Anti-CD3 antibody

Abstract

The present invention provides antibodies that specifically bind to human CD3. The antibodies can be monospecific (each binding site binds to the same target, i.e., CD3), bispecific (having at least two binding sites for two targets, including human CD3), or multispecific (having multiple binding sites for multiple targets, including human CD3). The antibodies can be used to treat various diseases, such as cancer, infectious diseases, and immune disorders.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 426,626, filed November 18, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains sequences within the 66,000 byte XML file 603846SEQLST, created on November 12, 2023, which are incorporated by reference. [Background technology]

[0003] Antigen-specific immune responses by the adaptive immune system are complex biological processes regulated by multiple positive and negative regulators. Naive T cells first recognize peptide antigens presented by major histocompatibility complex (MHC, also known as HLA for human proteins) molecules on antigen-presenting cells (APCs) and are stimulated via the T cell receptor (TCR) complex, which contains the TCR α and β (or γ and δ) chains and CD3 molecules. The initial interaction between the TCR and MHC (or HLA) in T cell activation is called signal 1. For optimal T cell activation and proliferation, a second signal (signal 2) is required, triggered by the interaction of costimulatory molecules, such as CD28 and ICOS, of the CD28 superfamily expressed on T cells with their respective counter-receptors expressed on APCs. Additionally, the immune system is positively regulated by other costimulatory molecules, such as CD40, OX40, GITR, CD27, HVEM, and 4-1BB, which belong to the TNF receptor superfamily, and negatively regulated by checkpoint molecules, such as PD-1, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD96, and CD112R. These costimulatory and checkpoint molecules are expressed depending on the cell type and developmental stage, delicately controlling the immune response in the body. Furthermore, various secreted proteins, such as cytokines and chemokines, are involved in regulating the immune response by promoting the activation, differentiation, proliferation, maintenance, and suppression of specific immune cell subsets. The action of cytokines on T cells is well known as signal transduction 3, a third mechanism required for T cell activation, differentiation, and proliferation.For reviews, see Curtsinger et al., Curr. Opin. Immunol. 22:333-340, 2010;Mahoney et al., Nat. Rev. Drug Discov. 14:561-584, 2015;Mercier et al., Front. Immunol. 6:418, 2015;Baumeister et al., Annu. Rev. Immunol. 34:539-573, 2016;Hurton et al., Proc. Natl. Acad. Sci. 113:E7788-E7797, 2016;Torphy et al., Int. J. Mol. Sci. 18:2642, 2017;Punt et al., Kuby Immunology, Eighth Edition. WH Freeman and Co., New York, See 2018.

[0004] There are two major groups of mature T cells: CD4+ helper T cells and CD8+ cytotoxic T cells. CD4+ helper T cells are further divided into T H 1. T H 2. T H 9. T H 17, T H 22, T FHCD4+ helper T cells are classified into CD4+ T cells, CD8+ cytotoxic T cells, and Treg cells, each with specific functions and characteristic cytokine expression patterns. The primary function of CD4+ helper T cells is to regulate other immune cells, such as B cells and CD8+ cytotoxic T cells, to ensure appropriate and timely responses in the immune defense system. Meanwhile, the primary function of CD8+ cytotoxic T cells is to destroy cells infected or transformed by pathogens in an antigen-specific manner. Upon activation by signals 1 and 2, CD8+ cytotoxic T cells secrete perforin and granzymes, which act synergistically to induce apoptosis of target cells. CD8+ cytotoxic T cells also play an important role in the elimination of tumor cells. For reviews, see Taniuchi, Annu. Rev. Immunol. 36:579-601, 2018; Punt et al., supra; Saravia et al., Cell. Mol. Immunol. 16:634-643, 2019; Raskov et al., Br. J. Cancer 124:359-367, 2021.

[0005] Bispecific antibodies are recombinant monoclonal antibodies that can bind to two different antigens. Recent studies have shown that bispecific antibodies that bind to CD3 expressed on T cells and cancer-associated surface molecules can bridge T cells and cancer cells, inducing T cell-mediated cytotoxicity against cancer cells. This type of bispecific antibody is called a T cell engager. Several T cell engagers have been approved for human use in the treatment of cancer, including Blincyto® (blinatumomab, anti-CD19 / CD3) for acute lymphoblastic leukemia, Tecvayli® (teclistamab, anti-BCMA / CD3) for multiple myeloma, and Lunsumio® (mosunetuzumab, anti-CD20 / CD3) for follicular lymphoma. Many other T cell engagers are being evaluated in clinical trials for the treatment of cancer. For reviews, see Middelburg et al., Cancers, 13:287, 2021, Ma et al., Front. Immunol. 12:Article 6266116, 2021, Wang et al., EMBO Mol. Med. 13:e14291, 2021; Arvedson et al., Annu. Rev. Cancer Biol. 6:17-34, 2022. Summary of the Invention

[0006] The present invention provides an antibody that specifically binds to human CD3, comprising a mature heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 derived from SEQ ID NO: 1, and a mature light chain variable region comprising CDRL1, CDRL2, and CDRL3 derived from SEQ ID NO: 2. Optionally, CDRH1, CDRH2, and CDRH3 comprise SEQ ID NOs: 43-45, respectively, and CDRL1, CDRL2, and CDRL3 comprise SEQ ID NOs: 46-48, respectively. Optionally, the antibody is a murine antibody. Optionally, the antibody is chimerized or veneered. Optionally, the antibody is a humanized antibody and comprises a humanized mature heavy chain variable region and a humanized mature light chain variable region. Optionally, positions 30, 49, 93, and 94 according to Kabat numbering of the humanized mature heavy chain variable region are occupied by N, A, V, and R, respectively. Optionally, positions 36, 46, 49, 66, and 71 of the humanized mature light chain variable region are occupied by V, G, G, L, and A, respectively. Optionally, the mature heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 1, and the mature light chain variable region has an amino acid sequence comprising SEQ ID NO: 2. Optionally, the mature heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 4, and the mature light chain variable region has an amino acid sequence comprising any of SEQ ID NOs: 6, 17, or 27. Optionally, the mature heavy chain variable region has an amino acid sequence consisting of or essentially consisting of SEQ ID NO: 4, and the mature light chain variable region has an amino acid sequence consisting of or essentially consisting of SEQ ID NO: 6, 17, or 27. Optionally, the antibody further comprises a heavy chain constant region fused to the mature heavy chain variable region and a light chain constant region fused to the mature light chain variable region.

[0007] Optionally, the antibody is a multispecific antibody comprising multiple pairs of mature heavy chain variable regions and mature light chain variable regions, one of the pairs comprising the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 derived from SEQ ID NO: 1, and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 derived from SEQ ID NO: 2, and the other of the pair binds to the target antigen.

[0008] Optionally, the antibody is a bispecific antibody comprising two pairs of mature heavy chain variable regions and mature light chain variable regions, one of which comprises the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 derived from SEQ ID NO: 1, and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 derived from SEQ ID NO: 2, and the other of the pair binds to the target antigen.

[0009] Optionally, the target antigen is a cancer-associated antigen, an immune cell antigen, or an antigen on a pathogen or pathogen-infected cell.

[0010] Optionally, one of the pair of mature heavy chain variable region and mature light chain variable region is an scFv, and the other of the pair of mature heavy chain variable region and mature light chain variable region further comprises a heavy chain constant region linked to the mature heavy chain variable region and a light chain constant region linked to the mature light chain variable region. Optionally, the mature heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 derived from SEQ ID NO: 1 and the mature light chain variable region comprising CDRL1, CDRL2, and CDRL3 derived from SEQ ID NO: 2 are linked as an scFv, and the other mature heavy chain variable region and mature light chain variable region of the pair are linked to heavy chain constant region and light chain constant region, respectively.

[0011] Optionally, the scFv is linked to the heavy chain constant region. Optionally, the scFv is linked to the heavy chain constant region via the mature light chain variable region of the scFv. Optionally, the scFv has a sequence comprising SEQ ID NO: 31 or 33. Optionally, the scFv has a sequence consisting of, or consisting essentially of, SEQ ID NO: 31 or 33. Optionally, the scFv is linked to the heavy chain constant region via the mature heavy chain variable region of the scFv. Optionally, the scFv has a sequence comprising SEQ ID NO: 29. Optionally, the scFv has a sequence consisting of, or consisting essentially of SEQ ID NO: 29. Optionally, the scFv is linked to the N-terminus of the other mature heavy chain variable region or mature light chain variable region of the pair.

[0012] The present invention further provides antibodies in the form of or comprising an scFv. An scFv comprises a mature heavy chain variable region fused as a single chain to a mature light chain variable region via a linker. Optionally, the antibody has a sequence comprising any of SEQ ID NOs: 29, 31, or 33. Optionally, the antibody has a sequence consisting of, or consisting essentially of, any of SEQ ID NOs: 29, 31, or 33.

[0013] The present invention further provides pharmaceutical compositions comprising any of the antibodies described above or disclosed elsewhere herein.

[0014] The present invention further provides a method for treating cancer, comprising administering to a patient with cancer an antibody as described above or disclosed elsewhere herein, wherein the target antigen is a cancer-associated antigen.

[0015] The present invention further provides a method for treating an immune disease, comprising administering to a patient having said immune disease an antibody as described above or disclosed elsewhere herein.

[0016] The present invention further provides a method for treating a pathogen infection, comprising administering to a patient infected with said pathogen an antibody as described above or disclosed elsewhere herein, regardless of whether the target antigen is an antigen of said pathogen or a pathogen-infected cell. [Brief explanation of the drawings]

[0017] [Figure 1]Alignment of the amino acid sequences of mature SP34 VH, HuSP34 VH1, and human acceptor M24236 VH. Residue numbers are assigned according to Kabat et al. (Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991). CDR sequences as defined by Kabat et al. (supra) are underlined in SP34 VH. The symbol "-" indicates the absence of an amino acid residue at the corresponding position.

[0018] [Figure 2] Alignment of the amino acid sequences of mature SP34 VL, HuSP34 VL1, and human acceptor Y14738 VL. Residue numbers are assigned according to Kabat et al. (supra). CDR sequences as defined by Kabat et al. (supra) are underlined in SP34 VL. The symbol "-" indicates the absence of an amino acid residue at the corresponding position.

[0019] [Figure 3] Nucleotide sequence (SEQ ID NO: 57) of the HuSP34 VH1 gene flanked by SpeI and HindIII sites (underlined), and the deduced amino acid sequence (SEQ ID NO: 58).

[0020] [Figure 4] Nucleotide sequence (SEQ ID NO: 59) of the HuSP34 VL1 gene flanked by NheI and EcoRI sites (underlined), and the deduced amino acid sequence (SEQ ID NO: 60).

[0021] [Figure 5A-C] Schematic diagram of pHuSP34A (A), pHuSP34C (B), and pJB554 (C).

[0022] [Figure 6] Flow cytometry analysis of binding of HuSP34A, HuSP34C, and HuSP34V to Jurkat Dual cells.

[0023] [Figure 7] Alignment of the amino acid sequences of mature SP34 VL, HuSP34 VL3, HuSP34 VL4, and human acceptor L37309 VL. Residue numbers are assigned according to Kabat et al. (supra). CDR sequences as defined by Kabat et al. (supra) are underlined in SP34 VL. The symbol "-" indicates the absence of an amino acid residue at the corresponding position.

[0024] [Figure 8] Nucleotide sequence (SEQ ID NO: 61) of the HuSP34 VL3 gene flanked by NheI and EcoRI sites (underlined), and the deduced amino acid sequence (SEQ ID NO: 62).

[0025] [Figure 9] Nucleotide sequence (SEQ ID NO: 63) of the HuSP34 VL4 gene flanked by NheI and EcoRI sites (underlined), and the deduced amino acid sequence (SEQ ID NO: 64).

[0026] [Figure 10] Schematic diagram of JB554 and JB559 bispecific antibodies.

[0027] [Figure 11A-B] T cell-mediated cytotoxicity by JB554 against Ramos cells (A) and T cell-mediated cytotoxicity by JB559 against EGFR-positive HT-29 cells (B).

[0028] [Figure 12A-B] Activation of Jurkat Dual cells by JB554 (A) and T cell-mediated cytotoxicity by JB554 against Ramos cells (B).

[0029] [Figure 13A-B] T cell-mediated cytotoxicity by JB559 against HT-29 cells (A and B).

[0030] [Figure 14] T cell-mediated cytotoxicity by JB564 against HL-60 cells.

[0031] definition The antibodies of the present invention are typically provided in isolated form. This means that the antibody is typically at least 50% w / w pure from interfering proteins and other impurities resulting from its production or purification, although this does not exclude the possibility that the antibody may be combined with an excess of pharmaceutically acceptable carriers or other vehicles intended to facilitate its use. Bispecific antibodies may be at least 60, 70, 80, 90, 95, or 99% w / w pure from interfering proteins and impurities resulting from its production or purification. In many cases, the antibody will be the predominant macromolecular species remaining after its purification.

