Novel multispecific antibody format

Abstract

The present invention relates to novel multispecific antigen-binding proteins capable of binding to multiple targets. Pharmaceutical compositions comprising the multispecific antigen-binding proteins and methods for producing them are also disclosed.

Description

[Technical Field]

[0001] The present invention relates to a multispecific antibody, a polynucleotide encoding a multispecific antibody, and a method for producing a multispecific antibody.

[0002] This application contains an ASCII “txt” compliant sequence listing suitable for both computer-readable format (CRF) and paper copies as required by 37 CFR sections 1.821(c) and 1.821(e), and which is incorporated herein by reference in its entirety. The name of the “txt” file created on November 18, 2020, is A-2536-WO-PCT_SEQ_LIST_20201118_ST25, and it is 36.1kb in size. [Background technology]

[0003] The clinical potential of multispecific antibodies (molecules that target multiple targets simultaneously), such as bispecific and tripspecific antibodies, is very promising for targeting complex diseases. However, generating molecules that exhibit the desired activity presents a significant challenge. Here, the inventors describe a novel format that allows for fine-tuning of the binding of two antigen-binding domains by linking them via linkers of different lengths and sequences, thereby optimizing antigen binding (Figure 1).

[0004] While over 100 bispecificity formats have been reported, many have failed to deliver the intended bispecificity biology. Such disappointing results are often associated with a lack of knowledge regarding the spatial location of epitopes on the cell surface relative to each therapeutic target. With this knowledge, it is possible to tailor these bispecificity formats to meet specific requirements for on-target activity while minimizing off-target activity, which has significant implications for delivering effective and safe therapeutics. [Overview of the Initiative] [Means for solving the problem]

[0005] In one aspect, the present invention provides: a) a first polypeptide comprising an antibody Fc region comprising a first hinge region, a first CH2 region, and a first CH3 region; b) a second polypeptide comprising an antibody heavy chain construct, the antibody heavy chain construct comprising: i) a scFv comprising 1) a first VH and a first VL that bind to form a first antigen-binding domain; 2) a first linker peptide linking the first VH and the first VL; and ii) an antibody heavy chain comprising a second VH, a second CH1 region, a second hinge region, a second CH2 region, and a second CH3 region; wherein the scFv is bound at its C-terminus to the N-terminus of the second VH region of the antibody heavy chain; c) a third polypeptide comprising an antibody light chain comprising a second VL and a CL; wherein the second VH of the antibody heavy chain and the second VL of the antibody light chain bind to form a second antigen-binding domain, relating to a multispecific antibody construct.

[0006] In another aspect, the present invention provides: a) a first polypeptide comprising an antibody Fc region comprising a first hinge region, a first CH2 region, and a first CH3 region; b) a second polypeptide comprising an antibody light chain construct, the antibody light chain construct comprising: i) a scFv comprising 1) a first VH and a first VL that bind to form a first antigen-binding domain; 2) a first linker peptide linking the first VH and the first VL; and ii) an antibody light chain comprising a second VL and a CL; wherein scFv, at its C-terminus, is bound to the second polypeptide at the N-terminus of the second VL region of the antibody light chain; c) A third polypeptide comprising an antibody heavy chain including a second VH, a second CH1 region, a second hinge region, a second CH2 region, and a second CH3 region. Includes, This invention relates to a multispecific antibody construct in which the second VH of the antibody heavy chain and the second VL of the antibody light chain are bound together to form a second antigen-binding domain.

[0007] In one embodiment, the first linker is: (Gly3Ser)2 (SEQ ID NO: 1), (Gly4Ser)2 (SEQ ID NO: 2), (Gly3Ser)3 (SEQ ID NO: 3), (Gly4Ser)3 (SEQ ID NO: 4), (Gly3Ser)4 (SEQ ID NO: 5), (Gly4Ser)4 (SEQ ID NO: 6), (Gly3Ser)5 (SEQ ID NO: 7), (Gly4Ser)5 (SEQ ID NO: 8), (Gly3Ser)6 (SEQ ID NO: 9), (Gly4Ser)6 (SEQ ID NO: 10), GSADDAKKDAAKKDAAKKDDAKKDDAGS (SEQ ID NO: 11), GSADDA The sequence includes sequences selected from the group consisting of KKDAAKKDAAKKDDAKKDDAKKDAGS (SEQ ID NO: 12), (Gly3Gln)2 (SEQ ID NO: 13), (Gly4Gln)2 (SEQ ID NO: 14), (Gly3Gln)3 (SEQ ID NO: 15), (Gly4Gln)3 (SEQ ID NO: 16), (Gly3Gln)4 (SEQ ID NO: 17), (Gly4Gln)4 (SEQ ID NO: 18), (Gly3Gln)5 (SEQ ID NO: 19), (Gly4Gln)5 (SEQ ID NO: 20), (Gly3Gln)6 (SEQ ID NO: 21), and (Gly4Gln)6 (SEQ ID NO: 22).

[0008] In one embodiment, scFv is bound to the antibody heavy chain via a second linker. In one embodiment, the second linker includes a sequence selected from the group consisting of (Gly3Ser)2 (SEQ ID NO: 1), (Gly4Ser)2 (SEQ ID NO: 2), (Gly3Ser)3 (SEQ ID NO: 3), (Gly4Ser)3 (SEQ ID NO: 4), (Gly3Ser)4 (SEQ ID NO: 5), (Gly4Ser)4 (SEQ ID NO: 6), (Gly3Ser)5 (SEQ ID NO: 7), (Gly4Ser)5 (SEQ ID NO: 8), (Gly3Ser)6 (SEQ ID NO: 9), and (Gly4Ser)6 (SEQ ID NO: 10).

[0009] In one embodiment, scFv includes a first VH bonded at its C-terminus to the N-terminus of a first linker, and the first linker is bonded at its C-terminus to the N-terminus of a first VL. In another embodiment, scFv includes a first VL bonded at its C-terminus to the N-terminus of a first linker, and the first linker is bonded at its C-terminus to the N-terminus of a first VH.

[0010] In one embodiment, the first antigen-binding domain and the second antigen-binding domain bind to different epitopes on different polypeptides. In another embodiment, the first antigen-binding domain and the second antigen-binding domain bind to different epitopes on the same polypeptide. In one embodiment, the multispecific antibody construct is a biparatopic antibody construct.

[0011] In one embodiment, the Fc region consists of a hinge region, a CH2 region, and a CH3 region.

[0012] In one embodiment, the N-terminus of the Fc region is linked to the C-terminus of the heavy chain via a third linker through its N-terminus.

[0013] In one embodiment, the third linker is: (Gly3Ser)2 (SEQ ID NO: 1), (Gly4Ser)2 (SEQ ID NO: 2), (Gly3Ser)3 (SEQ ID NO: 3), (Gly4Ser)3 (SEQ ID NO: 4), (Gly3Ser)4 (SEQ ID NO: 5), (Gly4Ser)4 (SEQ ID NO: 6), (Gly3Ser)5 (SEQ ID NO: 7), (Gly4Ser)5 (SEQ ID NO: 8), (Gly3Ser)6 (SEQ ID NO: 9), (Gly4Ser)6 (SEQ ID NO: 10), (Gly The sequence includes sequences selected from the group consisting of (3Gln)2 (SEQ ID NO: 13), (Gly4Gln)2 (SEQ ID NO: 14), (Gly3Gln)3 (SEQ ID NO: 15), (Gly4Gln)3 (SEQ ID NO: 16), (Gly3Gln)4 (SEQ ID NO: 17), (Gly4Gln)4 (SEQ ID NO: 18), (Gly3Gln)5 (SEQ ID NO: 19), (Gly4Gln)5 (SEQ ID NO: 20), (Gly3Gln)6 (SEQ ID NO: 21), and (Gly4Gln)6 (SEQ ID NO: 22).

[0014] In one embodiment, the first polypeptide consists of an antibody Fc region.

[0015] In one embodiment, the multispecific antibody construct is a bispecific antibody construct.

[0016] In one embodiment, one CH3 domain contains the mutations F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y, and the other CH3 domain contains the K409R mutation, with amino acid residue numbering following the EU index described in Kabat.

[0017] In another embodiment, one CH3 domain contains the T366W mutation, and the other CH3 domain contains the T366S, L368A, Y407V mutations, and the amino acid residue numbering follows the EU index described in Kabat.

[0018] In yet another embodiment, one CH3 domain contains the K / R409D and K392D mutations, and the other CH3 domain contains the D399K mutation, with amino acid residue numbering following the EU index described in Kabat. In one embodiment, the CH3 domain containing the D399K mutation also contains the E356K mutation, with amino acid residue numbering following the EU index described in Kabat. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 shows the crystal structures of two Fabs bound to two different domains within the same target protein. This provides a rational design for designing novel formats of multispecificity. [Figure 2] Figure 2 shows a schematic diagram of a multispecific molecule composed of three polypeptide chains. The scFv module can be linked to the N-terminus of the Fab VH via a flexible linker and / or rigid linker through its C-terminal VH. [Figure 3] Figure 3 shows the primary structure of a multispecific molecule that demonstrates a novel design implemented in chain A. [Figure 4] Figure 4 shows a schematic diagram of a multispecific molecule composed of three polypeptide chains. The scFv module can be linked to the N-terminus of the Fab VL via a flexible linker and / or rigid linker through its C-terminal VH. [Figure 5] Figure 5 shows a schematic diagram of a multispecific molecule composed of three polypeptide chains. The scFv module can be linked to the N-terminus of the Fab VL via a flexible linker and / or rigid linker through its C-terminal VH. [Figure 6] Figure 6 shows the expression and purification of the multispecific construct. [Figure 7] Figure 7 shows the binding assay for the multispecific construct. [Figure 8] Figure 8 shows cell binding assays for multispecific molecules. [Modes for carrying out the invention]

[0020] As used herein, the term “antigen-binding protein” refers to a protein that specifically binds to one or more target antigens. Antigen-binding proteins may include antibodies and their functional fragments. A “functional antibody fragment” is a portion of an antibody that lacks at least some of the amino acids present in the full-length heavy and / or light chain, but is still capable of specifically binding to an antigen. Examples of functional antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments, and may originate from any mammalian source, such as humans, mice, rats, rabbits, or camelids. Functional antibody fragments may be comparable to intact antibodies in terms of target antigen binding, and the fragments may be produced by modifying intact antibodies (e.g., enzymatic or chemical cleavage) or newly synthesized using recombinant DNA technology or peptide synthesis.

