Manipulation of the hinge region to promote antibody dimerization

Abstract

To provide multispecific antigen binding proteins comprising two distinct heavy chains in the hinge region that utilize charge pair mutations in order to both facilitate heterodimer formation while inhibiting homodimer formation.SOLUTION: Provided is an isolated heteromultimer, comprising: a first immunoglobulin hinge domain polypeptide comprising a plurality of specific amino acid substitutions; and a second immunoglobulin hinge domain polypeptide comprising a plurality of different specific amino acid substitutions.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals. In particular, the present invention relates to a multispecific antigen-binding protein that can specifically bind to at least two target antigens. The multispecific antigen-binding protein comprises two different heavy chains in a hinge region that utilize charge pair mutations to both promote heterodimerization and simultaneously inhibit homodimerization. [Background technology]

[0002] Antibodies have become a preferred mode of development within the biopharmaceutical industry due to several properties that attract those developing therapeutic molecules. Besides their ability to target specific structures or cells, antibodies make their targets more susceptible to Fc receptor cell-mediated phagocytosis and cell death (Raghavan and Bjorkman 1996). Furthermore, the ability of antibodies to interact with neonatal Fc receptors (FcRn) in a pH-dependent manner extends the serum half-life of antibodies (Ghetie and Ward 2000). This unique characteristic of antibodies makes it possible to extend the half-life of therapeutic proteins or peptides in serum by manipulating Fc fusion molecules.

[0003] In some cases, it is desirable to create molecules that contain the Fc portion of an antibody but also include a heterodimer. An important application of Fc heterodimer molecules is the production of multispecific antibodies, such as bispecific antibodies. A bispecific antibody is an antibody that is specific to at least two different antigens (Nolan and O'Kennedy 1990; de Leij, Molema et al. 1998; Carter 2001). Bispecific antibodies do not have identical sequences in both Fabs, but rather carry different sequences in the two Fabs, so that each arm of the Y-shaped molecule can bind to a different antigen. Another application of Fc heterodimers is to add half-life extension portions to therapeutic molecules. In such cases, one or both of the two different Fc portions can be fused to one or more therapeutic molecules that require half-life extension.

[0004] The classical method for producing Fc heterodimers was developed by Carter and his collaborators when they manipulated the heavy chain using a "knobs-into-holes" strategy for heterodimerization (Ridgway, Presta et al. 1996; Atwell, Ridgway et al. 1997; Merchant, Zhu et al. 1998; Carter 2001). The knob-into-hole concept was originally proposed by Crick as a model for packing amino acid side chains between adjacent α-helices (Crick 1952). Carter and his collaborators created knobs at the CH3 domain interface of the first chain by substituting smaller amino acid side chains with larger ones (e.g., T366Y), and created holes at parallel positions at the CH3 interface of the second chain by substituting larger amino acid side chains with smaller ones (e.g., Y407T). The fundamental principle for creating knobs and holes in parallel positions is that the interaction between knobs and holes is favorable for heterodimer formation, while the knob-knob and hole-hole interactions inhibit homodimer formation due to steric collisions and the loss of favorable interactions, respectively. Knob-into-hole mutations have also been combined with CH3 domain disulfide bond manipulation to enhance heterodimer formation (Sowdhamini, Srinivasan et al. 1989; Atwell, Ridgway et al. 1997). In addition to these mutations, the DNA input ratio has also been altered to maximize yield (Merchant, Zhu et al. 1998). The "knob-into-hole" technique is disclosed in U.S. Patent No. 5,731,168 and No. 7,183,076. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent No. 5,731,168 [Patent Document 2] U.S. Patent No. 7,183,076

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[0006]

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Summary of the Invention

Problems to be Solved by the Invention

[0007] The clinical potential of multispecific antibodies (molecules that simultaneously target multiple targets), such as bispecific and trispecific antibodies, and therapeutically active proteins with an extended half-life is highly promising for targeting complex diseases. However, in many cases, there are significant challenges in generating these molecules because specifically promoting the pairing of multiple polypeptide chains present in solution is desired. Here, the manipulation of the hinge region with a small number of mutations that can alone successfully lead to the promotion of Fc dimerization is described. [Means for solving the problem]

[0008] In one embodiment, the present invention relates to a heteropolymer that exists alone, the heteropolymer comprising a heterodimer immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, (i) The first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243K, A244K, P245K, N / E246K and L247K, and (ii) The second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243D, A244D, P245D, N / E246D and L247D, The numbering of amino acid residues follows the EU index listed in Kabat. This study focuses on heteromultimers that exist independently.

[0009] In another embodiment, the present invention relates to a heteropolymer that exists alone, the heteropolymer comprising a heterodimer immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, (i) The first immunoglobulin hinge domain polypeptide contains the following amino acid substitution: A244H, and (ii) The second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: N / E246D and L247D, The numbering of amino acid residues follows the EU index listed in Kabat. This study focuses on heteromultimers that exist independently.

[0010] In another embodiment, the present invention relates to a heteropolymer that exists alone, the heteropolymer comprising a heterodimer immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, (i) The first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: H237K, T238K, A244K and N / E246K, and (ii) The second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: H237D, T238D, A244D and N / E246D, The numbering of amino acid residues follows the EU index listed in Kabat. This study focuses on heteromultimers that exist independently.

[0011] In certain embodiments, each hinge domain polypeptide of the heteromultimer further comprises an L248C substitution.

[0012] In certain embodiments, each immunoglobulin hinge domain polypeptide further comprises a CH3 domain. In one embodiment, one CH3 domain contains the mutations F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y, and the other CH3 domain contains the K409R mutation, with the amino acid residue numbering following the EU index described in Kabat. In another embodiment, one CH3 domain contains the T366W mutation, and the other CH3 domain contains the mutations T366S, L368A, or Y407V, with the amino acid residue numbering following the EU index described in Kabat. In one embodiment, one CH3 domain contains the K / R409D and K392D mutations, and the other CH3 domain contains the D399K and E356K mutations, and the amino acid residue numbering follows the EU index described in Kabat. In another embodiment, one CH3 domain contains the Y349C mutation, and the other CH3 domain contains either the E356C or S354C mutation, and the amino acid residue numbering follows the EU index described in Kabat. In yet another embodiment, one CH3 domain contains the Y349C and T366W mutations, and the other CH3 domain contains the E356C, T366S, L368A and Y407V mutations, and the amino acid residue numbering follows the EU index described in Kabat. In one embodiment, one CH3 domain contains the Y349C and T366W mutations, and the other CH3 domain contains the S354C, T366S, L368A, and Y407V mutations, and the amino acid residue numbering follows the EU index described in Kabat.

[0013] In a particular embodiment, the immunoglobulin hinge region is the IgG1 hinge region.

[0014] In certain embodiments, the heteromultimer is a bispecific or multispecific antibody. [Brief explanation of the drawing]

[0015] [Figure 1] This represents the targeting of the hinge region for heterodimer formation. [Figure 2] The CPPC motif represents a human IgG1 crystal structure that does not show a second disulfide bond, which may be an artificial result introduced by irradiation damage during X-ray data acquisition. Furthermore, the IgG1 mouse crystal structure (1IGY) and "in-house" mass spectrometry data strongly suggest that disulfide C242 should be intact. Therefore, using the mIgG1 and huIgG2 structures as guides will deepen our understanding of the rotational isomer positions of the residues downstream of the CPPC motif. [Figure 3] This shows the sequence alignment of IgG1, IgG2, and IgG4. [Figure 4] This shows a summary table of hinge design and quality control evaluation (MSQC). [Figure 5] The charged zipper hinge design CZH01 is represented. According to the crystal structure, C239 is the only C that forms a disulfide bridge within the IgG1 hinge (Saphire & Wilson, Science, 2001 (anti-HIV-1 B12 antibody)). However, other data suggest that a second Cys (C242) can also form a disulfide bond, and that P241 also appears to be important for the same bond to occur. Therefore, in principle, we would design a CPM chain downstream of this second disulfide (see the mutation in the orange line) and then insert a novel disulfide into L248C (indicated by the dotted orange line). [Figure 6] This represents CEX and CZH01 for analytical spectrometry. [Figure 7] Charged zipper hinge - representing design CZH09. [Figure 8] This represents CEX and CZH09 for analytical spectrometry. [Figure 9] This represents structural guidance for charged zipper hinges in IgG2. [Figure 10] This represents CEX and CZH11 for analytical spectrometry. [Figure 11] This represents the targeting of the hinge region for heterodimer formation + CH3 CPM v11. [Figure 12] This table shows a summary of the hinge design +CH3-CH3'CPM v11. [Figure 13] This shows the thermal stability analysis of the hinge design. Hinge mutations do not adversely affect Ab's stability, while the CH3 CPM v11 mutation appears to lower Tm by approximately 2 degrees. [Modes for carrying out the invention]

[0016] In one embodiment, the present invention relates to a heteropolymer that exists alone, the heteropolymer comprising a heterodimer immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, (i) The first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243K, A244K, P245K, N / E246K and L247K, and (ii) The second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243D, A244D, P245D, N / E246D and L247D, The numbering of amino acid residues follows the EU index listed in Kabat. This study focuses on heteromultimers that exist independently.

[0017] In another embodiment, the present invention relates to a heteropolymer that exists alone, the heteropolymer comprising a heterodimer immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, (i) The first immunoglobulin hinge domain polypeptide contains the following amino acid substitution: A244H, and (ii) The second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: N / E246D and L247D, The numbering of amino acid residues follows the EU index listed in Kabat. This study focuses on heteromultimers that exist independently.

[0018] In another embodiment, the present invention relates to a heteropolymer that exists alone, the heteropolymer comprising a heterodimer immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, (i) The first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: H237K, T238K, A244K and N / E246K, and (ii) The second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: H237D, T238D, A244D and N / E246D, The numbering of amino acid residues follows the EU index listed in Kabat. This study focuses on heteromultimers that exist independently.

[0019] In certain embodiments, each hinge domain polypeptide of the heteromultimer further comprises an L248C substitution.