[0032] Specific binding of an antibody to a target antigen, or of a bispecific or multispecific antibody to a target antigen, is at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 The term "specific binding" refers to the affinity of an antibody to a target. An antibody that specifically binds to a target can also be called an antibody against the target. Affinity can vary for different targets. Specific binding is detectably high enough to be distinguishable from nonspecific binding to at least one unrelated target. Specific binding can be the result of specific functional group bonds or the formation of a specific spatial fit (e.g., a lock-and-key shape), while nonspecific binding is typically the result of van der Waals forces. However, specific binding does not necessarily mean that an antibody with two identical binding sites binds only to one target, or that a bispecific antibody with two different binding sites binds only to the targets of these two binding sites.

[0033] The basic structural unit of an antibody is a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region comprising approximately 100-110 amino acids primarily involved in antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. A variable region without a signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region refers to a light chain variable region without a light chain signal peptide. However, when referring to a variable region, a signal sequence is not necessarily present; in fact, the signal sequence is cleaved upon expression and secretion of the antibody of the present invention. The pair of heavy and light chain variable regions defines the binding region of the antibody. The carboxy-terminal portions of the light and heavy chains define the light chain constant region and heavy chain constant region, respectively. The heavy chain constant region is primarily responsible for effector function. In IgG antibodies, the heavy chain constant region is divided into CH1, hinge, CH2, and CH3 regions. In IgA, the heavy chain constant region is divided into CH1, CH2, and CH3 regions. The CH1 region is linked to the light chain constant region by disulfide bonds and noncovalent bonds. The hinge region provides flexibility between the binding and effector regions of the antibody and also provides a site for intermolecular disulfide bonds between the two heavy chain constant regions within the tetrameric subunit. The CH2 and CH3 regions are the primary sites of effector function and FcRn binding.

[0034] Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. In light and heavy chains, the variable and constant regions are joined by a "J" segment containing about 12 or more amino acids, while the heavy chain contains a "D" segment containing about 10 or more amino acids (see generally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989), Ch. 7) (incorporated by reference in its entirety for all purposes).

[0035] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites, i.e., is bivalent. In natural antibodies, the binding sites are identical. The binding sites of bispecific or multispecific antibodies may be identical or different, depending on the configuration (see, for example, Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). All variable regions exhibit the same typical structure, consisting of relatively conserved framework regions (FRs) joined by three hypervariable regions, also called complementarity-determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N- to C-terminus, both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain follows the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989), or other definitions of CDRs as set forth in Table 1 below. [Table 1]

[0036] CDR-H1 according to the Chothia definition can end at H32, H33, or H34 depending on the length of the loop. This is because the Kabat numbering places the insertion of additional residues at 35A and 35B, while Chothia numbering places them at 31A and 31B. If H35A and H35B (Kabat numbering) are absent, CDR-H1 in Chothia ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34.

[0037] Kabat also provides a widely used numbering scheme (Kabat numbering) that assigns the same number to corresponding residues between different heavy chain variable regions or different light chain variable regions. Kabat numbering can be used for antibody heavy chain constant regions, although, as in the present application, the EU index (also referred to as EU numbering) is more commonly used. When an antibody is described as containing a CDR according to a particular definition of CDR (e.g., Kabat), that definition specifies the minimum number of CDR residues present in the antibody (i.e., Kabat CDRs). This does not exclude the presence of other residues that fall within other conventional CDR definitions but do not fall within the particular definition. For example, an antibody containing a CDR defined by Kabat potentially includes an antibody in which the CDR contains Kabat CDR residues but no other CDR residues, and an antibody in which the CDR H1 is a composite Chothia-Kabat CDR H1 and the other CDRs contain Kabat CDR residues but no additional CDR residues according to other definitions.

[0038] The term "antibody" includes intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they are derived for specific binding to a target and include individual heavy and light chains, Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and scFv. Fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes bispecific and multispecific antibodies.

[0039] The term "epitope" refers to a site on an antigen to which an antibody or an arm of a bispecific or multispecific antibody binds. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also called linear epitopes) typically persist when exposed to denaturing solvents, whereas epitopes formed by tertiary folding (also called conformational epitopes) typically are lost when treated with denaturing solvents. Some antibodies bind end-specific epitopes; that is, they preferentially bind to polypeptides with free ends compared to identical polypeptides that have lost their free ends due to fusion to another polypeptide. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids that adopt a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0040] The term "antigen" or "target antigen" refers to the target molecule bound by one binding site of an antibody or bispecific antibody. Antigens can be proteins of any length (natural, synthetic, or recombinantly expressed), nucleic acids, carbohydrates, and other molecules. Antigens include receptors, ligands, counter-receptors, coat proteins, and the like.

[0041] Antibodies that recognize the same or overlapping epitopes can be identified by simple immunoassays that demonstrate the ability of one antibody to compete with the binding of another antibody to a target antigen. Antibody epitopes can also be defined by X-ray crystallography of the antibody bound to the antigen, identifying contact residues. Two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

[0042] Competition between antibodies is determined by an assay in which the antibody under test inhibits the specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits binding of the reference antibody by at least 50%, preferably 75%, 90%, or 99%, as measured in a competitive binding assay. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close together to sterically hinder the epitope bound by the reference antibody.

[0043] The term "subject" includes humans and other mammalian subjects receiving prophylactic or therapeutic treatment. Other mammalian subjects include animal models of the human condition (e.g., rodents, non-human primates) and veterinary subjects.

[0044] For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve the replacement of amino acids from the same class with each other. Non-conservative substitutions involve the exchange of a member of one of these classes for a member of another class.

[0045] Percentage sequence identity is determined using maximally aligned antibody sequences according to Kabat numbering for variable regions and EU numbering for constant regions. After alignment, when a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by identical amino acids in both the subject and reference antibody regions, without considering gaps, divided by the total number of aligned positions in these two regions, and multiplied by 100 to convert to a percentage.

[0046] A composition or method "comprising" one or more listed elements may also include other elements not specifically listed. For example, a composition that includes an antibody may contain only the antibody, or may contain the antibody in combination with other components.

[0047] The term "antibody-dependent cellular cytotoxicity" (ADCC) refers to a mechanism of cell death induction that depends on the interaction of antibody-coated target cells (i.e., antibody-bound cells) with lytic immune cells (also called effector cells). Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. ADCC is initiated by the interaction of the Fc region of a cell-bound antibody with Fcγ receptors (particularly FcγRI and FcγRIII) present on immune effector cells such as neutrophils, macrophages, and natural killer cells. Target cells are eliminated by phagocytosis or lysis, depending on the type of effector cell involved. Death of antibody-coated target cells occurs as a result of effector cell activity.

[0048] The term opsonization, also known as "antibody-dependent cellular phagocytosis" or ADCP, refers to the process by which antibody-coated cells are internalized, in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulin.

[0049] The term "complement-dependent cytotoxicity" or CDC (also called CMC) refers to a mechanism by which the Fc effector domain of a target-bound antibody activates a series of enzymatic reactions that result in the formation of holes in the target cell membrane, inducing cell death. Generally, antigen-antibody complexes (e.g., on antibody-coated cells) bind and activate complement component C1q, which activates the complement cascade, leading to target cell death. Complement activation may also result in the deposition of complement components on the target cell surface, which promotes ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0050] pH-dependent binding of an antibody to the FcRn receptor means that the antibody binds to such a receptor more strongly at pH 60 than at pH 75. Binding to FcRn at low pH in endosomes after pinocytic internalization rescues IgG antibodies from catabolic degradation in lysosomes. The rescued IgG antibodies are then released from FcRn at neutral pH and recycled into the blood circulation. Such pH-dependent FcRn binding provides the molecular mechanism underlying the long serum half-life of IgG antibodies (and the bispecific antibodies of the present invention) (Ghetie et al., Annu. Rev. Immunol. 18:739-766, 2000). For example, human IgG antibodies bind to human neonatal Fc receptor (FcRn) at pH 6.0, but only weakly at pH 7.5. The FcRn-binding site in IgG antibodies is located at the junction between the CH2 and CH3 domains. Because the mu heavy chain does not bind to FcRn at pH 6.0 or pH 7.5, native IgM generally has a shorter half-life than native IgG antibodies because it cannot utilize the FcRn-mediated pathway to rescue the antibody from degradation in the lysosome.

[0051] Protein A is a 40-60 kDa surface protein originally discovered in the cell wall of Staphylococcus aureus. Protein A specifically binds with high affinity to mouse IgG2a and IgG2b as well as human IgG1, IgG2, and IgG4. Protein A does not bind to human IgG3, IgA, or IgM. Protein A is used for affinity purification of antibodies.

[0052] Protein G is a 65 kDa (G148 protein G) and 58 kDa (C40 protein G) streptococcal cell surface protein. Protein G contains a serum albumin-binding domain, but this domain is not required for IgG binding and is often deleted. Protein G specifically binds all isotypes of human IgG, but does not bind IgA or IgM. Protein G is also used in antibody purification. DETAILED DESCRIPTION OF THE INVENTION

[0053] I. General matters The present invention provides antibodies that specifically bind to human CD3. The antibodies may be monospecific (each binding site binds to the same target, i.e., CD3), bispecific (having at least two binding sites for two targets, one of which is human CD3), or multispecific (having multiple binding sites for multiple targets, one of which is human CD3). These antibodies can be used to treat diseases such as cancer, infectious diseases, and immune disorders.

[0054] II. target Human CD3 is a complex comprising CD3 delta (e.g., Swiss Prot P04234), CD3 gamma (e.g., Swiss Prot PO9693), two molecules of CD3 epsilon (e.g., Swiss Prot P07766), and two molecules of CD3 zeta (e.g., Swiss Prot P20963). Reference to human CD3 and its subunits includes the exemplified human forms and other known allelic variants in humans as shown in the Swiss Prot database.

[0055] One class of proteins that can serve as second targets for bispecific or multispecific antibodies are cancer-associated antigens, which are expressed by cancers and typically at higher levels (overexpression) than in control normal tissues. Examples of cancer-associated antigens include alpha-folate receptor (ovarian cancer and epithelial cancer), CAIX (renal carcinoma), CD19 (B-cell malignancies, CLL, ALL), CD20 (B-cell malignancies, lymphoma), CD22 (B-cell malignancies), CD23 (CLL), CD24 (pancreatic cancer), CD30 (lymphoma), CD33 (AML), CD38 (NHL), CD44v7 / 8 (cervical cancer), CEA (colorectal cancer), EGFRvIII (glioblastoma), EGP-2 (multiple malignancies), EGP-40 (colorectal cancer), EphA2 (glioblastoma), Erb-B2 (breast cancer, prostate cancer, colorectal cancer), FBP (ovarian cancer), G D2(neuroblastoma, melanoma), G D3(melanoma), HER2 (pancreatic cancer, ovarian cancer, glioblastoma, osteosarcoma), HMW-MAA (melanoma), IL-11Rα (osteosarcoma), IL-13Rα2 (glioma, glioblastoma), KDR (tumor vasculature), kappa light chain (B-cell malignancies), Lewis Y (various cancers), L1 (neuroblastoma), MAGE-A1 (melanoma), mesothelin (mesothelioma), MUC1 (breast cancer and ovarian cancer), MUC16 (ovarian cancer), NKG2D (myeloma, ovarian cancer), NY-ESO-1 (multiple myeloma), oncofetal antigen (various tumors), PSCA (prostate cancer), PSMA (prostate cancer), ROR1 (B-CLL), TAG-72 (adenocarcinoma), and VEGF-R2 (tumor angiogenesis). (Sadelain et al., Cancer Discov 3:388-98, 2013). Examples of other cancer-associated antigens that can be targeted include alpha-fetoprotein (AFP), alpha-actinin-4, A3, ART-4, B7, and Ba. 733, BAGE, BCMA, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCLl9, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD52, CD54, CD5 5, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123,CD126, CD132, CD133, CD138, CD147, CD154, CD155 , CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEA (CEACAM-5), CEACAM-6, c-Met, claudin-6, claudin-18.2, DAM, DLL3, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, EphA10, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, GD2, gpA33, GPC3, gp100, GRO-beta, GUCY2C, HLA-DR, HLA-A*02:01:gp100, HM1.24, human chorionic gonadotropin (HCG) and its subunits unit, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, IGF-1R, IFN-γ, IFN-, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), integrin beta 4, KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUC17, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, NY-ESO1 pancreatic cancer mucin, p-cadherin, PD1 receptor, placenta growth factor, p53, PLAGL2, prolactin receptor, prostatic acid phosphatase, PSA, PSCA, PRAME , PSMA, PIGF, ILGF, ILGF-R, IL-6, IL-25, ROR1, RS5, RANTES, T101, SAGE, S100, SSTR2, STEAP1, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, transferrin receptor, TNF-α, Tn antigen, 5T4, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, Kras, oncogene markers or oncogene products (e.g., Sensi et al.(See also: Parmiani et al., J Immunol 2007, 178:1975-79; Novellino et al., Cancer Immunol Immunother. 2005, 54:187-207). CD33 binds sialic acid and is overexpressed primarily in bone marrow-derived cancers such as acute myeloid leukemia. EGFR binds EGF and is overexpressed primarily in gastric, breast, endometrial, colorectal, head and neck, ovarian, cervical, bladder, and esophageal cancers. PD-L1 binds PD1 and is overexpressed primarily in gastric, hepatocellular carcinoma, renal cell carcinoma, esophageal, pancreatic, ovarian, and bladder cancers.