[0021] The "heavy" and "light" chains refer to two polypeptides containing IgG. The heavy chain can be broken down from the N-terminus to the C-terminus into the following domains: VH, CH1, hinge, CH2, and CH3. The light chain can be broken down from the N-terminus to the C-terminus into the following domains: VL and CL. The CH1 and CL domains interact with the VH and VL domains to form a functional higher-order structure that binds to the antigen.

[0022] Furthermore, antigen-binding proteins may also include proteins containing one or more functional antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. A "multispecific antibody construct" is one or more polypeptides containing one or more functional antibody moieties that bind to two or more different antigens. A multispecific antibody construct may include polypeptides containing scFv linked to an antibody heavy chain or an antibody light chain.

[0023] In certain embodiments, the antigen-binding protein of the present invention is "multispecific," meaning that it can specifically bind to two or more different antigens. In another embodiment, the antigen-binding protein of the present invention is "bispecific," meaning that it can specifically bind to two different antigens.

[0024] As used herein, an antigen-binding protein has a significantly higher binding affinity for a target antigen compared to its affinity for other irrelevant proteins under similar binding assay conditions, and as a result, can distinguish that antigen, then it "specifically binds" to the target antigen. An antigen-binding protein that specifically binds to an antigen has an equilibrium dissociation constant (K D ) that can be ≦ 1 × 10 -6 M. An antigen-binding protein specifically binds to an antigen with "high affinity" when K D is ≦ 1 × 10 -8 M. In one embodiment, the antigen-binding protein of the present invention binds to the target antigen with a K -7 of ≦ 5 × 10 D . In another embodiment, the antigen-binding protein of the present invention binds to the target antigen with a K -7 of ≦ 1 × 10 D

[0025] Affinity is determined using various techniques, and as an example, there is an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). Using this methodology, the association rate constant (k a units: M -1 s -1 ) and the dissociation rate constant (k d units: s -1 ) can be measured. Then, the equilibrium dissociation constant (K D units: M) is calculated as the ratio of the kinetic rate constants (k d / k aThe affinity can be calculated from the following. In some embodiments, affinity is determined by kinetic methods such as the binding equilibrium exclusion method (KinExA) as described in Rathanaswami et al. Analytical Biochemistry, Vol.373:52-60, 2008. Using the KinExA assay, the equilibrium dissociation constant (K D Units: M) and coupling rate constant (k a Unit: M -1 s -1 ) can be measured. Dissociation rate constant (k d Unit: s -1 ) are these values ​​(K D ×k a ) can be calculated from. In other embodiments, affinity is determined by equilibrium / solution method. In specific embodiments, affinity is determined by FACS binding assay. In specific embodiments of the present invention, antigen-binding protein is determined by binding equilibrium exclusion method performed by Rathanaswami et al. Analytical Biochemistry, Vol.373:52-60, 2008 to a target antigen expressed by mammalian cells (e.g., CHO, HEK 293, Jurkat), with a concentration of 20 nM (2.0 × 10⁻¹⁰). -8 M) K below D So, 10nM (1.0 × 10 -8 M) K below D So, 1nM(1.0×10 -9 M) K below D So, 500 pM (5.0 × 10 -10 M) K below D So, 200 pM (2.0 × 10 -10 M) K below D So, 150 pM (1.50 × 10 -10 M) K below D So, 125 pM (1.25 × 10 -10 M) K below D So, 105 pM (1.05 × 10 -10 M) K below D So, 50 pM (5.0 × 10 -11 M) K below D Or, 20 pM (2.0 × 10-11 M) K below D And it binds specifically. In some embodiments, the multispecific antibody constructs described herein are k d The binding affinity to the target antigen, measured by the (dissociation rate constant), is approximately 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 s -1 The following conditions must be met (lower values ​​indicate higher bond affinity), and / or K D The binding affinity to the target antigen, measured by the equilibrium dissociation constant, is approximately 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 It exhibits desired characteristics such as being M or less (a lower value indicates higher binding affinity).

[0026] As used herein, the term "binding domain" and, interchangeably, "antigen-binding domain" refer to a region of an antigen-binding protein containing amino acid residues that interact with the antigen and confer specificity and affinity to the antigen to the antigen-binding protein. As used herein, the term "target antigen" refers, for example, to the first and / or second target antigen of a bispecific molecule, and also to the first, second, third, and / or fourth target antigen of a quadruspecific molecule.

[0027] In certain embodiments of the multispecific antibody constructs of the present invention, the binding domain may originate from the antibody or a functional fragment thereof. For example, the binding domain of the multispecific antibody construct of the present invention may include one or more complementarity-determining regions (CDRs) derived from the light chain variable region and heavy chain variable region of an antibody that specifically binds to a target antigen. As used herein, the term "CDR" refers to a complementarity-determining region (also referred to as the "minimum recognition unit" or "hypervariable region") within an antibody variable sequence. There are three heavy chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2, and CDRL3). As used herein, the term "CDR region" refers to a group of three CDRs present within 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 a framework region to form a structure that specifically binds to a specific epitope or domain of the target protein. From the N-terminus to the C-terminus, both the naturally occurring light-chain and heavy-chain variable regions typically correspond to the following order of elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0028] Both the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), and the AHo numbering scheme (Honegger A. and Plueckthun AJ Mol Biol. 2001 Jun 8;309(3):657-70) can be used in the present invention. The amino acid positions of a given antibody, as well as the complementarity-determining regions (CDRs) and framework regions (FRs), can be identified using either system. For example, EU heavy chain positions 39, 44, 183, 356, 357, 370, 392, 399, and 409 are equivalent to AHo heavy chain positions 46, 51, 230, 484, 485, 501, 528, 535, and 551, respectively. Similarly, EU light chain positions 38, 100, and 176 are equivalent to AHO light chain positions 46, 141, and 230, respectively.

[0029] When an antibody is digested with papain, two identical antigen-binding fragments called "Fab" fragments (each possessing a single antigen-binding site) and the remainder as an "Fc" fragment (containing the immunoglobulin constant region) are produced. The Fab fragment contains the variable domain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, the "Fab fragment" consists of one immunoglobulin light chain (variable region (VL) and constant region (CL)) and the CH1 region and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. The Fc fragment represents a carbohydrate and is responsible for many antibody effector functions (such as complement binding and cell receptors) that distinguish one class of antibody from another. The "Fd fragment" contains the VH domain and CH1 domain derived from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0030] A "Fab fragment" is a Fab fragment that has one or more cysteine ​​residues derived from the antibody hinge region at the C-terminus of the CH1 domain.

[0031] The "F(ab')2 fragment" is a divalent fragment containing two Fab' fragments linked at the hinge region by disulfide bridges between heavy chains.

[0032] The "Fv" fragment is the smallest fragment containing a complete antigen-recognition binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in a tightly covalently bound state. In this configuration, the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single light or heavy chain variable region (or half of the Fv fragment containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, but has lower affinity than the entire binding site containing both VH and VL.

[0033] In this specification, the terms "variable region" (variable region of the light chain (VL), variable region of the heavy chain (VH)), used interchangeably with "variable domain," refer to regions in the immunoglobulin light chain and immunoglobulin heavy chain, respectively, that are directly involved in the binding of antibodies to antigens. As discussed earlier, the variable light chain and variable heavy chain regions have the same general structure, and each region contains four framework (FR) regions whose sequences are widely conserved and linked by three CDRs. The framework regions employ a beta-sheet structure, and the CDRs can form loops linking the beta-sheet structures. The CDRs within each chain are held in a three-dimensional structure by the framework regions and, together with CDRs from the other chain, form an antigen-binding site.

[0034] An "immunoglobulin domain" refers to a peptide containing approximately 100 amino acid residues, including an amino acid sequence similar to that of an immunoglobulin and containing at least two cysteine ​​residues. Examples of immunoglobulin domains include VH, CH1, CH2, and CH3 of immunoglobulin heavy chains, and VL and CL of immunoglobulin light chains. Immunoglobulin domains are also found in proteins other than immunoglobulins. Examples of immunoglobulin domains in proteins other than immunoglobulins include immunoglobulin domains contained in proteins belonging to the immunoglobulin superfamily, such as major histocompatibility complex (MHC), CD1, B7, and T cell receptor (TCR). Any of these immunoglobulin domains can be used as immunoglobulin domains for the multivalent antibodies of the present invention.

[0035] In human antibodies, CH1 refers to the region containing the amino acid sequence at positions 118-215 of the EU index. A highly flexible amino acid region called the "hinge region" exists between CH1 and CH2. CH2 represents the region containing the amino acid sequence at positions 231-340 of the EU index, and CH3 represents the region containing the amino acid sequence at positions 341-446 of the EU index.

