Engineering hinge regions to drive antibody dimerization

Abstract

The invention relates to engineering hinge regions to drive antibody dimerization. The clinical potential of multispecific antibodies, such as bispecific and trispecific antibodies, shows a huge hope for targeting complex diseases. However, the production of these molecules puts forward a huge challenge because, in many cases, it is desirable to specifically drive the specific pairing of a plurality of polypeptide chains present in solution. In the case of a heavy chain, there are two major regions forming a dimer interface. One of the regions is a CH3 region, which has been broadly utilized by inserting charge pair mutations (CPM) to manipulate the dimer interface, or inserting large volume residues into cavities (pestle-mortar) to physically facilitate or disfacilitate the formation of dimers. However, each of these policies may not be applicable to each molecule, thus requiring more tools. Here, we describe engineering of the hinge region with a small amount of mutations that can successfully drive heavy chain dimerization individually.

Description

[0001] This application is a divisional application. The application date of the original application is May 29, 2020, the application number is 202080054199.X (PCT / US2020 / 035196), and the name of the invention is “Engineering hinge regions to drive antibody dimerization”. Technical Field

[0002] The present invention relates to the field of biopharmaceuticals. In particular, the present invention relates to a multispecific antigen-binding protein capable of specifically binding to at least two target antigens. The multispecific antigen-binding protein comprises two different heavy chains in the hinge region, and the two different heavy chains utilize charge pair mutations to promote heterodimer formation while inhibiting homodimer formation. Background Art

[0003] Antibodies have become the modality of choice in the biopharmaceutical industry because they possess several properties that are attractive to those developing therapeutic molecules. In addition to their ability to target specific structures or cells, antibodies can render their targets susceptible to Fc receptor cell-mediated phagocytosis and killing (Raghavan and Bjorkman 1996). Furthermore, the ability of antibodies to interact with the neonatal Fc receptor (FcRn) in a pH-dependent manner gives them a prolonged serum half-life (Ghetie and Ward 2000). This unique property of antibodies allows for the engineering of Fc fusion molecules to extend the half-life of therapeutic proteins or peptides in serum.

[0004] In some cases, it is desirable to produce a molecule that contains the Fc portion of an antibody but contains a heterodimer. An important application of Fc heterodimer molecules is to produce multispecific antibodies, for example, bispecific antibodies. Bispecific antibodies refer to antibodies that are specific for at least two different antigens (Nolan and O'Kennedy 1990; de Leij, Molema et al. 1998; Carter 2001). Instead of having the same sequence in two Fabs, bispecific antibodies have different sequences in 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 a half-life extension moiety to a therapeutic molecule. In this case, one or both of the two different Fc parts can be fused to one or more therapeutic molecules that need to extend the half-life.

[0005] The classic approach to generating Fc heterodimers was developed by Carter and coworkers, who used a "knobs-into-holes" strategy to engineer heavy chains 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 the packing of amino acid side chains between adjacent α-helices (Crick 1952). Carter and coworkers generated knobs at the CH3 domain interface of the first chain by replacing smaller amino acid side chains with larger ones (e.g., T366Y); and holes at juxtaposed positions at the CH3 interface of the second chain by replacing larger amino acid side chains with smaller ones (e.g., Y407T). The basis for producing knobs and mortars in juxtaposed positions is that the interaction of knobs and mortars favors the formation of heterodimers, while the interactions of knobs-knobs and mortars-mortars hinder homodimer formation due to steric conflicts and deletions of favorable interactions, respectively. Knob-mortar mutations are also combined with CH3 interdomain disulfide bonds engineered to enhance heterodimer formation (Sowdhamini, Srinivasan et al. 1989; Atwell, Ridgway et al. 1997). In addition to these mutations, the input DNA ratio is also altered to maximize yield (Merchant, Zhu et al. 1998). The "knob-mortar" technology is disclosed in U.S. Patent Nos. 5,731,168 and 7,183,076.

[0006] The clinical potential of multispecific antibodies (molecules that target multiple targets simultaneously, such as bispecific and trispecific antibodies) as well as therapeutic proteins with extended half-lives show great promise for targeting complex diseases. However, the generation of these molecules presents a great challenge, as in many cases we wish to specifically drive the pairing of multiple polypeptide chains present in solution. Here, we describe engineering the hinge region with a small number of mutations that are able to successfully drive Fc dimerization alone. Summary of the invention

[0007] In one aspect, the present invention relates to an isolated heteromultimer comprising a heterodimeric immunoglobulin hinge domain, wherein the heterodimeric immunoglobulin hinge domain comprises a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, wherein:

[0008] (i) the first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243K, A244K, P245K, N / E246K and L247K; and

[0009] (ii) the second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243D, A244D, P245D, N / E246D and L247D;

[0010] The numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0011] In another aspect, the present invention relates to an isolated heteromultimer comprising a heterodimeric immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, wherein:

[0012] (i) the first immunoglobulin hinge domain polypeptide comprises the following amino acid substitution: A244H; and

[0013] (ii) the second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: N / E246D and L247D;

[0014] The numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0015] In another aspect, the present invention relates to an isolated heteromultimer comprising a heterodimeric immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, wherein:

[0016] (i) the first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: H237K, T238K, A244K and N / E246K; and

[0017] (ii) the second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: H237D, T238D, A244D and N / E246D;

[0018] The numbering of amino acid residues is according to the EU index as set forth in Kabat.

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

[0020] In certain embodiments, each immunoglobulin hinge domain polypeptide further comprises a CH3 domain. In one embodiment, one CH3 domain comprises a F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y mutation; and the other CH3 domain comprises a K409R mutation; wherein the numbering of the amino acid residues is according to the EU index set forth in Kabat. In one embodiment, one CH3 domain comprises a T366W mutation; and the other CH3 domain comprises a T366S, L368A, Y407V mutation; wherein the numbering of the amino acid residues is according to the EU index set forth in Kabat. In one embodiment, one CH3 domain comprises K / R409D and K392D mutations; and the other CH3 domain comprises D399K and E356K mutations; wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. In one embodiment, one CH3 domain comprises Y349C mutation; and the other CH3 domain comprises E356C or S354C mutations; wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. In one embodiment, one CH3 domain comprises Y349C and T366W mutations; and the other CH3 domain comprises E356C, T366S, L368A, and Y407V mutations; wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. In one embodiment, one CH3 domain comprises Y349C and T366W mutations; and the other CH3 domain comprises S354C, T366S, L368A, Y407V mutations; wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0021] In certain embodiments, the immunoglobulin hinge region is an IgG1 hinge region.

[0022] In certain embodiments, the heteromultimer is a bispecific or multispecific antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Targeting the hinge region to form heterodimers is described.

[0024] Figure 2A human IgG1 crystal structure was described that does not show a second disulfide bond at the CPPC, although this may be an artifact introduced by radiation damage during X-ray data collection. Furthermore, the IgG1 murine crystal structure (1IGY), as well as “in-house” mass spectrometry data, strongly suggest that disulfide C242 should be intact. Thus, using the mIgG1 and huIgG2 structures as a guide may provide a better understanding of the rotamer positions of residues downstream of the CPPC motif.

[0025] Figure 3 Sequence alignments of IgG1, IgG2, and IgG4 are depicted.

[0026] Figure 4 Summary table describing hinge design and quality control assessment (MSQC).

[0027] Figure 5 Charged zipper hinge - design CZH01 is depicted. According to the crystal structure, C239 is the only C that forms a disulfide bridge within the IgG1 hinge (Saphire and Wilson, Science, 2001 (anti-HIV-1 B12 antibody). However, other data suggest that a second Cys (C242) can still form a disulfide bond, and P241 appears to be important for the same bond to occur. It would then be reasonable to design the CPM string downstream of this second disulfide bond (see mutations in the orange line) and then insert a new disulfide bond at L248C (orange dashed line).

[0028] Figure 6 Analytical CEX and mass spectrometry-CZH01 is described.

[0029] Figure 7 Described is the live zipper hinge - design CZH09.

[0030] Figure 8 Analytical CEX and mass spectrometry-CZH09 is described.

[0031] Fig. 9 Structural guidance for the charged zipper hinge in IgG2 is described.

[0032] Fig.10 Analytical CEX and mass spectrometry-CZH11 is described.

[0033] Fig.11 Targeting the hinge region to form the heterodimer +CH3 CPM v11 is described.

[0034] Fig.12 Summary table describing the hinge design +CH3-CH3'CPM v11.

[0035] Fig.13Thermostability analysis of the hinge design is described. The hinge mutations had no negative impact on the stability of the Ab, and the CH3 CPM v11 mutation appeared to reduce the Tm + / - 2 deg. DETAILED DESCRIPTION

[0036] In one aspect, the present invention relates to an isolated heteromultimer comprising a heterodimeric immunoglobulin hinge domain, wherein the heterodimeric immunoglobulin hinge domain comprises a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, wherein:

[0037] (i) the first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243K, A244K, P245K, N / E246K and L247K; and

[0038] (ii) the second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: P243D, A244D, P245D, N / E246D and L247D;

[0039] The numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0040] In another aspect, the present invention relates to an isolated heteromultimer comprising a heterodimeric immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, wherein:

[0041] (i) the first immunoglobulin hinge domain polypeptide comprises the following amino acid substitution: A244H; and

[0042] (ii) the second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: N / E246D and L247D;

[0043] The numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0044] In another aspect, the present invention relates to an isolated heteromultimer comprising a heterodimeric immunoglobulin hinge domain comprising a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, wherein:

[0045] (i) the first immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: H237K, T238K, A244K and N / E246K; and

[0046] (ii) the second immunoglobulin hinge domain polypeptide comprises the following amino acid substitutions: H237D, T238D, A244D and N / E246D;

[0047] The numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0048] In certain embodiments, each hinge domain polypeptide of the heteromultimer further comprises a L248C substitution.