[0020] In certain embodiments, each immunoglobulin hinge domain polypeptide further comprises a CH3 domain. In one embodiment, one CH3 domain contains the mutations F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y, and the other CH3 domain contains the K409R mutation, with the amino acid residue numbering following the EU index described in Kabat. In another embodiment, one CH3 domain contains the T366W mutation, and the other CH3 domain contains the mutations T366S, L368A, or Y407V, with the amino acid residue numbering following the EU index described in Kabat. In one embodiment, one CH3 domain contains the K / R409D and K370E mutations, and the other CH3 domain contains the D399K and E357K mutations, and the amino acid residue numbering follows the EU index described in Kabat.

[0021] In certain embodiments, the heterodimer antibody comprises a first heavy chain containing charged amino acids at positions 392 and 409 (e.g., substitutions of K392D and K409D), and a second heavy chain containing positively charged amino acids at positions 356 and 399 (e.g., substitutions of E356K and D399K). In other specific embodiments, the heterodimer antibody comprises a first heavy chain containing charged amino acids at positions 392, 409 and 370 (e.g., substitutions of K392D, K409D and K370D), and a second heavy chain containing positively charged amino acids at positions 356, 399 and 357 (e.g., substitutions of E356K, D399K and E357K). In related embodiments, the first heavy chain is derived from an anti-CGRP receptor antibody, and the second heavy chain is derived from an anti-PAC1 receptor antibody. In other related embodiments, the first heavy chain is derived from an anti-PAC1 receptor antibody, and the second heavy chain is derived from an anti-CGRP receptor antibody.

[0022] In one embodiment, one CH3 domain contains the Y349C mutation, and the other CH3 domain contains either the E356C or S354C mutation, and the amino acid residue numbering follows the EU index described in Kabat. In another embodiment, one CH3 domain contains the Y349C and T366W mutations, and the other CH3 domain contains the E356C, T366S, L368A, and Y407V mutations, and the amino acid residue numbering follows the EU index described in Kabat. In yet another embodiment, one CH3 domain contains the Y349C and T366W mutations, and the other CH3 domain contains the S354C, T366S, L368A, and Y407V mutations, and the amino acid residue numbering follows the EU index described in Kabat.

[0023] In a particular embodiment, the immunoglobulin hinge region is the IgG1 hinge region.

[0024] In certain embodiments, the heteromultimer is a bispecific or multispecific antibody.

[0025] As used herein, the term “antigen-binding protein” refers to a protein that specifically binds to one or more target antigens. Antigen-binding proteins may include antibodies and their functional fragments. A “functional antibody fragment” is a portion of an antibody that lacks at least some of the amino acids present in the full-length heavy and / or light chain, but is still capable of specifically binding to an antigen. Examples of 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 may originate from any mammalian source, such as humans, mice, rats, rabbits, or camelids. Functional antibody fragments can be comparable to intact antibodies in terms of target antigen binding, and fragments may be produced by modifying intact antibodies (e.g., enzymatic or chemical cleavage) or newly synthesized using recombinant DNA technology or peptide synthesis.

[0026] Antigen-binding proteins may also include proteins containing one or more functional antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, antigen-binding proteins include, but are not limited to, single-chain Fv (scFv), diabodies (see, e.g., European Patent No. 404,097, International Publication No. 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 linked by a peptide linker, see Ward et al., Nature, Vol. 341:544-546, 1989); and maxibodies (two scFv 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 Sciences). See Methods, Vol.251:123-135, 2001); triabody; tetrabody; minibody (scFv fused to the CH3 domain, see Olafsen et al., Protein Eng Des Sel., Vol.17:315-23, 2004); peptidebody (one or more peptides attached to the Fc region, see International Publication No. 00 / 24782); linear antibody (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide, see Zapata et al., Protein Examples include small modular immunotherapies (see Eng., Vol.8:1057-1062, 1995); and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0027] "Multispecificity" means that an antigen-binding protein can specifically bind to two or more different antigens. "Bispecificity" means that an antigen-binding protein can specifically bind to two different antigens. As used herein, an antigen-binding protein has a significantly higher binding affinity for a target antigen compared to its affinity for other unrelated proteins under similar binding assay conditions, and as a result, can distinguish the antigen, and thus "specifically binds" to the target antigen. An antigen-binding protein that specifically binds to an antigen can have an equilibrium dissociation constant (K D ) ≤ 1×10 -6 M. An antigen-binding protein specifically binds to an antigen with "high affinity" when K D is ≤ 1×10 -8 M.

[0028] Affinity is determined using various techniques, an example of which is an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., an assay using BIAcore®). Using this method, the association rate constant (k a , units: M -1 s -1 ) and the dissociation rate constant (k d , units: s -1 ) can be measured. Then, the equilibrium dissociation constant (K D , units: M) can be calculated from the ratio of the kinetic rate constants (k d / k a ). In some embodiments, affinity is determined by a kinetic method such as the Kinetic Exclusion Assay (KinExA) described in Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008. Using the KinExA assay, the equilibrium dissociation constant (K D , units: M) and the association rate constant (k a , units: M -1 s -1 ) can be measured. The dissociation rate constant (k d , units: s-1 ) are these values ​​(K D ×k a It can be calculated from ). In other embodiments, affinity is determined by the equilibrium / solution method. In certain embodiments, affinity is determined by the FACS binding assay.

[0029] In some embodiments, the bispecific antigen-binding protein described herein is k d The bond affinity, measured by the (dissociation rate constant), is approximately 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 s -1 The following (lower values ​​indicate higher bond affinity), and / or K D The binding affinity, measured by the equilibrium dissociation constant, is approximately 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 It exhibits desirable characteristics such as M or less (a lower value indicates higher binding affinity).

[0030] As used herein, the term "antigen-binding domain," used interchangeably with "binding domain," refers to a region of an antigen-binding protein containing amino acid residues that interact with the antigen and confer specificity and affinity to that antigen to the antigen-binding protein.

[0031] As used herein, the term "CDR" refers to the complementarity-determining region (also called the "minimum 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 a framework region to form a structure that specifically binds to a specific epitope or domain of the target protein. From the N-terminus to the C-terminus, both naturally occurring light-chain and heavy-chain variable regions typically correspond to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering scheme has been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering scheme is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity-determining region (CDR) and framework region (FR) of a given antibody can be identified using this scheme.

[0032] In some embodiments of the bispecific antigen-binding proteins of the present invention, the binding domain comprises Fab, Fab', F(ab')2, Fv, a single-chain variable fragment (scFv), or a nanobody. In one embodiment, both binding domains are Fab fragments. In another embodiment, one binding domain is a Fab fragment and the other binding domain is an scFv.

[0033] When an antibody is digested with papain, two identical antigen-binding fragments called "Fab" fragments (each possessing a single antigen-binding site) and the remainder being an "Fc" fragment (containing the immunoglobulin constant region) are produced. The Fab fragment contains the variable domain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the "Fab fragment" consists of one immunoglobulin light chain (variable region (VL) and constant region (CL)) and the CH1 region and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. The Fc fragment is a carbohydrate and is involved in many antibody effector functions (such as complement binding and cell receptors) that distinguish one class of antibody from another. The "Fd fragment" contains the VH domain and CH1 domain derived from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0034] A "Fab fragment" is a Fab fragment having one or more cysteine ​​residues derived from the antibody hinge region at the C-terminus of the CH1 domain.

[0035] The "F(ab')2 fragment" is a divalent fragment containing two Fab' fragments linked by disulfide bridges between heavy chains in the hinge region.

[0036] The "Fv" fragment is the smallest fragment containing a complete antigen recognition and binding site derived from the antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in a tight, non-cohesive 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 the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, but its affinity is lower than that of the entire binding site containing both VH and VL.

[0037] A "single-chain variable antibody fragment" or "scFv fragment" comprises a VH region and a VL region of the antibody, which are present on a single polypeptide chain and optionally include a peptide linker between the VH and VL regions, allowing Fv to form a desired structure for antigen binding (see, for example, Bird et al., Science, Vol.242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol.85:5879-5883, 1988).

[0038] A "nanobody" is the heavy chain variable region of a heavy chain antibody. Such a variable domain is the smallest fully functional antigen-binding fragment of such a heavy chain antibody, with a molecular mass of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking a light chain occur naturally in certain animal species, e.g., nurse sharks, sable sharks, and camelids (Camelidae), e.g., camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is a single-domain VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding site; that is, these functional antibodies are homodimers of the heavy chain (referred to as "heavy chain antibodies" or "HCAbs") that have only the structure H2L2. Camelized VHH, according to reports, contains a hinge domain, a CH2 domain, and a CH3 domain, and recombines with the IgG2 and IgG3 constant regions, which lack a CH1 domain. Camelized VHH domains have been shown to bind to antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276: 26285-90, 2001) and exhibit high stability in solution (Ewert et al., Biochemistry, Vol. 41: 3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Publication Nos. 2005 / 0136049 and 2005 / 0037421. Another scaffold can be fabricated from human variable-like domains that more closely conform to the shark V-NAR scaffold and may provide a framework of long, penetrating loop structures.

[0039] In certain embodiments of the bispecific antigen-binding protein of the present invention, the binding domain comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) of an antibody or antibody fragment that specifically binds to a desired antigen.

[0040] In this specification, the term "variable region," used interchangeably with "variable domain" (variable region of the light chain (VL), variable region of the heavy chain (VH)), refers to a region in the immunoglobulin light chain and immunoglobulin heavy chain, respectively, that is directly involved in the binding of the antibody to the 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 whose sequences are widely conserved and linked by three CDRs. The framework regions employ a β-sheet structure, and the CDRs can form loops linking the β-sheet structures. The CDRs in each chain are held in their three-dimensional structure by the framework regions and, together with the CDRs of the other chain, form an antigen-binding site.

[0041] The binding domain that specifically binds to the target antigen may be derived from a) known antibodies against these antigens, or b) novel antibodies or antibody fragments obtained by novel immunization methods using the antigen protein or a fragment thereof, by phage display, or by other conventional methods. The antibody from which the binding domain of the bispecific antigen-binding protein originates may be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the antibody from which the binding domain originates is a monoclonal antibody. In these or other embodiments, the antibody may be a human antibody or a humanized antibody, and may be of type IgG1, IgG2, IgG3, or IgG4.