[0036] "CL" represents the constant region of the light chain. In the case of the κ chain of human antibodies, CL represents the region with an amino acid sequence at positions 108-214 of the EU index. In the λ chain, CL represents the region with an amino acid sequence at positions 108-215.

[0037] The binding domain that specifically binds to the target antigen may be derived from a) a known antibody against the antigen, or b) a novel antibody or antibody fragment obtained by a novel immunization method using the antigen protein or a fragment thereof, by phage display, or by other routine methods. The antibody from which the binding domain of the multispecific antibody construct is derived may be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the antibody from which the binding domain is derived is a monoclonal antibody. In these, or other embodiments, the antibody may be a human antibody or a humanized antibody, and may be of type IgG1, IgG2, IgG3, or IgG4.

[0038] As used herein, the term “monoclonal antibody” (or “mAb”) refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are directed to individual antigen sites or epitopes, in contrast to polyclonal antibody preparations, which typically contain a variety of antibodies directed to various epitopes and are highly specific. Monoclonal antibodies may be produced, for example, by immortalizing spleen cells harvested from transgenic animals after completion of an immunization schedule, using any technique known in the art. Spleen cells can be immortalized, for example, by fusing spleen cells with myeloma cells to produce hybridomas, using any technique known in the art. Myeloma cells used in the fusion procedure to produce hybridomas are non-antibody-producing, have high fusion efficiency, and possess enzyme deficiencies that prevent them from growing in specific selective media that support the proliferation of only the desired fusion cells (hybridoms). Examples of cell lines suitable for use in mouse fusion include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7, and S194 / 5XXO Bul; examples of cell lines used in rat fusion include R210.RCY3, Y3-Ag1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion include U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0039] In some cases, hybridoma cells are generated by immunizing animals (e.g., transgenic animals with human immunoglobulin sequences) with a target antigen immunogen; harvesting spleen cells from the immunized animals; fusing the harvested spleen cells with myeloma cell lines to generate hybridoma cells; establishing hybridoma cell lines from the hybridoma cells; and identifying hybridoma cell lines that produce antibodies binding to the target antigen.

[0040] Monoclonal antibodies secreted by hybridoma cell lines can be purified using any technique known in this art, such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Hybridomas or mAbs can be further screened to identify mAbs with specific properties, such as the ability to bind to cells expressing a target antigen, the ability to block or interfere with the binding of the target antigen to its respective receptor or ligand, or the ability to functionally block any of the target antigens.

[0041] In some embodiments, the binding domain of the multispecific antibody construct of the present invention may be derived from a humanized antibody against a target antigen. A "humanized antibody" refers to an antibody in which a region (e.g., a framework region) is modified to include a corresponding region derived from human immunoglobulin. Generally, humanized antibodies can be generated from monoclonal antibodies initially produced in non-human animals. Typically, certain amino acid residues within this monoclonal antibody, derived from the non-antigen recognition portion of the antibody, are modified to be homologous to the corresponding residues in the corresponding isotype of human antibody. Humanization can be carried out using various methods, for example, by substituting at least a portion of the rodent variable region with the corresponding region of a human antibody (see, for example, U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., Nature, Vol. 321:522-525, 1986; Riechmann et al., Nature, Vol. 332:323-27, 1988; Verhoeyen et al., Science, Vol. 239:1534-1536, 1988). The CDRs of the light chain and heavy chain variable regions of antibodies produced in another species can be grafted onto consensus human FRs. To create consensus human FRs, several FRs derived from human heavy chain amino acid sequences or human light chain amino acid sequences may be aligned to identify a consensus amino acid sequence.

[0042] Novel antibodies produced against target antigens from which the binding domain of the multispecific antibody construct of the present invention may originate can be made into fully human antibodies. A "fully human antibody" is an antibody that includes a variable region and a constant region derived from a human germline immunoglobulin sequence. One specific means provided for producing fully human antibodies is the "humanization" of the mouse humoral immune system. The introduction of a human immunoglobulin (Ig) locus into a mouse inactivated with an endogenous Ig gene is one means of producing fully human monoclonal antibodies (mAbs) in mice, which are animals that can be immunized with any desired antigen. Using fully human antibodies can minimize immunogenic and allergic responses that may occur when mouse mAbs or mouse-derived mAbs are administered to humans as therapeutic agents.

[0043] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that can produce a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more consecutive amino acids and, in some cases, are conjugated to a carrier such as a hapten. See, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551-2555, Jakobovits et al., 1993, Nature 362:255-258, and Bruggermann et al., 1993, Year in Immunol. 7:33. In one example of such a method, the transgenic animal is produced by inactivating the endogenous mouse immunoglobulin loci encoding the mouse immunoglobulin heavy chain and immunoglobulin light chain, and inserting a large fragment of human genomic DNA containing loci encoding human heavy chain and light chain proteins into the mouse genome. Next, partially modified animals having less complement than the complete complement of the human immunoglobulin locus are crossed to obtain animals possessing all the desired immune system modifications. Upon administration of the immunogen, the transgenic animals produce antibodies that are immunospecific to the immunogen but have a human amino acid sequence, including the variable region, rather than a mouse amino acid sequence. For further details of such methods, see, for example, International Publication No. 96 / 33735 and International Publication No. 94 / 02602.Further methods related to transgenic mice for producing human antibodies are described in U.S. Patent Nos. 5,545,807, 6,713,610, 6,673,986, 6,162,963, 5,939,598, 5,545,807, 6,300,129, 6,255,458, and 5,877,397. This is described in the Book, U.S. Patent No. 5,874,299, and U.S. Patent No. 5,545,806, PCT Publications International Publication Brochures No. 91 / 10741, No. 90 / 04036, No. 94 / 02602, No. 96 / 30498, and No. 98 / 24893, and European Patent No. 546073B1 and European Patent Application Publication No. 546073A1.

[0044] The transgenic mice referred to herein as "HuMab" mice contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (mu and gamma) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous mu and kappa chain gene loci (Lonberg et al., 1994, Nature 368:856-859). Consequently, the mice exhibit reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgG kappa monoclonal antibodies (Lonberg et al., op. cit.; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764:536-546). HuMab mouse preparation was performed by Taylor et al.,1992,Nucleic Acids Research 20:6287-6295;Chen et al.,1993,International Immunology 5:647-656;Tuaillon et al.,1994,J.Immunol.152:2912-2920;Lonberg et al.,1994,Nature 368:856-859;Lonberg,1994,Handbook of Exp.Pharmacology 113:49-101;Taylor et al.,1994,International Immunology 6:579-591;Lonberg and Huszar,1995,Intern.Rev.Immunol.13:65-93;Harding and Lonberg, 1995, Ann.NY This is described in detail in Acad.Sci.764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851, and these documents are incorporated herein by reference in their entirety for all purposes.Furthermore, see U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, as well as U.S. Patent No. 5,545,807, International Publication No. 93 / 1227, International Publication No. 92 / 22646, and International Publication No. 92 / 03918 (all of these disclosures are incorporated herein by reference in their entirety for all purposes). The techniques used to generate human antibodies in these genetically modified mice are disclosed in International Publication No. 98 / 24893 and in Mendez et al., 1997, Nature Genetics 15:146-156, which are incorporated herein by reference.

[0045] Furthermore, human-derived antibodies can also be generated using phage display technology. Phage display is described, for example, in Dower et al., International Publication No. 91 / 17271, McCafferty et al., International Publication No. 92 / 01047, and Caton and Koprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each of which is incorporated herein by reference in its entirety. Antibodies generated by phage technology typically lack effector function because they are generated in bacteria as antigen-binding fragments, such as Fv or Fab fragments. Effector function can be introduced by one of two strategies. The fragments can be manipulated to, if necessary, become complete antibodies expressed in mammalian cells, or to become multispecific antibody fragments having a second binding site that can trigger effector function. Typically, the Fd fragment (VH-CH1) and light chain (VL-CL) of an antibody can be cloned separately by PCR, randomly recombined in a combinatorial phage display library, and then selected for binding to a specific antigen. The antibody fragment is expressed on the phage surface, and selection by antigen binding (i.e., the phage containing the DNA encoding the antibody fragment) of either Fv or Fab is achieved by performing several rounds of antigen binding and re-amplification, a procedure called panning. Antigen-specific antibody fragments are enriched and finally isolated. Phage display technology can also be used in an approach for humanizing rodent monoclonal antibodies called "guided selection" (see Jespers, LS, et al., Bio / Technology 12, 899-903 (1994)). In this approach, the Fd fragment of a mouse monoclonal antibody can be presented in combination with a human light chain library, and the resulting hybrid Fab library can then be selected by antigen. This allows the mouse Fd fragments to provide a template to guide the selection process. Subsequently, the selected human light chains are combined with the human Fd fragment library.By selecting the resulting libraries, a complete human Fab can be obtained.

[0046] As used herein, the term “identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by aligning and comparing their sequences. As used herein, “identity percentage” means the percentage of identical residues between amino acids or nucleotides in the molecules being compared, and is calculated based on the smallest size of the molecules being compared. For this calculation, any gaps in the alignment must be addressed by a specific mathematical model or computer program (i.e., “algorithm”). Methods that may be used to determine the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined by standard methods commonly used to compare the similarity of amino acid positions in two polypeptides.Using a computer program such as BLAST or FASTA, two polypeptide or polynucleotide sequences are aligned so that their respective residues are optimally matched (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences). The program applies default opening and gap penalties. A scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) can be used with the computer program. The identity percentage can then be calculated, for example, by multiplying the total number of perfect matches by 100 and dividing by the sum of the length of the longer sequence within the matched span and the number of gaps introduced in the longer sequence to align the two sequences. In calculating the identity percentage, the sequences being compared are aligned to maximize the number of matches between them.

