided for achieving the production of fully human antibodies is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated is one way to generate fully human monoclonal antibodies (mAbs) in mice, animals that can be immunized with any desired antigen. The use of fully human antibodies can minimize immunogenic and allergic responses that can occur when mouse mAbs or mouse-derived mAbs are administered to humans as therapeutic agents.

[0055] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that lack endogenous immunoglobulin production and are capable of producing a repertoire of human antibodies. Antigens for this purpose typically have six or more consecutive amino acids and are optionally conjugated to a carrier (such as a hapten). See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; and Bruggermann et al., 1993, Year in Immunol. 7:33. In one example of such a method, a transgenic animal is generated by disabling the endogenous mouse immunoglobulin loci encoding the mouse's immunoglobulin heavy and light chains and inserting into the mouse genome a large fragment of human genomic DNA containing loci encoding human heavy and light chain proteins. The partially modified animals, which have less than the full complement of human immunoglobulin loci, are then crossbred to obtain animals with all the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that are immunospecific for the immunogen but have human, rather than murine, amino acid sequences, including the variable regions. For further details of such methods, see, e.g., WO 96 / 33735 and WO 94 / 02602.Additional methods relating to transgenic mice for making human antibodies are described in U.S. Patent Nos. 5,545,807, 6,713,610, 6,673,986, 6,162,963, 5,939,598, 5,545,807, 6,300,129, 6,255,458, 5,877,397, 5,877,397, and 5,877,498. 74,299 and 5,545,806, PCT publications WO 91 / 10741, WO 90 / 04036, WO 94 / 02602, WO 96 / 30498, WO 98 / 24893, and EP 546073B1 and EP 546073A1.

[0056] These transgenic mice, referred to herein as "HuMab" mice, contain a human immunoglobulin gene minilocus encoding unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous mu and kappa chain loci (Lonberg et al., 1994, Nature 368:856-859). Thus, the mice exhibit reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG kappa monoclonal antibodies (Lonberg et al., supra; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764:536-546). HuMab mice were created by Taylor et al.,1992, Nucleic Acids Research 20:6287-6295;Chen et al.,1993,International Immunology 5:647-656;Tuaillon et al.,1994,J.Immunol.152:2912-2920;Lonberg et al.,1994,Nature 368:856-859;Lonberg,1994,Handbook of Exp.Pharmacology 113:49-101;Taylor et al.,1994,International Immunology 6:579-591;Lonberg and Huszar,1995,Intern.Rev.Immunol.13:65-93;Harding and Lonberg, 1995, Ann.NY Acad. Sci. 764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851, which are incorporated herein by reference in their entireties for all purposes.See also U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, as well as U.S. Patent No. 5,545,807, WO 93 / 1227, WO 92 / 22646, and WO 92 / 03918, the disclosures of all of which are incorporated herein by reference in their entireties for all purposes. Techniques utilizing the production of human antibodies in such transgenic mice are also disclosed in WO 98 / 24893 and Mendez et al., 1997, Nature Genetics 15:146-156, which are incorporated herein by reference.

[0057] Human-derived antibodies can also be produced using phage display technology. Phage display is described, for example, in Dower et al., WO 91 / 17271; McCafferty et al., WO 92 / 01047; and Caton and Koprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each of which is incorporated herein by reference in its entirety. Antibodies produced by phage technology are typically produced in bacteria as antigen-binding fragments, such as Fv or Fab fragments, and therefore lack effector function. Effector function can be introduced by one of two strategies: the fragment can be engineered into a complete antibody expressed in mammalian cells, if desired, or into an antibody fragment with a second binding site capable of eliciting effector function. Typically, antibody Fd fragments (VH-CH1) and light chains (VL-CL) are cloned separately by PCR, randomly recombined in a combinatorial phage display library, and then selected for specific antigen binding. Antibody fragments are expressed on the surface of phage, and antigen-binding selection of Fv or Fab fragments (and thus phage containing DNA encoding antibody fragments) is achieved through several rounds of antigen binding and reamplification, a procedure called panning. Antigen-specific antibody fragments are enriched and ultimately isolated. Phage display technology can also be used in an approach for humanizing rodent monoclonal antibodies called "guided selection" (see Jespers, LS, et al., Bio / Technology 12, 899-903 (1994)). For this, Fd fragments of mouse monoclonal antibodies can be displayed in combination with a human light chain library, and the resulting hybrid Fab library can then be selected using antigen. The mouse Fd fragments thus provide a template to guide the selection. The selected human light chains are then combined with a human Fd fragment library.Selection of the resulting library yields fully human Fabs.

[0058] In certain embodiments, antigen-binding proteins of the present invention comprise antibodies. As used herein, the term "antibody" refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (each approximately 25 kDa) and two heavy chain polypeptides (each approximately 50-70 kDa). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising, from amino to carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be kappa (κ) or lambda (λ). The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG class antibodies and IgA class antibodies are further divided into subclasses, i.e., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domain can be derived from any immunoglobulin isotype, including its subtypes. Antibody chains are linked via interpolypeptide disulfide bonds between the CL and CH1 domains (ie, between the light and heavy chains) and between the hinge regions of the antibody heavy chains.

[0059] In certain embodiments, the antigen binding proteins of the invention are heterodimeric antibodies (used interchangeably herein as "heteroimmunoglobulins" or "hetero-Igs"), which refers to antibodies comprising two different light chains and two different heavy chains.

[0060] Heterodimeric antibodies can include any immunoglobulin constant region. The term "constant region," as used herein, refers to all domains of an antibody other than the variable region. The constant region is not directly involved in antigen binding but exerts various effector functions. As described above, antibodies are divided into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) depending on the amino acid sequence of the constant region of their heavy chain. The light chain constant region can be, for example, a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region, which is found in all five antibody isotypes. Examples of human immunoglobulin light chain constant region sequences are shown in Table 1 below.

[0061] [Table 1]

[0062] The heavy chain constant region of the heterodimeric antibody can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In some embodiments, the heterodimeric antibody comprises a heavy chain constant region derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In one embodiment, the heterodimeric antibody comprises a heavy chain constant region derived from a human IgG1 immunoglobulin. In another embodiment, the heterodimeric antibody comprises a heavy chain constant region derived from a human IgG2 immunoglobulin. Exemplary human IgG1 and IgG2 heavy chain constant region sequences are shown in Table 2 below.

[0063] [Table 2]

[0064] The variable regions can be attached to the light and heavy chain constant regions described above to form complete antibody light and heavy chains, respectively. Furthermore, the heavy and light chain polypeptides thus generated can each be combined to form a complete bispecific antibody structure, e.g., a heterodimeric antibody. It should be understood that the heavy and light chain variable regions provided herein can also be attached to other constant domains having sequences different from the exemplary sequences listed above.

[0065] In certain embodiments of the present invention, two different heavy chains are used to form heterodimeric molecules of the present invention. To facilitate assembly of the light and heavy chains into heterodimeric antibodies, the light and / or heavy chains from each antibody can be engineered to reduce the formation of mismatched molecules. For example, one approach to promoting heterodimer formation over homodimer formation is the so-called "knobs-into-hole" method, which involves introducing mutations into the CH3 domains of two different antibody heavy chains at the contact interface. Specifically, one or more bulky amino acids in one heavy chain are replaced with amino acids with short side chains (e.g., alanine or threonine) to generate a "hole," while one or more amino acids with large side chains (e.g., tyrosine or tryptophan) are introduced into the other heavy chain to generate a "knob." When the engineered heavy chains are co-expressed, heterodimers (knobs-holes) form at a greater rate than homodimers (hole-hole or knob-knob). The "knob-into-hole" method is described in detail in WO 96 / 027011, Ridgway et al., Protein Eng., Vol. 9:617-621, 1996, and Merchant et al., Nat. Biotechnol., Vol. 16:677-681, 1998, all of which are incorporated herein by reference in their entireties.

[0066] Another approach to promoting heterodimer formation and eliminating homodimer formation involves utilizing an electrostatic steering mechanism (see Gunasekaran et al., J. Biol. Chem., Vol. 285:19637-19646, 2010, which is incorporated herein by reference in its entirety). This approach involves introducing or utilizing charged residues in the CH3 domains of each heavy chain so that two different heavy chains associate due to opposite charges that cause electrostatic attraction. Homodimerization of identical heavy chains is disfavored because identical heavy chains have the same charge and are therefore repulsive. This same electrostatic steering technique can be used to prevent mispairing of light chains with non-cognate heavy chains by introducing oppositely charged residues into the binding interface for correct light-heavy chain pairs. Electrostatic steering techniques and suitable charge pair mutations to promote heterodimerization and correct light / heavy chain pairing are described in WO 2009 / 089004 and WO 2014 / 081955, both of which are incorporated herein by reference in their entireties.