[0056] Another class of proteins are antigens expressed on the surface of pathogens or pathogen-infected cells. Another class of proteins are antigens expressed on immune cells associated with diseases such as autoimmune diseases.

[0057] III. Antibodies to CD3 An exemplary antibody against CD3 is SP34. This antibody is characterized by the mature heavy chain variable region of SEQ ID NO: 1 and the mature light chain variable region of SEQ ID NO: 2. The Kabat CDRs H1, H2, and H3 of the mature heavy chain variable region are provided by SEQ ID NOs: 43-45, respectively, and the Kabat CDRs L1, L2, and L3 of the mature light chain variable region are provided by SEQ ID NOs: 46-48, respectively. SP34 is an agonistic antibody that specifically binds to the extracellular domain of the epsilon chain of human CD3. Cross-linking of CD3 by SP34 induces T cell activation. SP34 specifically binds to CD3 of humans and a wide range of non-human primates (e.g., chimpanzees, cynomolgus monkeys, and rhesus monkeys). In contrast, other anti-CD3 antibodies (e.g., M291, OKT3) widely used in T cell engagers have been reported not to specifically bind to cynomolgus monkeys or rhesus monkeys. The ability to specifically bind to CD3 from non-human primates is advantageous in preclinical trials in animal models, and other antibodies of the invention preferably share this and other properties of SP34.

[0058] Some antibodies of the invention bind to the same or overlapping epitope on human CD3 as the antibody designated SP34 and / or compete for binding to human CD3 with SP34 or other antibodies sharing its mature variable region. Other antibodies with such binding specificities can be produced by immunizing mice with human CD3 or a portion thereof containing the desired epitope and screening for binding to human CD3, optionally in competition with SP34. Antibodies can be screened against mutant forms of the human CD3 antigen to identify antibodies that exhibit a binding profile identical or similar to that of SP34. Mutations can be systematic substitutions of alanine (or serine, if alanine is already present) throughout the entire extracellular domain of human CD3 or a portion thereof where the epitope is known to be located, one residue at a time or at more widely spaced intervals.

[0059] Antibodies with the binding specificity of SP34 can also be produced using a modified version of the phage display method. See Winter, WO 92 / 20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy or light chain variable region of a selected mouse antibody is used as the starting material. For example, if a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the mouse starting material) but different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. Phages that exhibit strong specific binding to human CD3 (e.g., at least 10 and preferably at least 10 M) are selected. The heavy chain variable region from this phage then serves as the starting material for constructing a further phage library, in which each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) but a different light chain variable region. The light chain variable region can be obtained, for example, from a library of rearranged human variable light chain regions. Again, phage are selected that show strong specific binding to CD3. The resulting antibodies typically have the same or similar epitope specificity as the murine starting material.

[0060] Other antibodies can be obtained by mutagenesis of cDNA encoding the heavy and light chains of exemplary antibodies, such as SP34. Also included in the present invention are monoclonal antibodies that are at least 90%, 95%, or 99% identical in amino acid sequence to SP34 in the mature heavy and / or light chain variable regions and retain its functional properties, and / or that differ from the respective antibodies by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Also included are monoclonal antibodies having at least one, and preferably all six, CDRs according to the Kabat definition that are 90%, 95%, 99%, or 100% identical to the corresponding CDRs of SP34.

[0061] The antibodies of the present invention can be provided in a conventional tetrameric format comprising two pairs of heavy and light chain variable regions, or in the form of fragments such as scFvs, or in a form comprising either of these. The antibodies may comprise, consist of, or consist essentially of the disclosed mature heavy and light chain variable regions, and combinations thereof.

[0062] A. Non-human Antibodies Other non-human monoclonal antibodies against human CD3, e.g., mouse, guinea pig, primate, rabbit, or rat monoclonal antibodies, can be produced by immunizing animals with, for example, human CD3 or a fragment thereof, or cells bearing human CD3, optionally coexpressed with its co-receptor protein, as described above. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such immunogens can be obtained from natural sources, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused or complexed with a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvants can be used, including: For immunization of laboratory animals, Freund's complete adjuvant followed by incomplete adjuvant is preferred; rabbits and guinea pigs are typically used for polyclonal antibody production; and mice are typically used for monoclonal antibody production. Antibodies are screened for specific binding to human CD3. Optionally, the antibodies are further screened for binding to a specific region of human CD3. Such screening can be performed by measuring the binding of the antibodies to a collection of deletion variants of human CD3. Binding can be assessed, for example, by Western blot, FACS, or ELISA.

[0063] B. Humanized Antibodies Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. Nos. 5,859,205, 6,881,557; Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequences can be, for example, mature human antibody sequences, composites of such sequences, consensus sequences of human antibody sequences, or germline region sequences. Thus, a humanized antibody is an antibody having some or all of the CDRs derived entirely or substantially from the donor antibody, and constant region (if present) and variable region framework sequences derived entirely or substantially from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor antibody heavy chain, and a heavy chain constant region (if present) and heavy chain variable region framework sequences derived substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor antibody light chain, and a light chain constant region (if present) and light chain variable region framework sequences derived substantially from human light chain variable region framework and constant region sequences. Excluding nanobodies and dAbs, humanized antibodies comprise humanized heavy chains and humanized light chains. CDRs in a humanized antibody are substantially derived from corresponding CDRs in a non-human antibody when at least 85%, 90%, 95%, or 100% of corresponding residues (as defined by Kabat) between corresponding CDRs are identical. An antibody chain variable region framework sequence or antibody chain constant region is substantially derived from a human variable region framework sequence or human constant region, respectively, if at least 85%, 90%, 95% or 100% of the corresponding residues according to the Kabat definition are identical.

[0064] Humanized antibodies often incorporate all six CDRs (preferably according to the Kabat definition) from a murine antibody, although humanized antibodies can also be generated with fewer than all CDRs from a murine antibody (e.g., at least three, four, or five CDRs) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0065] In some antibodies, only a portion of the CDRs, i.e., a subset of CDR residues required for binding, called SDRs, are required in humanized antibodies to retain binding. CDR residues that do not contact antigen and are not present in the SDRs can be identified by molecular modeling and / or empirical analysis from regions of the Kabat CDRs outside the Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987) based on previous studies (e.g., residues H60-H65 in CDR H2 are often not required), or as described in Gonzales et al., Mol. Immunol. 41:863, 2004. In such humanized antibodies, where one or more donor CDR residues are absent or where the entire donor CDR has been removed, the amino acid occupying that position can be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. The number of donor-to-acceptor amino acid substitutions in the CDRs reflects a balance of competing considerations. Such substitutions are advantageous in that they potentially reduce the number of murine amino acids in the humanized antibody, thereby reducing potential immunogenicity. However, substitutions may also result in changes in affinity, and it is preferable to avoid significant decreases in affinity. The position of substitution within the CDR and the amino acid to be substituted can also be selected empirically.

[0066] The human acceptor antibody sequence can be optionally selected from among many known human antibody sequences to provide a high degree of sequence identity (e.g., 65-85% identity) between the variable region framework of the human acceptor sequence and the corresponding variable region framework of the donor antibody chain. The human acceptor antibody sequence can be derived from a human antibody, or a composite of two or more human antibodies, or a consensus of human antibodies. The human acceptor sequence can be a germline sequence. The heavy chain human acceptor sequence and the light chain human acceptor sequence can be of the same or different origins, e.g., the heavy and light chains of the same antibody, or the heavy and light chains of different antibodies.

[0067] Specific amino acids from the human variable region framework residues can be selected for substitution based on their potential effect on CDR conformation and / or binding to antigen, which can be studied by modeling, examining the characteristics of the amino acid at a particular position, or empirically observing the effects of substituting or mutagenesing specific amino acids.

[0068] For example, if an amino acid differs between the murine variable region framework residue and the selected human variable region framework residue, that amino acid may be: (1) Direct non-covalent binding to antigens; (2) adjacent to the CDR regions; (3) otherwise interacts with the CDR region (e.g., is within about 6 angstroms of the CDR region). Where it is reasonably expected to be the case, a human framework amino acid can be substituted with the equivalent framework amino acid from the murine antibody.

[0069] Other substitution candidates are acceptor human framework amino acids that are unusual for human immunoglobulins at that position. These amino acids can be substituted with amino acids from the equivalent positions in the mouse donor antibody or from more typical human immunoglobulins. Other substitution candidates are acceptor human framework amino acids that are unusual for human immunoglobulins at that position. The VH sequence encoded by the human M24236 cDNA is an exemplary acceptor sequence for heavy chain humanization. The human Vλ region encoded by the Y14738 cDNA or the human mature Vκ region encoded by the L37309 cDNA are exemplary acceptor sequences for light chain humanization.

[0070] An exemplary humanized heavy chain variable region of SP34 is designated HuSP34 VH1 and assigned SEQ ID NO: 4. At framework positions 30, 49, 93, and 94 (Kabat numbering), where a three-dimensional model of the SP34 variable region indicates that these amino acids may play important roles in forming the antigen-binding site, amino acid residues in M24236 VH can be substituted with the corresponding residues in murine SP34 VH, i.e., N, A, V, and R. An exemplary humanized light chain variable region designated HuSP34 VL1 is assigned SEQ ID NO: 6. This light chain variable region contains backmutations to murine residues at positions 36, 46, and 49 (Kabat numbering), which are occupied by V, G, and G, respectively. Another exemplary humanized light chain variable region based on the kappa acceptor sequence is designated HuSP34 VL3 and assigned SEQ ID NO: 17. This humanized light chain has back mutations at positions 36, 46, 49, 58, 66, 67, 69, 70, and 71 according to the Kabat numbering system, which are occupied by V, G, G, V, L, I, D, K, and A, respectively. Another humanized light chain variable region has been designated HuSP34 VL4 and assigned SEQ ID NO:27. SEQ ID NO:27 is identical to SEQ ID NO:17 except for the deletion of some back mutations that are not necessary for binding but may increase immunogenicity. The remaining back mutations still present in SEQ ID NO:27 are at positions 36, 46, 49, 66, and 71, which are occupied by V, G, G, L, and A, respectively.

[0071] An exemplary humanized antibody of the invention is characterized by a mature heavy chain variable region of SEQ ID NO: 4 and a mature light chain variable region of any of SEQ ID NOs: 6, 17, or 27, preferably SEQ ID NO: 27. The invention also includes humanized antibodies comprising a mature heavy chain variable region comprising SEQ ID NO: 4 and a mature light chain variable region comprising any of SEQ ID NOs: 6, 17, or 27, preferably SEQ ID NO: 27. The invention also provides a variant of the humanized anti-CD3 IgG1 / kappa antibody designated HuSP34V, comprising HuSP34 VH1 (SEQ ID NO: 4) and HuSP34 VL4 (SEQ ID NO: 27). Such variants typically differ from the variable region sequence of HuSP34V by a small number of substitutions, deletions, or insertions (e.g., usually no more than 1, 2, 3, 5, 7, 8, 9, or 10). Such differences are usually in the framework but can also occur in the CDRs. For example, only a subset of substitutions can be made: positions 30, 49, 93, and 94 in the heavy chain, and positions 36, 46, 49, 66, and 71 in the light chain. Many of the framework residues not in contact with the CDRs of a humanized mAb can accommodate amino acid substitutions from the corresponding positions in the donor murine mAb or other murine or human antibodies; even many potential CDR-contacting residues are amenable to substitution, and even amino acids within the CDRs may change. One example of a CDR substitution is to replace a residue within a CDR with a residue occupying the corresponding position in the human acceptor sequence used to provide the variable region framework.

[0072] In many cases, substitutions made in the variable regions of the variant HuSP34V sequence are conservative with respect to the substituted HuSP34V amino acids. Preferably, HuSP34V substitutions (whether conservative or not) do not substantially affect the binding affinity or potency of the humanized mAb, i.e., its ability to specifically bind and agonize human CD3. Preferably, the mature variant light and heavy chain V region sequences are at least 90%, more preferably at least 95%, and most preferably at least 98% identical to the respective HuSP34V mature light and heavy chain V regions. Additionally, other human antibody acceptor sequences, particularly sequences with high sequence identity to the variable region framework sequences of HuSP34, are also suitable for providing humanized antibody variable region framework sequences.

[0073] In some variants of HuSP34V, at least 1, 2, 3, 4, 5, 7, or all 8 of the acceptor to donor substitution positions described in connection with the exemplified antibodies, i.e., Kabat positions 30, 49, 93, and 94 in the heavy chain and Kabat positions 36, 46, 49, 66, and 71 in the light chain, are occupied by residues occupying the corresponding positions in the heavy or light chain variable region of SP34.