[0047] The GCG program package is a computer program that can be used to determine the percentage of identity, and this package includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or two polynucleotides whose sequence identity percentage is to be determined. The sequences are aligned to obtain the best possible matching of each amino acid or nucleotide (the "matched span" determined by the algorithm). A gap opening penalty (calculated as 3 × average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix to be used; where the "diagonal" is the score or number assigned to each complete amino acid match by a particular comparison matrix) and a gap extension penalty (usually 1 / 10th of the gap opening penalty), as well as comparison matrices such as PAM 250 or BLOSUM 62, are used with the algorithm. In certain embodiments, standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919 for the BLOSUM 62 comparison matrix) are also used by the algorithm.

[0048] The following are recommended parameters for determining the identity percentage of polypeptide or nucleotide sequences using the GAP program: Algorithm: Needleman et al., 1970, J.Mol.Biol.48:443-453; Comparison matrix: Henikoff et al., 1992, BLOSUM 62 cited above; Gap penalty: 12 (however, no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0

[0049] A specific alignment scheme for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this aligned small region may have very high sequence identity despite the absence of a significant relationship between the two full-length sequences. Therefore, the selected alignment method (GAP program) can be adjusted as needed to obtain alignment across at least 50 consecutive amino acids of the target polypeptide.

[0050] As used herein, the term “antibody” refers to a tetrameric immunoglobulin protein comprising two light-chain polypeptides (each approximately 25 kDa) and two heavy-chain polypeptides (each approximately 50–70 kDa). The term “light chain” or “immunoglobulin light chain” refers to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus to the carboxyl terminus. The immunoglobulin light chain constant domain (CL) may be kappa (κ) or lambda (λ). The term “heavy chain” or “immunoglobulin heavy chain” refers to a polypeptide comprising a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus to the carboxyl terminus. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG and IgA class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The constant domains of the immunoglobulin heavy chain can originate from any immunoglobulin isotype for which subtypes are listed. The antibody chains are linked via interpolypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light chain and the heavy chain), and between the hinge regions of the antibody heavy chain.

[0051] Multispecific antibody constructs can contain any immunoglobulin constant region. As used herein, the term "constant region" refers to all domains of an antibody other than the variable region. The constant region does not directly participate in antigen binding but exerts various effector functions. As described above, antibodies are classified into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) depending on the amino acid sequence of their heavy chain constant region. The light chain constant region can be, for example, a kappa-type or lambda-type light chain constant region, such as the human kappa-type or lambda-type light chain constant region found in all five antibody isotypes. Examples of human immunoglobulin light chain constant region sequences are shown in the table below.

[0052] [Table 1]

[0053] The heavy chain constant region of a multispecific antibody construct can be, for example, an alpha-type, delta-type, epsilon-type, gamma-type, or mu-type heavy chain constant region, such as a human alpha-type, delta-type, epsilon-type, gamma-type, or mu-type heavy chain constant region. In some embodiments, the multispecific antibody construct includes a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In one embodiment, the multispecific antibody construct includes a heavy chain constant region derived from human IgG1 immunoglobulin. In another embodiment, the multispecific antibody construct includes a heavy chain constant region derived from human IgG2 immunoglobulin. Examples of human IgG1 and IgG2 heavy chain constant region sequences are shown in Table 5 below.

[0054] [Table 2]

[0055] The variable region may be bound to the aforementioned light chain constant region and heavy chain constant region to form a complete antibody light chain and antibody heavy chain, respectively. Furthermore, each of the heavy chain polypeptides and light chain polypeptides thus produced may be combined to form a multispecific antibody construct. It should be understood that the heavy chain variable region and light chain variable region provided herein may also be bound to other constant domains having sequences different from the exemplary sequences listed above.

[0056] In certain embodiments of the present invention, two different Fc domains are used to form the heterodimer molecule of the present invention. To facilitate the assembly of Fc chains and heavy chains into a multispecific antibody construct, the Fc domains derived from each of the Fc-containing polypeptides of the multispecific antibody construct and the Fc domains of the heavy chain can be manipulated to reduce the formation of mis-base pairing molecules. For example, one approach to promote heterodimer Fc formation over homodimer Fc formation is the so-called "knob-into-hole" method, which involves introducing mutations into the CH3 domains of two different antibody heavy chain Fc regions at a contact interface. Specifically, one or more bulky amino acids in the Fc region of one antibody heavy chain are substituted with amino acids having short side chains (e.g., alanine or threonine) to create a "hole," while one or more amino acids having large side chains (e.g., tyrosine or tryptophan) are introduced into the other heavy chain to create a "knob." When the modified heavy chain Fc region is co-expressed, a larger percentage of heterodimers (knob-hole) are formed compared to homodimers (hole-hole or knob-knob). The "knob-into-hole" methodology is detailed in International Publication No. 96 / 027011; Ridgway et al., Protein Eng., Vol.9:617-621, 1996; and Merchant et al., Nat, Biotechnol., Vol.16:677-681, 1998, all of which are incorporated herein by reference in their entirety.

[0057] Another approach to promote heterodimer formation while excluding homodimer formation involves utilizing an electrostatic steering mechanism (see Gunasekaran et al., J. Biol. Chem., Vol. 285: 19637-19646, 2010, which is incorporated herein by reference in its entirety). This approach involves introducing or utilizing charged residues within the CH3 domains of each Fc region so that two different Fc regions are related via opposite charges that cause electrostatic attraction. Homodimerization of the same Fc region is undesirable because identical Fc regions repel each other due to having the same charge. Electrostatic steering techniques, as well as appropriate charge pair mutations to promote heterodimer and accurate light / heavy chain pair formation, are described in International Publication Nos. 2009089004 and International Publication Nos. 2014081955, both of which are incorporated herein by reference in their entirety.

[0058] In certain embodiments, one or more amino acids (e.g., lysine) at positions selected from 370, 392, and 409 (EU numbering system) of one CH3 domain are substituted with positively charged amino acids (e.g., aspartic acid and glutamic acid), and one or more amino acids (e.g., aspartic acid or glutamic acid) at positions selected from 356, 357, and 399 (EU numbering system) of the other CH3 domain are substituted with positively charged amino acids (e.g., lysine, histidine, and arginine).

[0059] In certain embodiments, the multispecific antibody construct comprises a first CH3-containing polypeptide (heavy chain or Fc domain) containing charged amino acids (e.g., substitutions of K392D and K409D) at positions 392 and 409, and a second CH3-containing polypeptide containing positively charged amino acids (e.g., substitutions of E356K and D399K) at positions 356 and 399. In other specific embodiments, the multispecific antibody construct comprises a first CH3-containing polypeptide containing charged amino acids (e.g., substitutions of K392D, K409D, and K370D) at positions 392, 409, and 370, and a second CH3-containing polypeptide containing positively charged amino acids (e.g., substitutions of E356K, D399K, and E357K) at positions 356, 399, and 357.

[0060] In one embodiment, the problem of mis-base pairing is avoided by linking the Fc domain to the heavy chain via a linker. In such an example, the heavy chain and the Fc domain can form a single-chain Fc(scFc).

[0061] Any of the constant domains can be modified to contain one or more of the aforementioned charge pair mutations to facilitate the accurate assembly of the multispecific antibody construct.

[0062] Furthermore, the multispecific antibody construct of the present invention includes an antibody comprising a heavy chain and / or a light chain, wherein with respect to either the heavy chain or the light chain, one, two, three, four, or five amino acid residues are missing from the N-terminus, C-terminus, or both, due to post-translational modifications resulting from, for example, the type of host cell on which the antibody is expressed. For example, Chinese hamster ovary (CHO) cells frequently cleave the C-terminal lysine from the antibody heavy chain.

[0063] As used herein, the term “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain that can be produced by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally comprises two constant domains, namely the CH2 domain and the CH3 domain, and possibly a hinge domain. In certain embodiments, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises the CH2 and CH3 domains derived from human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector functions, as described in further detail herein.

[0064] In certain embodiments of the multispecific antibody constructs of the present invention, the binding domain located at the amino terminus of the Fc region (i.e., the amino-terminal binding domain) is a Fab fragment fused to the amino terminus of the Fc region via a peptide linker as described herein or via an immunoglobulin hinge region. The “immunoglobulin hinge region” refers to the amino acid sequence linking the CH1 and CH2 domains of an immunoglobulin heavy chain. The hinge region of human IgG1 is typically defined as the amino acid sequence from approximately Glu216 or Cys226 to approximately Pro230. The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by positioning the first and last cysteine ​​residues to form an inter-heavy-chain disulfide bond at the same position, and this is determinable to those skilled in the art. In some embodiments, the amino-terminal binding domain is bound to the amino terminus of the Fc region via the human IgG1 hinge region. In other embodiments, the amino-terminal binding domain is bound to the amino terminus of the Fc region via the human IgG2 hinge region. In one embodiment, the amino-terminal binding domain (e.g., Fab fragment) is fused to the Fc region via the carboxyl terminus of the CH1 region of Fab.

[0065] As used herein, the term "modified heavy chain" refers to a fusion protein comprising an immunoglobulin heavy chain, particularly a human IgG1 or human IgG2 heavy chain, and a functional antibody fragment (e.g., scFv), wherein the fragment or portion is fused to the N-terminus or C-terminus of the heavy chain, possibly via a peptide linker.

[0066] As used herein, the term "modified light chain" refers to a fusion protein comprising an immunoglobulin light chain and a functional antibody fragment (e.g., scFv), wherein the fragment or portion is fused to the N-terminus or C-terminus of the light chain, possibly via a peptide linker.