[0067] In embodiments in which the bispecific antigen-binding protein of the invention is a heterodimeric antibody comprising a first light chain (LC1) and a first heavy chain (HC1) from a first antibody that specifically binds to a first target antigen, and a second light chain (LC2) and a second heavy chain (HC2) from a second antibody that specifically binds to target 2, HC1 or HC2 may comprise one or more amino acid substitutions that replace a positively charged amino acid with a negatively charged amino acid. For example, in one embodiment, the CH3 domain of HC1 or the CH3 domain of HC2 comprises an amino acid sequence that differs from the wild-type human IgG amino acid sequence such that one or more positively charged amino acids (e.g., lysine, histidine, and arginine) in the wild-type IgG amino acid sequence are substituted with one or more negatively charged amino acids (e.g., aspartic acid and glutamic acid) at the corresponding positions in the CH3 domain. In these and other embodiments, an amino acid (e.g., lysine) at one or more positions selected from 370, 392, and 409 (EU numbering system) is substituted with a negatively charged amino acid (e.g., aspartic acid and glutamic acid). Amino acid substitutions in an amino acid sequence are generally designated herein by a single-letter abbreviation for the amino acid residue at a particular position, followed by the number of the amino acid position relative to the original sequence of interest, followed by the single-letter abbreviation for the substituted amino acid residue. For example, "T30D" represents the substitution of a threonine residue with an aspartic acid residue at amino acid position 30 relative to the original sequence of interest. Another example, "S218G" represents the substitution of a serine residue with a glycine residue at amino acid position 218 relative to the original amino acid sequence of interest.

[0068] In certain embodiments, the HC1 or HC2 of a heterodimeric antibody may contain one or more amino acid substitutions that replace a negatively charged amino acid with a positively charged amino acid. For example, in one embodiment, the CH3 domain of HC1 or the CH3 domain of HC2 contains an amino acid sequence that differs from the wild-type human IgG amino acid sequence such that one or more negatively charged amino acids in the wild-type human IgG amino acid sequence are substituted with one or more positively charged amino acids at corresponding positions in the CH3 domain. In these and other embodiments, an amino acid (e.g., aspartic acid or glutamic acid) at one or more positions selected from 356, 357, and 399 (EU numbering system) of the CH3 domain is substituted with a positively charged amino acid (e.g., lysine, histidine, and arginine).

[0069] In certain embodiments, the heterodimeric antibody comprises a first heavy chain comprising negatively charged amino acids at positions 360, 370, 392, and 409 (e.g., K360E / D, K370E / D, K392E / D, and K409E / D substitutions), and a second heavy chain comprising positively charged amino acids at positions 356 and 399 (e.g., E356K and D399K substitutions). In other certain embodiments, the heterodimeric antibody comprises a first heavy chain comprising negatively charged amino acids at positions 392, 409, and 370 (e.g., K392D, K409D, and K370D substitutions), and a second heavy chain comprising positively charged amino acids at positions 356, 399, and 357 (e.g., E356K, D399K, and E357K substitutions). In a related embodiment, the first heavy chain is derived from an anti-first antigen antibody and the second heavy chain is derived from an anti-second antigen antibody.

[0070] The present invention relates to an isolated heteromultimer comprising a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, wherein the first CH3 domain polypeptide comprises an amino acid modification at position K360, wherein (i) the first CH3 domain polypeptide further comprises an amino acid modification at position K370, and (ii) the second CH3 domain polypeptide comprises an amino acid modification at position E357, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0071] In another aspect, the invention is directed to a method for stabilizing an isolated heteromultimer at about pH 5.0, wherein the heteromultimer comprises a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, wherein (i) the first CH3 domain polypeptide comprises an amino acid modification at position K370 and (ii) the second CH3 domain polypeptide comprises an amino acid modification at position E357, the method comprising introducing an amino acid modification at position K360 of the first CH3 domain, wherein the modification is a substitution of K360 with glutamic acid or aspartic acid, wherein amino acid residue numbering is according to the EU index as set forth in Kabat.

[0072] In one embodiment, the amino acid modification at position K360 is selected from the group consisting of K360E and K360D.

[0073] In one embodiment, the amino acid modification at position K370 is selected from the group consisting of K370E and K370D.

[0074] In one embodiment, the amino acid modification at position E357 is selected from the group consisting of E357K, E357H and E357R.

[0075] In one embodiment, the amino acid modification at position K360 is K360E, the amino acid modification at position K370 is K370D, and the amino acid modification at position E357 is E357K.

[0076] In one embodiment, one CH3 domain polypeptide further comprises an amino acid modification at position K409, and the other CH3 domain polypeptide further comprises an amino acid modification at position D399.

[0077] In one embodiment, the first CH3 domain polypeptide comprises an amino acid modification at position K409 and the second CH3 domain polypeptide comprises an amino acid modification at position D399.

[0078] In one embodiment, the first CH3 domain polypeptide comprises an amino acid modification at position D399 and the second CH3 domain polypeptide comprises an amino acid modification at position K409.

[0079] In one embodiment, the amino acid modification at position K409 is selected from the group consisting of K409E and K409D, and the amino acid modification at position D399 is selected from the group consisting of D399K, D399H and D399R.

[0080] In one embodiment, the amino acid modification at position K409 is K409D and the amino acid modification at position D399 is D399K.

[0081] In one embodiment, the CH3 domain polypeptide comprising an amino acid modification at position K409 further comprises an amino acid modification at position K392.

[0082] In one embodiment, the amino acid modification at position K392 is selected from the group consisting of K392E and K392D.

[0083] In one embodiment, one CH3 domain polypeptide further comprises an amino acid modification at position K439, and the other CH3 domain polypeptide further comprises an amino acid modification at position E356.

[0084] In one embodiment, the first CH3 domain polypeptide comprises an amino acid modification at position K439 and the second CH3 domain polypeptide comprises an amino acid modification at position E356.

[0085] In one embodiment, the first CH3 domain polypeptide comprises an amino acid modification at position E356 and the second CH3 domain polypeptide comprises an amino acid modification at position K439.

[0086] In one embodiment, the amino acid modification at position K439 is selected from the group consisting of K439E and K439D, and the amino acid modification at position E356 is selected from the group consisting of E356K, E356H, and E356R.

[0087] In one embodiment, the amino acid modification at position K439 is K439E and the amino acid modification at position E356 is E356K.

[0088] In one embodiment, the first CH3 domain polypeptide comprises K360E, K370D, K409D and K392D mutations, and the second CH3 domain polypeptide comprises E357K and D399K mutations.

[0089] In one embodiment, the first CH3 domain polypeptide further comprises a K439E mutation and the second CH3 domain polypeptide further comprises an E356K mutation.

[0090] In one embodiment, the heterodimeric CH3 domain is comprised in an Fc region based on an IgG Fc region.

[0091] In one embodiment, the IgG Fc region is an IgG1 Fc region.

[0092] In one embodiment, the heteromultimer is a bispecific or multispecific antibody.

[0093] To promote association of a particular heavy chain with its cognate light chain, both the heavy and light chains may contain complementary amino acid substitutions. As used herein, "complementary amino acid substitutions" refers to the pairing of a positively charged amino acid substitution in one chain with a negatively charged amino acid substitution in the other chain. For example, in some embodiments, a heavy chain contains at least one amino acid substitution that introduces a charged amino acid, and the corresponding light chain contains at least one amino acid substitution that introduces a charged amino acid, the charged amino acid introduced in the heavy chain having the opposite charge of the amino acid introduced in the light chain. In certain embodiments, at the first light chain (LC1) / pairing heavy chain (HC1) binding interface, one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into LC1 and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into HC1, while at the second light chain (LC2) / pairing heavy chain (HC2) binding interface, one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into LC2 and one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into HC2. Electrostatic interactions attract oppositely charged residues (polarity) at the interface, thereby inducing LC1 to pair with HC1 and inducing LC2 to pair with HC2. Heavy / light chain pairs with similarly charged residues (polarity) at the interface (eg, LC1 / HC2 and LC2 / HC1) are repulsed, resulting in the suppression of undesired HC / LC pairings.