[0049] In certain embodiments, each immunoglobulin hinge domain polypeptide further comprises a CH3 domain. In one embodiment, one CH3 domain comprises a F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y mutation; and the other CH3 domain comprises a K409R mutation; wherein the numbering of the amino acid residues is according to the EU index set forth in Kabat. In one embodiment, one CH3 domain comprises a T366W mutation; and the other CH3 domain comprises a T366S, L368A, Y407V mutation; wherein the numbering of the amino acid residues is according to the EU index set forth in Kabat. In one embodiment, one CH3 domain comprises K / R409D and K370E mutations; and the other CH3 domain comprises D399K and E357K mutations; wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0050] In a specific embodiment, the heterodimeric antibody comprises a first heavy chain comprising negatively charged amino acids at positions 392 and 409 (e.g., K392D and K409D substitutions), and a second heavy chain comprising positively charged amino acids at positions 356 and 399 (e.g., E356K and D399K substitutions). In other specific embodiments, the heterodimeric antibody comprises a first heavy chain comprising negatively charged amino acids at positions 392, 409 and 370 (e.g., K392D, K409D and K370D substitutions), and a second heavy chain comprising positively charged amino acids at positions 356, 399 and 357 (e.g., E356K, D399K and E357K substitutions). In a related embodiment, the first heavy chain is from an anti-CGRP receptor antibody, and the second heavy chain is from an anti-PAC1 receptor antibody. In other related embodiments, the first heavy chain is from an anti-PAC1 receptor antibody and the second heavy chain is from an anti-CGRP receptor antibody.

[0051] In one embodiment, one CH3 domain comprises a Y349C mutation; and the other CH3 domain comprises an E356C or S354C mutation; wherein the numbering of the amino acid residues is according to the EU index as set forth in Kabat. In one embodiment, one CH3 domain comprises a Y349C and a T366W mutation; and the other CH3 domain comprises an E356C, a T366S, a L368A, and a Y407V mutation; wherein the numbering of the amino acid residues is according to the EU index as set forth in Kabat. In one embodiment, one CH3 domain comprises a Y349C and a T366W mutation; and the other CH3 domain comprises an S354C, a T366S, a L368A, a Y407V mutation; wherein the numbering of the amino acid residues is according to the EU index as set forth in Kabat.

[0052] In certain embodiments, the immunoglobulin hinge region is an IgG1 hinge region.

[0053] In certain embodiments, the heteromultimer is a bispecific or multispecific antibody.

[0054] 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 functional fragments thereof. "Functional antibody fragments" are at least a portion of the amino acids present in the antibody that do not contain the full-length heavy chain and / or light chain, but are still able to specifically bind to the antigen. Functional antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementary determining region (CDR) fragments, and may be derived from any mammalian source, such as humans, mice, rats, rabbits, or camels. Functional antibody fragments can compete with intact antibodies for binding to target antigens, and these fragments can be obtained by modifying intact antibodies (e.g., enzymatic or chemical cleavage) or synthesizing de novo using recombinant DNA technology or peptide synthesis.

[0055] Antigen binding proteins may also include proteins comprising one or more functional antibody fragments incorporated into a single polypeptide chain or into multiple polypeptide chains. For example, antigen binding proteins can include, but are not limited to, single-chain Fvs (scFvs), diabodies (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, Vol. 90: 6444-6448, 1993); intrabodies; domain antibodies (single VL or VH domain or two or more VH domains joined by a peptide linker; see Ward et al., Nature, Vol. 341: 544-546, 1989); macrobodies (fusions of two scFvs with an Fc region, see Fredericks et al., Protein Engineering, Design & Selection, Vol. 17: 95-106, 2004; and Powers et al., Journal of Immunological Biology, Vol. 1996: 115-123, 2008). Methods [Journal of Immunology], Vol. 251: 123-135, 2001); triabody; tetrabody; minibody (a fusion of scFv and CH3 domain; see Olafsen et al., Protein Eng Des Sel [Protein Engineering Design and Selection], Vol. 17: 315-23, 2004); peptibody (one or more peptides attached to the Fc region, see WO 00 / 24782); linear antibody (a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions, see Zapata et al., Protein Eng. [Protein Engineering], Vol. 8: 1057-1062, 1995); small modular immunopharmaceuticals (small modular immunopharmaceuticals). immunopharmaceutical) (see U.S. Patent Publication No. 20030133939); immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH and Fab-scFv-Fc).

[0056] "Multispecific" refers to the ability of an antigen binding protein to specifically bind to two or more different antigens. "Bispecific" refers to the ability of an antigen binding protein to specifically bind to two different antigens. As used herein, an antigen binding protein "specifically binds to" an antigen when its binding affinity for the target antigen is significantly greater than its affinity for other unrelated proteins under similar binding assay conditions and can therefore be distinguished. The equilibrium dissociation constant (K) of an antigen binding protein that specifically binds to an antigen is D )≤1x 10 -6 M. When K D ≤1x 10 -8 When M, the antigen binding protein specifically binds the antigen with "high affinity".

[0057] Affinity is determined using a variety of techniques, such as affinity ELISA assays. In various embodiments, affinity is determined by surface plasmon resonance assays (e.g., based on Using this technique, the association rate constant (expressed in M -1 s -1 k a ) and the dissociation rate constant (in s -1 k d ). Equilibrium dissociation constant (K expressed in M) D ) can be expressed by the ratio of the kinetic rate constants (k d / k a ) calculation. 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 expressed in M) can be measured. D ) and the association rate constant (in M -1 s -1 k a ). Dissociation rate constant (in s -1 k d ) can be calculated from these values ​​(K D xk a In other embodiments, affinity is determined by equilibrium / solution methods. In certain embodiments, affinity is determined by FACS binding assays.

[0058] In some embodiments, the bispecific antigen binding proteins described herein exhibit desirable properties, such as those indicated by K d (dissociation rate constant) measured about 10 -2 , 10 -3 , 10-4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 s -1 or lower binding affinity (lower values ​​indicate higher binding affinity), and / or by K D (equilibrium dissociation constant) measured about 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 The binding affinity of M is expressed as (lower values ​​indicate higher binding affinity).

[0059] As used herein, the term "antigen binding domain" is used interchangeably with "binding domain" and refers to the region of an antigen binding protein containing the amino acid residues that interact with the antigen and confer specificity and affinity to the antigen binding protein for the antigen.

[0060] As used herein, the term "CDR" refers to the complementary determining region (also referred to as "minimum recognition unit" or "hypervariable region") within the variable sequence of an antibody. There are three heavy chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2, and CDRL3). The term "CDR region" as used herein refers to a group of three CDRs that appear in a single variable region (i.e., three light chain CDRs or three heavy chain CDRs). The CDRs in each of the two chains are typically aligned by the framework region to form a structure that specifically binds to a specific epitope or domain on the target protein. From the N-terminus to the C-terminus, naturally occurring light and heavy chain variable regions typically follow the following order of these components: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The numbering system is obtained by assigning numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Maryland) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system.

[0061] In some embodiments of the bispecific antigen-binding proteins of the present invention, the binding domains include Fab, Fab', F(ab')2, Fv, single-chain variable fragments (scFv) or nano antibodies. 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 a scFv.

[0062] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site and a residual "Fc" fragment containing the constant region of an immunoglobulin. The Fab fragment contains all variable domains, as well as the constant domains of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" consists of an immunoglobulin light chain (the variable region of the light chain (VL) and the constant region (CL)) and the CH1 region and the variable region (VH) of an immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The Fc fragment displays carbohydrates and is responsible for many antibody effector functions (such as binding to complement and cell receptors) that distinguish one class of antibodies from another. The "Fd fragment" contains the VH and CH1 domains from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0063] A "Fab' fragment" is a Fab fragment that has one or more cysteine ​​residues from the antibody hinge region at the C-terminus of the CH1 domain.

[0064] A "F(ab')2 fragment" is a bivalent fragment comprising two Fab' fragments connected by a disulfide bridge between the heavy chains at the hinge region.

[0065] The "Fv" fragment is the smallest fragment that contains a complete antigen recognition and binding site from an antibody. The fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in tight non-covalent association. It is in this configuration that the three CDRs of each variable region interact to define an antigen binding site on the surface of the VH-VL dimer. A single light or heavy chain variable region (or half of an Fv fragment containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the entire binding site containing VH and VL.

[0066] A “single-chain variable antibody fragment” or “scFv fragment” comprises the VH and VL regions of an antibody, wherein these regions are present in a single polypeptide chain, and optionally comprises a peptide linker between the VH and VL regions which enables the Fv to form the desired structure for antigen binding (see, e.g., Bird et al., Science, 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988).

[0067] "Nano antibodies" are the heavy chain variable regions of heavy chain antibodies. Such variable domains are the smallest fully functional antigen-binding fragments in such heavy chain antibodies, with a molecular weight of only 15 kDa. See Cortez-Retamozo et al., Cancer Research [Cancer Research] 64: 2853-57, 2004. Functional heavy chain antibodies without light chains are naturally present in certain animal species, such as nurse sharks, wobbegong sharks and camelids, such as camels, dromedaries, alpacas and llamas. In these animals, the antigen binding site is reduced to a single domain, i.e., the VHH domain. These antibodies use only the heavy chain variable region to form an antigen binding region, i.e., these functional antibodies are heavy chain homodimers (referred to as "heavy chain antibodies" or "HCAbs") having only the structure H2L2. It is reported that camelized VHH is recombined with IgG2 and IgG3 constant regions, which contain hinges, CH2 and CH3 domains and lack the CH1 domain. Camelized VHH domains have been found to bind antigen with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and have 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. Alternative scaffolds can be made from human variable-like domains that are closer to the shark V-NAR scaffold and may provide a framework for long penetrating loop structures.

[0068] In particular, in embodiments of the bispecific antigen binding proteins of the present invention, the binding domains comprise 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.

[0069] "Variable region" (variable region of light chain (VL), variable region of heavy chain (VH)), which can be used interchangeably with "variable domain" herein, refers to the region of each of the light and heavy immunoglobulin chains that is directly involved in antibody binding to antigen. As described above, the regions of the variable light chain and the variable heavy chain have the same general structure, and each region comprises four framework (FR) regions, the sequences of which are widely conserved and connected by three CDRs. The framework region is in a β-pleated configuration and the CDRs can form loops connecting the β-pleated structure. The CDRs in each chain maintain their three-dimensional structure through the framework region, and together with the CDRs from the other chain, form an antigen binding site.

[0070] The binding domain that specifically binds to the target antigen can be derived from a) known antibodies to these antigens, or b) new antibodies or antibody fragments obtained by phage display or other conventional methods by de novo immunization methods using antigenic proteins or fragments thereof. The antibody from which the binding domain of the bispecific antigen-binding protein is derived can 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 is derived is a monoclonal antibody. In these and other embodiments, the antibody is a human antibody or a humanized antibody, and can be an IgG1-, IgG2-, IgG3- or IgG4-type.