[0067] The heavy chain constant region or Fc region of the multispecific antibody constructs described herein may contain one or more amino acid substitutions that affect the glycosylation and / or effector function of antigen-binding proteins. One function of the Fc region of immunoglobulins is to transmit the immunoglobulin to the immune system when it is bound to its target. This is commonly referred to as “effector function.” Transmission can lead to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated cytotoxicity (CDC). ADCC and ADCP are mediated through the binding of the Fc region to Fc receptors on the surface of immune system cells. CDC is mediated through the binding of Fc to complement system proteins, such as C1q. In some embodiments, the multispecific antibody constructs of the present invention contain one or more amino acid substitutions within the constant region that enhance effector functions, including ADCC activity, CDC activity, ADCP activity, and / or the clearance or half-life of antigen-binding proteins. 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, S Examples include 298D, S298V, S298G, S298T, T299A, Y300L, V305I, Q311M, K326A, K326E, K326W, A330S, A330L, A330M, A330F, I332E, D333A, E333S, E333A, K334A, K334V, A339D, A339Q, P396L, or any combination of the above.

[0068] In other embodiments, the multispecific antibody constructs of the present invention include one or more amino acid substitutions within the constant region that reduce effector function. Examples of amino acid substitutions (EU numbering) that can reduce effector function include, but are not limited to, C220S, C226S, C229S, E233P, L234A, L234V, V234A, L234F, L235A, L235E, G237A, P238S, S267E, H268Q, N297A, N297G, V309L, E318A, L328F, A330S, A331S, P331S, or any combination thereof.

[0069] Glycosylation can contribute to the effector function of antibodies, particularly IgG1 antibodies. Therefore, in some embodiments, the multispecific antibody constructs of the present invention may contain one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. Polypeptide glycosylation is typically either N-linked or O-linked. N-linking refers to the binding of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic binding of a carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0070] In certain embodiments, glycosylation of the multispecific antibody constructs described herein is increased by adding one or more glycosylation sites, for example, to the Fc region of a binding protein. Addition of glycosylation sites to antigen-binding proteins can be easily achieved by modifying the amino acid sequence to include one or more of the above tripeptide sequences (in the case of N-linked glycosylation sites). Alternatively, the modification may be made by adding or substituting one or more serine or threonine residues to the start sequence (in the case of O-linked glycosylation sites). For ease of modification, the amino acid sequence of the antigen-binding protein may be modified at the DNA level, particularly by mutating the DNA encoding the target polypeptide with pre-selected bases so that codons that will be translated to desired amino acids are generated.

[0071] The present invention also encompasses the production of bispecific antigen-binding protein molecules in which the carbohydrate structure is modified, resulting in altered effector activity, such as antigen-binding proteins with no or reduced fucosylation that exhibit enhanced ADCC activity. Various methods for reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by the binding of antibody molecules to the FcγRIII receptor, which has been shown to depend on the carbohydrate structure of N-linked glycosylation at the N297 residue of the CH2 domain. Non-fucosylated antibodies bind to this receptor with high affinity and trigger FcγRIII-mediated effector function more efficiently than naturally occurring fucosylated antibodies. For example, recombinant generation of non-fucosylated antibodies in CHO cells in which the alpha-1,6-fucosyltransferase enzyme is knocked out results in antibodies with 100-fold increased ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. 87(5):614-22, 2004). Similar effects can be achieved by reducing the activity of alpha-1,6-fucosyltransferase enzyme or other enzymes in the fucosylation pathway, for example, by siRNA or antisense RNA treatment, manipulation of cell lines to knock out the enzyme, or culture using selective glycosylation inhibitors (see Rothman et al., Mol Immunol. 26(12):1113-23, 1989). Some host cell lines, such as Lec13 or the rat hybridoma YB2 / 0 cell line, naturally produce antibodies with lower levels of fucosylation (see Shields et al., J Biol Chem. 277(30):26733-40, 2002 and Shinkawa et al., J Biol Chem. 278(5):3466-73, 2003). Furthermore, it has been found that ADCC activity is increased by increasing the level of bifurcated glycans through recombinant antibody generation in cells that overexpress the GnTIII enzyme (see Umana et al., Nat Biotechnol. 17(2):176-80, 1999).

[0072] In other embodiments, glycosylation of the multispecific antibody constructs described herein is reduced or eliminated, for example, by removing one or more glycosylation sites from the Fc region of the binding protein. N-linked glycosylation of the antigen-binding protein can be reduced or eliminated by amino acid substitutions that eliminate or modify the N-linked glycosylation site. In certain embodiments, the multispecific antibody constructs described herein include mutations at the N297 position (EU numbering), such as N297Q, N297A, or N297G. In a particular embodiment, the multispecific antibody construct of the present invention includes an Fc region derived from a human IgG1 antibody having the N297G mutation. To improve the stability of the molecule containing the N297 mutation, the Fc region of the molecule may be further manipulated. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine ​​to promote disulfide bond formation in the dimeric state. Therefore, residues corresponding to V259, A287, R292, V302, L306, V323, or I332 (EU numbering) in the IgG1 Fc region may be substituted with cysteine. In one embodiment, disulfide bond scrambling is limited or prevented by substituting specific pairs of residues with cysteine ​​so as to preferentially form disulfide bonds with each other. In certain embodiments, the pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In certain embodiments, the multispecific antibody constructs described herein include an Fc region derived from a human IgG1 antibody having the R292C and V302C mutations. In such embodiments, the Fc region may also include the N297G mutation. In certain embodiments, the multispecific antibody constructs described herein include Fc regions derived from human IgG1 antibody having L234A and L235A mutations. In certain embodiments, the multispecific antibody constructs described herein include Fc regions derived from human IgG1 antibody having N297G, R292C, V302C, L234A, and L235A mutations.

[0073] Furthermore, modifications to the multispecific antibody constructs of the present invention to extend the serum half-life may be desired, for example, by incorporating or adding a salvage receptor-binding epitope (e.g., by mutation in an appropriate region, or by incorporating the epitope into a peptide tag and subsequently fusing it to an antigen-binding protein at any terminal or center, for example, by DNA or peptide synthesis; see, for example, International Publication No. 96 / 32478), or by adding molecules such as polysaccharide polymers, PEG, or other water-soluble polymers. The salvage receptor-binding epitope preferably constitutes a region to which one or more amino acid residues derived from one or two loops of the Fc region are transferred to a similar position in the antigen-binding protein. In one embodiment, three or more residues derived from one or two loops of the Fc region are transferred. In one embodiment, the epitope is taken from the CH2 domain of the Fc region (e.g., the IgG Fc region) and transferred to the CH1, CH3, or VH region, or multiple such regions, of the antigen-binding protein. In addition, epitopes are taken from the CH2 domain of the Fc region and transferred to the CL region, VL region, or both of the antigen-binding protein. For a description of Fc variants and their interactions with salvage receptors, see International Publication Nos. 97 / 34631 and 96 / 32478.

[0074] The present invention comprises one or more isolated nucleic acids encoding multispecific antibody constructs and their components as described herein. Examples of nucleic acid molecules of the present invention include DNA and RNA in both single-stranded and double-stranded forms, and corresponding complementary sequences. Examples of DNA include cDNA, genomic DNA, chemically synthesized DNA, PCR-amplified DNA, and combinations thereof. Examples of nucleic acid molecules of the present invention include full-length genes or cDNA molecules, and combinations of their fragments. In one embodiment, the nucleic acids of the present invention are derived from human sources, but the present invention also includes those derived from non-human species.

[0075] The relevant amino acid sequence derived from immunoglobulins or their regions (e.g., variable regions, Fc regions, etc.) or polypeptides of interest can be determined by direct protein sequencing, and an appropriate coding nucleotide sequence can be designed according to a universal codon table. In addition, genomic DNA or cDNA encoding monoclonal antibodies from which the binding domain of the multispecific antibody construct of the present invention may be derived can be isolated and sequenced from cells producing such antibodies using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody).

[0076] In this specification, "isolated nucleic acid," as used interchangeably with "isolated polynucleotide," refers to a nucleic acid isolated from adjacent gene sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. For example, in the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, cDNA molecule, or oligonucleotide, the nucleic acid derived from such a process is understood to be an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a distinct fragment, or a nucleic acid molecule as a component of a larger nucleic acid construct. In one embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is derived from DNA or RNA that has been isolated at least once in a substantially pure form and in an amount or concentration that allows for the identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (e.g., those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are typically provided and / or constructed in the form of an open reading frame that is not interrupted by internal untranslated sequences or introns, which are present within eukaryotic genes. The sequence of untranslated DNA may be located 5' or 3' from the open reading frame, and at this location, the sequence does not interfere with the manipulation or expression of the coding region. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence considered herein is the 5' end, and the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of production of a nascent RNA transcript from 5' to 3' is referred to as the transcription direction, the sequence region on the DNA strand having the same sequence as the RNA transcript at the 5' end of the RNA transcript is referred to as the “upstream sequence”, and the sequence region on the DNA strand having the same sequence as the RNA transcript at the 3' end of the RNA transcript is referred to as the “downstream sequence”.