[0094] In these and other embodiments, the heavy chain CH1 domain or the light chain CL domain comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more positively charged amino acids in the wild-type IgG amino acid sequence are substituted with one or more negatively charged amino acids. Alternatively, the heavy chain CH1 domain or the light chain CL domain comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are substituted with one or more positively charged amino acids. In some embodiments, one or more amino acids in the CH1 domain of the first and / or second heavy chain in the heterodimeric antibody at EU positions selected from F126, P127, L128, A141, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S183, V185, and K213 are substituted with charged amino acids. In certain embodiments, the heavy chain residue substituted with a negatively or positively charged amino acid is S183 (EU numbering system). In some embodiments, S183 is substituted with a positively charged amino acid. In alternative embodiments, S183 is substituted with a negatively charged amino acid. For example, in one embodiment, S183 is substituted with a negatively charged amino acid in the first heavy chain (e.g., S183E) and in the second heavy chain, S183 is substituted with a positively charged amino acid (e.g., S183K).

[0095] In embodiments where the light chain is a kappa light chain, one or more amino acids in the CL domain of the first and / or second light chain in the heterodimeric antibody at a position selected from F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174 and S176 (EU numbering for a kappa light chain) are replaced with a charged amino acid. In embodiments where the light chain is a lambda light chain, one or more amino acids in the CL domain of the first and / or second light chain in the heterodimeric antibody at a position selected from T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176, and Y178 (EU numbering for the lambda chain) are replaced with a charged amino acid. In some embodiments, the residue replaced with a negatively or positively charged amino acid is S176 (EU numbering system) in the CL domain of a kappa or lambda light chain. In certain embodiments, S176 in the CL domain is replaced with a positively charged amino acid. In alternative embodiments, S176 in the CL domain is replaced with a negatively charged amino acid. In one embodiment, S176 is substituted with a positively charged amino acid in the first light chain (e.g., S176K) and in the second light chain, S176 is substituted with a negatively charged amino acid (e.g., S176E).

[0096] In addition to, or as an alternative to, complementary amino acid substitutions in the CH1 and CL domains, the light and heavy chain variable regions of the heterodimeric antibody may include one or more complementary amino acid substitutions introducing charged amino acids. For example, in some embodiments, the heavy chain VH region or the light chain VL region comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more positively charged amino acids in the wild-type IgG amino acid sequence are substituted with one or more negatively charged amino acids. Alternatively, the heavy chain VH region or the light chain VL region comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are substituted with one or more positively charged amino acids.

[0097] V region interface residues in the VH region (i.e., amino acid residues that mediate assembly of the VH and VL regions) include EU positions 1, 3, 35, 37, 39, 43, 44, 45, 46, 47, 50, 59, 89, 91, and 93. One or more of these interface residues in the VH region may be substituted with a charged (positively or negatively charged) amino acid. In a specific embodiment, the amino acid at EU position 39 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, e.g., lysine. In an alternative embodiment, the amino acid at EU position 39 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In some embodiments, the amino acid at EU position 39 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G39E) and the amino acid at EU position 39 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G39K). In some embodiments, the amino acid at EU position 44 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at EU position 44 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In certain embodiments, the amino acid at EU position 44 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G44E) and the amino acid at EU position 44 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G44K).

[0098] V region interface residues in the VL region (i.e., amino acid residues that mediate assembly of the VH and VL regions) include EU positions 32, 34, 35, 36, 38, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 85, 87, 89, 90, 91, and 100. One or more interface residues in the VL region may be substituted with a charged amino acid, preferably an amino acid with an opposite charge to that introduced into the VH region of the cognate heavy chain. In some embodiments, the amino acid at EU position 100 in the VL region of the first and / or second light chain is substituted with a positively charged amino acid, e.g., lysine. In an alternative embodiment, the amino acid at EU position 100 in the VL region of the first and / or second light chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In certain embodiments, the amino acid at EU position 100 in the VL region of the first light chain is substituted with a positively charged amino acid (e.g., G100K) and the amino acid at EU position 100 in the VL region of the second light chain is substituted with a negatively charged amino acid (e.g., G100E).

[0099] Any of the constant domains may be modified to contain one or more of the charge-pair mutations described above to facilitate the correct assembly of heterodimeric antibodies.

[0100] Heterodimeric antibodies of the invention also include antibodies comprising a heavy and / or light chain in which 1, 2, 3, 4, or 5 amino acid residues have been deleted from the N-terminus, C-terminus, or both, for either one of the heavy and light chains due to, for example, post-translational modifications resulting from the type of host cell the antibody is expressed in. For example, Chinese hamster ovary (CHO) cells often cleave C-terminal lysines from antibody heavy chains.

[0101] In certain embodiments, the antigen-binding proteins of the present invention comprise (i) a first binding domain that specifically binds to a first target antigen, (ii) a second binding domain that specifically binds to a second target antigen, and (iii) a human immunoglobulin Fc region, wherein one binding domain is located at the amino terminus of the Fc region and the other binding domain is located at the carboxyl terminus of the Fc region. In some such embodiments, the first and second binding domains each comprise an immunoglobulin variable region. For example, in certain embodiments, the first binding domain comprises a first light chain variable region (VL1) and a first heavy chain variable region (VH1) derived from an anti-first target antigen antibody, and the second binding domain comprises a second light chain variable region (VL2) and a second heavy chain variable region (VH2) derived from an anti-second target antigen antibody.

[0102] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, which can be generated by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. In certain embodiments, the Fc region is an Fc region derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises the CH2 domain and the CH3 domain derived from a human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector function, as described in more detail herein.

[0103] In certain embodiments of the bispecific antigen-binding proteins of the present invention, the binding domain located at the amino terminus of the Fc region (i.e., the amino-terminal binding domain) is a Fab fragment fused to the amino terminus of the Fc region via a peptide linker described herein or via an immunoglobulin hinge region. An "immunoglobulin hinge region" refers to the amino acid sequence connecting the CH1 and CH2 domains of an immunoglobulin heavy chain. The hinge region of human IgG1 is generally defined as the amino acid sequence from about Glu216 or about Cys226 to about Pro230. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form inter-heavy chain disulfide bonds in the same positions, and this can be determined by one of skill in the art. In some embodiments, the amino-terminal binding domain is linked to the amino terminus of the Fc region via a human IgG1 hinge region. In other embodiments, the amino-terminal binding domain is linked to the amino terminus of the Fc region via a human IgG2 hinge region. In one embodiment, an amino-terminal binding domain (eg, a Fab fragment) is fused to an Fc region via the carboxyl terminus of the CH1 region of the Fab.

[0104] As used herein, the term "modified heavy chain" refers to a fusion protein comprising an immunoglobulin heavy chain, in particular a human IgG1 or human IgG2 heavy chain, and a functional antibody fragment (e.g., a Fab) or portion thereof (e.g., an immunoglobulin light chain or an Fd fragment), wherein the functional antibody fragment or portion thereof is fused at its N-terminus, optionally via a peptide linker, to the C-terminus of the heavy chain.

[0105] In some embodiments of the antigen binding proteins of the invention, the binding domain located at the carboxyl terminus of the Fc region (i.e., the carboxyl-terminal binding domain) is a Fab fragment. In such embodiments, the Fab is fused or linked via the amino terminus of the VH region of the Fab fragment to the carboxyl terminus of the Fc region (e.g., the carboxyl terminus of the CH3 domain) via a peptide linker. Thus, in one embodiment, a Fab is fused to an Fc region via the amino terminus of the VH region of the Fab, such that the resulting fusion protein comprises, from N- to C-terminus, a CH2 domain, a CH3 domain, a peptide linker, a VH region, and a CH1 domain.

[0106] The peptide linker linking the Fc region to the carboxyl-terminal Fab fragment can be any of the peptide linkers described herein. In certain embodiments, the peptide linker linking the Fc region to the carboxyl-terminal Fab fragment is at least 5 amino acids in length. In other embodiments, the peptide linker linking the Fc region to the carboxyl-terminal Fab fragment is at least 8 amino acids in length. A particularly suitable peptide linker for linking the Fc region to the carboxyl-terminal Fab fragment is (Gly x Ser) n (where x=3 or 4, and n=2, 3, 4, 5, or 6). In one embodiment, the peptide linker linking the Fc region to the carboxyl-terminal Fab fragment is an L10(G4S)2 linker (SEQ ID NO: 10). In another embodiment, the peptide linker linking the Fc region to the carboxyl-terminal Fab fragment is an L9 or G3SG4S linker (SEQ ID NO: 11).