[0071] As used herein, the term "monoclonal antibody" (or "mAb") refers to an antibody obtained from a substantially homologous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations present in trace amounts. Relative to polyclonal antibody preparations that typically include different antibodies for different epitopes, monoclonal antibodies are highly specific for individual antigenic sites or epitopes. Monoclonal antibodies can be produced using any technique known in the art, such as by immortalizing spleen cells collected from transgenic animals after completing an immunization program. Any technique known in the art can be used, such as by immortalizing spleen cells by fusing them with myeloma cells to produce hybridomas. Myeloma cells used in the fusion program for producing hybridomas are preferably non-antibody-producing, have high fusion efficiency and enzyme defects, and are such that they cannot grow in certain selective culture media that only support the growth of the desired fused cells (hybridomas). Examples of cell lines suitable for mouse fusion include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 41, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XXO Bul; Examples of cell lines used for rat fusion include R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines that can be used for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0072] In some cases, hybridoma cell lines are produced by: immunizing an animal (e.g., a transgenic animal having human immunoglobulin sequences) with a target antigen; collecting spleen cells from the immunized animal; fusing the collected spleen cells with a myeloma cell line, thereby producing hybridoma cells; establishing hybridoma cell lines from the hybridoma cells, and identifying hybridoma cell lines that produce antibodies that bind to the target antigen.

[0073] Monoclonal antibodies secreted by hybridoma cell lines can be purified using any technique known in the art, such as protein A-Sepharose, hydroxylapatite 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 the target antigen, the ability to block or interfere with the binding of the target antigen ligand to its respective receptor, or the ability to functionally block either receptor, such as a cAMP assay.

[0074] In some embodiments, the binding domain of the bispecific antigen-binding protein of the present invention may be derived from a humanized antibody. "Humanized antibody" refers to an antibody in which a region (e.g., a framework region) has been modified to include a corresponding region from a human immunoglobulin. Typically, a humanized antibody can be produced from a monoclonal antibody initially produced in a non-human animal. Certain amino acid residues in the monoclonal antibody, typically amino acid residues from the non-antigen recognition portion of the antibody, are modified to be homologous to the corresponding residues in a human antibody of the corresponding isotype. Humanization can be carried out, for example, using various methods by replacing at least a portion of a rodent variable region with a corresponding region of a human antibody (see, e.g., U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., Nature [Nature], Vol. 321: 522-525, 1986; Riechmann et al., Nature [Nature], Vol. 332: 323-27, 1988; Verhoeyen et al., Science [Science], Vol. 239: 1534-1536, 1988). The CDRs of the light chain variable region and heavy chain variable region of an antibody produced in another species can be grafted onto the common human FRs. To determine the common human FRs, the FRs from several human heavy or light chain amino acid sequences can be aligned to identify the common amino acid sequence.

[0075] The new antibodies produced against the target antigen (from which the binding domains of the bispecific antigen-binding proteins of the present invention are derived) can be fully human antibodies. "Fully human antibodies" are antibodies comprising variable and constant regions derived from or indicative of human germline immunoglobulin sequences. A specific approach provided for producing fully human antibodies is the "humanization" of the mouse humoral immune system. Introducing human immunoglobulin (Ig) loci into mice with inactivated endogenous Ig genes is a way to produce fully human monoclonal antibodies (mAbs) in mice, which are animals that can be immunized with any desired antigen. The use of fully human antibodies can minimize immunogenicity and allergic responses, which are sometimes caused by administering mice or mouse-derived mAbs to humans as therapeutic agents.

[0076] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that are capable of producing a human antibody repertoire without producing endogenous immunoglobulins. Antigens used for this purpose typically have six or more consecutive amino acids and are optionally combined with a carrier, such as a hapten. See, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 90: 2551-2555; Jakobovits et al., 1993, Nature [Nature] 362: 255-258; and Bruggermann et al., 1993, Year in Immunol. [Annual Review of Immunology] 7: 33. In one example of such a method, a transgenic animal is produced by disabling the endogenous mouse immunoglobulin loci encoding mouse heavy and light chain immunoglobulin chains and inserting a larger fragment of a locus containing human genomic DNA encoding human heavy and light chain proteins into the mouse genome. Animals with partial modifications of less than the full complement of human immunoglobulin loci are then cross-bred to obtain animals with all the desired immune system modifications. When an immunogen is administered, these transgenic animals produce antibodies that are immunospecific for that immunogen, while having human rather than murine amino acid sequences, including variable regions. For further details of such methods, see, for example, WO 96 / 33735 and WO 94 / 02602. Other methods for producing transgenic mice for human antibodies are described in U.S. Pat. Nos. 5,545,807, 6,713,610, 6,673,986, 6,162,963, 5,939,598, 5,545,807, 6,300,129, 6,255,458, 5,877,397, 5,874,299, and 5,545,806; and PCT Publications WO 91 / 10741, WO 90 / 04036, WO 94 / 02602, WO 96 / 30498, WO 98 / 24893, and EP 546073B1 and EP 546073A1.

[0077] The transgenic mice are referred to herein as "HuMab" mice, containing human immunoglobulin gene mini-locus encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences and targeted mutations that inactivate endogenous μ and κ chain loci (Lonberg et al., 1994, Nature 368: 856-859). Thus, the mice exhibit reduced mouse IgM or κ expression and immunization responses, and the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGκ monoclonal antibodies (Lonberg et al., supra; Lonberg and Huszar, 1995, Intern. Rev. Immunol. [International Immunology Review] 13: 65-93; Harding and Lonberg, 1995, Ann. N. Y Acad. Sci. [Annual of the New York Academy of Sciences] 764: 536-546). The preparation of HuMab mice is described in detail in the following: 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. N. Y Acad. Sci. 764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851; the above references are incorporated herein by reference in their entirety for all purposes.See also U.S. Pat. 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; and U.S. Pat. No. 5,545,807; International Publication Nos. WO 93 / 1227, WO 92 / 22646; and WO 92 / 03918, the disclosures of all references are incorporated herein by reference in their entirety for all purposes. Techniques for producing human antibodies in these transgenic mice are also disclosed in WO 98 / 24893 and Mendez et al., 1997, Nature Genetics 15:146-156, which are incorporated herein by reference.

[0078] Antibodies of human origin can also be produced using phage display technology. Phage display is described in, for example, Dower et al., WO 91 / 17271; McCafferty et al., WO 92 / 01047; and Caton and Koprowski, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 87: 6450-6454 (1990), each of which is incorporated herein by reference in its entirety. Antibodies produced by phage technology are usually produced in bacteria as antigen-binding fragments, such as Fv or Fab fragments, and therefore lack effector functions. Effector functions can be introduced by one of the following two strategies: if necessary, these fragments can be engineered into complete antibodies for expression in mammalian cells, or engineered into bispecific antibody fragments with a second binding site capable of triggering effector functions. Typically, the Fd fragment (VH-CH1) and light chain (VL-CL) of the cloned antibody are separated by PCR and randomly recombined in a combinatorial phage display library, and then selection can be made for binding to a specific antigen. These antibody fragments are expressed on the phage surface, and antigen binding is used to select Fv or Fab (and therefore the phage containing the DNA encoding the antibody fragment is selected) through several rounds of antigen binding and re-amplification. This procedure is called panning. Antibody fragments with specificity for antigen are enriched and finally separated. Phage display technology can also be used in methods for humanizing rodent monoclonal antibodies, referred to as "guided selection" (see Jespers, LS, et al., Bio / Technology [Biology / Technology] 12, 899-903 (1994)). For this reason, the Fd fragment of a mouse monoclonal antibody can be displayed in combination with a human light chain library, and then the hybrid Fab library obtained thereby can be selected with an antigen. The mouse Fd fragment thus provides a template for guiding selection. Subsequently, the selected human light chain is combined with a human Fd fragment library. Selection of the resulting library yielded fully human Fabs.

[0079] In certain embodiments, the bispecific antigen-binding proteins of the present invention are antibodies. As used herein, the term "antibody" refers to a tetrameric immunoglobulin 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) can 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), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3) and optionally an immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus to the carboxyl terminus. Heavy chains are classified into mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε) chains, and they define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Antibodies of the IgG and IgA categories are further subdivided into several subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains in IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains in IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via inter-polypeptide 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 chains.

[0080] In certain embodiments, the bispecific antigen binding proteins of the invention are heterodimeric antibodies (used interchangeably herein with "heteroimmunoglobulin" or "heteroIg"), which refers to antibodies comprising two different light chains and two different heavy chains.

[0081] Heterodimeric antibodies may include any immunoglobulin constant region. As used herein, the term "constant region" refers to all domains in an antibody except the variable region. The constant region does not directly participate in the binding of an antigen, but exhibits various effector functions. As described above, antibodies are divided into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) depending on the amino acid sequence of their heavy chain constant region. The light chain constant region may be, for example, a κ-type or λ-type light chain constant region found in all five antibody isotypes, such as a human κ-type or λ-type light chain constant region.

[0082] The heavy chain constant region of the heterodimeric antibody can be, for example, an α, Δ, ε, γ or μ type heavy chain constant region, such as a human α, Δ, ε, γ or μ type heavy chain constant region. In some embodiments, the heterodimeric antibody comprises a heavy chain constant region from an IgG1, IgG2, IgG3 or IgG4 immunoglobulin. In one embodiment, the heterodimeric antibody comprises a heavy chain constant region from a human IgG1 immunoglobulin. In another embodiment, the heterodimeric antibody comprises a heavy chain constant region from a human IgG2 immunoglobulin.

[0083] In one embodiment, the bispecific antibody of the present disclosure is a Duobody TM , which may be formed by a DuoBody as described below TM Technology platform (Genmab A / S) preparation: for example, International Publication Nos. WO 2008 / 119353, WO 2011 / 131746, WO 2011 / 147986, and WO 2013 / 060867, Labrijn AF et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 110(13):5145-5150 (2013), Gramer et al., mAbs [monoclonal antibodies], 5(6):962-973 (2013) and Labrijn et al., Nature Protocols [Natural Experimental Program], 9(10):2450-2463 (2014). This technology can be used to combine half of a first monospecific antibody containing two heavy chains and two light chains with half of a second monospecific antibody containing two heavy chains and two light chains. The resulting heterodimer comprises one heavy chain and one light chain from the first antibody paired with one heavy chain and one light chain from the second antibody. When two monospecific antibodies recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

[0084] For DuoBody TMThe present invention relates to a bispecific antibody comprising a heavy chain constant region with a single point mutation in the CH3 domain. The interaction between the CH3 domains in the bispecific antibody produced by these point mutations is stronger than the interaction between the CH3 domains in any monospecific antibody without mutation. The single point mutation in each monospecific antibody can be at residues 366, 368, 370, 399, 405, 407 or 409 (EU numbering) of the heavy chain constant region CH3 domain (see WO 2011 / 131746). In addition, the single point mutation is located at a different residue in one monospecific antibody relative to another monospecific antibody. For example, one monospecific antibody may comprise a mutation F405L (EU numbering; a phenylalanine to leucine mutation at residue 405), or one of the F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y mutations, while another monospecific antibody may comprise a mutation K409R (EU numbering; a lysine to arginine mutation at residue 409). The heavy chain constant region of the monospecific antibody may be of the IgG1, IgG2, IgG3, or IgG4 isotype (e.g., human IgG1 isotype), and is expressed by the DuoBody TM Bispecific antibodies produced using the technology can be modified to alter (e.g., reduce) Fc-mediated effector function and / or improve half-life. TM One method includes the following: (i) separately expressing two parental IgG1s containing single matching point mutations in the CH3 domain (i.e., K409R and F405L (or one of the F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y mutations) (EU numbering)); (ii) mixing the parental IgG1s under permissive redox conditions in vitro to enable reassembly of the half molecules; (iii) removing the reducing agent to allow reoxidation of the interchain disulfide bonds; and (iv) analyzing the exchange efficiency and final products using chromatography-based or mass spectrometry (MS)-based methods (see Labrijn et al., Nature Protocols, 9(10):2450-2463 (2014)).