[0074] C. Chimeric and Veneered Antibodies The present invention further provides chimerized and veneered forms of SP34.

[0075] Chimerized antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., murine) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the murine antibody and contain approximately two-thirds human sequences.

[0076] A veneered antibody is a type of humanized antibody that retains some, usually all, of the CDRs and non-human variable region framework residues of a non-human antibody, but replaces other variable region framework residues that may contribute to B-cell or T-cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues from the corresponding positions in a human antibody sequence. The result is an antibody in which the CDRs are completely or substantially derived from a non-human antibody and the variable region framework of the non-human antibody has been made human-like by the substitutions. veneered form of SP34.

[0077] D. Human Antibodies Human antibodies against human CD3 can be obtained by various techniques, as described below. Some human antibodies are selected by competitive binding experiments, Winter's phage display method, or other methods to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the Examples. Also, human antibodies can be screened for a particular epitope specificity by using only fragments of human CD3.

[0078] Methods for producing human antibodies include the trioma method (Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666), the use of transgenic mice containing human immunoglobulin genes (e.g., Lonberg et al., WO93 / 12227 (1993); U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Neuberger, Nat. Biotechnol. 14:826 (1996), and Kucherlapati, WO 91 / 10741 (1991)), and phage display methods (see, e.g., Dower et al., WO 91 / 17271, McCafferty et al., WO 92 / 01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743 and 5,565,332) and the methods described in WO 2008 / 081008 (e.g., immortalization of memory B cells isolated from humans using, for example, EBV, screening for the desired properties, and cloning and expression of the recombinant forms).

[0079] IV. Bispecific antibodies Bispecific or multispecific antibodies are formed from pairs of heavy and light chain variable regions of constituent antibodies. The constituent antibodies can be rodent, chimeric, veneered, humanized, primatized, primate, or human, among others. The constituent antibodies can be of the same or different species. For example, one can be humanized and the other human.

[0080] One of the constituent antibodies of the bispecific or multispecific antibody is the aforementioned antibody against human CD3. The other constituent antibody or antibodies bind to antigens other than human CD3. For example, the other constituent antibody or antibodies can bind to targets present on cells to be eliminated. Such cells include cancer cells, pathogens such as viruses, bacteria, or fungi, and cells infected with these pathogens, as well as immune cells associated with immune disorders, particularly autoimmune disorders.

[0081] Methods for producing nonhuman, humanized, chimeric, veneered, and human antibodies targeting human CD3 have been reported. The same methods can be applied to the production of other antibodies targeting related targets instead of human CD3. Many antibodies approved for therapeutic use as monospecific antibodies can be combined into bispecific antibodies. Table 2 below lists examples of antibodies approved for the treatment of cancer. [Table 2] JPEG2025540657000004.jpg213166JPEG2025540657000005.jpg223166JPEG2025540657000006.jpg223166JPEG2025540657000007.jpg102166

[0082] More than 100 formats for bispecific or multispecific antibodies have been described (e.g., Kontermann et al., Drug Discovery Today 20, 838-847 (2015); Sedykh et al., Drug Des. Devel. Ther. 2, 195-209 (2018)). Such formats contain at least one binding site for each of at least two targets. Some formats contain two or more binding sites for each target.

[0083] Some formats have a tetrameric structure similar to that of conventional antibodies, with two binding regions, one for each target. Each binding region is formed from paired heavy and light chain variable regions, which are linked to heavy and light chain constant regions, respectively. Such bispecific antibodies differ from conventional antibodies in that the two binding sites and the heavy and light chain pairs that form them are different. Therefore, such antibodies require the association of two different heavy and light chain pairs.

[0084] The "knob-into-hole" approach has been employed to reduce homodimer formation and heavy-chain mispairing by substituting small amino acids for large ones in the CH3 domain of one antibody ("knob") and vice versa ("hole") in the other antibody (Ridgway et al., Protein Eng 9:617-21, 1996; Atwell et al., J Mol Biol 270:26-35, 1997; and US Pat. No. 7,695,936). Light-chain mispairing in such formats can be reduced by several strategies. One strategy is to use a common light-chain variable region for two different heavy-chain variable regions. However, this is only applicable to some antibodies. Another approach involves expressing the knob-containing half-molecule and the hole-containing half-molecule in separate bacteria. Another approach, called CrossMab, involves swapping the CH1 domain of one heavy chain with the constant CL domain of the corresponding light chain to induce correct pairing between the designed heavy and light chains (Schaefer et al., Proc Natl Acad Sci USA 108:11187-92, 2011; WO 2009 / 080251; WO 2009 / 080252; WO 2009 / 080253). Another approach involves introducing additional mutations at the VH-VL and CH1-CL interfaces (Lewis et al., Nat. Biotechnol., 32 (2014), pp. 191-198). These mutations promote preferential pairing of the heavy chain with the light chain. Another approach is to introduce mutations into one of the Fc regions that enhance Protein A binding and select heterodimer pairs with intermediate Protein A binding from homodimers with higher or lower Protein A binding by affinity chromatography (Tusdian et al, MAbs. 2016 May-Jun;8(4):828-38).

[0085] Other bispecific antibodies circumvent the mispairing problem by combining multiple binding specificities in the same heavy and light chain pair. One approach to this, called dual variable domains, is to link two different heavy chain variable regions in tandem to a heavy chain constant region and two different light chain variable regions in tandem to a light chain constant region (Correia et al., MAbs. 2013 May 1;5(3):364-372). Such antibodies can be assembled as tetramers by associating two identical paired heavy and light chains. The assembled antibody contains two different binding sites for each target. Another approach is to attach an scFv, which provides a binding site for one target, to the N-terminus of the heavy or light chain variable region of the heavy or light chain, which provides a binding site for another target. Such bispecific antibodies can also be assembled as tetramers.

[0086] Another approach, employed in embodiments of the present invention, is to incorporate a second binding specificity by linking a single-chain Fv (scFv) to the C-terminus of the heavy chain constant region, optionally omitting the C-terminal lysine residue. Similar to standard antibodies, such bispecific antibodies contain a first binding site formed by the heavy and light chain variable regions linked to the N-termini of the heavy and light chain constant regions. The C-terminus of the heavy chain is linked to an scFv, which provides a second binding site. The scFv is typically linked via a linker, which further connects the heavy and light chain variable regions of the scFv. The scFv can be linked to an Fc region via a linker from either its light chain variable region or the end of its heavy chain variable region. When assembled by complexing two identical paired heavy and light chains, such bispecific antibodies contain two binding sites, each for two different specificities. The antigen-binding arms of such bispecific antibodies for CD3 and other targets can be attached in either orientation. That is, the antigen-binding arm for CD3 can be in the form of an scFv, while the antigen-binding arm for other targets can be in a standard antibody format, or vice versa. The arms binding to the N-terminus of the heavy and light chain constant regions are provided separately as heavy and light chain variable regions, and the arms binding to the C-terminus are provided as scFv fragments. The advantage of this format is that the two different binding spaces are separated by the entire heavy chain constant region, which may facilitate cell-cell bridging. To enhance stability, cysteine ​​substitutions can be optionally introduced into the heavy and light chain variable regions of the scFv (e.g., position 44 in the heavy chain variable region and position 100 in the light chain variable region according to the Kabat numbering system).

[0087] In another format, an scFv that specifically binds one target is linked to the heavy chain constant region, and an scFv that specifically binds another target is linked to the light chain constant region. Such antibodies are assembled as tetramers containing two copies of each binding site (Bs(scFv)4-IgG) (Zuo et al., Protein Eng 13:361-367, 2000).

[0088] Other formats link scFv binding regions in a single chain without constant regions. For example, the BiTe format links two scFv fragments via a linker (see, e.g., Ross et al., PLoS ONE 12(8):e0183390, 2017). While such formats tend to lack effector function and have short half-lives, they may offer the advantage of easy accessibility and production due to their small size.

[0089] Many of the above formats include a linker peptide between the heavy and light chain variable regions or between the variable and constant regions. Linkers are short peptides that provide flexibility and are often composed primarily of Gly, Ala, and / or Ser. Some exemplary linkers are Gly-Gly-Ala-Ala, Gly-Gly-Gly-Gly-Ser, Leu-Ala-Ala-Ala-Ala, and multimers thereof.

[0090] Any combination of heavy and light chain variable regions described above for the SP34 antibody, and chimeric, veneered, and humanized forms thereof, or other CD3 antibodies described herein, can be incorporated into scFvs in the above formats. Exemplary scFvs have a sequence comprising, consisting of, or consisting essentially of any of SEQ ID NOs: 29, 31, or 33. An exemplary bispecific antibody comprises an scFv linked via the mature light chain variable region of the scFv to the heavy chain constant region of another antibody, wherein the scFv has a sequence comprising SEQ ID NO: 31 or 33. Another exemplary bispecific antibody comprises an scFv linked via the mature light chain variable region of the scFv to the heavy chain constant region of another antibody, wherein the scFv has a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 29.

[0091] Selection of constant regions Many monospecific, bispecific, or multispecific antibody formats contain at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity is desired. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. The light chain constant region can be lambda or kappa. Furthermore, human IgG1 and IgG3 induce stronger cell-mediated effector functions than human IgG2 and IgG4. Here, ADCC, ADCP, and CDC are useful for providing an additional mechanism of action against cancer cells or infected cells bound by one arm of the bispecific antibody, but are not useful for agonizing CD3 and activating immune cells by the other arm.

[0092] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains, e.g., the C-terminal lysine of the heavy chain, may be deleted or derivatized in some or all of the molecules. A counter ion may or may not be present to form a pharmaceutically acceptable salt. Water or other solvent may or may not be present in association with the antibody (e.g., as a hydrate or solvate). Amino acid substitutions can be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006) or to increase half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). For example, there are many known mutations in IgG Fc that increase FcRn binding. Exemplary substitutions include Gln at position 250 and / or Leu at position 428, Ser or Asn at position 434, Tyr at position 252, Thr at position 254, Glu at position 256, and Ala at position 434 (EU numbering). Increased FcRn binding is advantageous because the hybrid proteins of the present invention can more strongly compete with endogenous IgG for binding to FcRn. Numerous mutations are known to reduce ADCC, ADCP, or CDC (see, e.g., Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006). For example, substitution of any of the amino acid residues at positions 234, 235, 236 and / or 237 reduces affinity for Fcγ receptors, particularly FcγRI receptors (see, eg, US Pat. No. 6,624,821).Optionally, the amino acid residues at positions 234, 236, and / or 237 of human IgG2 are substituted with Ala, and position 235 is substituted with Gln or Glu (see, e.g., US 5,624,821). Other substitutions that reduce effector function include Ala at position 268, Gly or Ala at position 297, Leu at position 309, Ala at position 322, Gly at position 327, Ser at position 330, Ser at position 331, Ser at position 238, Ala at position 268, and Leu at position 309.

[0093] Human constant regions exhibit allotypic and isoallotypic variation among different individuals. That is, the constant region may differ at one or more polymorphic positions in different individuals. Isoallotypes differ from allotypes in that sera that recognize an isoallotype bind to non-polymorphic regions of one or more other isotypes.

[0094] Expression of recombinant antibodies Monospecific, bispecific, or multispecific antibodies are typically produced by recombinant expression. Depending on the format, expression of one, two, or more antibody chains may be required. When multiple chains are expressed, they can be expressed from the same vector or different vectors. Recombinant polynucleotide constructs typically contain expression control sequences, including naturally occurring or heterologous expression control elements, such as a promoter, operably linked to the coding sequences of the antibody chains. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences and the collection and purification of the bispecific antibody.

[0095] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors typically contain selectable markers, such as ampicillin-resistance or hygromycin-resistance, to permit detection of those cells transformed with the desired DNA sequences.

[0096] Escherichia coli (E. coli) is one prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Saccharomyces is a yeast host with suitable vectors containing expression control sequences, an origin of replication, termination sequences, etc., as desired. Typical promoters include glycolytic enzymes such as 3-phosphoglycerate kinase. Inducible yeast promoters include promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, among others.

[0097] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). Numerous suitable host cell lines capable of secreting intact heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas, including Sp2 / 0 and NS0. The cells may also be non-human. Expression vectors for use in these cells may contain expression control sequences, such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Expression control sequences may include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0098] Alternatively, the antibody coding sequence can be incorporated into a transgene that is introduced into the genome of the transgenic animal and then expressed in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for the light and / or heavy chains operably linked to a promoter and enhancer derived from a mammary gland-specific gene, such as casein or beta-lactoglobulin.

[0099] Vectors containing the desired DNA segment can be introduced into host cells by methods appropriate for the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, gene guns, or viral-based transfection can be used for other cellular hosts. Methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To generate transgenic animals, transgenes can be injected into fertilized oocytes by microinjection or integrated into the genome of embryonic stem cells, the nuclei of which can then be transferred into enucleated oocytes.