[0042] The term “monoclonal antibody” (or “mAb”), as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for any innate variations that may be present in small amounts. Monoclonal antibodies are highly specific and are typically directed at individual antigen sites or epitopes, in contrast to polyclonal antibody preparations, which typically contain various antibodies against various epitopes. Monoclonal antibodies can be produced using any technique known in the art, for example, by immortalizing spleen cells isolated from transgenic animals after completion of an immunization schedule. Spleen cells can be immortalized using any technique known in the art, for example, by fusing spleen cells with myeloma cells to produce hybridomas. Myeloma cells for use in the fusion procedure to produce hybridomas are preferably non-antibody-producing, highly fusion-efficient, and enzyme-deficient, making them unable to grow in certain selective media that support the growth of only the desired fusion cells (hybridoms). Examples of cell lines suitable for use in mouse fusion 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 / 5XXOBul. Examples of cell lines used in rat fusion include R210.RCY3, Y3-Ag1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion include U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0043] In some cases, hybridoma cell lines are produced by immunizing animals (e.g., transgenic animals possessing human immunoglobulin sequences) with a target antigen, collecting spleen cells from the immunized animals, fusing the collected spleen cells with myeloma cell lines, thereby generating hybridoma cells, establishing hybridoma cell lines from the hybridoma cells, and identifying hybridoma cell lines that produce antibodies binding to the target antigen.

[0044] Monoclonal antibodies secreted by hybridoma cell lines can be purified using any technique known in the art, such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Hybridomas or mAbs can be further screened to identify mAbs with specific properties, such as the ability to bind to cells expressing a target antigen, the ability to block or interfere with the binding of a target antigen ligand to its respective receptor, or the ability to functionally block either receptor, using, for example, a cAMP assay.

[0045] In some embodiments, the binding domain of the bispecific antigen-binding protein of the present invention may be derived from a humanized antibody. A "humanized antibody" refers to an antibody modified so that a region (e.g., a framework region) includes a region derived from a corresponding human immunoglobulin. Generally, humanized antibodies can be produced from monoclonal antibodies initially made in non-human animals. Typically, certain amino acid residues of this monoclonal antibody, derived from the non-antigen-recognition portion of the antibody, are modified to be homologous to the corresponding residues in the corresponding isotype of human antibody. Humanization can be carried out, for example, by replacing at least a portion of the variable region of a rodent with the corresponding region of a human antibody using various methods (see, for example, U.S. Patent 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). CDRs of the light chain and heavy chain variable regions of antibodies produced in another species can be transplanted into consensus human FRs. To create consensus human FRs, a consensus amino acid sequence can be identified by aligning FRs derived from multiple human heavy chain amino acid sequences or human light chain amino acid sequences.

[0046] Novel antibodies produced against target antigens that can be the origin of the binding domain of the bispecific antigen-binding protein of the present invention can be fully human antibodies. A "fully human antibody" is an antibody that includes a variable region and a constant region that are derived from or represent a human germline immunoglobulin sequence. One specific means provided for carrying out the production of fully human antibodies is the "humanization" of the mouse humoral immune system. Introducing a human immunoglobulin (Ig) locus into a mouse in which the endogenous Ig gene has been inactivated is one means of producing fully human monoclonal antibodies (mAbs) in a mouse, an animal that can be immunized with any desired antigen. Using fully human antibodies can minimize immunogenic and allergic responses that may occur when mouse mAbs or mouse-derived mAbs are administered to humans as therapeutic agents.

[0047] 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, for example, 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, the transgenic animal is created by deactivating the endogenous mouse immunoglobulin loci encoding the mouse immunoglobulin heavy and light chains and inserting a large fragment of human genomic DNA containing loci encoding human heavy and light chain proteins into the mouse genome. Next, partially modified animals having less complement than the complete complement of the human immunoglobulin locus are crossbred to obtain animals possessing all the desired immunosystem modifications. Upon administration of the immunogen, these transgenic animals produce antibodies that are immunospecific to the immunogen but have a human amino acid sequence, including the variable region, rather than a mouse amino acid sequence. For further details of such methods, see, for example, International Publication No. 96 / 33735 and International Publication No. 94 / 02602.Further methods related to transgenic mice for producing 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, and 5,87 This is described in Patent No. 4,299 and No. 5,545,806, in the PCT publications International Publication Brochures 91 / 10741, 90 / 04036, 94 / 02602, 96 / 30498, and 98 / 24893, and in the specifications of European Patent No. 546073B1 and European Patent Application Publication EP546073A1.

[0048] The transgenic mice described above, referred to herein as "HuMab" mice, contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (mu and gamma) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous mu and kappa chain gene loci (Lonberg et al., 1994, Nature 368:856-859). Consequently, the mice exhibit reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy chain and light chain transgenes undergo class switching and somatic mutations to produce high-affinity human IgG kappa monoclonal antibodies (Lonberg et al., previously cited; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764:536-546). HuMab mouse preparation was performed 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 This is described in detail in Acad.Sci.764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851, and these documents are incorporated herein by reference in their entirety for all purposes.Furthermore, please refer to 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, International Publication No. 93 / 1227, International Publication No. 92 / 22646, and International Publication No. 92 / 03918. All of their disclosures are incorporated herein by reference in their entirety for all purposes. The techniques used to produce human antibodies in these transgenic mice are disclosed in International Publication No. 98 / 24893 and in Mendez et al., 1997, Nature Genetics 15:146-156, which are incorporated herein by reference.

[0049] Human-derived antibodies can also be produced using phage display technology. Phage display is described, for example, in Dower et al., International Publication No. 91 / 17271, McCafferty et al., International Publication No. 92 / 01047, and in Caton and Koprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each of which is incorporated herein by reference in whole. Antibodies produced by phage technology are typically produced in bacteria as antigen-binding fragments, such as Fv fragments or Fab fragments, and therefore lack effector function. Effector function can be introduced by one of two strategies: that is, the fragments can be manipulated to become complete antibodies that can be expressed in mammalian cells as needed, or to become bispecific antibody fragments having a second binding site that can induce 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 to bind to a specific antigen. The antibody fragments are expressed on the phage surface, and selection by antigen binding of Fv or Fab (and therefore the phage containing the DNA encoding the antibody fragment) is achieved by performing several rounds of antigen binding and re-amplification, a procedure called panning. Antigen-specific antibody fragments are enriched and finally isolated. Phage display technology can also be used in a technique for humanizing rodent monoclonal antibodies called "guided selection" (see Jespers, LS, et al., Bio / Technology 12, 899-903 (1994)). In this case, the Fd fragment of a mouse monoclonal antibody can be presented in combination with a human light chain library, and the resulting hybrid Fab library can then be selected using an antigen. Thus, the mouse Fd fragment provides a template for inducing selection. Next, the selected human light chain is combined with a human Fd fragment library.By selecting the obtained libraries, a complete human Fab can be obtained.

[0050] In certain embodiments, the bispecific antigen-binding protein of the present invention is an antibody. As used herein, the term “antibody” refers to a tetrameric immunoglobulin protein comprising two light-chain polypeptides (each about 25 kDa) and two heavy-chain polypeptides (each about 50–70 kDa). The term “light chain” or “immunoglobulin light chain” refers to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus to the carboxyl terminus. The immunoglobulin light chain constant domain (CL) may be kappa (κ) or lambda (λ). The term “heavy chain” or “immunoglobulin heavy chain” refers to a polypeptide comprising 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) from the amino terminus to the carboxyl terminus. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG and IgA class antibodies are further divided into subclasses, namely 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 constant domain of the immunoglobulin heavy chain can originate from any immunoglobulin isotype, including its subtypes. The antibody chains are linked via interpolypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light chain and the heavy chain), and between the hinge regions of the antibody heavy chain.

[0051] In certain embodiments, the bispecific antigen-binding protein of the present invention is a heterodimer antibody (used herein interchangeably with "heteroimmunoglobulin" or "heteroIg") which is an antibody comprising two different light chains and two different heavy chains.

[0052] Heterodimeric antibodies may contain any immunoglobulin constant region. The term "constant region," as used herein, refers to all domains of the antibody other than the variable region. The constant region does not directly participate in antigen binding but exerts various effector functions. As described above, antibodies are classified into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) depending on the amino acid sequence of their heavy chain constant region. The light chain constant region can be, for example, a kappa-type or lambda-type light chain constant region, such as the human kappa-type or lambda-type light chain constant region found in all five antibody isotypes.

[0053] The heavy chain constant region of a heterodimer antibody can be, for example, an alpha, delta, epsilon, gamma, or muon heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or muon heavy chain constant region. In some embodiments, the heterodimer antibody contains a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In one embodiment, the heterodimer antibody contains a heavy chain constant region derived from human IgG1 immunoglobulin. In another embodiment, the heterodimer antibody contains a heavy chain constant region derived from human IgG2 immunoglobulin.

[0054] In one embodiment, the bispecific antibody of this disclosure is Duobody®. Duobody can be prepared using the DuoBody® technology platform (Genmab A / S) as described, for example, in International Publication No. 2008 / 119353, International Publication No. 2011 / 131746, International Publication No. 2011 / 147986, and International Publication No. 2013 / 060867, Labrijn AF et al., PNAS, 110(13):5145-5150 (2013), Gramer et al., mAbs, 5(6):962-973 (2013), and Labrijn et al., Nature Protocols, 9(10):2450-2463 (2014). Using this technique, half of a first monospecific antibody containing two heavy chains and two light chains can be combined with half of a second monospecific antibody containing two heavy chains and two light chains. The resulting heterodimer contains a pair of one heavy chain and one light chain from the first antibody and one heavy chain and one light chain from the second antibody. If both monospecific antibodies recognize different epitopes of different antigens, the resulting heterodimer is a bispecific antibody.

[0055] For the DuoBody® platform, each monospecific antibody contains a heavy chain constant region with a single point mutation in the CH3 domain. These point mutations enable stronger interactions between the CH3 domains of the resulting bispecific antibodies than between the CH3 domains of either monospecific antibody without mutations. The single point mutation in each monospecific antibody can be located at residue positions 366, 368, 370, 399, 405, 407, or 409 (EU numbering) in the CH3 domain of the heavy chain constant region (see International Publication No. 2011 / 131746). Furthermore, the single point mutation is located at a different residue within one monospecific antibody relative to the other monospecific antibody. For example, one monospecific antibody may contain mutation F405L (EU numbering; mutation from phenylalanine to leucine at residue 405), or one of the mutations F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y, while the other monospecific antibody may contain mutation K409R (EU numbering; mutation from lysine to arginine at residue 409). The heavy chain constant region of the monospecific antibody may be an isotype of IgG1, IgG2, IgG3, or IgG4 (e.g., human IgG1 isotype), and bispecific antibodies produced by DuoBody® technology can be modified to alter (e.g., reduce) Fc-mediated effector function and / or improve half-life.One method for producing a Duobody (trademark) includes: (i) separating the expression of two parental IgG1 cells containing a pair of point mutations (i.e., K409R and F405L (or one of the mutations F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y) (EU numbering)) in the CH3 domain; (ii) mixing the parental IgG1 cells in vitro under permissible redox conditions to enable recombination of the halves; (iii) removing the reducing agent to reoxidize the interchain disulfide bond; and (iv) analyzing the exchange efficiency and final product using chromatographic or mass spectrometry (MS) methods (Labrijn et al., Nature). See Protocols, 9(10):2450-2463 (2014).