[0107] In some embodiments of antigen binding proteins of the invention in which the carboxyl-terminal binding domain is a Fab fragment, the binding domain located amino-terminal to the Fc region (i.e., the amino-terminal binding domain) is also a Fab fragment. The amino-terminal Fab fragment can be fused to the amino terminus of the Fc region via a peptide linker or an immunoglobulin hinge region as described herein. In some embodiments, the amino-terminal Fab fragment is linked to the amino terminus of the Fc region via a human IgG1 hinge region. In other embodiments, the amino-terminal Fab fragment is linked to the amino terminus of the Fc region via a human IgG2 hinge region. In one embodiment, the amino-terminal Fab fragment is fused to the Fc region via the carboxyl terminus of the CH1 region of the Fab.

[0108] In some embodiments, the bispecific antigen-binding proteins of the invention comprise a first antibody that specifically binds to a first target, and one polypeptide chain (e.g., heavy chain (VH2-CH1)) of a Fab fragment from a second antibody that specifically binds to a second target is fused to the carboxyl terminus of the heavy chain of the first antibody. The bispecific antigen-binding protein in such embodiments also comprises a polypeptide chain comprising the other half of the Fab fragment (e.g., light chain (VL2-CL)) from the second antibody. This format is referred to herein as the "IgG-Fab" format, and one embodiment of this type of molecule is shown schematically in Figure 1. Thus, in certain embodiments, the present invention comprises bispecific, multivalent antigen-binding proteins comprising: (i) a light chain from the first antibody, (ii) a heavy chain from the first antibody (wherein the heavy chain is fused at its carboxyl terminus via a peptide linker to a first polypeptide comprising the VH-CH1 domain of the second antibody, to form a modified heavy chain), and (iii) a second polypeptide comprising the VL-CL domain of the second antibody. When dimerized, the bispecific antigen-binding protein is a homohexamer comprising two polypeptide chains comprising two modified heavy chains, two light chains from a first antibody, and the other half of the Fab fragment (Fd fragment) from a second antibody. In another embodiment, the first polypeptide fused to the carboxyl terminus of the heavy chain comprises the VH domain and CL domain from the second antibody, and the second polypeptide comprises the VL domain and CL domain from the second antibody.

[0109] Charge-pair mutations or complementary amino acid substitutions as described herein can be introduced into the Fab region of a first antibody (Fab1) or the Fab region of a second antibody (Fab2) to promote correct heavy-light chain pairing. For example, in some embodiments, the amino acid at EU position 38 of the VL domain of Fab1 is substituted with a negatively charged amino acid (e.g., glutamic acid) and the amino acid at EU position 39 of the VH domain of Fab1 is substituted with a positively charged amino acid (e.g., lysine). In other embodiments, the amino acid at EU position 38 of the VL domain of Fab1 is substituted with a positively charged amino acid (e.g., lysine) and the amino acid at EU position 39 of the VH domain of Fab1 is substituted with a negatively charged amino acid (e.g., glutamic acid). In certain embodiments, the amino acid at EU position 38 of the VL domain of Fab2 is substituted with a negatively charged amino acid (e.g., glutamic acid) and the amino acid at EU position 39 of the VH domain of Fab2 is substituted with a positively charged amino acid (e.g., lysine). In other embodiments, the amino acid at EU position 38 of the VL domain of Fab2 is substituted with a positively charged amino acid (e.g., lysine) and the amino acid at EU position 39 of the VH domain of Fab2 is substituted with a negatively charged amino acid (e.g., glutamic acid).

[0110] In embodiments in which a VH-CH1 region (i.e., an Fd fragment) from a second antibody is fused to the heavy chain of a first antibody, the heavy chain from the first antibody comprises a S183E mutation (EU numbering), the light chain from the first antibody comprises a S176K mutation (EU numbering), the light chain from the second antibody comprises a S176E mutation (EU numbering), and the Fd region from the second antibody (which is fused to the C-terminus of the heavy chain from the first antibody) comprises a S183K mutation (EU numbering). In other embodiments, the heavy chain from a first antibody comprises a G44E mutation (EU) and a S183E mutation (EU numbering), the light chain from a first antibody comprises a G100K mutation (EU) and a S176K mutation (EU numbering), the light chain from a second antibody comprises a G100E mutation (EU) and a S176E mutation (EU numbering), and the Fd region from the second antibody (which is fused to the C-terminus of the heavy chain from the first antibody) comprises a G44K mutation (EU) and a S183K mutation (EU numbering). The charges in the foregoing examples can also be reversed, as long as the charges on the corresponding light or heavy chain are also reversed so that the correct heavy / light chain pair has the opposite charge.

[0111] "Corresponding to" in reference to the VH2 and second CH1 domain means that, if no linker is present, the amino acid residues of the VH2 and second CH1 domain are counted from the C-terminus of the first heavy chain. If a peptide linker is present, the amino acid residues of the VH2 and second CH1 domain are counted from the C-terminus of the peptide linker. In either case, amino acid residues are not counted from the N-terminus of the first heavy chain. Rather, in the case of the VH2 and second CH1 domain, counting begins with the first amino acid residue of the VH2 domain. Counting of amino acid residues is done according to EU or AHo conventions.

[0112] In certain embodiments, a) VH1 comprises a Q39E mutation and the first CH1 domain comprises a S183K mutation (using EU numbering); b) VH2 comprises a Q39K mutation and the second CH1 domain comprises a S183E mutation (using EU numbering); c) VL1 comprises a Q38K mutation and the first CL domain comprises a S176E mutation (using EU numbering); and d) VL2 comprises a Q38E mutation and the second CL domain comprises a S176K mutation (using EU numbering).

[0113] In certain embodiments, a) the first CH1 domain comprises G44E and S183K mutations (using EU numbering); b) the second CH1 domain comprises G44K and S183E (using EU numbering); c) the first CL domain comprises G100K and S176E mutations (using EU numbering); and d) the second CL domain comprises G100E and S176K mutations (using EU numbering).

[0114] In certain embodiments, a) VH1 comprises a Q39K mutation and the first CH1 domain comprises a S183E mutation (using EU numbering); b) VH2 comprises a Q39E mutation and the second CH1 domain comprises a S183K mutation (using EU numbering); c) VL1 comprises a Q38E mutation and the first CL domain comprises a S176K mutation (using EU numbering); and d) VL2 comprises a Q38K mutation and the second CL domain comprises a S176E mutation (using EU numbering).

[0115] In certain embodiments, a) the first CH1 domain comprises G44K and S183E mutations (using EU numbering); b) the second CH1 domain comprises G44E and S183K (using EU numbering); c) the first CL domain comprises G100E and S176K mutations (using EU numbering); and d) the second CL domain comprises G100K and S176E mutations (using EU numbering).

[0116] In certain embodiments, the first heavy chain is fused to VH2 via a peptide linker, hi certain embodiments, the peptide linker comprises a sequence selected from the group consisting of (Gly3Ser)2, (Gly4Ser)2, (Gly3Ser)3, (Gly4Ser)3, (Gly3Ser)4, (Gly4Ser)4, (Gly3Ser)5, (Gly4Ser)5, (Gly3Ser)6, and (Gly4Ser)6. These sequences can also be written as GGGSGGGS (SEQ ID NO: 12), GGGGSGGGGS (SEQ ID NO: 13), GGGSGGGSGGGS (SEQ ID NO: 14), GGGGSGGGGSGGGGS (SEQ ID NO: 15), GGGSGGGSGGGSGGGS (SEQ ID NO: 16), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 18), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 19), GGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 20) and GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 21).

[0117] Additionally, or alternatively, correct heavy-light chain pairing can be promoted by swapping the CH1 and CL domains in the carboxyl-terminal Fab-binding domain. As an example, a first polypeptide fused to the carboxyl terminus of a heavy chain may comprise the VL and CH1 domains from a second antibody, and a second polypeptide may comprise the VH and CL domains from a second antibody. In another embodiment, a first polypeptide fused to the carboxyl terminus of a heavy chain may comprise the VH and CL domains from a second antibody, and a second polypeptide may comprise the VL and CH1 domains from a second antibody.