[0100] After introducing vector(s) encoding the antibody heavy and light chains into the cell culture, the cell pools can be screened for antibody productivity and quality in serum-free medium. The highest producing cell pools can then be screened by FACS. TM Monoclonal lines can be generated by single-cell cloning based on the antibody-specific antibody cloning method. Specific production rates of 50 pg / cell or over 100 pg / cell per day, equivalent to over 7.5 g per liter of culture medium, can be used. Antibodies produced by single-cell clones can be analyzed by turbidity, filtration characteristics, PAGE, IEF, UV scanning, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and ELISA or BIACORE. TMSelected clones can then be stored in multiple vials and frozen for later use.

[0101] After expression, antibodies can be purified according to standard methods in the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, etc. (See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0102] Methodologies for commercial production of antibodies can be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single cell cloning, cell banking, use of selectable markers for copy number amplification, CHO terminators, or increased protein titer (see, e.g., US 5,786,464; US 6,114,148; US 6,063,598; US 7,569,339; WO2004 / 050884; WO2008 / 012142; WO2008 / 012142; WO2005 / 019442; WO2008 / 107388; WO2009 / 027471; and US 5,888,809).

[0103] nucleic acid The present invention further provides nucleic acids encoding any of the heavy and light chains described above. Optionally, such nucleic acids may further encode a signal peptide, which may be linked to the constant region-encoding sequence of the nucleic acid for expression, and may be operably linked to regulatory sequences ensuring expression of the coding sequence, such as a promoter, enhancer, ribosome binding site, or transcription termination signal. The nucleic acids encoding the heavy and light chains may be generated in isolated form or cloned into one or more vectors. The nucleic acids may be synthesized, for example, by solid-phase synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may be joined as a single contiguous nucleic acid, for example, within an expression vector, or may be cloned separately, for example, each into its own expression vector.

[0104] Therapeutic methods and pharmaceutical compositions The bispecific or multispecific antibodies of the present invention can be used to treat cancers in which one arm of the bispecific or multispecific antibody binds to a target expressed or overexpressed in cancer, as disclosed above. The bispecific or multispecific antibodies can be used to treat solid tumors and hematological malignancies. Hematological malignancies include leukemia (e.g., T-cell large granular lymphocyte leukemia), lymphoma (Hodgkin's or non-Hodgkin's), or multiple myeloma. Solid tumors include those of the skin (e.g., melanoma), ovary, endometrium, kidney, liver, pancreas, bladder, breast, ovary, prostate, rectum, colon, stomach, intestine, pancreas, lung, thymus, thyroid, kidney, and brain.

[0105] Bispecific antibodies of the present invention can be used to treat pathogenic infections when the bispecific antibody has one arm that specifically binds to an antigen present on a pathogen or an antigen expressed in cells infected with the pathogen but not in corresponding uninfected cells. Such antigens can be encoded by the pathogen or expressed by cells in response to infection by the pathogen. Examples of antigens expressed on infected cells include glycoproteins gp41 and gp120 of human immunodeficiency virus (HIV), Env protein of human T-cell leukemia virus type 1 (HTLV-1), glycoproteins gB and gH of herpes simplex virus (HSV), influenza hemagglutinin (HA) and neuraminidase (NA), and F protein of respiratory syncytial virus (RSV). Examples of pathogenic infections that can be treated with bispecific antibodies include viral, bacterial, protozoan, and fungal infections. Examples of viral infections include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, Epstein-Barr virus), adenovirus, XMRV, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, MLV-related virus, papillomavirus, molescum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus. Some examples of bacterial infections include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospirosis, lyme burgdorferi, streptococci, or neisseria. Examples of pathogenic fungi include candida, aspergillus, cryptococcus, histoplasmosis, pneumocystis, and stachybotrys. Examples of protozoa include cryptosporidium, giardia lamblia, and plasmodium.

[0106] Bispecific antibodies, like monospecific antibodies against human CD3, can also be used to treat immune diseases. Examples of immune diseases include autoimmune diseases such as type 1 diabetes, Crohn's disease, ulcerative colitis, multiple sclerosis, stiff-person syndrome, rheumatoid arthritis, myasthenia gravis, and lupus erythematosus. In these diseases, the body mounts a cellular and / or humoral immune response against one of its autoantigens, destroying that antigen and, in some cases, resulting in bodily dysfunction and / or death. Autoimmune diseases are treated by administering one of the monoclonal antibodies of the present invention. Other immune diseases that can be treated with the monoclonal antibodies of the present invention include asthma, allergies, celiac disease, psoriasis, and uveitis. Celiac disease, psoriasis, and uveitis are autoimmune diseases.

[0107] The monospecific, bispecific, or multispecific antibody is administered in an effective regimen, meaning a dosage, route of administration, and frequency of administration that delays onset, reduces severity, prevents further deterioration, and / or improves at least one sign or symptom of the condition. If the subject already has a disease, the regimen can be referred to as a therapeutically effective regimen. If the subject is at increased risk for the disease compared to the general population but has not yet experienced symptoms, the regimen can be referred to as a prophylactically effective regimen. In some instances, therapeutic or prophylactic efficacy can be observed in an individual subject compared to a historical control or previous experience in the same subject. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a population of treated subjects compared to a control population of untreated subjects in preclinical or clinical studies.

[0108] Preferably, a bispecific or multispecific antibody exhibits at least additive activity against cancer or infected cells, and more preferably synergistic activity, when its constituent antibodies are compared individually. Synergistic effects are preferably assessed quantitatively, as described in Tallarida, Genes Cancer. 2011 Nov;2(11):1003-1008. Also preferably, a bispecific antibody exhibits increased activity compared to a mixture of its constituent antibodies, each at equimolar concentrations with the bispecific antibody. Such activity can be measured, for example, as a cytotoxic or cytostatic effect against cancer cells, infected cells, or immune cells expressing an antigen specifically bound to one arm of the bispecific antibody in the presence of CD3-expressing immune cells.

[0109] Exemplary dosages of monospecific, bispecific, or multispecific antibodies are 0.01-20, or 0.5-5, or 0.01-1, or 0.01-0.5, or 0.05-0.5 mg / kg body weight (e.g., 0.1, 0.5, 1, 2, 3, 4, or 5 mg / kg), or 10-1500 mg as a fixed dose. Dosage will depend on the patient's condition and response to prior treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic, etc.

[0110] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Administration into the systemic circulation is preferably intravenous or subcutaneous. Intravenous administration can be carried out by infusion over a period of time, for example, 30 to 90 minutes.

[0111] The frequency of administration depends on, among other factors, the half-life of the antibody in the circulatory system, the condition of the subject, and the route of administration. Administration can be daily, weekly, monthly, quarterly, or irregular depending on changes in the patient's condition or the progression of the disorder being treated. For intravenous administration, an exemplary frequency is once a week to once a quarter during a continuous treatment period, although more or less frequent administration is also possible. For subcutaneous administration, an exemplary frequency is daily to monthly, although more or less frequent administration is also possible.

[0112] The number of doses depends on whether the disorder is acute or chronic and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, one to ten doses are often sufficient. A single bolus dose (optionally divided) may be sufficient during acute disease or acute exacerbations of chronic disease. Treatment can be repeated for recurrence of acute disease or acute exacerbations. For chronic diseases, the bispecific antibody can be administered periodically, e.g., weekly, biweekly, monthly, quarterly, or every six months, for at least one year, five years, ten years, or for the life of the subject.

[0113] The pharmaceutical composition is preferably suitable for parenteral administration to humans (e.g., in accordance with FDA standards). Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and produced under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a dosage for a single administration). The pharmaceutical composition can be formulated using one or more pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Pharmaceutically acceptable means suitable for administration to humans, e.g., approved or approvable by the FDA. The formulation will vary depending on the chosen route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, physiological saline, or acetate buffer (to reduce discomfort at the injection site). The solution can contain formulating agents such as suspending agents, stabilizers, and dispersing agents. Alternatively, the antibody can be in lyophilized form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0114] Treatment with the antibodies of the invention can be combined with other treatments that are effective against the disorder being treated. When used to treat cancer, the antibodies of the invention can be combined with chemotherapy, radiation, stem cell therapy, surgery, or Herceptin against the HER2 antigen. TM (trastuzumab), Avastin for VEGF TM (bevacizumab), or antibodies against the EGF receptor, e.g., (Erbitux TM , cetuximab), and Vectibix TM It can be combined with treatment with other biologics such as fluticasone (panitumumab) or other antibodies shown in Table 2. Chemotherapeutic agents include chlorambucil, cyclophosphamide or melphalan, carboplatinum, daunorubicin, doxorubicin, idarubicin, and mitoxantrone, methotrexate, fludarabine, and cytarabine, etoposide or topotecan, vincristine, and vinblastine. Infections can be treated with a combination of antibiotics, antiviral agents, antifungal agents, antiparasitic agents, and the like.

[0115] Other methods The antibodies of the present invention can also be used in diagnostic, prognostic, and experimental methods. They can measure the levels of antigens expressed by cancer or in the circulation of patients with cancer to determine whether the levels are measurable or elevated, and thus can be used to monitor and guide cancer treatment. This is because cancers associated with measurable or elevated levels of antigen are most susceptible to treatment with bispecific antibodies containing a cancer-binding arm. The antibodies can be used, inter alia, in ELISA assays, radioimmunoassays, or immunohistochemistry. The antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes and can be provided in the form of kits containing all the reagents necessary to perform the assay.

[0116] All patent applications, websites, other publications, accession numbers, etc., cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with accession numbers at different times, the version associated with the accession number as of the effective filing date of this application is meant. The effective filing date refers to the earlier of the actual filing date or the filing date of the priority application, with reference to the accession number, if applicable. Similarly, where different versions of a publication, website, etc. are published at different times, the version most recently published as of the effective filing date of this application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention can be used in combination with any other, unless specifically indicated otherwise. While the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0117] Example 1: General Procedures, Methods and Materials Gene cloning, mutagenesis, plasmid construction, protein expression and purification, cell culture, ELISA, and flow cytometry were performed according to standard laboratory techniques described in Green and Sambrook (Molecular Cloning, A Laboratory Manual, 4th ed., 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Greenfield (Antibodies, A Laboratory Manual, 2nd ed., 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Kostelny et al. (Int. J. Cancer 93:556-565, 2001), Cole et al. (J. Immunol. 159:3613-3621, 1997), and Tsurushita et al. (Methods 36:69-83, 2005), or according to vendor protocols. For the positions of amino acid residues in chimeric, humanized, and human IgG1 / kappa (or lambda) antibodies, the numbering system of Kabat et al. (Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991) was used.

[0118] SP34 is a murine IgG3 / lambda monoclonal agonistic antibody that binds to human and cynomolgus monkey CD3 epsilon protein. Cross-linking of CD3 epsilon protein on the surface of T cells by SP34 activates the T cells. (Passano et al., EMBO J. 4:337-344, 1985; Yang et al., J. Immunol. 137:1097-1100, 1986; Salmeron et al., J. Immunol. 147:3047-3052, 1991; Perez-Aciego et al., J. Exp. Med. 174:319-326, 1991; Conrad et al., Cytom. A 71A:925-933, 2007; US Patents 8,236,308 and 10,066,015). The amino acid sequence of the mature heavy chain variable region (VH) of SP34 is EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 1). The amino acid sequence of the mature light chain variable region (VL) of SP34 is QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 2).

[0119] Example 2: Humanized anti-human CD3 antibody [I] The VH and VL amino acid sequences of humanized SP34 were designed according to the general procedure described in Tsurushita et al. (supra). Briefly, a three-dimensional molecular model of the mouse SP34 variable region was first constructed using appropriate software. The molecular model was then used to identify framework amino acid residues important for the formation of the CDR structure. In parallel, cDNA-derived human VH and VL amino acid sequences with high homology to SP34 VH and VL, respectively, were selected. Finally, the CDR sequences, along with the framework amino acid residues important for the proper formation of the antigen-binding site, were grafted from SP34 VH and VL onto the corresponding selected human framework sequences.

[0120] To design the humanized SP34 VH, the human VH sequence encoded by the M24236 cDNA (GenBank accession number: Sanz et al., J. Immunol. 142:883-887, 1989) (M24236 VH; SEQ ID NO: 3) was selected as the acceptor for humanization. M24236 VH, which belongs to the IGHV3-15 subgroup of human germline VH segments, has no somatic hypermutations in the amino acid sequence of the framework regions. The framework amino acid sequence of M24236 VH shares 85.2% identity with the sequence of SP34 VH. The CDR sequences of SP34 VH were first transferred to the corresponding positions of M24236 VH. Next, amino acid residues in M24236 VH were substituted with the corresponding residues in murine SP34 VH at framework positions 30, 49, 93, and 94, where the three-dimensional model of the SP34 variable region indicated that these amino acid residues may play important roles in forming the antigen-binding site. The resulting amino acid sequence of the mature humanized VH, designated HuSP34 VH1 (SEQ ID NO: 4), is shown in Figure 1, along with the amino acid sequences of the mature SP34 and M24236 VH.