[0077] The present invention also includes nucleic acids that hybridize to nucleic acids encoding polypeptides described herein under moderately stringent conditions or highly stringent conditions. Basic parameters influencing the selection of hybridization conditions, and guidance for devising appropriate conditions, are shown by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), which can be readily determined by those skilled in the art, for example, based on the length and / or base composition of the DNA. One method for achieving moderately stringent conditions involves using a pre-wash solution containing 5×SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide and 6×SSC, a hybridization temperature of about 55°C (or other similar hybridization solutions, e.g., one containing about 50% formamide at a hybridization temperature of about 42°C), and washing conditions of about 60°C in 0.5×SSC and 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions similar to those described above, except for washing at about 68°C with 0.2×SSC and 0.1% SDS. SSPE (1 × SSPE is 0.15M NaCl, 10mM NaH2PO4, and 1.25mM EDTA, pH 7.4) can be replaced with SSC (1 × SSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization buffer and wash buffer, and after hybridization is complete, a 15-minute wash is performed.Of course, the washing temperature and washing salt concentration can be adjusted as needed to achieve the desired degree of stringency by applying the basic principles that are known to those skilled in the art and that determine the hybridization reaction and double-strand stability, as will be further described below (see, for example, Sambrook et al., 1989). When a nucleic acid is hybridized to a target nucleic acid of an unknown sequence, the hybrid length is assumed to be the length of the nucleic acid being hybridized. When a nucleic acid of a known sequence is hybridized, the hybrid length can be determined by aligning the sequence of the nucleic acid and identifying the region of optimal sequence complementarity. The hybridization temperature of a hybrid expected to be less than 50 base pairs long must be 5-10°C lower than the melting temperature (Tm) of the hybrid, where Tm is calculated according to the following formula: For a hybrid less than 18 base pairs long, Tm(°C) = 2(number of A bases + T bases) + 4(number of G bases + 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 ([Na+] of 1×SSC = 0.165M).In one embodiment, each such hybridizing nucleic acid has a length of at least 15 nucleotides (or at least 18 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, or at least 50 nucleotides), or a length of at least 25% (or at least 50%, at least 60%, at least 70%, or at least 80%) of the length of the nucleic acid of the present invention that it hybridizes, and has at least 60% sequence identity (or at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%). Here, sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when they are aligned in such a way that duplication and identity are maximized while sequence gaps are minimized, as will be described in more detail later.

[0078] The antigen-binding protein variants described herein can be prepared by generating mutant-encoding DNA using site-directed mutagenesis of nucleotides in the polypeptide-encoding DNA, by cassette or PCR mutagenesis, or by other techniques known in the art, and then expressing the recombinant DNA in a cell culture as outlined herein. However, antigen-binding proteins containing mutant CDRs having up to approximately 100–150 residues may also be prepared by in vitro synthesis using established techniques. The variants typically exhibit the same qualitative biological activity as naturally occurring analogs, e.g., 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. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final construct, provided that the final construct retains the desired properties. Furthermore, changes in amino acids may alter the post-translational processes of the antigen-binding protein, such as changing the number or location of glycosylation sites. In certain embodiments, antigen-binding protein variants are prepared with the aim of modifying amino acid residues directly involved in epitope binding. In other embodiments, modifications of residues not directly involved in epitope binding, or residues not involved in epitope binding at all, are desired for purposes considered herein. Mutagenesis is intended within any CDR region and / or framework region. Those skilled in the art can use analysis of covariance techniques to design useful modifications to the amino acid sequence of antigen-binding proteins.See, for example, Choulier, et al., Proteins 41:475-484, 2000; Demarest et al., J.Mol.Biol.335:41-48, 2004; Hugo et al., Protein Engineering 16(5):381-86, 2003; Aurora et al., U.S. Patent Application Publication No. 2008 / 0318207A1; Glaser et al., U.S. Patent Application Publication No. 2009 / 0048122A1; Urech et al., International Publication No. 2008 / 110348A1; Borras et al., International Publication No. 2009 / 000099A2. Such modifications, determined by analysis of covariance, can improve the potency, pharmacokinetic properties, pharmacodynamic properties, and / or manufacturability properties of antigen-binding proteins.

[0079] The nucleic acid sequence of the present invention. As will be understood by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be produced, all of which encode the CDR of the present invention (as well as the heavy and light chains of antigen-binding proteins, or other components, as described herein). Therefore, once a particular amino acid sequence is identified, those skilled in the art will be able to produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the encoded protein.

[0080] The present invention also includes a vector comprising one or more nucleic acids encoding one or more components of the multispecific antibody construct of the present invention (e.g., a variable region, a light chain, a heavy chain, a modified heavy chain, and an Fd fragment). The term “vector” refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information to a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. As used herein, the terms “expression vector” or “expression construct” refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid regulatory sequences necessary for the expression of said coding sequence in a particular host cell, operably ligated thereto. An expression vector may, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, sequences that affect RNA splicing of the coding region operably ligated thereto. Nucleic acid sequences necessary for expression in prokaryotes include promoters, possibly operator sequences, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. The secreted signal peptide sequence may, in some cases, be encoded by an expression vector operably linked to the coding sequence of interest, thereby allowing recombinant host cells to secrete the expressed polypeptide, if necessary, making it easier to isolate the polypeptide of interest from the cell. For example, in some embodiments, the signal peptide sequence may be added to / fused to the amino terminus of any of the polypeptide sequences of the present invention. In a particular embodiment, a signal peptide having the amino acid sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 32) is fused to the amino terminus of any of the polypeptide sequences of the present invention. In other embodiments, a signal peptide having the amino acid sequence MAWALLLLTLLTQGTGSWA (SEQ ID NO: 33) is fused to the amino terminus of any of the polypeptide sequences of the present invention.In yet another embodiment, a signal peptide having the amino acid sequence MTCSPLLLTLLIHCTGSWA (SEQ ID NO: 34) is fused to the amino terminus of any of the polypeptide sequences of the present invention. 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), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 38), and MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 39). Other signal peptides are known to those skilled in the art and may be fused to any of the polypeptide chains of the present invention, for example, to promote or optimize their expression in a particular host cell.

[0081] Typically, the expression vector used in host cells to generate the bispecific antigen protein of the present invention will contain a sequence for plasmid maintenance, as well as a sequence for cloning and expressing the exogenous nucleotide sequence encoding the components of the bispecific antigen-binding protein. Such sequences, collectively referred to as “flanking sequences,” will, in certain embodiments, typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and one or more selective marker elements. Each of these sequences will be discussed below.

[0082] In some cases, 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, wherein the oligonucleotide tag sequence encodes polyHis (e.g., hexaHis), FLAG, HA (hemagglutinin influenza virus), myc, or another “tag” molecule for which a commercially available antibody exists. Typically, upon expression of the polypeptide, the tag fuses to the polypeptide and can function as a means for affinity purification or detection of the polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as an affinity matrix. In some cases, the tag can then be removed from the purified polypeptide by various means, such as using a specific cleavage peptidase.

[0083] Flanking sequences can be homogeneous (i.e., derived from the same species and / or strain as the host cell), heterogeneous (i.e., derived from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from multiple sources), synthetic, or natural. Thus, the sources of flanking sequences may be any prokaryotes or eukaryotes, any vertebrates or invertebrates, or any plants, provided that the flanking sequences are functional in host cellular mechanisms and can be activated by such mechanisms.

[0084] Flanking sequences useful in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and / or restriction endonuclease digestion and can therefore be isolated from a suitable tissue source using a suitable restriction endonuclease. In some cases, the entire nucleotide sequence of the flanking sequence may be known. Here, the flanking sequence can be synthesized using conventional methods of nucleic acid synthesis or cloning.

[0085] Whether all or only some of the flanking sequences are known, flanking sequences can be obtained by screening a genomic library using polymerase chain reaction (PCR) and / or appropriate probes such as oligonucleotides and / or flanking sequence fragments from the same or different species. If the flanking sequences are unknown, fragments of DNA containing the flanking sequences can be isolated from larger DNA fragments that may also contain coding sequences or even other genes. Isolation can be achieved by generating appropriate DNA fragments by restriction endonuclease digestion, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to those skilled in the art. The selection of appropriate enzymes to achieve this objective will be readily apparent to those skilled in the art.

[0086] The origin of replication is typically a component of commercially available prokaryotic expression vectors, and this origin assists in the amplification of the vector within the host cell. If the selected vector does not contain an origin of replication site, the site may be chemically synthesized based on a known sequence and ligated into the vector. For example, the origin of replication derived from plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, varicella-stomatitis virus (VSV), or papillomavirus, e.g., HPV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of replication component is not required for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the viral initial promoter).

[0087] Transcription termination sequences are typically located 3' to the end of the polypeptide coding region and function to terminate transcription. In prokaryotic cells, transcription termination sequences are usually GC-rich fragments followed by poly-T sequences. These sequences can be readily cloned from libraries or even commercially purchased as part of vectors, but they can also be readily synthesized using known nucleic acid synthesis methods.

[0088] Selection marker genes encode proteins necessary for the survival and proliferation of host cells grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, to prokaryotic host cells; (b) compensate for deficiencies in the cellular nutritional requirements; or (c) supply essential nutrients unavailable from complex or standard media. Specific selection markers include kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, neomycin resistance genes can also be used for selection in both prokaryotic and eukaryotic host cells.

[0089] Other select genes may be used to amplify the genes that will be expressed. Amplification is the process by which genes required for the production of proteins important for proliferation or cell survival are repeated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable select markers for mammalian cells include the dihydrofolate reductase (DHFR) gene and the promoterless thymidine kinase gene. Mammalian cell transformants are placed under selective pressure such that only the transformant is adapted to survive by the select gene present in the vector. Selective pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selector in the culture medium is continuously increased, leading to amplification of both the selectable gene and the DNA encoding another gene, such as one or more components of the multispecific antibody constructs described herein. As a result, large amounts of polypeptides are synthesized from the amplified DNA.