[0085] Another exemplary method for preparing bispecific antibodies is by the knob-hole technique (Ridgway et al., Protein Eng. [Protein Engineering], 9: 617-621 (1996); WO 2006 / 028936). In this technique, the Ig heavy chain mispairing problem, which is a major drawback in preparing bispecific antibodies, is reduced by mutating selected amino acids that form the CH3 domain interface in IgG. At the position where the two heavy chains directly interact within the CH3 domain, amino acids with small side chains are introduced into the sequence of one heavy chain (knob-hole), and amino acids with large side chains are introduced into the corresponding interacting residue position on the other heavy chain (knob). In some cases, the antibodies of the present disclosure have immunoglobulin chains in which the CH3 domain has been modified by mutating selected amino acids that interact at the interface between the two polypeptides so as to preferentially form bispecific antibodies. Bispecific antibodies can be composed of immunoglobulin chains of the same subclass or different subclasses. In one example, a bispecific antibody that binds gp120 and CD3 comprises a T366W (EU numbering) mutation in the "knob chain" and T366S, L368A, Y407V (EU numbering) mutations in the "hole chain". In certain embodiments, additional interchain disulfide bridges are introduced between the CH3 domains by, for example, introducing a Y349C mutation into the "knob chain" and an E356C mutation or an S354C mutation into the "hole chain". In certain embodiments, R409D, K370E mutations are introduced into the "knob chain", and D399K, E357K mutations are introduced into the "hole chain". In other embodiments, Y349C, T366W mutations are introduced into one of the chains, and E356C, T366S, L368A, Y407V mutations are introduced into the corresponding chain. In some embodiments. In one chain, Y349C, T366W mutations are introduced, and in the corresponding chain, S354C, T366S, L368A, Y407V mutations are introduced. In other embodiments, Y349C, T366W mutations are introduced in one chain, and S354C, T366S, L368A, Y407V mutations are introduced in the corresponding chain. In yet other embodiments, Y349C, T366W mutations are introduced in one chain, and S354C, T366S, L368A, Y407V mutations are introduced in the corresponding chain (all according to EU numbering).

[0086] Another method for preparing bispecific antibodies is the CrossMab technology. CrossMab is a chimeric antibody consisting of half of two full-length antibodies. In order to achieve correct chain pairing, it combines two technologies: (i) a knob-hole that facilitates correct pairing between the two heavy chains; and (ii) an exchange between the heavy chain and the light chain of one of the two Fabs to introduce asymmetry that avoids light chain mispairing. See, Ridgway et al., Protein Eng. [Protein Engineering], 9: 617-621 (1996); Schaefer et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 108: 11187-11192 (2011). CrossMab can combine two or more antigen binding domains for targeting two or more targets, or for introducing bivalence to one target, such as a 2: 1 format.

[0087] In order to promote the association of a specific heavy chain with its cognate light chain, both the heavy chain and the light chain may include complementary amino acid substitutions. As used herein, "complementary amino acid substitutions" refer to the substitution pairing of positively charged amino acids in one chain with negatively charged amino acids in another chain. For example, in some embodiments, the heavy chain comprises at least one amino acid substitution to introduce a charged amino acid, and the corresponding light chain comprises at least one amino acid substitution to introduce a charged amino acid, wherein the charged amino acid introduced in the heavy chain has an opposite charge to the amino acid introduced in the light chain. In certain embodiments, one or more positively charged residues (e.g., lysine, histidine, or arginine) may be introduced into the first light chain (LC1), and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) may be introduced into the companion heavy chain (HC1) at the LC1 / HC1 binding interface, and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) may be introduced into the second light chain (LC2), and one or more positively charged residues (e.g., lysine, histidine, or arginine) may be introduced into the companion heavy chain (HC2) at the LC2 / HC2 binding interface. When oppositely charged residues (polarity) at the interface attract, electrostatic interactions will guide LC1 to pair with HC1 and LC2 to pair with HC2. Heavy / light chain pairs with identically charged residues (polarity) at the interface (e.g., LC1 / HC2 and LC2 / HC1) will repel, leading to inhibition of unwanted HC / LC pairing.

[0088] In these and other embodiments, the CH1 domain of the heavy chain or the CL domain of the light chain comprises an amino acid sequence 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 by one or more negatively charged amino acids. Alternatively, the CH1 domain of the heavy chain or the CL domain of the light chain comprises an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are replaced by one or more positively charged amino acids. In some embodiments, the CH1 domain of the first and / or second heavy chain in the heterodimeric antibody is selected from F126, P127, L128, A141, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S183, V185 and K213. One or more amino acids at EU positions are replaced by charged amino acids. In certain embodiments, the preferred residue substituted with negatively or positively charged amino acids is S183 (EU numbering system). In some embodiments, S183 is replaced by a positively charged amino acid. In alternative embodiments, S183 is replaced by a negatively charged amino acid. For example, in one embodiment, S183 is replaced by a negatively charged amino acid (e.g., S183E) in the first heavy chain, and S183 is replaced by a positively charged amino acid (e.g., S183K) in the second heavy chain.

[0089] In embodiments where the light chain is a kappa light chain, one or more amino acids in the CL domain of the first and / or second light chain in the heterodimeric antibody at positions selected from F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174 and S176 (in EU and Kabat numbering in the kappa light chain) are replaced with a charged amino acid. In the embodiment where the light chain is a lambda light chain, one or more amino acids in the CL domain of the first and / or second light chain in the heterodimeric antibody are replaced by a charged amino acid at a position selected from T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176 and Y178 (in the lambda chain with Kabat numbering). In some embodiments, the preferred residue substituted with a negatively or positively charged amino acid is S176 of the CL domain of a kappa or lambda light chain (EU and Kabat numbering systems). In certain embodiments, S176 of the CL domain is replaced by a positively charged amino acid. In an alternative embodiment, S176 of the CL domain is replaced by a negatively charged amino acid. In one embodiment, S176 is substituted with a positively charged amino acid (eg, S176K) in the first light chain, and S176 is substituted with a negatively charged amino acid (eg, S176E) in the second light chain.

[0090] In addition to the complementary amino acid substitutions in the CH1 and CL domains or as an alternative, the variable regions of the light and heavy chains in the heterodimeric antibody may include 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 the heterodimeric antibody comprises 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 by one or more negatively charged amino acids. Alternatively, the VH region of the heavy chain or the VL region of the light chain comprises an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are replaced by one or more positively charged amino acids.

[0091] The V region interface residues (i.e., amino acid residues mediating the assembly of VH and VL regions) in the VH region include Kabat positions 1, 3, 35, 37, 39, 43, 44, 45, 46, 47, 50, 59, 89, 91 and 93. One or more of these interface residues in the VH region can be replaced by charged (positively or negatively charged) amino acids. In certain embodiments, the amino acid at Kabat position 39 in the VH region of the first and / or second heavy chain is replaced by a positively charged amino acid (e.g., lysine). In an alternative embodiment, the amino acid at Kabat position 39 in the VH region of the first and / or second heavy chain is replaced by a negatively charged amino acid (e.g., glutamic acid). In some embodiments, the amino acid at Kabat position 39 in the VH region of the first heavy chain is replaced by a negatively charged amino acid (e.g., G39E), and the amino acid at Kabat position 39 in the VH region of the second heavy chain is replaced by a positively charged amino acid (e.g., G39K). In some embodiments, the amino acid at Kabat position 44 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid (e.g., lysine). In alternative embodiments, the amino acid at Kabat position 44 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid (e.g., glutamic acid). In certain embodiments, the amino acid at Kabat position 44 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G44E), and the amino acid at Kabat position 44 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G44K).

[0092] The V region interface residues (i.e., amino acid residues mediating the assembly of the VH and VL regions) in the VL region include Kabat positions 32, 34, 35, 36, 38, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 85, 87, 89, 90, 91 and 100. One or more interface residues in the VL region can be replaced with charged amino acids (preferably with amino acids with opposite charges to those in the VH region of the homologous heavy chain). In some embodiments, the amino acid at Kabat position 100 in the VL region of the first and / or second light chain is replaced by a positively charged amino acid (e.g., lysine). In an alternative embodiment, the amino acid at Kabat position 100 in the VL region of the first and / or second light chain is replaced by a negatively charged amino acid (e.g., glutamic acid). In certain embodiments, the amino acid at Kabat position 100 in the VL region of the first light chain is substituted with a positively charged amino acid (e.g., G100K), and the amino acid at Kabat position 100 in the VL region of the second light chain is substituted with a negatively charged amino acid (e.g., G100E).

[0093] In certain embodiments, the heterodimeric antibodies of the present invention comprise a first heavy chain and a second heavy chain and a first light chain and a second light chain, wherein the first heavy chain comprises amino acid substitutions at positions 44 (Kabat), 183 (EU), 392 (EU) and 409 (EU), wherein the second heavy chain comprises amino acid substitutions at positions 44 (Kabat), 183 (EU), 356 (EU) and 399 (EU), wherein the first and second light chains comprise amino acid substitutions at positions 100 (Kabat) and 176 (EU), and wherein the amino acid substitutions introduce charged amino acids at said positions. In related embodiments, the glycine at position 44 (Kabat) of the first heavy chain is replaced by glutamic acid, the glycine at position 44 (Kabat) of the second heavy chain is replaced by lysine, the glycine at position 100 (Kabat) of the first light chain is replaced by lysine, the glycine at position 100 (Kabat) of the second light chain is replaced by glutamic acid, the serine at position 176 (EU) of the first light chain is replaced by lysine, the serine at position 176 (EU) of the second light chain is replaced by glutamic acid, the serine at position 183 (EU) of the first heavy chain is replaced by glutamic acid, the lysine at position 392 (EU) of the first heavy chain is replaced by aspartic acid, the lysine at position 409 (EU) of the first heavy chain is replaced by aspartic acid, the serine at position 183 (EU) of the second heavy chain is replaced by lysine, the glutamic acid at position 356 (EU) of the second heavy chain is replaced by lysine, and / or the aspartic acid at position 399 (EU) of the second heavy chain is replaced by lysine.