[0056] Another exemplary method for producing bispecific antibodies is the knob-into-hole technique (Ridgway et al., Protein Eng., 9:617-621 (1996); International Publication No. 2006 / 028936). The major drawback of producing bispecific antibodies, the problem of mispairing of Ig heavy chains, is mitigated in this technique by mutating selected amino acids that form the interface of the CH3 domain in IgG. An amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain at a position within the CH3 domain where the two heavy chains directly interact, and an amino acid with a large side chain (knob) is introduced into the other heavy chain at a corresponding interacting residue position. In some examples, the antibodies of this disclosure have immunoglobulin chains in which the CH3 domain is modified by mutating selected amino acids that interact at the interface between the two polypeptides in order to preferentially form bispecific antibodies. Bispecific antibodies may consist of immunoglobulin chains of the same or different subclasses. In one example, a bispecific antibody that binds to gp120 and CD3 contains the T366W (EU numbered) mutation in the "knob chain" and the T366S, L368A, and Y407V (EU numbered) mutations in the "hole chain". In a particular embodiment, additional interchain disulfide crosslinks are introduced between the CH3 domains, for example, by introducing the Y349C mutation into the "knob chain" and the E356C or S354C mutation into the "hole chain". In a particular embodiment, the R409D and K370E mutations are introduced into the "knob chain" and the D399K and E357K mutations are introduced into the "hole chain". In another embodiment, the Y349C and T366W mutations are introduced into one of the chains, and the E356C, T366S, L368A, and Y407V mutations are introduced into the other chain. In some embodiments, the Y349C and T366W mutations are introduced into one strand, while the S354C, T366S, L368A, and Y407V mutations are introduced into the other strand. In some embodiments, the Y349C and T366W mutations are introduced into one strand, while the S354C, T366S, L368A, and Y407V mutations are introduced into the other strand.In yet another embodiment, the Y349C and T366W mutations are introduced into one strand, and the S354C, T366S, L368A, and Y407V mutations are introduced into the other strand (all EU numbered).

[0057] Another method for producing bispecific antibodies is the CrossMab technique. A CrossMab is a chimeric antibody composed of each half of two full-length antibodies. This technique combines two techniques to ensure accurate chain pairing: (i) a knob-in-to-hole mechanism that favors accurate pairing between the two heavy chains; and (ii) an exchange between one heavy chain and one light chain of the two Fabs to introduce asymmetry and avoid mispairing of the light chain. See Ridgway et al., Protein Eng., 9:617-621 (1996); Schaefer et al., PNAS, 108:11187-11192 (2011). CrossMab can combine two or more antigen-binding domains to target two or more targets, or to introduce bivalence, such as a 2:1 configuration, for a single target.

[0058] To facilitate the association of a particular heavy chain with a light chain of its cognate, both the heavy and light chains may contain complementary amino acid substitutions. As used herein, “complementary amino acid substitution” refers to a pairing of a positively charged amino acid substitution in one chain with an uncharged amino acid substitution in the other chain. For example, in some embodiments, the heavy chain contains at least one amino acid substitution to introduce a charged amino acid, and the corresponding light chain contains at least one amino acid substitution to introduce a charged amino acid, wherein the charged amino acid introduced into the heavy chain has the opposite charge to the amino acid introduced into the light chain. In certain embodiments, one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the first light chain (LC1), and one or more uncharged residues (e.g., aspartic acid or glutamic acid) can be introduced into the paired heavy chain (HC1) at the LC1 / HC1 binding interface. Conversely, one or more uncharged residues (e.g., aspartic acid or glutamic acid) can be introduced into the second light chain (LC2), and one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the paired heavy chain (HC2) at the LC2 / HC2 binding interface. Electrostatic interactions induce LC1 to pair with HC1 and LC2 to pair with HC2 because the oppositely charged residues (polarity) at the interface attract each other. At the interface, heavy / light chain pairs with the same charged residue (polarity) (e.g., LC1 / HC2 and LC2 / HC1) will repel each other, and as a result, the formation of undesirable HC / LC pairs is suppressed.

[0059] In these and other embodiments, the CH1 domain of the heavy chain or the CL domain of the light chain contains an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more positively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more uncharged amino acids. Alternatively, the CH1 domain of the heavy chain or the CL domain of the light chain contains an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more uncharged amino acids in the wild-type IgG amino acid sequence are replaced with one or more positively charged amino acids. In some embodiments, one or more amino acids in the CH1 domain of the first and / or second heavy chain in the heterodimer antibody at EU positions selected from F126, P127, L128, A141, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S183, V185 and K213 are replaced with charged amino acids. In certain embodiments, the preferred residue to be substituted with an uncharged or positively charged amino acid is S183 (EU numbering scheme). In some embodiments, S183 is substituted with a positively charged amino acid. In alternative embodiments, S183 is substituted with an uncharged amino acid. For example, in one embodiment, S183 is substituted with an uncharged amino acid (e.g., S183E) in the first heavy chain, and S183 is substituted with a positively charged amino acid (e.g., S183K) in the second heavy chain.

[0060] 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 heterodimer antibody at positions selected from F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174, and S176 (EU and Kabat numbering in the kappa light chain) are substituted with charged amino acids. 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 heterodimer antibody at positions selected from T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176, and Y178 (Kabat numbering in the lambda chain) are substituted with charged amino acids. In some embodiments, a preferred residue to be substituted with an uncharged or positively charged amino acid is S176 (EU and Kabat numbering scheme) in the CL domain of either the kappa light chain or the lambda light chain. In certain embodiments, S176 of the CL domain is substituted with a positively charged amino acid. In alternative embodiments, S176 of the CL domain is substituted with an uncharged amino acid. In one embodiment, S176 is substituted with a positively charged amino acid (e.g., S176K) in the first light chain, and S176 is substituted with an uncharged amino acid (e.g., S176E) in the second light chain.

[0061] In addition to, or as an alternative to, complementary amino acid substitutions in the CH1 and CL domains, the variable regions of the light and heavy chains in a heterodimer antibody may contain one or more complementary amino acid substitutions to introduce charged amino acids. For example, in some embodiments, the VH region of the heavy chain or the VL region of the light chain of a heterodimer antibody contains an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more positively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more uncharged amino acids. Alternatively, the VH region of the heavy chain or the VL region of the light chain contains an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more uncharged amino acids in the wild-type IgG amino acid sequence are replaced with one or more positively charged amino acids.

[0062] The V-region interface residues within the VH region (i.e., amino acid residues that mediate the assembly of the VH and VL regions) include positions 1, 3, 35, 37, 39, 43, 44, 45, 46, 47, 50, 59, 89, 91, and 93 of Kabat. One or more of these interface residues in the VH region can be substituted with charged (positively charged or uncharged) amino acids. In certain embodiments, the amino acid at position 39 of Kabat in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, such as lysine. In alternative embodiments, the amino acid at position 39 of Kabat in the VH region of the first and / or second heavy chain is substituted with an uncharged amino acid, such as glutamic acid. In some embodiments, the amino acid at position 39 of Kabat in the VH region of the first heavy chain is substituted with a positively charged amino acid (e.g., G39E), and the amino acid at position 39 of Kabat 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 position 44 of Kabat 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 position 44 of Kabat in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, e.g., glutamic acid. In a particular embodiment, the amino acid at position 44 of Kabat in the VH region of the first heavy chain is substituted with a positively charged amino acid (e.g., G44E), and the amino acid at position 44 of Kabat in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G44K).

[0063] The V-region interface residues within the VL region (i.e., amino acid residues mediating the assembly of the VH region and the VL region) include 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 of Kabat. One or more interface residues in the VL region can be substituted with a charged amino acid, preferably an amino acid with the opposite charge to that introduced in the VH region of the cognate heavy chain. In some embodiments, the amino acid at position 100 of Kabat in the VL region of the first and / or second light chain is substituted with a positively charged amino acid, such as lysine. In alternative embodiments, the amino acid at position 100 of Kabat in the VL region of the first and / or second light chain is substituted with a positively charged amino acid, such as glutamic acid. In certain embodiments, the amino acid at position 100 of Kabat 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 position 100 of Kabat in the VL region of the second light chain is substituted with a negatively charged amino acid (e.g., G100E).

[0064] In certain embodiments, the heterodimer antibody of the present invention comprises a first heavy chain and a second heavy chain, and a first light chain and a second light chain, wherein the first heavy chain contains amino acid substitutions at positions 44 (Kabat), 183 (EU), 392 (EU), and 409 (EU), the second heavy chain contains amino acid substitutions at positions 44 (Kabat), 183 (EU), 356 (EU), and 399 (EU), and the first and second light chains contain amino acid substitutions at positions 100 (Kabat) and 176 (EU), the amino acid substitutions introducing charged amino acids at the aforementioned positions. In related embodiments, the glycine at position 44 (Kabat) of the first heavy chain is replaced with glutamic acid, the glycine at position 44 (Kabat) of the second heavy chain is replaced with lysine, the glycine at position 100 (Kabat) of the first light chain is replaced with lysine, the glycine at position 100 (Kabat) of the second light chain is replaced with glutamic acid, the serine at position 176 (EU) of the first light chain is replaced with lysine, and the serine at position 176 (EU) of the second light chain is replaced with glutamic acid. Substitutions occur, with serine at position 183 (EU) of the first heavy chain being replaced by glutamic acid, lysine at position 392 (EU) of the first heavy chain being replaced by aspartic acid, lysine at position 409 (EU) of the first heavy chain being replaced by aspartic acid, serine at position 183 (EU) of the second heavy chain being replaced by lysine, glutamic acid at position 356 (EU) of the second heavy chain being replaced by lysine, and / or aspartic acid at position 399 (EU) of the second heavy chain being replaced by lysine.