[0118] The heavy chain constant region or Fc region of the bispecific antigen-binding proteins described herein may contain one or more amino acid substitutions that affect the glycosylation and / or effector function of the antigen-binding protein. One of the functions of the Fc region of an immunoglobulin is to signal to the immune system when the immunoglobulin binds to its target. This is commonly referred to as "effector function." Signaling leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through binding of the Fc region to Fc receptors on the surface of cells of the immune system. CDC is mediated through binding of Fc to proteins of the complement system, such as C1q. In some embodiments, the bispecific antigen-binding proteins of the invention contain one or more amino acid substitutions in the constant region that enhance effector function, including ADCC activity, CDC activity, ADCP activity, and / or the clearance or half-life of the antigen-binding protein. Exemplary amino acid substitutions (EU numbering) that can enhance effector function include, but are not limited to, E233L, L234I, L234Y, L235S, G236A, S239D, F243L, F243V, P247I, D280H, K290S, K290E, K290N, K290Y, R292P, E294L, Y296W, S298A, S29 8D, S298V, S298G, S298T, T299A, Y300L, V305I, Q311M, K326A, K326E, K326W, A330S, A330L, A330M, A330F, I332E, D333A, E333S, E333A, K334A, K334V, A339D, A339Q, P396L, or any combination thereof.

[0119] In other embodiments, the bispecific antigen binding proteins of the invention comprise one or more amino acid substitutions in the constant region that reduce effector function. Exemplary amino acid substitutions (EU numbering) that can reduce effector function include, but are not limited to, C220S, C226S, C229S, E233P, L234A, L234V, V234A, L234F, L235A, L235E, G237A, P238S, S267E, H268Q, N297A, N297G, V309L, E318A, L328F, A330S, A331S, P331S, or any combination thereof.

[0120] Glycosylation can contribute to the effector function of antibodies, particularly IgG1 antibodies. Thus, in some embodiments, bispecific antigen-binding proteins of the invention may contain one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, and xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0121] In certain embodiments, glycosylation of the bispecific antigen-binding proteins described herein is increased by adding one or more glycosylation sites, for example, to the Fc region of the binding protein. Addition of glycosylation sites to the antigen-binding protein can be conveniently achieved by modifying the amino acid sequence to contain one or more of the tripeptide sequences described above (in the case of N-linked glycosylation sites). Modifications can also be made by adding to or substituting one or more serine or threonine residues in the starting sequence (in the case of O-linked glycosylation sites). To facilitate this, the antigen-binding protein amino acid sequence can be modified by changes at the DNA level, in particular by mutating the DNA encoding the target polypeptide at preselected base positions to generate codons that translate into the desired amino acids.

[0122] The present invention also encompasses the production of bispecific antigen-binding protein molecules with altered effector activity as a result of modified carbohydrate structures, e.g., antigen-binding proteins with no or reduced fucosylation and improved ADCC activity. Various methods for reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by binding of antibody molecules to the FcγRIII receptor, which has been shown to depend on the carbohydrate structure of N-linked glycosylation at residue N297 in the CH2 domain. Nonfucosylated antibodies bind to this receptor with high affinity and elicit FcγRIII-mediated effector function more efficiently than naturally fucosylated antibodies. For example, recombinant production of nonfucosylated antibodies in CHO cells in which the alpha-1,6-fucosyltransferase enzyme has been knocked out results in antibodies with a 100-fold increase in ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. 87(5):614-22, 2004). A similar effect can be achieved by reducing the activity of alpha-1,6-fucosyltransferase enzymes or other enzymes in the fucosylation pathway, for example, by siRNA or antisense RNA treatment, engineering cell lines to knock out the enzymes, or culturing with selective glycosylation inhibitors (see Rothman et al., Mol Immunol. 26(12):1113-23, 1989). Some host cell lines, such as the Lec13 or rat hybridoma YB2 / 0 cell lines, naturally produce antibodies with lower fucosylation levels (see Shields et al., J Biol Chem. 277(30):26733-40, 2002 and Shinkawa et al., J Biol Chem. 278(5):3466-73, 2003). Increasing the level of bisected carbohydrate, for example by recombinantly producing antibodies in cells overexpressing the GnTIII enzyme, has also been shown to increase ADCC activity (see Umana et al., Nat Biotechnol. 17(2):176-80, 1999).

[0123] In other embodiments, glycosylation of the bispecific antigen-binding proteins described herein is reduced or eliminated by removing one or more glycosylation sites, for example, from the Fc region of the binding protein. N-linked glycosylation of antigen-binding proteins can be reduced or eliminated by amino acid substitutions that eliminate or alter N-linked glycosylation sites. In certain embodiments, the bispecific antigen-binding proteins described herein comprise a mutation at position N297 (EU numbering), such as N297Q, N297A, or N297G. In certain embodiments, the bispecific antigen-binding proteins described herein comprise mutations at positions L234 and L235 (EU numbering), such as L234A and L235A. In one particular embodiment, the bispecific antigen-binding proteins of the invention comprise an Fc region derived from a human IgG1 antibody with an N297G mutation. To improve the stability of molecules containing the N297 mutation, the Fc region of the molecule may be further engineered. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine to promote disulfide bond formation in the dimeric state. Thus, residues corresponding to V259, A287, R292, V302, L306, V323, or I332 (EU numbering) in the IgG1 Fc region may be substituted with cysteine. In one embodiment, specific pairs of residues are substituted with cysteine to preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond disruption. In certain embodiments, pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In certain embodiments, the bispecific antigen-binding proteins described herein comprise an Fc region derived from a human IgG1 antibody with the mutations R292C and V302C. In such embodiments, the Fc region may also comprise a N297G mutation.

[0124] It may also be desirable to modify the bispecific antigen binding proteins of the invention to extend their serum half-life, for example by incorporating or adding a salvage receptor binding epitope (e.g., by mutating the appropriate region, or by incorporating the epitope into a peptide tag which is then fused to the antigen binding protein at either end or in the middle, for example by DNA or peptide synthesis; see, for example, WO 96 / 32478), or by the addition of a molecule such as PEG or other water soluble polymer, e.g., a polysaccharide polymer. The salvage receptor binding epitope preferably constitutes a region in which any one or more amino acid residues from one or two loops of the Fc region are transferred to analogous positions in the antigen binding protein. In one embodiment, three or more residues from one or two loops of the Fc region are transferred. In one embodiment, the epitope is taken from the CH2 domain of the Fc region (e.g., an IgG Fc region) and transferred to the CH1, CH3 or VH region, or two or more such regions, of the antigen binding protein. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL or VL region, or both, of the antigen binding protein. See International Applications WO 97 / 34631 and WO 96 / 32478 for a description of Fc variants and their interaction with salvage receptors.

[0125] The present invention includes one or more isolated nucleic acids encoding the bispecific antigen-binding proteins and components thereof described herein. Nucleic acid molecules of the present invention include DNA and RNA in both single- and double-stranded form, as well as corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Nucleic acid molecules of the present invention include combinations of full-length genes or cDNA molecules and fragments thereof. In one embodiment, nucleic acids of the present invention are derived from human sources, although the present invention also includes those derived from non-human species.

[0126] The relevant amino acid sequence from an immunoglobulin or a region thereof (e.g., variable region, Fc region, etc.) or polypeptide of interest can be determined by direct protein sequencing, and suitable encoding nucleotide sequences can be designed according to a universal codon table. Alternatively, genomic DNA or cDNA encoding a monoclonal antibody, from which the binding domain of a bispecific antigen-binding protein of the invention may be derived, can be isolated from cells producing such an antibody and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody).