[0094] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of a complete antibody. The Fc region of an immunoglobulin generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. In certain embodiments, the Fc region is an Fc region from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In certain embodiments, the Fc region comprises CH2 and CH3 domains from human IgG1 or human IgG2 immunoglobulins. The Fc region can retain effector functions, such as C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region can be modified to reduce or eliminate effector functions, as described in more detail herein.

[0095] In some embodiments of the antigen-binding proteins of the present invention, the binding domain located at the carboxyl terminus of the Fc region (i.e., the carboxyl terminal binding domain) is a scFv. In certain embodiments, the scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL) connected by a peptide linker. The variable region can be oriented in a VH-VL or VL-VH orientation in the scFv. For example, in one embodiment, the scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In another embodiment, the scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. The VH and VL regions of the scFv may comprise one or more cysteine ​​substitutions to allow a disulfide bond to be formed between the VH and VL regions. Such a cysteine ​​clamp stabilizes the two variable domains in the antigen binding configuration. In one embodiment, position 44 (Kabat numbering) in the VH region and position 100 (Kabat numbering) in the VL region are each substituted with a cysteine ​​residue.

[0096] In certain embodiments, the scFv is fused to the carboxyl terminus of the Fc region (e.g., the carboxyl terminus of the CH3 domain) at its amino terminus by a peptide linker or otherwise connected. Therefore, in one embodiment, the scFv is fused to the Fc region so that the resulting fusion protein comprises a CH2 domain, a CH3 domain, a first peptide linker, a VH region, a second peptide linker, and a VL region from the N-terminus to the C-terminus. In another embodiment, the scFv is fused to the Fc region so that the resulting fusion protein comprises a CH2 domain, a CH3 domain, a first peptide linker, a VL region, a second peptide linker, and a VH region from the N-terminus to the C-terminus. A "fusion protein" is a protein comprising a polypeptide component derived from more than one parent protein or polypeptide. Typically, the fusion protein is expressed from a fusion gene, wherein the nucleotide sequence encoding the polypeptide sequence from one protein is attached to the nucleotide sequence encoding the polypeptide sequence in an open reading frame, and is optionally separated from the nucleotide sequence encoding the polypeptide sequence from a different protein by a linker. The fusion gene can then be expressed by a recombinant host cell to produce a single fusion protein.

[0097] "Peptide linker" refers to an oligopeptide of about 2 to about 50 amino acids that covalently connects one polypeptide to another polypeptide. Peptide linkers can be used to connect the VH and VL domains in scFv. Peptide linkers can also be used to connect scFv, Fab fragments or other functional antibody fragments to the amino terminus or carboxyl terminus of the Fc region to produce bispecific antigen-binding proteins described herein. Preferably, the length of the peptide linker is at least 5 amino acids. In certain embodiments, the length of the peptide linker is about 5 amino acids to a length of about 40 amino acids. In other embodiments, the length of the peptide linker is about 8 amino acids to a length of about 30 amino acids. In other embodiments, the length of the peptide linker is about 10 amino acids to a length of about 20 amino acids.

[0098] Preferably, but not necessarily, the peptide linker comprises an amino acid from the twenty standard amino acids, in particular cysteine, glycine, alanine, proline, asparagine, glutamine and / or serine. In certain embodiments, the peptide linker is composed of a majority of amino acids (e.g., glycine, serine and alanine) that are sterically unhindered. Therefore, in some embodiments, preferred linkers include polyglycine, polyserine and polyalanine, or any combination of these. 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 In certain embodiments, the peptide linker is (Gly x Ser) n , wherein x=3 or 4 and n=2, 3, 4, 5 or 6. 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 connecting the VH and VL regions within the scFv is an L15 or (G4S)3 linker (SEQ ID NO: 31). In these and other embodiments, the peptide linker connecting the carboxyl-terminal binding domain (e.g., scFv or Fab) to the C-terminus of the Fc region is a L9 or G3SG4S linker (SEQ ID NO: 28) or a L10(G4S)2 linker (SEQ ID NO: 29).

[0099] 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); GGDDDGG (SEQ ID NO: 12); GDDDG (SEQ ID NO: 13); GDDD (SEQ ID NO: 14). 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).

[0100] The heavy chain constant region or Fc region of the bispecific antigen-binding proteins described herein may comprise one or more amino acid substitutions that affect the glycosylation and / or effector function of the antigen-binding protein. One of the functions of the immunoglobulin Fc region is to communicate with the immune system when the immunoglobulin binds to its target. This is generally referred to as "effector function". Communication leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by binding to Fc receptors on the surface of immune system cells via the Fc region. CDC is mediated by binding to proteins of the complement system, such as C1q, via Fc. In some embodiments, the bispecific antigen-binding proteins of the present invention comprise one or more amino acid substitutions in the constant region that enhance the effector function (including ADCC activity, CDC activity, ADCP activity and / or clearance rate or half-life) of the antigen-binding protein. Exemplary amino acid substitutions (EU numbering) that can enhance effector function include, but are not limited to, E233L, L234I, L234Y, L235S, G236A, S239D, F243L, F243V, P247I, D280H, K290S, K290E, K290N, K290Y, R292P, E294L, Y296W, S298A, S298D, S 298V, 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 foregoing.

[0101] In other embodiments, the bispecific antigen-binding proteins of the present invention include one or more amino acid substitutions that reduce effector function in the constant region. Exemplary amino acid substitutions (EU numbering) that can reduce effector function include, but are not limited to, C220S, C226S, C229S, E233P, L234A, L234V, V234A, L234F, L235A, L235E, G237A, P238S, S267E, H268Q, N297A, N297G, V309L, E318A, L328F, A330S, A331S, P331S or any combination of the foregoing.

[0102] Glycosylation can promote the effector function of antibodies, particularly IgG1 antibodies. Therefore, in some embodiments, the bispecific antigen-binding proteins of the present invention comprise one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. The glycosylation of polypeptides is typically N-connected or O-connected. N-connection refers to the carbohydrate moiety being connected to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid other than proline) are recognition sequences for enzymatically connecting the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any one of these tripeptide sequences in a polypeptide produces a potential glycosylation site. O-linked glycosylation refers to connecting one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

[0103] In certain embodiments, the glycosylation of the binding protein is increased by adding one or more glycosylation sites to the Fc region of the bispecific antigen-binding protein, for example, as described herein. Adding glycosylation sites to the antigen-binding protein can generally be achieved by changing the amino acid sequence so that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Changes can also be made by adding one or more serine or threonine residues to the starting sequence or by replacing one or more serine or threonine residues (for O-linked glycosylation sites). For convenience, the amino acid sequence of the antigen-binding protein can be changed by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at a pre-selected base, thereby generating codons that will be translated into the desired amino acids.

[0104] The present invention also encompasses the production of bispecific antigen-binding protein molecules with altered carbohydrate structures that cause changes in effector activity, including antigen-binding proteins that exhibit improved ADCC activity without or with reduced fucosylation. Various methods of reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by binding antibody molecules to the FcγRIII receptor, which has been shown to depend on the glycosylated carbohydrate structure of the N-linked residue N297 of the CH2 domain. Compared to natural, fucosylated antibodies, non-fucosylated antibodies bind to this receptor with increased affinity and more effectively trigger FcγRIII-mediated effector function. For example, recombinant production of non-fucosylated antibodies in CHO cells in which α-1,6-fucosyltransferase has been knocked out gives antibodies a 100-fold increased ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. [Biotechnology and Bioengineering] 87 (5): 614-22, 2004). Similar effects can be achieved by reducing the activity of α-1,6-fucosyltransferase or other enzymes in the fucosylation pathway, for example by siRNA or antisense RNA treatment, engineering the cell line to knock out one or more enzymes, or culturing with 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 a low degree 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). It has also been determined that increasing the amount of bisected carbohydrate, for example by recombinant production of antibodies in cells overexpressing the GnTIII enzyme, can increase ADCC activity (see Umana et al., Nat Biotechnol. 17(2): 176-80, 1999).

[0105] In other embodiments, the glycosylation of the binding protein is reduced or eliminated by, for example, removing one or more glycosylation sites from the Fc region of the bispecific antigen-binding protein described herein. Amino acid substitutions that eliminate or change the glycosylation sites connected by N- can reduce or eliminate the glycosylation of the N-connections of the antigen-binding protein. In certain embodiments, the bispecific antigen-binding protein described herein includes a mutation at position N297 (EU numbering), such as N297Q, N297A or N297G. In a specific embodiment, the bispecific antigen-binding protein of the present invention includes an Fc region with an N297G mutation from a human IgG1 antibody. In order to improve the stability of the molecule containing the N297 mutation, the Fc region of the molecule can be further engineered. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine ​​to promote the formation of disulfide bonds in the dimer state. Residues corresponding to V259, A287, R292, V302, L306, V323 or I332 (EU numbering) in the IgG1 Fc region can therefore be substituted with cysteine. Preferably, specific residue pairs are substituted with cysteine ​​so that they preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond disorder. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In a specific embodiment, the bispecific antigen-binding proteins described herein comprise an Fc region from a human IgG1 antibody with R292C and V302C mutations. In such embodiments, the Fc region may also comprise an N297G mutation.

[0106] It may also be desirable to modify the bispecific antigen-binding proteins of the invention to increase serum half-life, for example by incorporating or adding a salvage receptor binding epitope (e.g., by mutating the appropriate region or by incorporating the epitope into a peptide tag, which is then fused to the antigen-binding protein at either end or in the middle, for example by DNA or peptide synthesis; see, e.g., WO96 / 32478) or adding molecules such as PEG or other water-soluble polymers, including polysaccharide polymers. The salvage receptor binding epitope preferably constitutes a region in which any one or more amino acid residues from one or two loops in the Fc region are transferred to a similar position in the antigen-binding protein. Even more preferably, three or more amino acid residues from one or two loops in the Fc region are transferred. Even more preferably, the epitope is taken from the CH2 domain of the Fc region (e.g., the IgG Fc region) and transferred to the CH1, CH3 or VH region of the antigen-binding protein, or more than one such region. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the antigen-binding protein. For a description of Fc variants and their interaction with salvage receptors, see International Publication Nos. WO 97 / 34631 and WO 96 / 32478.