[0121] To design the humanized SP34 VL, the human Vλ region encoded by the Y14738 cDNA (GenBank accession number: Paterson et al., Immunotechnol. 4:37-47, 1998) (Y14738 VL; SEQ ID NO: 5) was selected as the acceptor for humanization. Y14738 VL, which belongs to the IGLV8-61 subgroup of human germline Vλ segments, has no somatic hypermutations in its framework region sequences. The framework amino acid sequence of Y14738 VL shares 67.1% identity with that of SP34 VL. The CDR sequences of SP34 VL were first transferred to the corresponding positions of Y14738 VL. Next, the amino acid residues of Y14738 VL were replaced with the corresponding residues of mouse SP34 VL at framework positions 36, 46, and 49, where the three-dimensional model of the SP34 variable region indicated that these amino acid residues may play important roles in the formation of the antigen-binding site. The amino acid sequence of the resulting mature humanized VL, designated HuSP34 VL1 (SEQ ID NO: 6), is shown in FIG. 2, along with the amino acid sequences of the mature SP34 and Y14738 VLs.

[0122] The gene encoding HuSP34 VH1 was synthesized as an exon containing a signal peptide (SEQ ID NO: 7), a splice donor signal, a SpeI site at the 5' end, and a HindIII site at the 3' end (Figure 3). The gene encoding HuSP34 VL1 was similarly synthesized as an exon containing a signal peptide (SEQ ID NO: 8), a splice donor signal, an NheI site at the 5' end, and an EcoRI site at the 3' end (Figure 4). The HuSP34 VH1 and VL1 genes were cloned between the SpeI and HindIII sites (for VH) or the NheI and EcoRI sites (for VL) of a mammalian expression vector for the production of a humanized IgG1 / lambda antibody. A schematic diagram of the resulting expression vector, designated pHuSP34A, is shown in Figure 5A. The humanized anti-CD3 IgG1 / lambda antibody (HuSP34A) was produced from pHuSP34A in mammalian cells.

[0123] Proceeding clockwise from the SalI site in Figure 5A, pHuSP34A contains a heavy chain transcription unit beginning with the human cytomegalovirus (CMV) major immediate-early promoter and enhancer (CMV-P in the figure) to initiate transcription of the antibody heavy chain gene. The CMV promoter is followed by a VH exon encoding HuSP34 VH1, as well as a genomic sequence containing the human gamma-1 heavy chain constant region, including CH1, hinge, CH2, and CH3 exons with an intervening intron, and a polyadenylation site following the CH3 exon. The CH2 region contains amino acid substitutions at positions 234 and 235 (Eu numbering, Kabat et al., supra) substituting alanine for leucine to eliminate effector function (Hazareh et al., J. Virol. 75:12161-12168, 2001). Following the heavy chain gene sequence, the light chain transcription unit begins with a CMV promoter and is followed by an exon encoding HuSP34A VL1, a genomic sequence containing the human lambda-2 chain constant region (Cλ2) preceded by a portion of an intron, and a polyadenylation site following the Cλ2 exon. The light chain gene is followed by the SV40 early promoter (SV40-P), the puromycin N-acetyltransferase gene (puro) for puromycin resistance, and a segment containing the SV40 polyadenylation site (SV40-A). Finally, the plasmid contains a portion of the pUC19 plasmid, which contains a bacterial origin of replication (pUC ori) and the beta-lactamase gene (β-lactamase). The locations of the relevant restriction enzyme sites are indicated in Figure 5A. Arrows indicate the direction of transcription.

[0124] The amino acid sequences of the CH1, hinge, CH2, and CH3 regions of the human gamma-1 heavy chain encoded by pHuSP34A are ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 9), EPKSCDKTHTCPPCP (SEQ ID NO: 10), and APEAAGGPSVFLFPPKPKDTLMISRT, respectively. PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 11), and GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12).

[0125] The amino acid sequence of the human lambda-2 constant region (Cλ2) encoded by pHuSP34A is GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 13).

[0126] The amino acid sequence of the heavy chain encoded by pHuSP34A is MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14). The mature HuSP34A heavy chain begins with the glutamic acid residue at position 20 of SEQ ID NO: 14.

[0127] The amino acid sequence of the light chain encoded by pHuSP34A is MAWISLILSLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 15). The mature HuSP34A light chain begins with the glutamine residue at position 17 of SEQ ID NO: 15.

[0128] The expression vector pHuSP34A was introduced into the chromosome of the Chinese hamster ovary cell line CHO-K1. Stable transfection into CHO-K1 was performed by electroporation. Prior to transfection, pHuSP34A was linearized with the restriction enzyme FspI (Figure 5A). Approximately 2.5 x 10 6 Each cell was transfected with 20 μg of linearized plasmid and suspended in SFM4CHO medium (HyClone, Logan, UT). After appropriate dilution, the cells were seeded into a 96-well plate and cultured at 37°C in a 7.5% CO2 incubator. After 48 hours, 10 μg / ml puromycin was added to isolate stably transfected cells.

[0129] Approximately 10 days after the start of selection, culture supernatants from stable CHO-K1 transfectants in 96-well plates were assayed for antibody production by sandwich ELISA. In a typical experiment, wells of an ELISA plate were coated overnight at 4°C with a goat anti-human IgG, Fcγ-specific, polyclonal antibody in PBS, washed with wash buffer (PBS containing 0.05% Tween 20), and blocked with ELISA buffer (PBS containing 2% skim milk and 0.05% Tween 20). After washing the wells with wash buffer, the test antibody, appropriately diluted in ELISA buffer, was applied to the ELISA plate. An appropriate humanized IgG1 / lambda (or kappa, as appropriate) antibody was used as a standard. The ELISA plate was incubated for 1 hour at room temperature and washed with wash buffer. Bound antibodies were then detected using an HRP-conjugated goat anti-human lambda chain (or kappa chain, as appropriate) polyclonal antibody. After incubation at room temperature for 0.5 h and washing with wash buffer, color development was initiated with ABTS substrate (Sigma-Aldrich, St. Louis, MO) and stopped with 2% oxalic acid. Absorbance was measured at 405 nm.

[0130] CHO-K1 stably transfected cells producing HuSP34A (CHO-K1 / pHuSP34A) were cultured in SFM4CHO in roller bottles until cell viability reached less than 50%. After centrifugation and filtration, the culture supernatant was loaded onto a Protein A column (HiTrap MabSelect SuRe, GE Healthcare, Piscataway, NJ). The column was washed with PBS before eluting the antibody with 0.1 M glycine-HCl buffer (pH 3.0) containing 0.1 M NaCl. After neutralization with 1 M Tris-HCl (pH 8.0), the eluted antibody was buffer exchanged into PBS by dialysis. The antibody concentration was determined by measuring absorbance at 280 nm (1.4 OD = 1 mg / ml).

[0131] The binding of HuSP34A to human CD3 was analyzed by flow cytometry using the human T cell line Jurkat Dual (Invivogen, San Diego, CA). In a typical experiment, HuSP34A was incubated with Jurkat Dual cells at various concentrations in FACS buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.05% sodium azide) for 30 minutes. After washing with FACS buffer, the cells were incubated with PE-conjugated goat anti-human IgG antibody for 20 minutes. After washing and resuspending in FACS buffer, the cells were subjected to flow cytometry. As shown in Figure 6, HuSP34A bound to Jurkat Dual cells in a dose-dependent manner. The EC50 value for HuSP34A binding to Jurkat Dual cells, calculated using the software Prism (GraphPad, San Diego, CA), was 92 ng / ml.

[0132] Example 3: Humanization of anti-human CD3 antibody [II] The second humanized SP34 VL was designed using a different method. The human mature Vκ region encoded by the L37309 cDNA (GenBank accession number: Ohlin et al., Mol. Immunol. 33:47-56, 1996) (L37309 VL; SEQ ID NO: 16) was used as the acceptor for humanization. L37309 VL, which belongs to the IGKV3-11 subgroup of human germline Vκ segments, has no somatic hypermutations in the framework region sequences. The framework amino acid sequence of L37309 VL shares 51.3% identity with that of SP34 VL. The CDR sequences of SP34 VL were first transferred to the corresponding positions of L37309 VL. Next, amino acid residues in L37309 VL were substituted with the corresponding residues in murine SP34 VL at framework positions 36, 46, 49, 58, 66, 67, 69, 70, and 71, where the three-dimensional model of the SP34 variable region indicated that these amino acid residues may play important roles in forming the antigen-binding site. The resulting amino acid sequence of the mature humanized VL, designated HuSP34 VL3 (SEQ ID NO: 17), is shown in Figure 7, along with the sequences of SP34 and L37309 VL.

[0133] The gene encoding HuSP34 VL3 was synthesized as an exon containing a signal peptide (SEQ ID NO: 18), a splice donor signal, an NheI site at the 5' end, and an EcoRI site at the 3' end (Figure 8). The HuSP34 VL3 gene was cloned between the NheI and EcoRI sites, replacing HuSP34 VL1 in pHuSP34A. Furthermore, the coding sequence for the human lambda-2 chain constant region (Cλ2) was replaced with the coding sequence for the human kappa chain constant region (Cκ). The resulting plasmid for expressing a humanized anti-CD3 IgG1 / kappa antibody (HuSP34C) containing HuSP34 VH1 and VL3 was designated pHuSP34C. A schematic diagram of pHuSP34C is shown in 5B. The amino acid sequence of the human kappa chain constant region encoded by pHuSP34C is RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19). The amino acid sequence of the heavy chain encoded by pHuSP34C is identical to that encoded by pHuSP34A (SEQ ID NO: 14). The amino acid sequence of the light chain encoded by pHuSP34C is MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:20). The mature HuSP34C light chain begins with the glutamic acid residue at position 21 of SEQ ID NO:20.

[0134] The expression vector pHuSP34C was introduced into CHO-K1 cells by electroporation as described above. CHO-K1 stably transfected cells expressing HuSP34C (CHO-K1 / pHuSP34C) were grown in SFM4CHO as described above. HuSP34C was purified from the culture supernatant of CHO-K1 / pHuSP34C using protein A as described above. Binding of HuSP34C to human CD3 was measured by flow cytometry using Jurkat Dual cells as described above. The results are shown in Figure 6. HuSP34C bound to Jurkat Dual cells in a dose-dependent manner. The EC50 value for HuSP34C binding to Jurkat Dual cells was 209 ng / ml.

[0135] Example 4: Mutagenesis of HuSP34 VL3 To reduce the potential for immunogenicity in humans, an attempt was made to reduce the number of murine-derived framework residues in HuSP34 VL3 (Figure 7). At positions 36, 58, 66, 67, 69, and 70 of the HuSP34 VL3 framework, murine-derived amino acid residues were replaced with the corresponding residues from the human L37309 VL acceptor by site-directed mutagenesis using overlap extension PCR. These HuSP34 VL3 variants are obtained by substituting valine at position 36 with tyrosine (V36Y; SEQ ID NO: 21), valine at position 58 with isoleucine (V58I; SEQ ID NO: 22), leucine at position 66 with glycine (L66G; SEQ ID NO: 23), isoleucine at position 67 with serine (I67S; SEQ ID NO: 24), aspartic acid at position 69 with threonine (D69T; SEQ ID NO: 25), and lysine at position 70 with aspartic acid (K70D; SEQ ID NO: 26) in HuSP34 VL3.

[0136] Six HuSP34 VL3 variants were each combined with HuSP34 VH1, replacing HuSP34VL3 in pHSP34C, and expressed as IgG1 / kappa antibodies. When the HuSP34 VL3 L66G variant was combined with HuSP34 VH1, antibody expression was significantly reduced. Combining HuSP34 VH1 with the HuSP34 VL3 V36Y variant significantly reduced antigen binding. When combined with HuSP34 VH1, the HuSP34 VL3 V58I, I67S, D69T, and K70D variants each exhibited antigen binding equivalent to that of the HuSP34 VH1 and VL3 combination (HuSP34C). Substitution of the mouse-derived hydrophobic isoleucine residue at position 67 with a human-derived hydrophilic serine residue (I67S) did not result in a decrease in antigen binding affinity and was tolerated by HuSP34 VL3. Similarly, neither (i) substitution of the mouse acidic aspartic acid residue at position 69 with a human neutral hydrophilic threonine residue (D69T) nor (ii) substitution of the mouse basic lysine residue at position 70 with a human acidic aspartic acid residue (K70D) resulted in a decrease in affinity.

[0137] The V58I, I67S, D69T, and K70D mutations were combined with HuSP34 VL3 to generate HuSP34 VL4. The sequence of mature HuSP34 VL4 is shown in Figure 7 (SEQ ID NO: 27). The gene encoding HuSP34 VL4 was generated by site-directed mutagenesis of HuSP34 VL3. The nucleotide sequence and deduced amino acid sequence of the HuSP34 VL4 gene are shown in Figure 9.