[0127] An "isolated nucleic acid," as used interchangeably herein with an "isolated polynucleotide," is a nucleic acid that, in the case of a nucleic acid isolated from a naturally occurring source, is separated from adjacent gene sequences present in the genome of the organism from which the nucleic acid is isolated. For example, in the case of a nucleic acid that is enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid resulting from such a process is understood to be an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In one embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is preferably derived from DNA or RNA that has been isolated at least once in a substantially pure form and in an amount or concentration that permits identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (e.g., those reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are provided and / or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are normally present in eukaryotic genes. Sequences of non-translated DNA can be present 5' or 3' from the open reading frame, provided that this does not interfere with manipulation or expression of the coding region. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence described herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of production of a nascent RNA transcript from 5' to 3' is referred to as the transcription direction, and the region of the DNA strand having the same sequence as the RNA transcript that is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand having the same sequence as the RNA transcript that is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0128] Variants of the antigen-binding proteins described herein can be prepared by site-directed mutagenesis of nucleotides in the DNA encoding the polypeptide, using cassette or PCR mutagenesis, or other techniques well known in the art, to generate DNA encoding the variant, followed by expressing the recombinant DNA in cell culture as outlined herein. However, antigen-binding proteins containing variant CDRs having up to about 100-150 residues can be prepared by in vitro synthesis using established techniques. The variants typically exhibit qualitative biological activity, e.g., antigen binding, similar to the natural analog. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antigen-binding protein. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct retains the desired properties. Amino acid changes may also alter post-translational processing of the antigen-binding protein, such as by changing the number or position of glycosylation sites. In certain embodiments, antigen-binding protein variants are prepared with the aim of modifying amino acid residues directly involved in epitope binding. In other embodiments, modification of residues not directly involved in epitope binding or not involved in epitope binding at all is desirable for the purposes described herein. Mutagenesis in either the CDR and / or framework regions is contemplated. To design useful modifications in the amino acid sequence of an antigen-binding protein, one skilled in the art can use covariance analysis techniques.See, e.g., Chourier, et al., Proteins 41:475-484, 2000; Demarest et al., J. Mol. Biol. 335:41-48, 2004; Hugo et al., Protein Engineering 16(5):381-86, 2003; Aurora et al., U.S. Patent Application Publication No. 2008 / 0318207A1; Glaser et al., U.S. Patent Application Publication No. 2009 / 0048122A1; Urech et al., WO 2008 / 110348A1; Borras et al., WO 2009 / 000099A2. Such modifications, as determined by covariance analysis, can improve the potency, pharmacokinetic, pharmacodynamic, and / or manufacturability properties of the antigen binding protein.

[0129] Nucleic Acid Sequences of the Invention. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a large number of nucleic acids can be generated, all of which encode the CDRs of the invention (as well as the heavy and light chains, or other components, of the antigen binding proteins described herein). Thus, having identified a particular amino acid sequence, one skilled in the art could generate any number of different nucleic acids by simply altering the sequence of one or more codons in a way that does not change the amino acid sequence of the encoded protein.

[0130] The present invention also includes vectors comprising one or more nucleic acids encoding one or more components of the bispecific antigen-binding proteins of the present invention (e.g., variable regions, light chains, heavy chains, modified heavy chains, and Fd fragments). The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information to a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. The term "expression vector" or "expression construct," as used herein, refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of an operably linked coding sequence in a particular host cell. Expression vectors can include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of the coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and optionally other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence, optionally encoded by the expression vector, can also be operably linked to the coding sequence of interest, allowing the recombinant host cell to secrete the expressed polypeptide, if desired, so that the polypeptide of interest can be more easily isolated from the cell. For example, in some embodiments, a signal peptide sequence can be added / fused to the amino terminus of any of the polypeptide sequences of the invention. In a specific embodiment, a signal peptide having the amino acid sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 22) is fused to the amino terminus of any of the polypeptide sequences of the invention. In other embodiments, a signal peptide having the amino acid sequence MAWALLLLTLLTQGTGSWA (SEQ ID NO: 23) is fused to the amino terminus of any of the polypeptide sequences of the invention.In yet other embodiments, a signal peptide having the amino acid sequence MTCSPLLLTLLIHCTGSWA (SEQ ID NO:24) is fused to the amino terminus of any of the polypeptide sequences of the invention. Other suitable signal peptide sequences that may be fused to the amino terminus of the polypeptide sequences described herein include MEAPAQLLFLLLLWLPDTTG (SEQ ID NO:25), MEWTWRVLFLVAAATGAHS (SEQ ID NO:26), METPAQLLFLLLLWLPDTTG (SEQ ID NO:27), METPAQLLFLLLLWLPDTTG (SEQ ID NO:28), MKHLWFFLLLVAAPRWVLS (SEQ ID NO:29), and MEWSWVFLFFLSVTTGVHS (SEQ ID NO:30). Other signal peptides are known to those of skill in the art and can be fused to any of the polypeptide chains of the invention to, for example, facilitate or optimize expression in a particular host cell.

[0131] Expression vectors used in host cells to produce the bispecific antigen binding proteins of the invention typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences encoding the components of the bispecific antigen binding protein. Such sequences, collectively referred to as "flanking sequences," typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

[0132] Optionally, the vector can contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence, which oligonucleotide tag sequence encodes polyHis (such as hexaHis), FLAG, HA (influenza virus hemagglutinin), myc, or another "tag" molecule for which a commercially available antibody exists. This tag is typically fused to the polypeptide upon expression and can serve as a means for affinity purification or detection of the polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as using specific cleavage peptidases.

[0133] Flanking sequences can be homologous (i.e., derived from the same species and / or strain as the host cell), heterologous (i.e., derived from a species other than the host cell's species or strain), hybrid (i.e., a combination of flanking sequences derived from two or more sources), synthetic, or natural. Thus, the source of the flanking sequences can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are functional in and can be activated by the host cell's machinery.

[0134] Flanking sequences useful in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and / or restriction endonuclease digestion. They can then be isolated from a suitable tissue source using appropriate restriction endonucleases. In some cases, the entire nucleotide sequence of the flanking sequence may be known. In this case, the flanking sequence can be synthesized using conventional methods of nucleic acid synthesis or cloning.

[0135] Whether all or only a portion of the flanking sequence is known, the flanking sequence can be obtained using polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes, such as oligonucleotides and / or flanking sequence fragments from the same or another species. If the flanking sequence is unknown, a fragment of DNA containing the flanking sequence can be isolated from a larger piece of DNA that may contain, for example, a coding sequence or another gene. Isolation can be achieved by generating the appropriate DNA fragment by restriction endonuclease digestion, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to those skilled in the art. The selection of appropriate enzymes to accomplish this purpose will be readily apparent to those skilled in the art.

[0136] Origins of replication are usually a part of commercially available prokaryotic expression vectors; this origin aids in the amplification of the vector within a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomavirus, such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (e.g., the SV40 origin is often used only because it also contains the viral early promoter).

[0137] A transcription termination sequence is typically located 3' to the end of a polypeptide coding region and functions to terminate transcription. In prokaryotic cells, this sequence is typically a GC-rich fragment followed by a poly-T sequence. This sequence can be easily cloned from a library or purchased commercially as part of a vector, or it can be easily synthesized using known nucleic acid synthesis methods.

[0138] A selectable marker gene encodes a protein necessary for the survival and growth of host cells grown in selective culture media. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, on prokaryotic host cells, (b) complement an auxotrophic deficiency of the cells, or (c) supply critical nutrients unavailable from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene can also be used for selection of both prokaryotic and eukaryotic host cells.

[0139] Other selection genes may be used to amplify the expressed gene. Amplification is the process by which genes required for the production of proteins important for growth or cell survival are tandemly repeated in the chromosomes of recombinant cells in successive generations. Examples of suitable selection markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure in which only the transformants are adapted to survive due to the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thereby leading to the amplification of both the selection gene and DNA encoding another gene, such as one or more components of the bispecific antigen-binding protein described herein. As a result, large amounts of polypeptide are synthesized from the amplified DNA.

[0140] A ribosome binding site is usually required for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions may be operably linked to an internal ribosome binding site (IRES), allowing translation of two open reading frames from a single RNA transcript.

[0141] In cases where glycosylation is desired in eukaryotic host cell expression systems, various pre- or pro-sequences can be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be modified or a pro-sequence can be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids at position -1 (relative to the first amino acid of the mature protein) that are incident to expression and may not be completely removed. For example, the final protein product may have one or two amino acid residues at the peptidase cleavage site attached to the amino terminus. Alternatively, the use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide when the enzyme cleaves at such a region within the mature polypeptide.

[0142] Expression and cloning vectors of the present invention typically contain a promoter that is recognized by the host organism and operably linked to a molecule encoding a polypeptide. The term "operably linked," as used herein, refers to the joining of two or more nucleic acid sequences to produce a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a control sequence in a vector "operably linked" to a protein-coding sequence is ligated to the protein-coding sequence such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence. More specifically, a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional control elements) is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0143] A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a structural gene (generally within about 100-1000 bp) and controls the transcription of the structural gene. Traditionally, promoters are grouped into two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, transcribe genes to which they are operably linked uniformly, i.e., with little or no control over gene expression. Numerous promoters recognized by a variety of potential host cells are well known. A suitable promoter is operably linked to DNA encoding, for example, the heavy chain, light chain, modified heavy chain, or other component of a bispecific antigen-binding protein of the invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into a vector.