[0107] The present invention includes one or more isolated nucleic acids encoding the bispecific antigen binding proteins and components thereof described herein. The nucleic acid molecules of the present invention include DNA and RNA in single-stranded and double-stranded forms, and corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the present invention include full-length genes or cDNA molecules, and combinations of fragments thereof. The nucleic acids of the present invention are preferably derived from human sources, but the present invention also includes nucleic acids derived from non-human species.

[0108] The relevant amino acid sequence or polypeptide of interest from an immunoglobulin or a region thereof (e.g., a variable region, an Fc region, etc.) can be determined by direct protein sequencing, and a suitable encoding nucleotide sequence can be designed according to a universal codon table. Alternatively, the genome or cDNA encoding the monoclonal antibody (from which the binding domain of the bispecific antigen-binding protein of the present invention can be derived) can be isolated and sequenced from cells producing the monoclonal antibody using conventional methods (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody).

[0109] "Isolated nucleic acid" is used interchangeably with "isolated polynucleotide" in this article, and in the case of isolating nucleic acid from naturally occurring sources, refers to nucleic acid separated from adjacent gene sequences present in the genome of an organism from which nucleic acid is isolated. In the case of nucleic acid synthesized by template enzymatic method or chemical synthesis, such as PCR product, cDNA molecule or oligonucleotide, it should be understood that the nucleic acid obtained by such method is an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of an independent fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, nucleic acid is substantially free of contaminating endogenous substances. The nucleic acid molecule is preferably derived from a substantially pure form and can utilize standard biochemical methods (such as Sambrook et al., Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual], 2nd edition, Cold Spring Harbor Laboratory [Cold Spring Harbor Laboratory], Cold Spring Harbor [Cold Spring Harbor], NY [New York] (1989) in the method of summarizing) to identify, operate and reclaim the amount or concentration of its component nucleotide sequence to separate at least once DNA or RNA. Such sequences are preferably provided and / or constructed in the form of an open reading frame that does not contain internal non-translated sequences or introns that are typically present in eukaryotic genes. Non-translated DNA sequences may be present at 5' or 3' of the open reading frame, where these sequences do not interfere with the operation or expression of the coding region. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as 5'. The 5' to 3' addition direction of a nascent RNA transcript is referred to as the transcription direction; a sequence region on a DNA strand that has the same sequence as an RNA transcript and is 5' relative to the 5' end of the RNA transcript is referred to as an "upstream sequence"; a sequence region on a DNA strand that has the same sequence as an RNA transcript and is 3' relative to the 3' end of the RNA transcript is referred to as a "downstream sequence".

[0110] The present invention also includes nucleic acids that hybridize to nucleic acids encoding polypeptides as described herein under conditions of moderate stringency, and more preferably under conditions of high stringency. Basic parameters that affect the selection of hybridization conditions and guidance on designing suitable conditions are set forth, for example, in 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., ed., John Wiley & Sons, Inc., Sections 2.10 and 6.3-6.4), and can be readily determined by one of ordinary skill in the art based on, for example, the length and / or base composition of the DNA. One way to achieve moderate stringency hybridization conditions involves using a pre-wash solution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer containing about 50% formamide, 6×SSC, and a hybridization temperature of about 55° C. (or other similar hybridization solutions, such as a hybridization solution containing about 50% formamide, and a hybridization temperature of about 42° C.), and a wash condition of about 60° C. and 0.5×SSC, 0.1% SDS. In general, high stringency conditions are defined as hybridization conditions as described above, but utilizing a wash condition of 0.2×SSC, 0.1% SDS at about 68° C. SSPE (1×SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can replace SSC (1×SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washing is performed after hybridization is complete and lasts for 15 minutes. It will be appreciated that the washing temperature and washing solution salt concentration may be adjusted, if necessary, to achieve the desired stringency by applying the basic principles governing hybridization reactions and duplex stability known to those of ordinary skill in the art and described further below (see, e.g., Sambrook et al., 1989). When a nucleic acid is hybridized to a target nucleic acid of unknown sequence, the hybrid length is assumed to be the length of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the nucleic acid sequences and identifying one or more regions of optimal sequence complementarity. The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5°C to 10°C lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equation.For hybrids less than 18 base pairs in length, 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), where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer (for 1×SSC, [Na+] = 0.165 M). Preferably, each such hybridizing nucleic acid 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 is at least 25% (more preferably at least 50%, or at least 60%, or at least 70% and most preferably at least 80%) the length of the nucleic acid of the invention to which it hybridizes, and has at least 60% (more preferably at least 70%, at least 75%, at least 80%) of the nucleic acid of the invention to which it hybridizes. %, 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%) sequence identity, wherein sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned for maximum overlap and identity while minimizing sequence gaps as described in more detail below.

[0111] As outlined herein, site-specific mutagenesis can be performed on nucleotides in the DNA encoding the polypeptide using cassette or PCR mutagenesis, or other techniques well known in the art, to produce DNA encoding variants, and then recombinant DNA is expressed in cell culture to prepare variants of the antigen-binding proteins described herein. However, determined techniques can also be used to prepare antigen-binding proteins comprising variant CDRs with up to about 100-150 residues by in vitro synthesis. These variants typically exhibit qualitative biological activity identical to naturally occurring analogs, such as binding to antigens. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antigen-binding protein. A combination of deletions, insertions and substitutions is performed to achieve the final construct, provided that the final construct has the desired characteristics. Amino acid changes may also change the post-translational processing of the antigen-binding protein, such as changing the number or position of glycosylation sites. In certain embodiments, the preparation of antigen-binding protein variants is intended to modify amino acid residues that are directly involved in epitope binding. In other embodiments, for the purposes discussed herein, residues that are not directly involved in epitope binding or residues that are not involved in epitope binding in any way need to be modified. Mutagenesis in any CDR region and / or framework region is contemplated. One of ordinary skill in the art can employ covariance analysis to design useful modifications in the amino acid sequence of an antigen binding protein. See, e.g., 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 Publication No. 2008 / 0318207A1; Glaser et al., U.S. Patent Publication No. 2009 / 0048122A1; Urech et al., WO 2008 / 110348A1; Borras et al., WO 2009 / 000099 A2. Such modifications, as determined by analysis of covariance, may improve the potency, pharmacokinetics, pharmacodynamics, and / or manufacturability characteristics of the antigen binding protein.

[0112] The present invention also includes vectors, which contain one or more nucleic acids encoding one or more components (e.g., variable regions, light chains, heavy chains, modified heavy chains, and Fd fragments) of the bispecific antigen-binding proteins of the present invention. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) used to transfer protein coding information to a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing the desired coding sequence and the appropriate nucleic acid control sequences required for expressing the operably connected coding sequence in a specific host cell. The expression vector may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, affect RNA splicing of the coding region operably connected thereto. The nucleic acid sequences required for expression in prokaryotes include promoters, optionally operator sequences, ribosome binding sites, and other sequences that may be present. It is known that eukaryotic cells can utilize promoters, enhancers, and termination and polyadenylation signals. The secretory signal peptide sequence operably linked to the coding sequence of interest may also be optionally encoded by the expression vector, so that the expressed polypeptide can be secreted by the recombinant host cell, making it easier to separate the polypeptide of interest from the cell when needed. For example, in some embodiments, the signal peptide sequence can be attached / fused to the amino terminus of any polypeptide sequence listed in Tables 6A, 6B, 7A, 7B, 9 and 10. In certain embodiments, the signal peptide having the amino acid sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 32) is fused to the amino terminus of any polypeptide sequence in Tables 6A, 6B, 7A, 7B, 9 and 10. In other embodiments, the signal peptide having the amino acid sequence of MAWALLLLTLLTQGTGSWA (SEQ ID NO: 33) is fused to the amino terminus of any polypeptide sequence in Tables 6A, 6B, 7A, 7B, 9 and 10. In yet other embodiments, a signal peptide having the amino acid sequence of 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 polypeptide chain listed in Tables 6A, 6B, 7A, 7B, 9 and 10, for example to promote or optimize expression in a particular host cell.

[0113] Typically, the expression vector used in the host cell to produce the bispecific antigen protein of the present invention will contain sequences for plasmid maintenance and for cloning and expressing the exogenous nucleotide sequences encoding the components of the bispecific antigen binding protein. In certain embodiments, collectively referred to as "flanking sequences", typically will include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker component. These sequences are discussed below.

[0114] 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; the oligonucleotide tag sequence encodes polyhistidine (e.g., hexa-histidine), FLAG, HA (hemagglutinin influenza virus), myc, or another "tag" molecule to which an existing commercially available antibody is directed. The tag is typically fused to the antibody when the polypeptide is expressed, and can be used as a means of affinity purification or detection of polypeptides from host cells. Affinity purification can be achieved, for example, by column chromatography, using antibodies to the tag as an affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as cleavage using certain peptidases.

[0115] The flanking sequences may be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species different from the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, as long as the flanking sequences function in and can be activated by the host cell machinery.

[0116] The flanking sequences that can be used in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, the flanking sequences that can be used herein will be identified in advance by positioning and / or by restriction endonuclease digestion and therefore can be separated from appropriate tissue sources using appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequence may be known. Here, the flanking sequence can be synthesized using conventional nucleic acid synthesis or cloning methods.

[0117] Whether all or only a portion of the flanking sequence is known, it can be obtained using the polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes, such as oligonucleotides and / or flanking sequence fragments from the same or another species. If the flanking sequence is unknown, a DNA fragment containing the flanking sequence can be isolated from a larger DNA fragment that may contain, for example, a coding sequence or even another gene or genes. Appropriate DNA fragments can be generated by restriction endonuclease digestion, followed by agarose gel purification, Separation can be accomplished by column chromatography (Chatsworth, CA) or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.

[0118] The replication origin is typically a part of a commercially available prokaryotic expression vector, and the starting point helps to amplify the vector in a host cell. If the selected vector does not contain a replication origin, it can be chemically synthesized based on a known sequence and connected to the vector. For example, the replication origin from plasmid pBR322 (New England Biolabs, Beverly, MA) in Beverly, Massachusetts) is suitable for most gram-negative bacteria, and various viral starting points (such as SV40, polyoma virus, adenovirus, vesicular stomatitis virus (VSV), or papillomavirus, such as HPV or BPV) can be used for cloning vectors in mammalian cells. In general, mammalian expression vectors do not need a replication origin component (for example, usually only using the SV40 starting point, because it also contains a viral early promoter).