[0090] The ribosome binding site is typically required for mRNA translation initiation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located at the 3' end of the promoter and at the 5' end of the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions may be operably ligated to an internal ribosome binding site (IRES), thereby enabling translation of two open reading frames from a single RNA transcript.

[0091] In cases where glycosylation is desired in a eukaryotic host cell expression system, various pre-sequences or pro-sequences may be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide may be modified, or a pro-sequence may be added, which may also affect glycosylation. The final protein product may have one or more additional amino acids associated with expression at position -1 (relative to the first amino acid of the mature protein), which do not necessarily have to be completely removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site attached to the amino terminus. In addition, by using several other enzyme cleavage sites, if the enzyme cleaves at such regions in the mature polypeptide, a slightly cleaved form of the desired polypeptide may be produced.

[0092] The expression vectors and cloning vectors of the present invention typically contain a promoter that is recognized by a host organism and operably ligated to a molecule encoding a polypeptide. As used herein, the term “operably ligated” refers to the ligation of two or more nucleic acid sequences such that a nucleic acid molecule is produced that can direct the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a regulatory sequence in a vector “operably ligated” to a protein-coding sequence is ligated to the protein-coding sequence such that the expression of the protein-coding sequence is achieved under conditions that are compatible with the transcriptional activity of the regulatory sequence. More specifically, a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional regulatory elements) is operably ligated to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system.

[0093] A promoter is a non-transcriptional sequence located upstream (i.e., 5') of the start codon of a structural gene (generally within approximately 100-1000 bp) and controls the transcription of that structural gene. Conventionally, promoters are grouped into one of two classes: inductive promoters and constitutive promoters. Inductive promoters initiate an increase in the transcription level from DNA under their control in response to any change in culture conditions, such as the presence or absence of nutrients or changes in temperature. Constitutive promoters, on the other hand, transcribe the gene to which they are operably ligated uniformly, i.e., with little or no control over gene expression. Numerous promoters recognized by various potential host cells are well known. By removing promoters from source DNA by restriction enzyme digestion and inserting the desired promoter sequence into a vector, the appropriate promoter is operably ligated to the DNA encoding, for example, the heavy chain, light chain, modified heavy chain, or other components of the multispecific antibody construct of the present invention.

[0094] Suitable promoters for use with yeast hosts are also well known in this art. Advantageously, yeast enhancers are used in conjunction with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (adenovirus type 2, etc.), bovine papillomavirus, arowana sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and Simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0095] Further promoters of note include, but are not limited to, the following: the early SV40 promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained within the long terminal repeat at the 3' end of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); the promoter and regulatory sequence derived from the metallothione gene (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al. Examples include al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731; or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Also noteworthy are the following animal transcriptional regulatory regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene regulatory region active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene regulatory region active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); and the immunoglobulin gene regulatory region active in lymphocytes (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444); Mouse mammary tumor virus regulatory region active in testes, mammary glands, lymphocytes, and mast cells (Leder et al., 1986, Cell 45:485-495); Albumin gene regulatory region active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); Alpha-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol.Cell.Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); Alpha-1 antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); Beta-globin gene regulatory region active in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene regulatory region active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0096] Enhancer sequences can be inserted into vectors to enhance the transcription of DNA encoding components of multispecific antibody constructs (e.g., light chain, heavy chain, modified heavy chain, Fd fragment) by higher eukaryotes. Enhancers are cis-acting elements of DNA, typically about 10–300 bp long, that act on promoters to increase transcription. Enhancers are relatively independent of direction and position and are found at both the 5' and 3' ends of the transcription unit. Several enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, viral enhancers are typically used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer, known in this technique, are exemplary enhancing elements for the activation of eukaryotic promoters. The enhancer may be located either 5' or 3' of the coding sequence in the vector, but is typically positioned 5' from the promoter. Sequences encoding appropriate native or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into the expression vector to promote extracellular secretion of antibodies. The choice of signal peptide or leader depends on the type of host cell from which the antibody will be produced, and heterologous signal sequences may replace native signal sequences. Examples of signal peptides have been previously described. Other signal peptides that function in mammalian host cells include the interleukin-7 (IL-7) signal sequence described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460 846.

[0097] The expression vector provided may be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not initially present in the vector, they may be obtained individually and ligated into the vector. Methods used to obtain each of the flanking sequences are well known to those skilled in the art. By introducing the expression vector into a host cell, proteins such as fusion proteins encoded by the nucleic acids described herein can be produced.

[0098] In certain embodiments, nucleic acids encoding different components of the multispecific antibody construct of the present invention may be inserted into the same expression vector. For example, the nucleic acid encoding the first antitarget antigen light chain can be cloned into the same vector as the nucleic acid encoding the first antitarget antigen heavy chain. In such embodiments, the two nucleic acids may be separated by an internal ribosome entry site (IRES) under the control of a single promoter so that the light and heavy chains are expressed from the same mRNA transcript. Alternatively, the two nucleic acids may be under the control of two separate promoters so that the light and heavy chains are expressed from two separate mRNA transcripts. In some embodiments, the nucleic acids encoding the first antitarget antigen light and heavy chains are cloned into one expression vector, and the nucleic acids encoding the second antitarget antigen light and heavy chains are cloned into a second expression vector.

[0099] After a vector is constructed and one or more nucleic acid molecules encoding components of the multispecific antibody constructs described herein are inserted into appropriate sites within the vector, the completed vector may be inserted into a host cell suitable for amplification and / or polypeptide expression. Thus, the present invention encompasses isolated host cells containing one or more expression vectors encoding components of bispecific antigen-binding proteins. As used herein, the term “host cell” refers to a cell that is or can be transformed with nucleic acid and thereby expresses the gene of interest. The term includes progeny of a parent cell, whether or not they are morphologically or genetically identical to the original parent cell, as long as the gene of interest is present. In one embodiment, a host cell containing the isolated nucleic acid of the present invention, operably ligated to at least one expression regulatory sequence (e.g., promoter or enhancer), is a “recombinant host cell.”

[0100] The transformation of selected host cells into antigen-binding protein expression vectors can be achieved by well-known methods, including translocation, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated translocation, or other known techniques. The chosen method will, to some extent, depend on the type of host cell to be used. Such methods and other suitable methods are well known to those skilled in the art and are shown, for example, in Sambrook et al., 2001.

[0101] When host cells are cultured under appropriate conditions, they synthesize antigen-binding proteins, which can then be collected from the culture medium (if the host cells secrete them into the medium) or directly from the producing host cells (if not secreted). The selection of appropriate host cells depends on various factors, including the desired expression level, polypeptide modifications (such as glycosylation or phosphorylation) desired or required for activity, and the ease of folding into biologically active molecules.

[0102] Examples of host cells include prokaryotic cells, yeast cells, or higher eukaryotic cells. Examples of prokaryotic host cells include eubacteria such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, for example Escherichia, for example Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, and Serratia, for example Serratia marcescens. Examples include the genera *Marcescens*, *Shigella*, and *Bacillus*, such as *B. subtilis* and *B. licheniformis*, as well as the genera *Pseudomonas* and *Streptomyces*. Eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for recombinant polypeptides. *Saccharomyces cerevisiae*, or common baker's yeast, are among the most commonly used lower eukaryotic host microorganisms.However, this does not apply to the genera Pichia, such as P. pastoris, Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, and Schwanniomyces, such as Schwanniomyces occidentalis. Hosts of Aspergillus species, such as A. nidulans and A. niger, and several other genera, species, and strains, are generally available and useful herein.

[0103] Host cells for the expression of glycosylated antigen-binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Several baculovirus strains and mutants, as well as corresponding insect-acceptable host cells derived from hosts such as the fall armyworm (Spodoptera frugiperda), Aedes aegypti (Aedes aegypti), Asian tiger mosquito (Aedes albopictus), fruit fly (Drosophila melanogaster), and silkworm (Bombyx mori), have been identified. Various virus strains for transduction into such cells, such as the L-1 mutant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available.

[0104] Vertebrate host cells are also suitable hosts, and the recombination of antigen-binding proteins from such cells is a common practice. Mammalian cell lines available as hosts for expression are well known in this technology and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but are not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cell / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36:59, 1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human hepatocellular carcinoma cells (Hep G2, HB 8065); Mouse mammary tumor cells (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC 5 cells or FS4 cells; Mammalian myeloma cells, and several other cell lines. In certain embodiments, cell lines may be selected by determining which cell lines constitutively express and produce the multispecific antibody construct of the present invention at high levels. In another embodiment, cell lines derived from B cell lines that do not produce antibodies themselves but have the ability to produce and secrete heterologous antibodies may be selected.In some embodiments, CHO cells are host cells for expressing the multispecific antibody construct of the present invention.

[0105] For the production of multispecific antibody constructs, host cells are transformed or transtransfected with the above-mentioned nucleic acids or vectors and cultured in conventional nutrient media modified to be suitable for promoter induction, transformant selection, or amplification of genes encoding desired sequences. In addition, novel vectors and transtransfected cell lines having multiple copies of transcription units separated by a selection marker are particularly useful for the expression of antigen-binding proteins. Therefore, the present invention also provides a method for preparing a bispecific antigen-binding protein as described herein, comprising culturing host cells containing one or more expression vectors as described herein in a culture medium under conditions that enable the expression of a bispecific antigen-binding protein encoded by the one or more expression vectors, and recovering the bispecific antigen-binding protein from the culture medium.