[0144] Suitable promoters for use with yeast hosts are also well known in the art. Advantageously, yeast enhancers are used in conjunction with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0145] Additional promoters of interest include the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310), the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445), the promoter and regulatory sequences from the metallothionine gene (Prinster et al., 1982, Nature 296:39-42), and prokaryotic promoters such as the β-lactamase promoter (Villa-Kamaroff et al., 1982, Nature 296:39-42). al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Also of interest are the following animal transcriptional regulatory regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene regulatory region, which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515), the insulin gene regulatory region, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), and the immunoglobulin gene regulatory region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region active in testis, breast, lymphocytes, and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58), alpha 1-antitrypsin gene control region active in liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta globin gene control region active in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), the myelin basic protein gene control region, which is active in oligodendrocyte cells of the brain (Readhead et al., 1987, Cell 48:703-712), the myosin light chain 2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and the gonadotropin-releasing hormone gene control region, which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0146] Enhancer sequences can be inserted into vectors to increase transcription of DNA encoding components of bispecific antigen-binding proteins (e.g., light chain, heavy chain, modified heavy chain, Fd fragment) by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase transcription. Enhancers are relatively independent of orientation and position and have been found at both the 5' and 3' ends of transcription units. Several enhancer sequences are known from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, viral enhancers are commonly used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, and adenovirus enhancers known in the art are exemplary enhancing elements for activation of eukaryotic promoters. An enhancer may be located either 5' or 3' to the coding sequence in a vector, but is generally located at a site 5' to the promoter. A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into an expression vector to promote extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cell in which the antibody is produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides are described above. Other signal peptides that function in mammalian host cells include the interleukin-7 (IL-7) signal sequence described in U.S. Pat. No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0 367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0 460 846.

[0147] The provided expression vector may be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not originally present in the vector, they may be obtained individually and ligated into the vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art. The expression vector can be introduced into a host cell, thereby producing a protein, such as a fusion protein, encoded by the nucleic acid described herein.

[0148] In certain embodiments, nucleic acids encoding different components of a bispecific antigen-binding protein of the invention may be inserted into the same expression vector. For example, a nucleic acid encoding an anti-first target antigen light chain can be cloned into the same vector as a nucleic acid encoding an anti-first target antigen heavy chain. In such embodiments, the two nucleic acids may be separated by an internal ribosome entry site (IRES) under the control of a single promoter so that the light and heavy chains are expressed from the same mRNA transcript. Alternatively, the two nucleic acids may be under the control of two separate promoters so that the light and heavy chains are expressed from two separate mRNA transcripts. In some embodiments, nucleic acids encoding the anti-first target antigen light and heavy chains are cloned into one expression vector, and nucleic acids encoding the anti-second target antigen light and heavy chains are cloned into a second expression vector.

[0149] Similarly, for an IgG-Fab bispecific antigen-binding protein, nucleic acids encoding each of the three components may be cloned into the same expression vector. In some embodiments, nucleic acids encoding the light chain of an IgG-Fab molecule and nucleic acids encoding the second polypeptide (including the other half of the C-terminal Fab domain) are cloned into one expression vector, while nucleic acids encoding the modified heavy chain (a fusion protein comprising the heavy chain and half of the Fab domain) are cloned into a second expression vector. In certain embodiments, all components of the bispecific antigen-binding proteins described herein are expressed from the same host cell population. For example, host cells are co-transfected with both expression vectors such that one cell produces all components of the bispecific antigen-binding protein, even if one or more components are cloned into separate expression vectors.

[0150] After the vectors have been constructed and one or more nucleic acid molecules encoding the components of the bispecific antigen-binding proteins described herein have been inserted into the appropriate sites in the vector(s), the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. Accordingly, the present invention encompasses isolated host cells comprising one or more expression vectors encoding the components of a bispecific antigen-binding protein. The term "host cell," as used herein, refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid and thereby expresses a gene of interest. The term includes the progeny of a parent cell, regardless of whether the morphology or genetic make-up of the progeny is identical to that of the original parent cell, so long as the gene of interest is present. In one embodiment, a host cell comprising an isolated nucleic acid of the invention operably linked to at least one expression control sequence (e.g., a promoter or enhancer) is a "recombinant host cell."

[0151] Transformation of the antigen-binding protein expression vector into the selected host cell can be achieved by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected will depend, in part, on the type of host cell used. These and other suitable methods are well known to those of skill in the art and are set forth, for example, in Sambrook et al., 2001, supra.

[0152] When cultured under appropriate conditions, the host cells will synthesize the antigen-binding protein, which can then be recovered from the culture medium (if the host cells secrete it into the medium) or directly from the host cells that produce it (if it is not secreted). The selection of an appropriate host cell depends on various factors, such as the desired expression level, modifications of the polypeptide that are desirable or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0153] Exemplary host cells include prokaryotic, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as gram-negative or gram-positive microorganisms, for example, Enterobacteriaceae, for example, Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescens, and the like. Examples of eukaryotic host microorganisms include Bacillus species, such as B. marcescens and Shigella, as well as Bacillus species, such as B. subtilis and B. licheniformis, Pseudomonas species, and Streptomyces species. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.However, Pichia species, such as P. pastoris, Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, Schwanniomyces species, such as Schwanniomyces occidentalis, Many other genera, species and strains of A. occidentalis, as well as filamentous fungi such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, e.g., A. nidulans, A. niger, etc., are commonly available and useful herein.

[0154] Host cells for expression of glycosylated antigen-binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many strains and variants of baculovirus have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various virus strains for transfection of such cells, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available.

[0155] Vertebrate host cells are also suitable hosts, and the recombinant production of antigen binding proteins from such cells is routine. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells 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); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells, and many other cell lines. In certain embodiments, cell lines may be selected by determining which cell lines have high expression levels and constitutively produce the bispecific antigen binding proteins of the invention.In another embodiment, a cell line from the B cell lineage can be selected that does not produce its own antibody but has the ability to produce and secrete heterologous antibodies, hi some embodiments, CHO cells are host cells for expressing the bispecific antigen binding proteins of the invention.

[0156] Host cells are transformed or transfected with the above-described nucleic acids or vectors for production of the bispecific antigen-binding proteins and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In addition, novel vectors and transfected cell lines carrying multiple copies of transcription units separated by a selectable marker are particularly useful for expressing antigen-binding proteins. Thus, the present invention also provides a method of preparing the bispecific antigen-binding proteins described herein, comprising culturing host cells containing one or more expression vectors described herein in a culture medium under conditions that allow expression of the bispecific antigen-binding proteins encoded by the one or more expression vectors, and recovering the bispecific antigen-binding proteins from the culture medium.

[0157] The host cells used to produce the antigen-binding proteins of the invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. Furthermore, any of the media described in Ham et al., Meth. Enz. 58:44, 1979; Barnes et al., Anal. Biochem. 102:255, 1980, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 103430, WO 87 / 00195, or U.S. Pat. Re. 30,985 can be used as a culture 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), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug Gentamicin™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary nutritional supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0158] When the host cells are cultured, the bispecific antigen-binding protein can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antigen-binding protein is produced intracellularly, the first step is to remove particulate host cell debris or lysed fragments, for example, by centrifugation or ultrafiltration. The bispecific antigen-binding protein can be purified using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, or affinity chromatography using the antigen of interest or protein A or protein G as the affinity ligand. Protein A can be used to purify proteins containing polypeptides 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 mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567-1575, 1986). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the protein contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the particular bispecific antigen-binding protein to be recovered, other techniques for protein purification, such as ethanol precipitation, reverse-phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also possible.

[0159] The bispecific antigen-binding proteins of the invention are useful for detecting target antigens in biological samples and for identifying cells or tissues that express the target antigen. The bispecific antigen-binding proteins described herein may be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with the target antigen. Also provided are methods for detecting the presence of a target antigen in a sample using classical immunohistological methods known to those skilled in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Vol. 15 (Eds. R.H. Burdon and P.H. van Knippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Target detection can be performed in vivo or in vitro.

[0160] Diagnostic applications provided herein include the use of antigen binding proteins to detect expression of target antigens. Examples of methods useful in detecting the presence of a receptor include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs).

[0161] For diagnostic applications, antigen binding proteins are typically labeled with a detectable labeling group. Suitable labeling groups include the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131Examples of secondary reporters include, but are not limited to, fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antigen-binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and may be used.