[0065] As used herein, the term “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain that can be produced by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally comprises two constant domains, namely the CH2 domain and the CH3 domain, and optionally, the CH4 domain. In certain embodiments, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises the CH2 and CH3 domains derived from human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cell-mediated 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 functions, as described further in this specification.

[0066] In some embodiments of the antigen-binding protein of the present invention, the binding domain located at the carboxyl terminus of the Fc region (i.e., the carboxyl-terminus binding domain) is scFv. In certain embodiments, scFv includes a heavy chain variable region (VH) and a light chain variable region (VL) linked by a peptide linker. The variable regions may be arranged within scFv in a VH-VL or VL-VH orientation. For example, in one embodiment, scFv includes a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In another embodiment, scFv includes a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. The VH and VL regions of scFv may contain one or more cysteine ​​substitutions to enable disulfide bond formation between the VH and VL regions. Such cysteine ​​clamping stabilizes the two variable domains in the antigen-binding configuration. In one embodiment, position 44 (Kabat numbered) in the VH region and position 100 (Kabat numbered) in the VL region are substituted with cysteine ​​residues.

[0067] In certain embodiments, the scFv is fused or otherwise linked at its amino terminus 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, the scFv is fused to the Fc region such that the resulting fusion protein contains, from N-terminus to C-terminus, a CH2 domain, a CH3 domain, a first peptide linker, a VH region, a second peptide linker, and a VL region. In another embodiment, the scFv is fused to the Fc region such that the resulting fusion protein contains, from N-terminus to C-terminus, a CH2 domain, a CH3 domain, a first peptide linker, a VL region, a second peptide linker, and a VH region. A "fusion protein" is a protein containing polypeptide components derived from more than one parent protein or polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is added in frame together with a nucleotide sequence encoding a polypeptide sequence from a different protein, and optionally separated from that sequence by a linker. This fusion gene can then be expressed by recombinant host cells to produce a single fusion protein.

[0068] A "peptide linker" refers to an oligopeptide of about 2 to about 50 amino acids that covalently bonds one polypeptide to another. The peptide linker is used to link the VH domain and the VL domain in the scFv. The peptide linker can also be used to link the scFv, Fab fragment, or other functional antibody fragment to the amino or carboxyl terminus of the Fc region to create the bispecific antigen-binding proteins described herein. Preferably, the peptide linker is at least 5 amino acids long. In certain embodiments, the peptide linker is about 5 to about 40 amino acids long. In other embodiments, the peptide linker is about 8 to about 30 amino acids long. In yet another embodiment, the peptide linker is about 10 to about 20 amino acids long.

[0069] Preferably, but not necessarily, the peptide linker comprises amino acids from the 20 standard amino acids, particularly cysteine, glycine, alanine, proline, asparagine, glutamine, and / or serine. In certain embodiments, the peptide linker is composed of a large number of sterically unhinged amino acids, such as glycine, serine, and alanine. Therefore, preferred linkers in some embodiments include polyglycine, polyserine, and polyalanine, or any combination thereof. Some exemplary peptide linkers include, but are not limited to, poly(Gly) 2~8 In particular, (Gly)3 (SEQ ID NO: 22), (Gly)4 (SEQ ID NO: 23), (Gly)5 (SEQ ID NO: 24), (Gly)6 (SEQ ID NO: 25), and (Gly)7 (SEQ ID NO: 26), as well as poly(Gly)4Ser, poly(Gly-Ala) 2~4 and poly(Ala) 2~8 Examples include: In a particular embodiment, the peptide linker is (Gly x Ser) n Here, x = 3 or 4, and n = 2, 3, 4, 5, or 6. Examples of such peptide linkers include "L5" (GGGGS or "G4S"; SEQ ID NO: 27), "L9" (GGGSGGGGS; or "G3SG4S"; SEQ ID NO: 28), "L10" (GGGGSGGGGS; or "(G4S)2"; SEQ ID NO: 29), "L15" (GGGGSGGGGSGGGGS; or "(G4S)3"; SEQ ID NO: 31), and "L25" (GGGGSGGGGSGGGGSGGGGSGGGGS; or "(G4S)5"; SEQ ID NO: 32). In some embodiments, the peptide linker linking the VH region and the VL region in scFv is L15 or the (G4S)3 linker (SEQ ID NO: 31). In these and other embodiments, the peptide linker that links the carboxyl-terminal binding domain (e.g., scFv or Fab) to the C-terminus of the Fc region is an L9 or G3SG4S linker (SEQ ID NO: 28) or an L10(G4S)2 linker (SEQ ID NO: 29).

[0070] Other specific examples of peptide linkers that can be used in the bispecific antigen-binding proteins of the present invention include (Gly)5Lys(SEQ ID NO: 1); (Gly)5LysArg(SEQ ID NO: 2); (Gly)3Lys(Gly)4(SEQ ID NO: 3); (Gly)3AsnGlySer(Gly)2(SEQ ID NO: 4); (Gly)3Cys(Gly)4(SEQ ID NO: 5); GlyProAsnGlyGly(SEQ ID NO: 6); GGEGGG(SEQ ID NO: 7); GGEEEGGG(SEQ ID NO: 8); GEEEG(SEQ ID NO: 9); GEEE(SEQ ID NO: 10); GGDGGG(SEQ ID NO: 11); GDDDDGG(SEQ ID NO: 12); GDDDG(SEQ ID NO: 13); GDDD(SEQ ID NO: 14); GGGGSDDSDEGSDGEDGGGGS(SEQ ID NO: 15); WEWEW(SEQ ID NO: 16); FEFEF(SEQ ID NO: 17); EEEWWW(SEQ ID NO: 18); EEEFFF(SEQ ID NO: 19); WWEEEWW(SEQ ID NO: 20); and FFEEEFF(SEQ ID NO: 21).

[0071] 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 function of the Fc region of immunoglobulins is to transmit the immunoglobulin to the immune system when it binds to its target. This is commonly referred to as “effector function.” This transmission leads to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated cytotoxicity (CDC). ADCC and ADCP are mediated through the binding of the Fc region to Fc receptors on the surface of immune system cells. CDC is mediated through the binding of a complement system protein, e.g., C1q, to ​​Fc. In some embodiments, the bispecific antigen-binding proteins of the present invention contain one or more amino acid substitutions in the constant region to enhance effector functions, including ADCC activity, CDC activity, and ADCP activity, and / or the clearance or half-life of the antigen-binding protein. Examples of amino acid substitutions (EU numbering) that can enhance effector functionality include, but are not limited to, E233L, L234I, L234Y, L235S, G236A, S239D, F243L, F243V, P247I, D280H, K290S, K290E, K290N, K290Y, R292P, E294L, Y296W, S298A, S298 Examples include D, 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 of the above.

[0072] In other embodiments, the bispecific antigen-binding proteins of the present invention include one or more amino acid substitutions in the constant region to reduce effector function. Examples of 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.

[0073] Glycosylation can be a factor in the effector function of antibodies, particularly IgG1 antibodies. Therefore, in some embodiments, the bispecific antigen-binding proteins of the present invention may contain one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. Polypeptide glycosylation is typically either N-linked or O-linked. N-linking refers to the binding 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 other than proline) are recognition sequences for the enzymatic binding of a carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0074] In certain embodiments, the glycosylation of the bispecific antigen-binding protein 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 easily achieved by modifying the amino acid sequence to contain one or more of the above-described tripeptide sequences (in the case of N-linked glycosylation sites). Modification can also be made by adding or substituting one or more serine or threonine residues to the start sequence (in the case of O-linked glycosylation sites). For ease of implementation, the antigen-binding protein amino acid sequence can be modified by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at pre-selected base positions so that codons translated to desired amino acids are generated.

[0075] The present invention also encompasses the production of bispecific antigen-binding protein molecules in which the carbohydrate structure is modified to alter effector activity, such as antigen-binding proteins exhibiting enhanced ADCC activity by lacking or reducing fucosylation. Various methods for reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by the binding of antibody molecules to the FcγRIII receptor, which has been shown to depend on the carbohydrate structure of N-linked glycosylation at the N297 residue of the CH2 domain. Non-fucosylated antibodies bind to this receptor with high affinity and induce FcγRIII-mediated effector function more efficiently than naturally occurring fucosylated antibodies. For example, recombinant production of non-fucosylated antibodies in CHO cells in which the alpha-1,6-fucosyltransferase enzyme is knocked out results in antibodies with 100-fold increased ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. 87(5):614-22, 2004). Similar effects can be achieved by reducing the activity of the alpha-1,6-fucosyltransferase enzyme or other enzymes in the fucosylation pathway, for example, by siRNA or antisense RNA treatment, cell line manipulation to knock out the enzyme, or culture using selective glycosylation inhibitors (see Rothman et al., Mol Immunol. 26(12):1113-23, 1989). Some host cell lines, such as Lec13 or the rat hybridoma YB2 / 0 cell line, naturally produce antibodies with lower levels of fucosylation (see Shields et al., J Biol Chem. 277(30):26733-40, 2002 and Shinkawa et al., J Biol Chem. 278(5):3466-73, 2003). For example, it has been found that increasing the levels of bifurcated carbohydrates by recombinant antibody production in cells overexpressing the GnTIII enzyme also increases ADCC activity (see Umana et al., Nat Biotechnol. 17(2):176-80, 1999).

[0076] 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 modify the N-linked glycosylation sites. In certain embodiments, the bispecific antigen-binding proteins described herein include mutations at position N297 (EU numbering), such as N297Q, N297A, or N297G. In a particular embodiment, the bispecific antigen-binding protein of the present invention includes an Fc region derived from a human IgG1 antibody having the N297G mutation. To improve the stability of the molecule containing the N297 mutation, the Fc region of the molecule may be further manipulated. 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. Therefore, residues corresponding to V259, A287, R292, V302, L306, V323, or I332 (EU numbering) in the IgG1 Fc region may be substituted with cysteine. Preferably, specific pairs of residues are substituted with cysteine ​​so as to preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond scrambling. Preferred 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 include an Fc region derived from a human IgG1 antibody having mutations in R292C and V302C. In such embodiments, the Fc region may also include the N297G mutation.