[0119] The transcription termination sequence is typically located at the 3' end of the polypeptide coding region and is used to terminate transcription. Usually, the transcription termination sequence in prokaryotes is a GC-rich fragment followed by a poly-T sequence. Although the sequence is easy to clone from a library or even commercially available as a part of a vector, it can also be easily synthesized using a nucleic acid synthesis method.

[0120] Selectable marker genes encode proteins required for survival and growth of host cells grown in selective media. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) supplement nutritional deficiencies of cells; or (c) provide important nutrients that are not available from complex media or defined media. Specific selectable markers are kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, the neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.

[0121] Other selectable genes can be used to amplify genes to be expressed. Amplification is the process of replicating genes in series in the chromosomes of consecutive generations of recombinant cells that produce proteins necessary for growth or cell survival. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure, wherein due to the presence of selectable genes in the vector, only transformants are uniquely suitable for survival. Selection pressure is by culturing transformed cells under conditions of continuously increasing the concentration of the selector in the culture medium, thereby applying DNA amplification of selectable genes and encoding another gene, such as one or more components of the bispecific antigen-binding proteins described herein. Therefore, a large amount of polypeptides are synthesized by amplified DNA.

[0122] The ribosome binding site is usually necessary for the initiation of mRNA translation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This component is typically located at the 3' end of the promoter and at the 5' end of the polypeptide coding sequence to be expressed. In certain embodiments, one or more coding regions may be operably linked to an internal ribosome binding site (IRES), thereby allowing translation of two open reading frames from a single RNA transcript.

[0123] In some cases, such as when glycosylation is needed in a eukaryotic host cell expression system, various pre-sequences or pro-sequences can be operated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide can be changed, or a pro-sequence can be added, which can also affect glycosylation. The final protein product can have one or more other amino acids that are easy to express at position-1 (relative to the first amino acid of the mature protein), which may not be completely removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site attached to the amino terminal. Alternatively, when the enzyme is cut in such regions in the mature polypeptide, some enzyme cleavage sites may be used to produce a slightly truncated form of the desired polypeptide.

[0124] The expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and is operably connected to the molecule encoding the polypeptide. As used herein, the term "operably connected" refers to two or more nucleic acid sequences that are connected in a manner that produces a nucleic acid molecule that can guide the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a control sequence that is "operably connected" to a protein coding sequence in a vector is connected to the coding sequence so that the expression of the protein coding sequence is achieved under conditions that are compatible with the transcriptional activity of the control sequence. More specifically, if a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional control components) stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system, it is operably connected to the coding sequence.

[0125] A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a structural gene that controls transcription of the structural gene (generally within about 100 to 1000 bp). Promoters are generally grouped into two categories: inducible promoters and constitutive promoters. Inducible promoters initiate transcription of DNA under their control at an increased level in response to a certain change in culture conditions (such as the presence or absence of nutrients, or temperature changes). On the other hand, constitutive promoters consistently transcribe the gene to which they are operably connected, i.e., have minimal or no control over gene expression. Many promoters recognized by a variety of potential host cells are well known. The source DNA promoter is removed by restriction enzyme digestion, and the desired promoter sequence is inserted into a vector, and a suitable promoter is operably connected to a DNA encoding, for example, a heavy chain, a light chain, a modified heavy chain or other components of a bispecific antigen-binding protein of the present invention.

[0126] Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are preferably used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from viral genomes such as polyoma virus, transmissible epithelioma virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0127] Other promoters of interest include, but are not limited to, the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the Rous sarcoma virus promoter (Rous sarcoma virus promoter). virus) (Yamamoto et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 78:1444-1445); the promoter and regulatory sequences from the metallothionein gene (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters, such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 80:21-25).Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been used in transgenic animals: the elastase I gene control region, which is active in pancreatic acinar cells (Swift et al., 1984, Cell, 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol., 50:399-409; MacDonald, 1987, Hepatology, 7:425-515); the insulin gene control region, which is active in pancreatic β cells (Hanahan, 1985, Nature, 315:115-122); the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell, 38:647-651); and the mitochondrial growth factor receptor (MTR) gene, which is active in pancreatic β cells. 658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); a mouse mammary tumor virus control region active in testicular, mammary, lymphoid, and mast cells (Leder et al., 1986, Cell 45:485-495); an albumin gene control region active in liver (Pinkert et al., 1987, Genes and Devel. [Genes and Development] 1: 268-276); the α-fetoprotein gene control region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. [Molecular Cell Biology] 5: 1639-1648; Hammer et al., 1987, Science [Science] 253: 53-58); the α1-antitrypsin gene control region active in the liver (Kelsey et al., 1987, Gene ... [Gene and Development] 1: 161-171); the beta-globin gene control region active in bone marrow cells (Mogram et al., 1985, Nature [Nature] 315: 338-340; Kollias et al., 1986, Cell [Cell] 46: 89-94); the myelin basic protein gene control region active in oligodendrocytes in the brain (Readhead et al., 1987, Cell [Cell] 48: 703-712); the myosin light chain-2 gene control region active in skeletal muscle (Sani, 1985, Nature [Nature] 314: 283-286); and the gonadotropin-releasing hormone gene control region active in the hypothalamus (Mason et al., 1986, Science [Science] 234: 1372-1378).

[0128] Enhancer sequences can be inserted into vectors to increase the transcription of DNA encoding components (e.g., light chains, heavy chains, modified heavy chains, Fd fragments) of bispecific antigen-binding proteins in higher eukaryotic cells. Enhancers are cis-acting elements of DNA, usually about 10-300bp in length, acting on promoters to increase transcription. Enhancers are relatively independent in direction and position, and have been found in the 5' and 3' positions of transcription units. Several enhancer sequences known to be available from mammalian genes (e.g., globulin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers from viruses are typically used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, and adenovirus enhancers known in the art are exemplary strengthening components for activating eukaryotic promoters. Although enhancers can be positioned at 5' or 3' of the coding sequence in the vector, they are typically located at the site of the promoter 5'. Sequences encoding appropriate natural or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into expression vectors to promote extracellular secretion of antibodies. The choice of signal peptide or leader sequence depends on the type of host cell to be produced for the antibody, and heterologous signal sequences can replace natural signal sequences. Examples of signal peptides are described above. Other signal peptides that work in mammalian host cells include the signal sequence of interleukin-7 (IL-7) described in U.S. Patent No. 4,965,195; Cosman et al., 1984, the signal sequence of the interleukin-2 receptor described in 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 EP Patent No. 0460846.

[0129] The provided expression vectors can be constructed from a starting vector (e.g., a commercially available vector). These vectors may or may not contain all desired flanking sequences. In the absence of one or more flanking sequences described herein in the vector, they can be independently obtained and connected to the vector. Methods for obtaining each flanking sequence are well known to those of ordinary skill in the art. The expression vector can be introduced into a host cell, thereby producing a protein encoded by a nucleic acid as described herein, including a fusion protein.

[0130] In certain embodiments, nucleic acids encoding different components of the bispecific antigen-binding proteins of the present invention may be inserted into the same expression vector. In such embodiments, the two nucleic acids may be separated by an internal ribosome entry site (IRES) and under the control of a single promoter, thereby expressing the light chain and heavy chain by the same mRNA transcript. Alternatively, the two nucleic acids may be under the control of two independent promoters, thereby expressing the light chain and heavy chain by two independent mRNA transcripts.

[0131] Similarly, for IgG-scFv bispecific antigen-binding proteins, the nucleic acid encoding the light chain can be cloned into the same expression vector as the nucleic acid encoding the modified heavy chain (comprising the fusion protein of heavy chain and scFv), wherein the two nucleic acids are controlled by a single promoter and separated by IRES, or wherein the two nucleic acids are controlled by two separate promoters. For IgG-Fab bispecific antigen-binding proteins, the nucleic acid encoding each of the three components can be cloned into the same expression vector. In certain embodiments, the nucleic acid encoding the light chain of the IgG-Fab molecule and the nucleic acid encoding the second polypeptide (which comprises the other half of the C-terminal Fab domain) are cloned into an expression vector, and the nucleic acid encoding the modified heavy chain (comprising the fusion protein of the heavy chain and half of the Fab domain) is cloned into the 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 a separate expression vector, the host cell is co-transfected with two expression vectors so that a cell produces all components of the bispecific antigen-binding proteins.

[0132] After constructing a vector and inserting one or more nucleic acid molecules encoding each component of the bispecific antigen-binding proteins described herein into one or more appropriate sites of one or more vectors, one or more complete vectors can be inserted into appropriate host cells for amplification and / or polypeptide expression. Therefore, the present invention includes isolated host cells, which include one or more expression vectors encoding bispecific antigen-binding protein components. As used herein, the term "host cell" refers to a cell transformed with nucleic acid or capable of being transformed with nucleic acid and expressing a gene of interest thereby. The term includes progeny of a parental cell, whether or not the progeny is consistent with the original parental cell in morphology or genetic composition, as long as there is a gene of interest. A host cell comprising an isolated nucleic acid of the present invention preferably operably connected to at least one expression control sequence (e.g., a promoter or enhancer) is a "recombinant host cell".

[0133] The expression vector of the antigen-binding protein can be transformed into the selected host cell by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, liposome transfection, DEAE-dextran mediated transfection or other known techniques. The selected method will vary in part with the type of host cell used. These methods and other suitable methods are well known to the technician and are described in, for example, Sambrook et al., 2001, supra.

[0134] The host cells, when cultured under appropriate conditions, synthesize antigen binding proteins, which can then be collected from the culture medium (if the host cells secrete it into the culture medium) or directly from the host cells that produced it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, polypeptide modifications (e.g., glycosylation or phosphorylation) required or necessary for activity, and the ease of folding into a biologically active molecule.

[0135] Exemplary host cells include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, and Streptomyces. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae, or commonly known as baker's yeast, is the most commonly used of the lower eukaryotic host microorganisms. However, a variety of other genera, species and strains are in common use and can be used herein, such as Pichia, e.g., P. pastoris, Schizosaccharomyces pombe; Kluyveromyces, Yarrowia; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0136] Host cells for expressing glycosylated antigen binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. A variety of baculovirus strains and variants from hosts and corresponding permissive insect host cells have been identified, such as Spodopterafrugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (flies) and Bombyx mori (Bombyx mori). A variety of virus strains for transfecting such cells are publicly available, such as the L-1 virus strain of Autographa californica NPV and the Bm-5 virus strain of Bombyx mori NPV.