[0138] The HuSP34 VL4 gene was cloned between the NheI and EcoRI sites to replace the HuSP34 VL3 in pHSP34C. The resulting expression vector, designated pHSP34V, expresses the humanized SP34 IgG1 / kappa antibody containing HuSP34 VH1 and VL4 (HuSP34V). The amino acid sequence of the heavy chain encoded by pHuSP34V is identical to that encoded by pHuSP34A (SEQ ID NO: 14). The amino acid sequence of the light chain encoded by pHuSP34V is MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:28). The mature HuSP34V light chain begins with the glutamic acid residue at position 21 of SEQ ID NO:28.

[0139] The expression vector pHuSP34V was introduced into CHO-K1 cells by electroporation as described above. CHO-K1 stable transfectants expressing HuSP34V (CHO-K1 / pHuSP34V) were selected and propagated in SFM4CHO as described above. HuSP34V was purified from the culture supernatant of CHO-K1 / pHuSP34V using protein A as described above. Binding of HuSP34V to human CD3 was measured by flow cytometry using Jurkat Dual cells as described above. The results are shown in Figure 6. HuSP34V bound to Jurkat Dual cells in a dose-dependent manner. The EC50 value for HuSP34V binding to Jurkat Dual cells was 234 ng / ml.

[0140] Example 5: Generation and characterization of anti-CD20 / CD3 bispecific antibodies The VH and VL domains of HuSP34A were converted into a single-chain Fv (scFv) with the following orientation: N'-VH-linker-VL-C'. Two amino acid substitutions were introduced into the scFv: glycine at position 44 of HuSP34 VH1 to cysteine ​​(Fig. 1) and glycine at position 100 of HuSP34 VL1 to cysteine ​​(Fig. 2) (Brinkman et al., Proc. Natl. Acad. Sci. 90:7538-7542, 1993). The resulting scFv form of HuSP34A (HuSP34A.scFv.HL.ds; SEQ ID NO: 29) was fused to the penultimate glycine residue of the CH3 domain via a flexible polypeptide linker (CH3-HuSP34A.scFv.HL.ds; SEQ ID NO: 30) (Figure 5B) in an antibody expression vector identical to pHuSP34C except that the VH and VL exons were replaced with those of an anti-CD20 antibody. The resulting expression vector was designated pJB509.

[0141] The VH and VL domains of HuSP34A were also converted into another scFv format with an N'-VL-linker-VH-C' orientation containing two amino acid substitutions: glycine at position 44 of HuSP34 VH1 to cysteine ​​and glycine at position 100 of HuSP34 VL1 to cysteine ​​(HuSP34A.scFv.LH.ds; SEQ ID NO: 31). HuSP34A.scFv.LH.ds fused to the penultimate glycine residue of the CH3 domain (CH3-HuSP34A.scFv.LH.ds; SEQ ID NO: 32) was used to replace CH3-HuSP34A.scFv.HL.ds in pJB509. The resulting expression vector was designated pJB510.

[0142] The expression vectors pJB509 and pJB510 were individually transfected into the human embryonic kidney cell line HEK293 using polyethyleneimine (Durocher et al. Nucl. Acids Res. 30:e9, 2002) to transiently express the recombinant antibodies. HEK293 cells were cultured in DME medium containing 10% fetal bovine serum (FBS; Life Technologies, Grand Island, NY) at 37°C in a 7.5% CO2 incubator. Expression of the anti-CD20 / CD3 bispecific antibody in the culture supernatant, as measured by sandwich ELISA using the method described above, was very low for both pJB509 and pJB510. When HuSP34A.scFv.HL and HuSP34A.scFv.LH were conjugated to the C-terminal region of the CH3 of other chimeric or humanized IgG1 antibodies, respectively, antibody expression was again very low.

[0143] Next, an anti-CD3 scFv antibody was constructed using HuSP34 VH1 and VL4 in the N'-VL-linker-VH-C' orientation. Two amino acid substitutions were then introduced into the resulting scFv: glycine at position 44 in HuSP34 VH1 to cysteine, and glycine at position 100 in HuSP34 VL4 to cysteine. The resulting scFv was designated HuSP34V.scFv.ds. The amino acid sequence of HuSP34V.scFv.ds is EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 33).

[0144] HuSP34V.scFv.ds was fused to the penultimate glycine residue of CH3 (CH3-HuSP34V.scFv.ds; SEQ ID NO: 34) via a flexible polypeptide linker separating the VH and VL of an IgG1 / kappa antibody expression vector (Figure 5B) identical to pHuSP34C, except that the VH and VL were derived from the chimeric anti-CD20 antibody C2B8 (Reff et al., Blood 83:435-445, 1994; Maloney et al., Blood 84:2457-2466, 1994). The resulting expression vector was designated pJB554. A schematic diagram of pJB554 is shown in Figure 5C.

[0145] The amino acid sequence of the mature C2B8 VH encoded by pJB554 is QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 35; https: / / go.drugbank.com / drugs / DB00073 ).

[0146] The amino acid sequence of the mature C2B8 VL encoded by pJB554 is QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 36; https: / / go.drugbank.com / drugs / DB00073).

[0147] The bispecific anti-CD20 / CD3 IgG1 / kappa antibody expressed from pJB554 was designated JB554. A schematic representation of JB554 is shown in Figure 10. The sequence of the heavy chain encoded by pJB554 is (SEQ ID NO:37). The mature JB554 heavy chain begins with the glutamine residue at position 20 of SEQ ID NO:37.

[0148] The sequence of the light chain encoded by pJB554 is MDFQVQIISFLLISASVIMSRGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:38). The mature JB554 light chain begins with the glutamine residue at position 23 of SEQ ID NO:38.

[0149] The expression vector pJB554 was transfected into HEK293 cells by the method described above. Expression of the anti-CD20 / CD3 bispecific antibody from pJB554 was improved compared to that of pJB509 and pJB510. Transiently expressed JB554 was purified by protein A by the method described above. The T cell-mediated cytotoxicity of JB554, a bispecific antibody capable of binding to CD20 and CD3, against CD20-positive human Burkitt's lymphoma Ramos cells was evaluated as follows. Purified human CD3+ pan T cells (Cat. # IQB-Hu1-T100, Lot # P19D0900, iQ Biosciences, Berkeley, CA) were incubated in RPMI 1640 medium containing 10% FBS, 1 mM sodium pyruvate, and 10 mM HEPES (RPMI 1640 complete medium) in the presence of 50 ng / ml recombinant human IL-2 (Acro Biosystems, Newark, DE) at 37°C in a 7.5% CO2 incubator for 4 days (activated T cells). One hundred thousand Ramos cells labeled with a fluorescent dye (Calcein AM, BioLegend, San Diego, CA) were incubated with one million activated T cells in 200 μl of RPMI 1640 complete medium in the presence or absence of 150 ng / ml JB554 in a 96-well plate for 4 hours. To monitor the degree of Ramos cell lysis, fluorescence in the culture medium was measured according to the manufacturer's instructions. As a 100% lysis control, Calcein AM-labeled Ramos cells were incubated alone as described above and then lysed with SDS, and the fluorescence of the culture supernatant was measured. As a background control, Calcein AM-labeled Ramos cells were incubated alone as described above and the fluorescence of the culture supernatant was measured.

[0150] The results of the cytotoxicity assay using Calcein AM-labeled Ramos cells are shown in Figure 11A. The relative fluorescence intensity (RFU) values ​​in the culture supernatants were (i) 23,911 for Ramos cells alone, (ii) 74,000 for SDS-treated Ramos cells, (iii) 33,668 for Ramos cells incubated with activated T cells, and (iv) 82,104 for Ramos cells incubated with activated T cells and JB554. The anti-CD20 / CD3 bispecific antibody JB554 efficiently induced lysis of Ramos cells in the presence of activated T cells.

[0151] Example 6: Generation and characterization of anti-EGFR / CD3 bispecific antibodies To construct an expression vector for a bispecific antibody that binds to CD3 and epidermal growth factor receptor (EGFR), the VH and VL genes of pJB554 were replaced with those derived from the murine anti-EGFR antibody 225 (Masui et al. Cancer Res. 44:1002-1007, 1984; Gill et al. J. Biol. Chem. 259:7755-7760, 1984). The resulting expression vector was named pJB559. The structure of pJB559 is identical to that of pJB554, except for the VH and VL sequences (Figure 5C).

[0152] The amino acid sequence of the mature 225 VH encoded by pJB559 is QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 39; https: / / go.drugbank.com / drugs / DB00002).

[0153] The amino acid sequence of the mature 225 VL encoded by pJB559 is DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 40; https: / / go.drugbank.com / drugs / DB00002).

[0154] The bispecific anti-EGFR / CD3 IgG1 / kappa antibody expressed from pJB559 was designated JB559. A schematic representation of JB559 is shown in Figure 10. The amino acid sequence of the JB559 heavy chain is (SEQ ID NO:41). The mature JB559 heavy chain begins with the glutamine residue at position 20 of SEQ ID NO:41.

[0155] The amino acid sequence of the JB559 light chain is MRAPAQFLGFLLFWIPASRSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:42). The mature JB559 light chain begins with the aspartic acid residue at position 21 of SEQ ID NO:42.

[0156] The expression vector pJB559 was transfected into HEK293 cells as described above. Transiently expressed JB559 was purified by protein A as described above. The T cell-mediated cytotoxicity of JB559, a bispecific antibody capable of binding to EGFR and CD3, against EGFR-positive human colorectal adenocarcinoma HT-29 cells was evaluated as follows. HT-29 cells were first seeded into wells of a 96-well plate at a concentration of 400,000 cells / ml in RPMI 1640 complete medium. After 48 hours, HT-29 cells were labeled with Calcein AM and incubated with activated T cells at 5 million cells / ml in the presence (or absence) of the test antibody (JB554 or JB559) as described above. As a control for 100% lysis, the fluorescence of the culture supernatant of Calcein AM-labeled HT-29 cells incubated alone and lysed with SDS was measured as described above. As a background control, Calcein AM-labeled HT-29 cells were incubated alone in the same manner as above, and the fluorescence of the culture supernatant was measured.

[0157] The results of the cytotoxicity assay using Calcein AM-labeled HT-29 cells are shown in Figure 11B. The RFU values ​​in the culture supernatants were (i) 5,119 for HT-29 cells alone, (ii) 75,928 for SDS-treated HT-29 cells, (iii) 7,945 for HT-29 cells incubated with activated T cells, (iv) 7,101 for HT-29 cells incubated with activated T cells and JB554, and (v) 38,523 for HT-29 cells incubated with activated T cells and JB559. The anti-EGFR / CD3 bispecific antibody JB559 induced HT-29 cell lysis in the presence of activated T cells. The anti-CD20 / CD3 bispecific antibody JB554, which does not bind to HT-29 cells, did not induce HT-29 cell lysis in the presence of activated T cells.

[0158] Example 7: Expression and purification of anti-CD20, anti-EGFR, and anti-CD33 IgG1 antibodies

[0159] The expression vector pChC2B8, which expresses the mouse-human chimeric anti-CD20 IgG1 / kappa antibody (ChC2B8), has the same structure as pJB554 (Figure 5C), except that (i) the CH3-HuSP34V.ScFv.ds region (SEQ ID NO: 34) is replaced with the wild-type CH3 sequence (SEQ ID NO: 12), and (ii) the CH2 region encodes the wild-type CH2 sequence of the human gamma 1 heavy chain (SEQ ID NO: 49). The amino acid sequence of the light chain encoded by pChC2B8 is identical to the light chain sequence encoded by pJB554 (SEQ ID NO: 38). The amino acid sequence of the heavy chain encoded by pChC2B8 is MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 50). The mature ChC2B8 heavy chain begins with the glutamine residue at position 20 of SEQ ID NO: 50.

[0160] The expression vector pCh225, which expresses the mouse-human chimeric anti-EGFR IgG1 / kappa antibody (Ch225), has the same structure as pJB559, except that (i) the CH3-HuSP34V.scFv.ds region (SEQ ID NO: 34) is replaced with the wild-type CH3 sequence (SEQ ID NO: 12), and (ii) the CH2 region encodes the wild-type CH2 sequence of the human gamma-1 heavy chain (SEQ ID NO: 49). The amino acid sequence of the light chain encoded by pCh225 is identical to the light chain sequence encoded by pJB559 (SEQ ID NO: 42). The amino acid sequence of the heavy chain encoded by pCh225 is: MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:51). The mature Ch225 heavy chain begins with the glutamine residue at position 20 of SEQ ID NO:51.

[0161] The expression vector pHuM195 (Co et al., J. Immunol. 148:1149-1154, 1992; U.S. Patent No. 5,693,761), which expresses the humanized anti-CD33 IgG1 antibody HuM195, has the same structure as pChC2B8, except that the VH and VL regions encode HuM195VH (SEQ ID NO: 52) and HuM195VL (SEQ ID NO: 53), respectively. The amino acid sequence of the heavy chain encoded by pHuM195 is MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54). The mature HuM195 heavy chain begins with the glutamine residue at position 20 of SEQ ID NO: 54. The sequence of the light chain encoded by pHuM195 is MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 55).The mature HuM195 light chain begins at the aspartic acid residue at position 21 of SEQ ID NO:55.