[0077] For example, modifications of the bispecific antigen-binding protein of the present invention to extend the serum half-life may also be desirable, such as by incorporating or adding a salvage receptor-binding epitope (e.g., by mutation in an appropriate region, or by incorporating the epitope into a peptide tag and subsequently fusing it to the antigen-binding protein at either terminal or central position, for example, by DNA or peptide synthesis; see, for example, International Publication No. 96 / 32478), or by adding molecules such as PEG or other water-soluble polymers, e.g., polysaccharide polymers. The salvage receptor-binding epitope preferably constitutes a region into which any one or more amino acid residues from one or two loops of the Fc region are transplanted to a similar position in the antigen-binding protein. More preferably, three or more residues from one or two loops of the Fc region are transplanted. Even more preferably, the epitope is taken from the CH2 domain of the Fc region (e.g., the IgG Fc region) and transplanted into the CH1, CH3, or VH region, or one or more such regions, of the antigen-binding protein. Alternatively, the epitope may be extracted from the CH2 domain of the Fc region and transferred to the CL region, the VL region, or both of the antigen-binding protein. For a description of the Fc variant and its interaction with salvage receptors, please refer to the international applications, International Publication Nos. 97 / 34631 and International Publication Nos. 96 / 32478.

[0078] The present invention comprises one or more single nucleic acids encoding bispecific antigen-binding proteins and their components as described herein. The nucleic acid molecules of the present invention include DNA and RNA in both single-stranded and double-stranded forms, as well as corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR-amplified DNA, and combinations thereof. The nucleic acid molecules of the present invention include full-length genes or cDNA molecules and combinations of their fragments. The nucleic acids of the present invention are preferably derived from human sources, but the present invention also includes those derived from non-human species.

[0079] The relevant amino acid sequence derived from immunoglobulin or its region (e.g., variable region, Fc region, etc.) or the polypeptide of interest can be determined by direct protein sequencing, and a suitable coding nucleotide sequence can be designed according to a universal codon table. Alternatively, genomic DNA or cDNA encoding a monoclonal antibody, which may be the origin of the binding domain of the bispecific antigen-binding protein of the present invention, can be isolated and sequenced from cells producing such antibodies using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody).

[0080] In this specification, "isolated nucleic acid," as used interchangeably with "isolated polynucleotide," refers to a nucleic acid isolated from a neighboring gene sequence present in the genome of the organism from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. For example, in the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, cDNA molecule, or oligonucleotide, the nucleic acid obtained 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 distinct fragment, or a nucleic acid molecule as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acid is substantially free of endogenous substances that would impure it. 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 allows for the identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and / or constructed in the form of an open reading frame that is not interrupted by internal untranslated sequences or introns, which are typically found in eukaryotic genes. The sequence of untranslated DNA may be located on the 5' or 3' side of the open reading frame, in which case this does not interfere with the manipulation or expression of the coding region. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence described herein is the 5' end, and the left direction of a double-stranded polynucleotide sequence is called the 5' direction. The direction of production of a nascent RNA transcript from 5' to 3' is called the transcription direction, the sequence region on the DNA strand having the same sequence as the RNA transcript on the 5' side of the 5' end of the RNA transcript is called the “upstream sequence”, and the sequence region on the DNA strand having the same sequence as the RNA transcript on the 3' side of the 3' end of the RNA transcript is called the “downstream sequence”.

[0081] The present invention also includes nucleic acids that hybridize to nucleic acids encoding polypeptides described herein under moderately stringent conditions, more preferably highly stringent conditions. Basic parameters influencing the selection of hybridization conditions and guidance for devising preferred conditions are shown by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), which can be readily determined by those skilled in the art, for example, based on the length and / or base composition of the DNA. One method to achieve moderately stringent conditions involves using a pre-wash solution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6×SSC, and a hybridization temperature of about 55°C (or other similar hybridization solutions, such as one containing about 50% formamide, and a hybridization temperature of about 42°C), as well as washing conditions of about 60°C in 0.5×SSC and 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions similar to those described above, except for washing at about 68°C with 0.2×SSC and 0.1% SDS. SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be replaced with SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization buffer and wash buffer, and after hybridization is complete, wash for 15 minutes.Naturally, as is known to those skilled in the art, and as will be further described below, the washing temperature and washing salt concentration can be adjusted as necessary to achieve the desired degree of stringency by applying the basic principles that determine the hybridization reaction and the stability of the double strand (see, e.g., Sambrook et al., 1989). When hybridizing a nucleic acid to a target nucleic acid of an unknown sequence, the hybrid length is assumed to be the length of the nucleic acid being hybridized. When a nucleic acid with a known sequence is hybridized, the hybrid length can be determined by aligning the sequences of both nucleic acids and identifying the region or group of regions with optimal sequence complementarity. The hybridization temperature of a hybrid expected to be less than 50 base pairs long must be 5–10°C lower than the melting temperature (Tm) of the hybrid, where Tm is determined by the following formula: For hybrids less than 18 base pairs long, Tm(°C) = 2(number of A+T bases) + 4(number of G+C bases). For hybrids longer than 18 base pairs, Tm(°C) = 81.5 + 16.6(log10[Na+]) + 0.41(%G+C) - (600 / N), (wherein N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer) ([Na+] of 1 × SSC = 0.165M).Preferably, each of the nucleic acids that hybridize is at least 15 nucleotides in length (or more preferably at least 18 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of the nucleic acid of the present invention that it hybridizes, and The nucleic acid of the present invention that hybridizes with this has at least 60% sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and most preferably at least 99.5%). Here, sequence identity is determined by comparing the sequences of the nucleic acids that hybridize with this nucleic acid when the sequences of the nucleic acids are aligned to maximize overlap and identity and minimize sequence gaps, as described in more detail above.

[0082] The antigen-binding protein variants described herein can be prepared by producing mutant-encoding DNA using site-directed mutagenesis of nucleotides in the polypeptide-encoding DNA, by cassette or PCR mutagenesis, or by other techniques known in the art, and then expressing the recombinant DNA in a cell culture as outlined herein. However, antigen-binding proteins, including mutant CDRs having up to approximately 100–150 residues, can be prepared by in vitro synthesis using established techniques. The variants typically exhibit qualitative biological activity similar to that of their native analogs, e.g., binding to antigens. Such variants include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antigen-binding protein. Any combination of deletions, insertions, and substitutions is performed to arrive at the final construct, provided that the final construct retains the desired properties. Amino acid changes may also alter the post-translational processes of the antigen-binding protein, such as changing the number or location 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, for the purposes described herein, modification of residues that are not directly involved in epitope binding, or residues that are not involved in epitope binding at all, is desirable. Mutagenesis in either the CDR region and / or the framework region is intended. Those skilled in the art can use analysis of covariance techniques to design useful modifications to the amino acid sequence of the antigen-binding protein.For example, see Choulier, 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., International Publication No. 2008 / 110348A1; Borras et al., International Publication No. 2009 / 000099A2. Such modifications, determined by analysis of covariance, can improve the potency, pharmacokinetic, pharmacodynamic and / or manufacturability properties of antigen-binding proteins.

[0083] The present invention also includes vectors comprising one or more nucleic acids encoding one or more components of the bispecific antigen-binding protein of the present invention (e.g., a variable region, a light chain, a heavy chain, a modified heavy chain, and an Fd fragment). The term “vector” refers to any molecule or entity (e.g., a nucleic acid, a plasmid, a bacteriophage, or a virus) used to transfer protein-coding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episome mammalian vectors, and expression vectors, e.g., recombinant expression vectors. The terms “expression vector” or “expression construct,” as used herein, refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid regulatory sequences necessary for the expression of a coding sequence operably ligated in a particular host cell. An expression vector may, but is not limited to, include sequences that affect or control transcription, translation, and, if introns are present, sequences that affect the RNA splicing of the coding region operably ligated to them. Nucleic acid sequences necessary for expression in prokaryotes include promoters, optionally operator sequences, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Secretory signal peptide sequences can also be optionally encoded by expression vectors and operably ligated to the desired coding sequence, thereby causing recombinant host cells to secrete the expressed polypeptide, which, if desired, can be more easily isolated from the cell. For example, in some embodiments, the signal peptide sequence may be added / fused to the amino terminus of any of the polypeptide sequences listed in Tables 6A, 6B, 7A, 7B, 9, and 10. In a particular embodiment, a signal peptide having the amino acid sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 32) is fused to the amino terminus of any of the polypeptide sequences in Tables 6A, 6B, 7A, 7B, 9, and 10.In other embodiments, a signal peptide having the amino acid sequence MAWALLLLTLLTQGTGSWA (SEQ ID NO: 33) is fused to the amino terminus of any of the polypeptide sequences in Tables 6A, 6B, 7A, 7B, 9, and 10. In yet another embodiment, a signal peptide having the amino acid sequence MTCSPLLLTLLIHCTGSWA (SEQ ID NO: 34) is fused to the amino terminus of any of the polypeptide sequences in Tables 6A, 6B, 7A, 7B, 9, and 10. Other suitable signal peptide sequences that can be fused to the amino terminus of the polypeptide sequences described herein include MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 35), MEWTWRVLFLVAAATGAHS (SEQ ID NO: 36), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 37), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 38), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 39), and MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 40). Other signal peptides are known to those skilled in the art and can be fused to any of the polypeptide chains listed in Tables 6A, 6B, 7A, 7B, 9, and 10, for example, to promote or optimize expression in specific host cells.

[0084] Typically, the expression vector used in host cells to produce the bispecific antigen protein of the present invention contains a sequence for plasmid maintenance, as well as a sequence for cloning and expression of exogenous nucleotide sequences encoding components of the bispecific antigen-binding protein. Such sequences, collectively referred to as “adjacent sequences,” typically in certain embodiments include 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 inserting the nucleic acid encoding the polypeptide to be expressed, and one or more of the following selection marker elements. Each of these sequences is described below.

[0085] Optionally, the vector may contain a “tag” coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence, which encodes polyHis (e.g., hexaHis), FLAG, HA (hemagglutinin influenza virus), 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 an antibody against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as using a specific cleavage peptidase.