[0106] The host cells used to produce the antigen-binding protein of the present invention can be cultured in various media. Commercial media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44, 1979; Barnes et al., Anal. Biochem. 102:255, 1980; U.S. Patent No. 4,767,704; U.S. Patent No. 4,657,866; U.S. Patent No. 4,927,762; U.S. Patent No. 4,560,655; or U.S. Patent No. 5,122,469; International Publication No. 90103430; International Publication No. 87 / 00195; or U.S. Reissue Patent No. 30,985 can be used as a culture medium for host cells. All of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin®), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary nutritional supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with host cells selected for expression and will be obvious to those skilled in the art.

[0107] When host cells are cultured, bispecific antigen-binding proteins can be produced intracellularly, in the perimembrane space, or directly secreted into the culture medium. If the antigen-binding protein is produced intracellularly, the first step is to remove granular fragments of host cells or lysed fragments, for example, by centrifugation or ultrafiltration. The bispecific antigen-binding protein can be purified, for example, using hydroxyapatite chromatography, cation or anion exchange chromatography, or affinity chromatography using the antigen of interest or protein A or protein G as an affinity ligand. Protein A can be used to purify proteins containing polypeptides based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J.Immunol.Meth.62:1-13, 1983). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J.5:15671575, 1986). The matrix to which affinity ligands bind is almost always agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene can achieve faster flow rates and shorter processing times than can be achieved using agarose. If the protein contains a CH3 domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Depending on the specific bispecific antigen-binding protein to be recovered, other techniques for protein purification such as ethanol precipitation, reverse-phase HPLC, chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation are also possible. [Examples]

[0108] A novel bispecific format, widely usable in both cis- and trans-action mechanisms, was rationally designed by determining the crystal structure of a ternary complex with two Fabs that bind to two different domains within the same target receptor molecule. This provided a template for conceptualizing a linker that ligates these two molecules into a single molecule during recombinant expression in a single cell (Figure 1). The use of this linker ligating these two warheads is crucial for implementing specific binding modes that can be tailored to the specific needs of particular therapeutic projects.

[0109] To bind the scFv module to Fab, we manipulated three flexible G4 linkers with multiple repeats and two semi-rigid helical linkers of different lengths (Figure 2). To extend the molecular half-life, we inserted an FC region into the C-terminus of the Fab CH1 domain (Figures 2 and 3) to produce a molecule with three polypeptide chains.

[0110] In addition, scFv can also be similarly ligated to the N-terminus of the light chain within Fab. This allows for molecular design that meets specific requirements for the desired binding mode and also balances the length of the polypeptide chain containing the molecule (Figure 3).

[0111] The molecule was expressed in HEK 293 6E cells, and ProA was purified. The total yield was approximately 75 mg / L (Figure 6).

[0112] Next, the molecules were tested in a binding assay that strictly enforced binding to the same receptor from both warheads. FG11 met the design objective and did not recognize / bind to epitopes within the two different receptor molecules (Figure 7).

[0113] To confirm that these bispecific molecules are functional while binding to targets on the cell surface, cell-based assays expressing human target proteins were performed. In this case, all bispecific molecules showed binding (Figure 8).

[0114] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. The disclosed invention is not limited to the specific methodologies, protocols, and materials described herein, and these are subject to change. It is also understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit the scope of the appended claims.

[0115] Those skilled in the art will recognize or be able to verify, through mere routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the subsequent claims.

Claims

1. a) A first polypeptide comprising an antibody Fc region including a first hinge region, a first CH2 region, and a first CH3 region, b) A second polypeptide comprising an antibody light chain construct, Here, the antibody light chain construct is i) 1) The first VH and the first VL, which are bound together to form the first antigen-binding domain, 2) The first linker peptide linking the first VH and the first VL scFv including, ii) Antibody light chain containing a second VL and CL Includes, The scFv is conjugated at its C-terminus to the N-terminus of the second VL region of the antibody light chain via a second linker. The second linker includes an ex selected from the group consisting of GSADDAKKDAAKKDAAKKDDAKKDDAGS (SEQ ID NO: 11) and GSADDAKKDAAKKDAAKKDDAKKDDAKKDDAGS (SEQ ID NO: 12). c) A third polypeptide comprising an antibody heavy chain including a second VH, a second CH1 region, a second hinge region, a second CH2 region, and a second CH3 region. Includes, A multispecific antibody construct in which the second VH of the antibody heavy chain and the second VL of the antibody light chain are bound together to form a second antigen-binding domain.

2. The first linker is (Gly ３ Ser) ２ (SEQ ID NO: 1), (Gly ４ Ser) ２ (SEQ ID NO: 2), (Gly ３ Ser) ３ (SEQ ID NO: 3), (Gly ４ Ser) ３ (SEQ ID NO: 4), (Gly ３ Ser) ４ (SEQ ID NO: 5), (Gly ４ Ser) ４ (SEQ ID NO: 6), (Gly ３ Ser) ５ (SEQ ID NO: 7), (Gly ４ Ser) ５ (SEQ ID NO: 8), (Gly ３ Ser) ６ (SEQ ID NO: 9), and (Gly ４ Ser) ６ (SEQ ID NO: 10), and the multispecific antibody construct according to claim 1, comprising a sequence selected from the group consisting of.

3. The multispecific antibody construct according to claim 1 or 2, wherein the scFv comprises the first VH, which is bound at its C-terminus to the N-terminus of the first linker, and the first linker is bound at its C-terminus to the N-terminus of the first VL.

4. The multispecific antibody construct according to claim 1 or 2, wherein the scFv comprises the first VL, which is bound at its C-terminus to the N-terminus of the first linker, and the first linker is bound at its C-terminus to the N-terminus of the first VH.

5. The multispecific antibody construct according to any one of claims 1 to 4, wherein the first antigen-binding domain and the second antigen-binding domain bind to epitopes on different polypeptides.

6. The multispecific antibody construct according to any one of claims 1 to 4, wherein the first antigen-binding domain and the second antigen-binding domain bind to different epitopes on the same polypeptide.

7. The multispecific antibody construct according to any one of claims 1 to 6, wherein the Fc region comprises a hinge region, a CH2 region, and a CH3 region.

8. The multispecific antibody construct according to any one of claims 1 to 7, wherein the N-terminus of the Fc region is linked to the C-terminus of the heavy chain via a third linker through its N-terminus.

9. The third linker is (Gly ３ Ser) ２ (Sequence ID 1), (Gly ４ Ser) ２ (Sequence ID 2), (Gly ３ Ser) ３ (Sequence ID 3), (Gly ４ Ser) ３ (Sequence No. 4), (Gly ３ Ser) ４ (Sequence No. 5), (Gly ４ Ser) ４ (Sequence No. 6), (Gly ３ Ser) ５ (Sequence ID 7), (Gly ４ Ser) ５ (Sequence No. 8), (Gly ３ Ser) ６ (Sequence No. 9), (Gly ４ Ser) ６ (Sequence No. 10), (Gly ３ GLN) ２ (Sequence No. 13), (Gly ４ GLN) ２ (Sequence No. 14), (Gly ３ GLN) ３ (Sequence No. 15), (Gly ４ GLN) ３ (Sequence No. 16), (Gly ３ GLN) ４ (Sequence No. 17), (Gly ４ GLN) ４ (Sequence No. 18), (Gly ３ GLN) ５ (Sequence No. 19), (Gly ４ GLN) ５ (Sequence ID 20), (Gly ３ GLN) ６ (Sequence ID 21), and (Gly ４ GLN) ６ A multispecific antibody construct according to claim 8, comprising a sequence selected from the group consisting of (Sequence ID 22).

10. The multispecific antibody construct according to any one of claims 1 to 7, wherein the first polypeptide comprises the antibody Fc region.

11. The multispecific antibody construct according to any one of claims 1 to 10, wherein the multispecific antibody construct is a bispecific antibody construct.

12. A multispecific antibody construct according to any one of claims 1 to 11, wherein one CH3 domain contains the mutation F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y, and the other CH3 domain contains the K409R mutation, and the numbering of amino acid residues follows the EU index described in Kabat.

13. A multispecific antibody construct according to any one of claims 1 to 12, wherein one CH3 domain contains the T366W mutation, and the other CH3 domain contains the T366S, L368A, Y407V mutations, and the amino acid residue numbering follows the EU index described in Kabat.

14. A multispecific antibody construct according to any one of claims 1 to 12, wherein one CH3 domain contains the K / R409D and K392D mutations, and the other CH3 domain contains the D399K mutation, and the amino acid residue numbering follows the EU index described in Kabat.

15. The multispecific antibody construct according to claim 14, wherein the CH3 domain containing the D399K mutation also contains the E356K mutation, and the amino acid residue numbering follows the EU index described in Kabat.

16. A linker for linking scFv to an antibody light chain, comprising the sequence GSADDDAKKDAAKKKDAAKKKDDAKKDDAGS (SEQ ID NO: 11) or GSADDDAKKDAAKKKDAAKKKDDAKKDDAKKDAGS (SEQ ID NO: 12), The scFv and the antibody light chain are included in the multispecific antibody construct, and the multispecific antibody construct is a) A first polypeptide comprising an antibody Fc region including a first hinge region, a first CH2 region, and a first CH3 region, b) A second polypeptide comprising an antibody light chain construct, Here, the antibody light chain construct is i) 1) The first VH and the first VL, which are bound together to form the first antigen-binding domain, 2) The first linker peptide linking the first VH and the first VL scFv including, ii) Antibody light chain containing a second VL and CL Includes, The scFv is conjugated at its C-terminus to the N-terminus of the second VL region of the antibody light chain via the linker. c) A third polypeptide comprising an antibody heavy chain including a second VH, a second CH1 region, a second hinge region, a second CH2 region, and a second CH3 region. Includes, The second VH of the antibody heavy chain and the second VL of the antibody light chain are bound together to form a second antigen-binding domain. Linker.

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