[0162] In another embodiment, the antigen binding proteins described herein can be used to identify one or more cells expressing a target antigen. In certain embodiments, the antigen binding protein is labeled with a labeling group, and binding of the labeled antigen binding protein to the target antigen is detected. In further specific embodiments, binding of the antigen binding protein to the target antigen is detected in vivo. In further specific embodiments, the antigen binding protein is isolated and measured using techniques known in the art. See, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor (ed. 1991 and periodic supplements); John E. Coligan, ed., 1993, Current Protocols in Immunology, New York: John Wiley & Sons. [Example]

[0163] Based on crystal structures of monoclonal antibodies with and without modified salt bridges, K360 has been identified as an interface residue between the two Fc chains. Initial experiments to screen for improved pairing and structure of hetero-Fc molecules were performed using a combination of a single-chain variable fragment (scFv) fused to an Fc chain lacking a warhead. When assembled into the intended hetero-Fc form, the molecular weight was approximately 75 kilodaltons (kDa), as shown in Figure 1. In contrast, when the molecule assembled into a homodimer with two scFv warheads or lacking all warheads, the molecular weights were approximately 100 kDa or 50 kDa, respectively. To generate these molecules, modified fragments were synthesized and cloned into plasmids using the Golden Gate cloning strategy. The resulting panel of recombinant molecules was produced using transient expression in HEK 293 EBNA1 cells in suspension, according to established and published methods by the National Research Council (NRCC). Six days after production, the culture medium (CM) was collected from the cell culture and subjected to Protein A purification using established procedures. In this analysis, the ratio of noncovalent aggregates to the desired species was measured using size-exclusion chromatography (SEC) according to established industrial methods. The ratio of homodimers to heterodimers was measured using a Caliper SDS microcapillary gel electrophoresis (mCE) instrument, based primarily on molecular size-dependent migration. From the initial screening of 44 variants, a set of molecules with favorable binding specificity and expression titers was selected for further optimization and analysis. The results of the lead variants in the screening are shown in Figure 2. Each of the selected variants had levels ranging from 89.4% to 94.6% compared to the WT (47.61% hetero-scFv-Fc), demonstrating improved binding specificity. The amount of hetero-scFv-Fc produced was calculated from the production titer and the percentage of the main peak calculated by mCE and shown under 75 kDa MP (mg / L) in Figure 2. By these measurements, many variants had more of the desired species than WT, and some had more of the desired species than the established variant CPMv1.

[0164] In a second experiment, the lead variants were tested with engineered monoclonal antibodies. As shown in Figure 3, the modifications included the addition of charge-pair mutations in the CH3 region and a DEVD cleavage site in the hinge between one of the Fab regions and the Fc region. This variant was used instead of heterodimerized IgG to eliminate variability introduced by nonspecific pairing between the light and heavy chains. In this experiment, production was performed using a CHO-K1 stable expression system coupled to an internal vector. Titers from these production runs ranged from 186 mg / L to 275 mg / L, demonstrating fairly low variance. Purification began with Protein A capture. This was followed by removal of HMW species by SEC according to established methods. The process was then completed with isolation from the monoclonal antibody by cation exchange (CEX) according to industry-standard techniques. Fractions were selected for pooling according to the purity of the desired product, as measured by SEC and MS. During the CEX step of purification, some variants were observed to split into multiple peaks. These peaks were analyzed by MS and were primarily species of consistent molecular weight. Furthermore, when samples from one peak were collected and reloaded onto the CEX column, they resolved into multiple peaks in the same pattern as previously observed (data not shown). This indicates that these variants possessed a degree of instability that led to conformational changes when exposed to the specific conditions of the CEX column. This phenomenon was particularly evident when the buffer used for CEX was pH 5.0, as shown in Figure 4, while the calculated pI of the molecules was in the range of 7.93. The peak distribution was more uniform when the buffer was pH 5.6 and 6.0. Furthermore, when the E357K CPM with K370D / E was used in combination with the K409D and D399K CPMs with K392D / E, the formation of multiple peaks was observed. As shown in Figure 5, when the E357K K370D / E charge pair was used in combination with the K360E variant, the degree of heterogeneous peak formation was substantially reduced, resulting in a main peak eluting at a time close to that observed at pH 5.6 and 6.0. These results identified CPMv531 and CPMv526, which contain K360E, as the most robust variants under standard purification conditions.

[0165] All publications, patents, and patent applications discussed and cited herein are hereby incorporated by reference in their entirety. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0166] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. An isolated heteromultimer comprising a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, wherein the first CH3 domain polypeptide comprises an amino acid modification at position K360; (i) the first CH3 domain polypeptide further comprises an amino acid modification at position K370; (ii) the second CH3 domain polypeptide comprises an amino acid modification at position E357; wherein the numbering of amino acid residues is according to the EU index as described in Kabat: Isolated heteromultimers.

2. 2. The isolated heteromultimer of claim 1, wherein the amino acid modification at position K360 is selected from the group consisting of K360E and K360D.

3. 3. The isolated heteromultimer of claim 1 or 2, wherein the amino acid modification at position K370 is selected from the group consisting of K370E and K370D.

4. The isolated heteromultimer according to any one of claims 1 to 3, wherein the amino acid modification at position E357 is selected from the group consisting of E357K, E357H and E357R.

5. The isolated heteromultimer according to any one of claims 1 to 4, wherein the amino acid modification at position K360 is K360E, the amino acid modification at position K370 is K370D, and the amino acid modification at position E357 is E357K.

6. The isolated heteromultimer of any one of claims 1 to 5, wherein one CH3 domain polypeptide further comprises an amino acid modification at position K409, and the other CH3 domain polypeptide further comprises an amino acid modification at position D399.

7. 7. The isolated heteromultimer of claim 6, wherein the first CH3 domain polypeptide comprises an amino acid modification at position K409 and the second CH3 domain polypeptide comprises an amino acid modification at position D399.

8. 7. The isolated heteromultimer of claim 6, wherein the first CH3 domain polypeptide comprises an amino acid modification at position D399 and the second CH3 domain polypeptide comprises an amino acid modification at position K409.

9. The amino acid modification at position K409 is selected from the group consisting of K409E and K409D, and the amino acid modification at position D399 is selected from the group consisting of D399K, D399H and D399R. The isolated heteromultimer according to any one of claims 6 to 8.

10. The isolated heteromultimer according to any one of claims 6 to 9, wherein the amino acid modification at position K409 is K409D, and the amino acid modification at position D399 is D399K.

11. The isolated heteromultimer according to any one of claims 6 to 10, wherein the CH3 domain polypeptide comprising the amino acid modification at position K409 further comprises an amino acid modification at position K392.

12. 12. The isolated heteromultimer of claim 11, wherein the amino acid modification at position K392 is selected from the group consisting of K392E and K392D.

13. The isolated heteromultimer of any one of claims 1 to 12, wherein one CH3 domain polypeptide further comprises an amino acid modification at position K439, and the other CH3 domain polypeptide further comprises an amino acid modification at position E356.

14. 14. The isolated heteromultimer of claim 13, wherein the first CH3 domain polypeptide comprises an amino acid modification at position K439 and the second CH3 domain polypeptide comprises an amino acid modification at position E356.

15. 14. The isolated heteromultimer of claim 13, wherein the first CH3 domain polypeptide comprises an amino acid modification at position E356 and the second CH3 domain polypeptide comprises an amino acid modification at position K439.

16. The amino acid modification at position K439 is selected from the group consisting of K439E and K439D, and the amino acid modification at position E356 is selected from the group consisting of E356K, E356H and E356R. The isolated heteromultimer according to any one of claims 13 to 15.

17. The isolated heteromultimer according to any one of claims 13 to 16, wherein the amino acid modification at position K439 is K439E, and the amino acid modification at position E356 is E356K.

18. 13. The isolated heteromultimer of claim 11 or 12, wherein the first CH3 domain polypeptide comprises K360E, K370D, K409D and K392D mutations, and the second CH3 domain polypeptide comprises E357K and D399K mutations.

19. 19. The isolated heteromultimer of claim 18, wherein the first CH3 domain polypeptide further comprises a K439E mutation and the second CH3 domain polypeptide further comprises an E356K mutation.

20. 20. The isolated heteromultimer of any one of claims 1 to 19, wherein the heterodimeric CH3 domain is comprised in an Fc region based on an IgG Fc region.

21. 21. The isolated heteromultimer of claim 20, wherein the IgG Fc region is an IgG1 Fc region.

22. 22. The isolated heteromultimer of any one of claims 1 to 21, wherein the heteromultimer is a bispecific or multispecific antibody.

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