[0086] Adjacent sequences can be homogeneous (i.e., derived from the same species and / or strain as the host cell), heterogeneous (i.e., derived from a species other than the host cell species or strain), hybrid (i.e., a combination of adjacent sequences derived from more than one source), synthetic, or native. Therefore, the source of the adjacent sequence can be any prokaryote or eukaryote, any vertebrate or invertebrate, or any plant, provided that the adjacent sequence functions in and can be activated by the host cell mechanism.

[0087] Flanking sequences useful for the vector of the present invention can be obtained by any of several methods well known in the art. Typically, the flanking sequences useful herein will have been identified in advance by mapping and / or restriction endonuclease digestion and can be isolated from a suitable tissue source using a suitable restriction endonuclease. 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.

[0088] Whether all or only some of the adjacent sequences are known, adjacent sequences can be obtained by polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes such as oligonucleotides and / or adjacent sequence fragments from the same or a different species. If the adjacent sequences are unknown, DNA fragments containing adjacent sequences can be isolated, for example, from larger DNA fragments that may contain coding sequences or even one or more other genes. Isolation can be achieved by producing suitable DNA fragments 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 suitable enzymes for achieving this objective will be readily apparent to those skilled in the art.

[0089] The origin of replication is typically a component of a commercially available prokaryotic expression vector, and this origin is useful for amplifying the vector in host cells. If the selected vector does not contain an origin of replication site, it may be chemically synthesized based on a known sequence and ligated into the vector. For example, the origin of replication derived from plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, varicella-stomatitis virus (VSV), or papillomavirus, e.g., HPV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of replication component is not necessary for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the initial viral promoter).

[0090] Transcription termination sequences are typically located at the 3' end of the polypeptide coding region and function to terminate transcription. In prokaryotic cells, the transcription termination sequence is usually a GC-rich fragment followed by a poly-T sequence. This sequence can be readily cloned from libraries, or even purchased commercially as part of a vector, but it can also be readily synthesized using known nucleic acid synthesis methods.

[0091] Selection marker genes encode proteins necessary for the survival and proliferation of host cells grown in a selective culture medium. Typical selection marker genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, to prokaryotic host cells; (b) proteins that compensate for deficiencies in the cellular nutritional requirements; or (c) proteins that supply essential nutrients unavailable from complex or limited media. Specific selection markers include kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, neomycin resistance genes can also be used for selection in both prokaryotic and eukaryotic host cells.

[0092] Other select genes may be used to amplify the expressed genes. Amplification is the process by which genes required for the production of proteins important for proliferation or cell survival are repeated in tandem within the chromosomes of recombinant cells over generations. Examples of suitable select markers for mammalian cells include the dihydrofolate reductase (DHFR) and promoter resthymidine kinase genes. Mammalian cell transformants are placed under selective pressure, in which only these transformants are adapted to survive by the select gene present in the vector. Selective pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selector in the culture medium is continuously increased, thereby leading to amplification of both the select gene and the DNA encoding another gene, such as one or more components of the bispecific antigen-binding proteins described herein. As a result, large amounts of polypeptides are synthesized from the amplified DNA.

[0093] The ribosome binding site is typically required for mRNA translation initiation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located at the 3' end of the promoter and at the 5' end of the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions may be operably ligated to an internal ribosome binding site (IRES), enabling translation of two open reading frames from a single RNA transcript.

[0094] In cases where glycosylation is desired in eukaryotic host cell expression systems, various pre-sequences or pro-sequences can be manipulated 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 associated with expression at position -1 (relative to the first amino acid of the mature protein), which do not necessarily have to be completely removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site, attached to the amino terminus. Alternatively, using several enzyme cleavage sites may result in a slightly cleaved form of the desired polypeptide when the enzyme cleaves at such regions within the mature polypeptide.

[0095] The expression vectors and cloning vectors of the present invention typically contain a promoter that is recognized by a host organism and operably ligated to a molecule encoding a polypeptide. The term "operably ligated," as used herein, refers to the ligation of two or more nucleic acid sequences such that a nucleic acid molecule is produced that can direct the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a regulatory sequence in a vector "operably ligated" to a protein-coding sequence is ligated to the protein-coding sequence such that the expression of the protein-coding sequence occurs under conditions compatible with the transcriptional activity of the regulatory sequence. More specifically, a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional regulatory elements) is operably ligated to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system.

[0096] A promoter is a non-transcriptional sequence located upstream (i.e., at the 5' end) of the start codon of a structural gene (generally within approximately 100–1000 bp) and controls the transcription of that structural gene. Promoters are typically classified into two classes: inductive promoters and constitutive promoters. Inductive promoters initiate an increase in the transcription level from DNA under their control in response to any change in culture conditions, such as the presence or absence of nutrients or changes in temperature. Constitutive promoters, on the other hand, transcribe the gene to which they are operably ligated uniformly, i.e., with little or no control over gene expression. Numerous promoters recognized by various potential host cells are well known. A suitable promoter is operably ligated to the DNA encoding, for example, the heavy chain, light chain, modified heavy chain, or other components of the bispecific antigen-binding protein of the present invention, by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into a vector.

[0097] Promoterians suitable for use with yeast hosts are also well known in the art. Yeast enhancers are advantageous when used with yeast promoters. Promoterians suitable for use with mammalian host cells are well known and include, but are not limited to, those derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (such as adenovirus type 2), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably Simian virus 40 (SV40). Other suitable mammalian promoters include heteromammalian promoters, such as heat shock promoters and actin promoters.

[0098] Further potential promoters include, but are not limited to, the SV40 initial promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); promoters contained in the long terminal repeats at the 3' end of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); promoters and regulatory sequences derived from the metallothionine gene (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al.) Examples include 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). The following animal transcriptional regulatory regions, which exhibit tissue specificity and are utilized in transgenic animals, are also included: the elastase I gene regulatory region 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 active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); and the immunoglobulin gene regulatory region 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 regulatory region active in testicular cells, mammary cells, lymphoid cells and mast cells (Leder et al., 1986, Cell 45:485-495); Albumin gene regulatory region active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); Alpha-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol.Cell.Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); Alpha-1 antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); Betaglobin gene regulatory 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 regulatory region active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain 2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0099] 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, typically about 10–300 bp in length, that act on promoters to increase transcription. Enhancers are relatively directional and position-independent and can be found at both the 5' and 3' ends of the transcription unit. Several enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, viral enhancers are typically used. The SV40 enhancer, cytomegalovirus initial promoter enhancer, polyoma enhancer, and adenovirus enhancer, known in the art, are exemplary enhancing elements for eukaryotic promoter activation. Enhancers may be located at either the 5' or 3' end of the coding sequence in the vector, but are typically located at the 5' end of the promoter. Sequences encoding appropriate native or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into expression vectors to promote extracellular antibody secretion. The choice of signal peptide or leader depends on the type of host cell in which the antibody will be produced, and heterologous signal sequences can replace native signal sequences. Examples of signal peptides are listed above. Other signal peptides that function in mammalian host cells include the interleukin-7 (IL-7) signal sequence described in U.S. Patent 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. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460846.

[0100] The expression vector provided 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 initially present in the vector, they may be obtained individually and ligated into the vector. 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 to produce a protein containing a fusion protein encoded by the nucleic acids described herein.

[0101] In certain embodiments, nucleic acids encoding different components of the bispecific antigen-binding protein of the present invention may be inserted into the same expression vector. In such embodiments, the two nucleic acids may be separated under the control of a single promoter by an intrasequence ribosome entry site (IRES) 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 distinct promoters so that the light and heavy chains are expressed from two distinct mRNA transcripts.

[0102] Similarly, for the IgG-scFv bispecific antigen-binding protein, the nucleic acid encoding the light chain may be cloned into the same expression vector as the nucleic acid encoding the modified heavy chain (a fusion protein containing the heavy chain and scFv), in which case the two nucleic acids are separated by IRES under the control of a single promoter, or the two nucleic acids are under the control of two separate promoters. For the IgG-Fab bispecific antigen-binding protein, the nucleic acids encoding each of the three components may be cloned into the same expression vector. In some embodiments, the nucleic acid encoding the light chain of the IgG-Fab molecule and the nucleic acid encoding the second polypeptide (containing the other half of the C-terminal Fab domain) are cloned into one expression vector, while the nucleic acid encoding the modified heavy chain (a fusion protein containing the heavy chain and half of the Fab domain) is 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, even if one or more components are cloned into separate expression vectors, both expression vectors can be simultaneously transfected into host cells so that a single cell produces all components of the bispecific antigen-binding protein.

[0103] After a vector is constructed and one or more nucleic acid molecules encoding components of the bispecific antigen-binding protein described herein are inserted into appropriate sites within one or more vectors, the completed vector can be inserted into a host cell suitable for amplification and / or polypeptide expression. Therefore, the present invention encompasses a single-presence host cell containing one or more expression vectors encoding components of the bispecific antigen-binding protein. The term “host cell,” as used herein, refers to a cell that is or can be transformed with nucleic acid to express the gene of interest. This term includes offspring of parental cells, regardless of whether the morphology or genetic structure of the offspring is identical to that of the original parental cell, as long as the gene of interest is present. A host cell containing a single-presence nucleic acid of the present invention, preferably operably linked to at least one expression regulatory sequence (e.g., a promoter or enhancer), is a “recombinant host cell.”

[0104] Transformation of selected host cells with antigen-binding protein expression vectors can be achieved by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The chosen method will, to some extent, depend on the type of host cell used. These methods and other suitable methods are well-known to those skilled in the art and are shown, for example, in Sambrook et al., 2001.

[0105] When cultured under appropriate conditions, host cells synthesize antigen-binding proteins, which can then be harvested from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of a suitable host cell will depend on various factors, including the desired expression level, polypeptide modifications desirable or required for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0106] Exemplary host cells include prokaryotes, yeasts, or higher eukaryotic cells. Prokaryotic host cells include eubacteria such as Gram-negative or Gram-positive microorganisms, such as Enterobacteriaceae, for example Escherichia, for example E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, and Serratia, for example Serratia marcescens. This includes the genera *Marcescens*, *Shigella*, and *Bacillus*, such as *B. subtilis* and *B. licheniformis*, *Pseudomonas*, and *Streptomyces*. Eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for recombinant polypeptides. *Saccharomyces cerevisiae*, or common baker's yeast, is the most commonly used lower eukaryotic host microorganism.However, genera such as Pichia (e.g., P. pastoris), Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, and Schwanniomyces (e.g., Schwanniomyces occidentalis) are not included. Aspergillus (occidentalis), as well as filamentous fungi, such as the genera Neurospora, Penicillium, Tolypocladium, and Aspergillus, several other genera, species, and strains such as A. nidulans and A. niger are generally available and useful here.