[0137] Vertebrate host cells are also suitable hosts, and recombinant production of antigen binding proteins from such cells has become routine procedure. Mammalian cell lines that can be used as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, including CHOK1 cells (ATCCCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 77:4216, 1980); monkey kidney CV1 cells transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture (Graham et al., J. Gen Virol. [Journal of General Virology] 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. [Reproduction Biology] 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 hepatoma cells (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals of N.YAcad.Sci. [Annals of the New York Academy of Sciences] 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells and a variety of other cell lines. In another embodiment, a cell line that does not produce autoantibodies but is capable of producing and secreting heterologous antibodies can be selected from the B cell lineage. In some embodiments, CHO cells are preferred host cells for expressing the bispecific antigen-binding proteins of the present invention.

[0138] Host cells are transformed or transfected with the above nucleic acids or vectors to produce bispecific antigen-binding proteins and cultured in conventional nutrient media appropriately modified to induce promoters, select transformants or amplify genes encoding desired sequences. In addition, novel vectors and transfected cell lines having multiple copies of transcription units separated by selectable markers are particularly useful for expressing antigen-binding proteins. Therefore, the present invention also provides a method for preparing the bispecific antigen-binding proteins described herein, the method comprising culturing a host cell comprising one or more expression vectors in a culture medium under conditions that allow expression of the bispecific antigen-binding proteins encoded by one or more expression vectors described herein; and recovering the bispecific antigen-binding proteins from the culture medium.

[0139] Host cells for producing antigen-binding proteins of the invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any medium described in Ham et al., Meth. Enz. [Enzymology] 58:44, 1979; Barnes et al., Anal. Biochem. [Analytical biochemistry] 102:255, 1980; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90103430; WO 87 / 00195; or U.S. Pat. No. 30,985 can be used as a culture medium for host cells. If necessary, any of these culture media can be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamycin TM ) drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those of ordinary skill in the art may also be included at appropriate concentrations. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to the skilled artisan.

[0140] After culturing the host cells, the bispecific antigen-binding proteins can be produced intracellularly, in the periplasmic space, or can be directly secreted into the culture medium. If the antigen-binding proteins are produced intracellularly, as a first step, the granular fragments of the host cells or dissolved fragments are removed, for example, by centrifugation or ultrafiltration. The bispecific antigen-binding proteins can be purified using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, or preferably affinity chromatography, using one or more target antigens or protein A or protein G as affinity ligands. Protein A can be used to purify proteins including polypeptides based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. [Journal of Immunological Methods] 62: 1-13, 1983). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. [Journal of the European Molecular Biology Association] 5: 15671575, 1986). The matrix to which the affinity ligand is connected is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene can increase flow rates and reduce processing times compared to agarose. In the case of proteins containing CH3 domains, BakerbondABX TM Resins (JT Baker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification such as ethanol precipitation, reverse phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also possible, depending on the specific bispecific antigen binding protein to be recovered.

[0141] Examples

[0142] CHZ01

[0143] The ability to generate multispecific antigen binding proteins and therapeutic proteins with extended half-lives is paramount in advancing many therapeutic candidates to the clinic. Often, this means extensive protein design with varying degrees of success. In either case, two different Fc moieties come together and form heterodimers. In order to do this, the protein native sequence must be altered. Traditionally, these changes have focused on the CH3-CH3' interface of the Fc moiety, where charge pairing mutations and knob-in-hole designs have been inserted.

[0144] It can be seen that the two heavy chains in a monoclonal antibody have two major contact points: Fc-CH3 and the hinge ( Figure 1). The hinge region connects the Fc to the two Fab domains, and it must subsequently display a flexible structure that allows the Fab to rotate freely, which is necessary for these warheads to adopt the correct angle of approach to engage their targets while the Fc region can interact with its multiple binding partners such as FcγR, FcRn, and C1q. Despite the flexibility around the hinge region, this interface is mediated by a strong and structurally rigid motif, the “CPPC” motif ( Figure 1-3 Here, the proline residues introduce a very specific and stable secondary structure that allows the -SH side chains of the cysteine ​​residues to meet their counterparts and form disulfide bonds. In addition, the same rigid framework is likely to extend upstream and downstream of the "CPP" motif, suggesting that the side chains of those residues near this motif may also assume a stable conformation.

[0145] The only crystal structure of a human full-length IgG1 antibody available in the Protein Data Bank ("PDB") (PDB 1HZH) shows only a partially complete CPPC motif, in which the second cysteine ​​(C242) does not form a disulfide bond and each side chain at position 242 in the two polypeptide chains points in opposite directions ( Figure 2 Although there is structural information for mouse IgG1 antibodies showing the complete structure of the CPPC motif, the sequence variation between the two species ( Figure 3 ) does not allow accurate prediction of the spatial position of the side chains of those residues downstream of Cys242 in human IgG1. Therefore, while maintaining the native sequence of the CPPC motif, a string of oppositely charged residues named 'charged zipper hinge 01' (CZH01) was inserted downstream in the same region ( Figure 4 ). This "charged zipper" is believed to both attract heavy chains with opposite charges and repel chains with the same charge.

[0146] To test this hypothesis, these mutations were inserted into Antibody X and transiently expressed in HEK293 cells, followed by a single-step protein purification (Protein A followed by CEX). To verify whether the newly generated antibody was composed of two different heavy chains (negatively and positively charged, respectively), extensive reducing and non-reducing mass spectrometry was performed. Indeed, the results showed that this strategy successfully drove heavy chain pairing without any additional mutations in the Fc-CH3 region ( Figure 4-6 ). Most likely, the lysine string (243 to 247) is facing the aspartic acid string (243 to 247) and interacting with it.

[0147] CHZ11

[0148] Furthermore, structural analysis of the mouse IgG2 molecule showed that the side chains of residues 246 and 247 in one chain and residue 244 in the corresponding chain point toward each other ( Fig. 9). These residues are located downstream of Cys242 and in close proximity to the CPPC motif, which indicates conformational stability. In addition, the charged residues have side chains that can fit into the structural spatial arrangement and therefore do not disrupt the CPPC interface. Asn246 and Leu247 are both replaced by negatively charged aspartate residues. In order to pair with these two residues, a single histidine replaces Ala244 in the opposite chain in a way that allows it to make productive contacts or show a repulsive effect with the opposite charged residues (Asp246 and Asp247) ( Fig. 9 and 10 )(CZH11). To test the rationality of the design, the variant was expressed and purified. Unexpectedly, mass spectrometry confirmed that these three residues indeed formed a tripartite and were sufficient to drive hinge dimerization ( Fig.10 ).

[0149] CHZ09

[0150] Next, the region upstream of the CPPC region was tested to verify whether engineering of this sequence could also drive heavy chain dimerization. While retaining the two charged pair mutations Ala244Lys / Asp and Glu246Lys / Asp downstream of the Cys242 residue, two additional charged pair mutations His237Lys / Asp and Thr246Lys / Asp (CZH09) were inserted upstream of the Cys239 residue ( Figure 7 ). The data suggest that CPM inserted upstream of the CPPC motif is also able to drive heavy chain dimerization.

[0151] CH3 CPM

[0152] Furthermore, the CPMs (charge pair mutations) inserted in the hinge region were compatible with the CPMs previously inserted in the Fc-CH3 region. The so-called 'v11' CH3 CPMs (D399K in one CH3 region and K409D / K392D in the other CH3 region) were added to the molecules containing the CZH01, CZH09 and CZH11 mutations ( Fig.11 and 12 The results showed that all three CPMs inserted into the hinge region (CZH01, CZH09 and CZH11) were also able to successfully drive heavy chain pairing in the presence of Fc-CH3CPM.

[0153] To obtain the impact of these new mutations engineered in the hinge region, stability assays were run to look at the thermal stability values ​​of these new variants compared to the wild-type molecule. The data showed that the Tm data for CZH01, CZH09, and CZH11 compared well with the control CHZ00 (73.3°C) in the absence of CH3-CPM v11 and with CHZ00 (70.7°C) in the presence of CH3-CPM v11 (Table 1).

[0154] Table 1.

[0155]

[0156]

Claims

1. An isolated heteromultimer comprising a heterodimeric immunoglobulin hinge domain, wherein the heterodimeric immunoglobulin hinge domain comprises a first immunoglobulin hinge domain polypeptide and a second immunoglobulin hinge domain polypeptide, wherein: (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 is according to the EU index as set forth in Kabat.

2. The isolated heteromultimer of claim 1, wherein each hinge domain polypeptide further comprises a L248C substitution.

3. An isolated heteromultimer according to any preceding claim, wherein each immunoglobulin hinge domain polypeptide further comprises a CH3 domain.

4. The isolated heteromultimer according to claim 3, wherein one CH3 domain comprises a F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W or F405Y mutation; and another CH3 domain comprises a K409R mutation; wherein the numbering of the amino acid residues is according to the EU index listed in Kabat.

5. The isolated heteromultimer according to claim 3, wherein one CH3 domain comprises a T366W mutation; and another CH3 domain comprises T366S, L368A, Y407V mutations; wherein the numbering of the amino acid residues is according to the EU index listed in Kabat.

6. The isolated heteromultimer according to claim 3, wherein one CH3 domain comprises K / R409D and K392D mutations; and the other CH3 domain comprises D399K and E356K mutations; wherein the numbering of the amino acid residues is according to the EU index listed in Kabat.

7. The isolated heteromultimer according to any one of claims 4 to 6, wherein one CH3 domain comprises a Y349C mutation; and another CH3 domain comprises an E356C or S354C mutation; wherein the numbering of the amino acid residues is according to the EU index listed in Kabat.

8. The isolated heteromultimer according to claim 3, wherein one CH3 domain comprises Y349C and T366W mutations; and the other CH3 domain comprises E356C, T366S, L368A and Y407V mutations; wherein the numbering of the amino acid residues is according to the EU index listed in Kabat.

9. The isolated heteromultimer according to claim 3, wherein one CH3 domain comprises Y349C and T366W mutations; and another CH3 domain comprises S354C, T366S, L368A, Y407V mutations; wherein the numbering of the amino acid residues is according to the EU index listed in Kabat.

10. The isolated heteromultimer according to any preceding claim, wherein the immunoglobulin hinge region is an IgG1 hinge region.

11. An isolated heteromultimer according to any preceding claim, wherein the heteromultimer is a bispecific or multispecific antibody.

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