[0107] Host cells for the expression of glycosylated antigen-binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and mutants, as well as corresponding insect-acceptable host cells derived from hosts such as the fall armyworm (Spodoptera frugiperda), Aedes aegypti (Aedes aegypti), Asian tiger mosquito (Aedes albopictus), fruit fly (Drosophila melanogaster), and silkworm (Bombyx mori), have been identified. Various virus strains for transfection of such cells, e.g., the L-1 mutant of Autographa californica NPV and the Bm-5 strain of silkworm (Bombyx mori) NPV, are publicly available.

[0108] Vertebrate host cells are also suitable hosts, and recombinant production of antigen-binding proteins from such cells is a common practice. 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 are not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cell / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell 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 cancer 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 tumor cells (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC 5 cells or FS4 cells; Mammalian myeloma cells, and several other cell lines. In another embodiment, a cell line derived from a B cell lineage can be selected that does not produce its own antibodies but has the ability to produce and secrete heterologous antibodies. In some embodiments, CHO cells are preferred host cells for expressing the bispecific antigen-binding protein of the present invention.

[0109] For the production of bispecific antigen-binding proteins, host cells are transformed or transfected with the nucleic acids or vectors described above and cultured in a conventional nutrient medium modified to be suitable for promoter induction, transformant selection, or amplification of a gene encoding a desired sequence. In addition, novel vectors and transfected cell lines having multiple copies of transcription units separated by a selection marker are particularly useful for the expression of antigen-binding proteins. Accordingly, the present invention also provides a method for preparing a bispecific antigen-binding protein as described herein, comprising the steps of culturing host cells containing one or more expression vectors as described herein in a culture medium under conditions that enable the expression of a bispecific antigen-binding protein encoded by the one or more expression vectors, and recovering the bispecific antigen-binding protein from the culture medium.

[0110] The host cells used to produce the antigen-binding protein of the present invention can be cultured in a variety of media. Commercial media such as Ham F10 (Sigma), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing 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. Patent No. 4,767,704; U.S. Patent No. 4,657,866; U.S. Patent No. 4,927,762; U.S. Patent No. 4,560,655; or U.S. Patent No. 5,122,469; International Publication No. 90103430; International Publication No. 87 / 00195; or U.S. Reissue Patent No. 30,985 can be used as the culture medium for host cells. Any of these culture media may 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 phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin®), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary nutritional supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with host cells selected for expression and will be obvious to those skilled in the art.

[0111] When host cells are cultured, bispecific antigen-binding proteins can be produced intracellularly, in the perimembrane space, or directly secreted into the culture medium. If the antigen-binding protein is produced intracellularly, the first step is to remove granular fragments of host cells or lysed fragments, for example, by centrifugation or ultrafiltration. The bispecific antigen-binding protein can be purified, for example, by hydroxyapatite chromatography, cation or anion exchange chromatography, or preferably by 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.5:15671575, 1986). The matrix to which the affinity ligand binds is most often agarose, but 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 those achievable with agarose. When the protein contains a CH3 domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Depending on the specific bispecific antigen-binding protein to be recovered, other techniques for protein purification, such as ethanol precipitation, reverse-phase HPLC, chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation, are also possible. [Examples]

[0112] CZH01 The ability to generate multispecific antigen-binding proteins and therapeutic proteins with extended half-lives is crucial for bringing many therapeutic candidates to clinical practice. This often means extensive protein design with varying degrees of success. In any case, two different Fc moieties combine to form a heterodimer. To achieve this, alterations to the protein's native sequence are necessary. Traditionally, such alterations have focused on the CH3-CH3' interface of the Fc moiety, where charge-pair mutation designs and knob-in-hole designs have been inserted.

[0113] The two heavy chains in the monoclonal antibody can be observed to have two main contact points: Fc-CH3 and a hinge (Figure 1). The hinge region must link Fc to the two Fab domains and subsequently exhibit a flexible structure that allows for the free rotation of the Fab, which is necessary for these warheads (Fab) to take the precise angle of approach to engage with their target, while the Fc domain can interact with multiple binding partners such as FcγR, FcRn, and C1q. Despite the flexibility around the hinge region, this interface is mediated by a strong, structurally rigid "CPPC" motif (Figures 1-3). Here, the proline residue introduces a highly specific and stable secondary structure that allows the -SH side chain of the cysteine ​​residue to contact its counterpart and form a disulfide bond. Furthermore, this same rigid frame is likely to extend upstream and downstream of the CPPC motif, suggesting that the side chains of those residues in the vicinity of this motif are also likely to exhibit stable three-dimensional structures.

[0114] The only available crystal structure of human full-length IgG1 antibody (PDB 1HZH) in the Protein Data Bank (PDB) shows only a partially intact CPPC motif, where the second cysteine ​​(C242) does not form a disulfide bond and the side chains of the two polypeptide chains point in opposite directions at position 242 (Figure 2). Although structural information exists for mouse IgG1 antibody showing the intact structure of the CPPC motif, the sequence differences between these two (Figure 3) made it impossible to accurately predict the spatial arrangement of the side chains of the residues downstream of Cys242 in human IgG1. Therefore, while maintaining the native sequence of the CPPC motif, a series of countercharged residues, named "Charged Zipper Hinge 01" (CZH01), were inserted downstream of the same region (Figure 4). This "charged zipper" is thought to both attract oppositely charged heavy chains together and repel like-charged chains together.

[0115] To test this hypothesis, these mutations were inserted into antibody X, transiently expressed in HEK293 cells, and then subjected to single-step protein purification (protein A, followed by CEX). Detailed reduced and non-reduced mass spec assays were performed to verify whether the newly generated antibody consisted of two different heavy chains (loaded and positively charged, respectively). Indeed, the results showed that this strategy successfully promoted heavy chain pairing without any additional mutations in the Fc-CH3 region (Figures 4-6). The lysine chain (243-247) is most likely to interact with the aspartic acid chain (243-247).

[0116] CZH11 Furthermore, structural analysis of the mouse IgG2 molecule shows that residues 246 and 247 on one chain and residue 244 on the other chain have corresponding side chains (Figure 9). These residues are located downstream of Cys242 and near the CPPC motif, suggesting steric stability. In addition, the charged residues have side chains that can fit into the structural and spatial arrangement, and therefore do not interfere with the CPPC interface. Both Asn246 and Leu247 were substituted with charged aspartate residues. To pair with these two residues, Ala244 on the opposite chain was substituted with a single histidine, allowing it to either produce productive contact with the opposite charged residues (Asp246 and Asp247) or exhibit a repulsive effect (Figures 9 and 10) (CZH11). To test whether the design was rational, this mutant was expressed and purified. Surprisingly, mass spectrometry confirmed that these three residues are indeed sufficient to form a triad and promote hinge dimerization (Figure 10).

[0117] CZH09 Next, we tested the upstream region of the CPPC domain to verify whether manipulation of this sequence could also promote heavy chain dimerization. Two additional charge-pair mutations, His237Lys / Asp and Thr238Lys / Asp, were inserted upstream of Cys239, while two charge-pair mutations, Ala244Lys / Asp and Glu246Lys / Asp, were maintained downstream of Cys242 (CZH09) (Figure 7). The data indicate that CPM inserted upstream of the CPPC motif can also promote heavy chain dimerization.

[0118] CH3 CPM Furthermore, the CPM (charge pair mutation) inserted into the hinge region is compatible with the CPM previously inserted into the Fc-CH3 region. The so-called "v11" CH3 CPM (D399K in one CH3 region and K409D / K392D in the other CH3 region) was added to molecules containing the CZH01, CZH09, and CZH11 mutations (Figures 11 and 12). The results showed that all three CPMs inserted into the hinge region (CZH01, CZH09, and CZH11) successfully promoted heavy chain pair formation, even in the presence of the Fc-CH3 CPM.

[0119] To assess the effects of these novel mutations manipulated in the hinge region, stability assays were performed to investigate the thermal stability of these novel mutants compared to the wild-type molecule. The data show that the Tm data for CZH01, CZH09, and CZH11 are comparable to those of the control CZH00 (73.3°C) without CH3-CPM v11 and CZH00 (70.7°C) with CH3-CPM v11 (Table 1).

[0120] [Table 1]

Claims

1. An isolated heteromultimer comprising a heterodimer immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, wherein the first and second immunoglobulins are human IgG1 or mouse IgG2. (i) The first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243K, A244K, P245K, N / E246K and L247K, and (ii) The second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243D, A244D, P245D, N / E246D and L247D, The numbering of amino acid residues follows Kabat. (iii) The heteropolymer further comprises at least two binding domains, the binding domains being a Fab fragment, a Fab' fragment, and F(ab') ２ These are fragments, Fv fragments, single-chain variable antibody fragments (scFv), or nanobodies. Isolated heteromultimer.

2. An isolated heteromultimer according to claim 1, comprising two Fab fragments.

3. The isolated heteromultimer according to claim 1, comprising a Fab fragment and an scFv fragment.

4. The isolated heteromultimer according to any one of claims 1 to 3, wherein the binding domain is located at the carboxyl terminus of the Fc region.

5. An isolated heteromultimer according to any one of claims 1, 3, and 4, comprising an scFv linked to the carboxyl terminus of the Fc region via a peptide linker at an amino or carboxyl terminus.

6. The isolated heteromultimer according to claim 5, comprising, from the N-terminus to the C-terminus, (i) a CH2 domain, a CH3 domain, a first peptide linker, a VH region, a second peptide linker, a VL region, or (ii) a CH2 domain, a CH3 domain, a first peptide linker, a VL region, a second peptide linker, and a VH region.

7. The first peptide linker is L9, or G ３ SG ４ S linker (Sequence No. 28), or L10 (G ４ S) ２ The isolated heteromultimer according to claim 6, wherein the linker is (SEQ ID NO: 29).

8. The second peptide linker is L15 or (G ４ S) ３ The isolated heteromultimer according to claim 6 or 7, wherein the linker is (SEQ ID NO: 30).

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