[0162] pChC2B8, pCh225, and pHuM195 were each stably transfected into CHO-K1 cells using the method described above. ChC2B8, Ch225, and HuM195 IgG1 antibodies were purified from the culture supernatant of each stable CHO-K1 transfectant using protein A. SDS-PAGE analysis of the ChC2B8, Ch225, and HuM195 antibodies under reducing conditions confirmed only two major bands: a heavy chain of approximately 50 kDa and a light chain of 25 kDa.

[0163] Example 8: Further characterization of JB554 binding to CD20 and CD3

[0164] A stable CHO-K1 cell line (CHO-K1 / pJB554) producing JB554 was generated by electroporation as described above. Growth of CHO-K1 / pJB554 cells in SFM4CHO medium and purification of JB554 with protein A were performed as described above. SDS-PAGE analysis of purified JB554 under reducing conditions revealed only two major bands: a heavy chain of approximately 75 kDa and a light chain of 25 kDa.

[0165] The biological activity of JB554 purified from CHO-K1 / pJB554 cells was examined using Jurkat Dual reporter cells (Invivogen, San Diego, CA). In Jurkat Dual cells, cross-linking of cell surface CD3 activates the intracellular NF-κB signaling pathway, resulting in the expression and secretion of recombinant Lucia luciferase. Approximately 400,000 Jurkat Dual cells were incubated in 200 μl of RPMI 1640 medium containing 10% FBS in wells of a 96-well plate in the presence (or absence) of 200,000 CD20+ Ramos cells and test antibodies in a 37°C, 7.5% CO2 incubator for 1 day. Luciferase activity in the culture supernatant was measured in triplicate using QUANTI-Luc reagent (Invivogen) according to the manufacturer's protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek, Winooski, VT). The mean relative luminescence unit (RLU) values ​​were: (i) 539 for Jurkat Dual cells alone; (ii) 1,940 for Jurkat Dual cells incubated with 1 μg / ml ChC2B8 (anti-CD20 IgG1 antibody) and 1 μg / ml HuSP34V (anti-CD3 IgG1 antibody); (iii) 827 for Jurkat Dual cells incubated with 1 μg / ml JB554 (anti-CD20 / CD3 bispecific antibody); (iv) 459 for Jurkat Dual cells incubated with 1 μg / ml JB559 (anti-EGFR / CD3 bispecific antibody); (v) 256 for Jurkat Dual cells incubated with Ramos cells; (vi) 2,212 for Jurkat Dual cells incubated with 1 μg / ml ChC2B8, 1 μg / ml HuSP34V, and Ramos cells; (vii) 1 (viii) 14,730 for Jurkat Dual cells incubated with 1 μg / ml JB554 and Ramos cells, and (viii) 691 for Jurkat Dual cells incubated with 1 μg / ml JB559 and Ramos cells. Figure 12A shows the RLU values ​​with standard deviation error bars for the Jurkat Dual assay.High levels of luciferase activity were observed in the culture supernatant only when Jurkat Dual cells were incubated with JB554 and Ramos cells.

[0166] JB554 purified from CHO-K / pJB554 cells was further analyzed for T cell-mediated cytotoxicity against Calcein AM-labeled Ramos cells as described above, except that each test antibody was used at 1 μg / ml. Activated T cells used in this experiment were human CD3 pan T cells (Cat. # IQB-Hu1-T100, iQ Biosciences, Berkeley, CA) from donor 3820. The relative fluorescence unit (RFU) values ​​in the culture supernatants were (i) 18,474 for Ramos cells alone, (ii) 79,395 for SDS-treated Ramos cells, (iii) 18,940 for Ramos cells incubated with activated T cells, (iv) 19,220 for Ramos cells incubated with activated T cells, ChC2B8, and HuSP34V, (v) 79,395 for Ramos cells incubated with activated T cells and JB554, and (vi) 20,663 for Ramos cells incubated with activated T cells and JB559 (Figure 12B). The anti-CD20 / CD3 bispecific antibody JB554 efficiently induced lysis of Ramos cells in the presence of activated T cells, whereas the anti-EGFR / CD3 bispecific antibody JB559 and the combination of the anti-CD20 antibody ChC2B8 and the anti-CD3 antibody HuSP34V did not efficiently induce lysis of Ramos cells.

[0167] Example 9: Further characterization of JB559 binding to EGFR and CD3

[0168] A stable CHO-K1 cell line (CHO-K1 / pJB559) producing JB559 was generated by electroporation as described above. Growth of CHO-K1 / pJB559 cells in SFM4CHO medium and purification of JB559 with protein A were performed as described above. SDS-PAGE analysis of purified JB559 under reducing conditions revealed only two major bands: a heavy chain of approximately 75 kDa and a light chain of 25 kDa.

[0169] JB559 purified from CHO-K1 / pJB559 cells was analyzed for its ability to induce T cell-mediated cytotoxicity against Calcein AM-labeled HT-29 cells using the method described above. Each test antibody was used at 1 μg / ml. Activated T cells used in the first cytotoxicity experiment were from donor 3820, a human CD3 pan T cell line (Cat. # IQB-Hu1-T100, iQ Biosciences, Berkeley, CA). In the second experiment, activated T cells from donor 3661, a human CD3 pan T cell line, were used. The relative fluorescence unit (RFU) values ​​of the culture supernatants in the first experiment were: (i) 6,112 for HT-29 cells alone; (ii) 75,173 for SDS-treated HT-29 cells; (iii) 6,482 for HT-29 cells incubated with activated T cells; (iv) 5,968 for HT-29 cells incubated with activated T cells, Ch225 (anti-EGFR IgG1 antibody), and HuSP34V (anti-CD3 IgG1 antibody); (v) 6,350 for HT-29 cells incubated with activated T cells and JB554 (anti-CD20 / CD3 bispecific antibody); and (vi) 56,634 for HT-29 cells incubated with activated T cells and JB559 (anti-EGFR / CD3 bispecific antibody) (Figure 13A). In the second experiment, the RFU values ​​in the culture supernatants were (vii) 5,046 for HT-29 cells alone, (viii) 72,990 for SDS-treated HT-29 cells, (ix) 6,114 for HT-29 cells incubated with activated T cells, (x) 5,391 for HT-29 cells incubated with activated T cells, Ch225, and HuSP34V, (xi) 5,662 for HT-29 cells incubated with activated T cells and JB554, and (xii) 35,443 for HT-29 cells incubated with activated T cells and JB559 (Figure 13B). In both experiments with HT-29 cells, the anti-EGFR / CD3 bispecific antibody JB559 efficiently induced lysis of HT-29 cells in the presence of activated T cells, whereas the anti-CD20 / CD3 bispecific antibody JB554 or the combination of Ch225 and HuSP34V did not efficiently induce lysis of HT-29 cells.

[0170] Example 10: Lack of EGFR-independent T cell activation by anti-EGFR / CD3 bispecific antibody JB559

[0171] To investigate whether circulating T cells are activated by JB559 in an EGFR-independent manner, we performed 10 5 Human peripheral blood mononuclear cells (PBMCs) were cultured in 200 μl of RPMI 1640 medium containing 10% FBS with either (i) no reagent, (ii) 1 μg / ml JB559, or (iii) 8 x 10 4 Human CD3 / CD28 T cell activation beads (anti-CD3 / CD28 beads, BioLegend, CA) were added to wells of a 96-well plate and incubated at 37°C in a 7.5% CO2 incubator for 3 days. This assay was performed in duplicate. JB559 is a bispecific antibody that binds to EGFR and CD3. EGFR has been reported to be either not expressed or only minimally expressed in PBMCs (http: / / proteinatlas.org).

[0172] The expression levels of IFN-γ and IL-2 in the culture supernatants were measured using the ELISA MAX Standard Set Human IFN-γ and ELISA MAX Standard Set Human IL-2 kits (BioLegend), respectively. The mean IFN-γ expression levels for duplicate samples were (a) 23.3 pg / ml without reagent, (b) 11.6 pg / ml with JB559, and (c) 2,670 pg / ml with anti-CD3 / CD28 beads. The mean IL-2 expression levels for duplicate samples were (d) 33.8 pg / ml without reagent, (e) 31.9 pg / ml with JB559, and (f) 11,500 pg / ml with anti-CD3 / CD28 beads. Incubation of PBMCs with JB559 for 3 days in the absence of EGFR-expressing cells did not result in any signs of T cell activation.

[0173] Example 11: Generation and characterization of anti-CD33 / CD3 bispecific antibodies

[0174] To create an expression vector for a bispecific antibody that binds to CD33 and CD3, the VH and VL genes of pJB554 were replaced with genes encoding HuM195 VH (SEQ ID NO: 52) and HuM195 VL (SEQ ID NO: 53), respectively. The resulting plasmid was designated pJB564. The structure of pJB564 is identical to that of pJB554, except for the VH and VL sequences (Figure 5C). The anti-CD33 / CD3 bispecific antibody expressed from pJB564 was designated JB564. A schematic diagram of JB564 is shown in Figure 10. The amino acid sequence of the heavy chain encoded by pJB564 is (SEQ ID NO: 56).The mature JB564 heavy chain begins at glutamine residue 20 of SEQ ID NO: 56. The amino acid sequence of the light chain encoded by pJB564 is identical to the sequence of the light chain encoded by pHuM195 (SEQ ID NO: 55).

[0175] A stable CHO-K1 cell line (CHO-K1 / pJB564) producing JB564 was generated by electroporation as described above. Growth of CHO-K1 / pJB564 cells in SFM4CHO medium and purification of JB564 with protein A were performed as described above. Analysis of purified JB564 by SDS-PAGE under reducing conditions revealed that it consisted of two polypeptides: a heavy chain of approximately 75 kDa and a light chain of 25 kDa.

[0176] JB564 was assayed for its ability to induce T cell-mediated cytotoxicity against Calcein AM-labeled human CD33-positive HL-60 cells (Cat. # CCL-240, American Type Culture Collection, Manassas, VA) in the presence (or absence) of activated T cells and 1 μg / ml of test antibody, as described above. The relative fluorescence unit (RFU) values ​​in the culture supernatants were: (i) 25,364 for HL-60 cells alone; (ii) 75,253 for SDS-treated HL-60 cells; (iii) 22,895 for HL-60 cells incubated with activated T cells; (iv) 25,756 for HL-60 cells incubated with activated T cells, HuM195 (anti-CD33 IgG1 antibody), and HuSP34V (anti-CD3 IgG1 antibody); (v) 21,928 for HL-60 cells incubated with activated T cells and JB559 (anti-EGFR / CD3 bispecific antibody); and (vi) 68,226 for HL-60 cells incubated with activated T cells and JB564 (anti-CD33 / CD3 bispecific antibody) (Figure 14). The anti-CD33 / CD3 bispecific antibody JB564 efficiently induced lysis of HL-60 cells in the presence of activated T cells, whereas the anti-EGFR / CD3 bispecific antibody JB559 and the combination of HuM195 and HuSP34V did not.

[0177] Sequence Listing SEQ ID NO: 1

[0178] Amino acid sequence of mature SP34 VH

[0179] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0180] SEQ ID NO: 2

[0181] Amino acid sequence of mature SP34 VL

[0182] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL

[0183] SEQ ID NO: 3

[0184] Amino acid sequence of mature M24236 VH

[0185] EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDSLPPHRVWGQGTLVTVSS

[0186] SEQ ID NO:4

[0187] Amino acid sequence of mature HuSP34 VH1

[0188] EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0189] SEQ ID NO:5

[0190] Amino acid sequence of mature Y14738 VL

[0191] QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYQQTPGQAAPRTLIYTTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCVLYMGGVWVFGGGTKLTVL

[0192] SEQ ID NO:6

[0193] Amino acid sequence of mature HuSP34 VL1

[0194] QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL

[0195] SEQ ID NO:7

[0196] Amino acid sequence of the signal peptide of HuSP34 VH1

[0197] MLLGLKWVFFVVFYQGVHC

[0198] SEQ ID NO:8

[0199] Amino acid sequence of the signal peptide of HuSP34 VL1

[0200] MAWISLILSLLALSSG

[0201] SEQ ID NO:9

[0202] Amino acid sequence of the CH1 region of the human gamma-1 heavy chain encoded by pHuSP34A

[0203] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0204] SEQ ID NO: 10

[0205] Amino acid sequence of the hinge region of the human gamma-1 heavy chain encoded by pHuSP34A

[0206] EPKSCDKTHTCPPCP

[0207] SEQ ID NO: 11

[0208] Amino acid sequence of the CH2 region of the human gamma-1 heavy chain encoded by pHuSP34A

[0209] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0210] SEQ ID NO: 12

[0211] Amino acid sequence of the CH3 region of the human gamma-1 heavy chain encoded by pHuSP34A

[0212] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0213] SEQ ID NO: 13

[0214] Amino acid sequence of the human lambda-2 constant region encoded by pHuSP34A

[0215] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 13)

[0216] SEQ ID NO: 14

[0217] Heavy chain amino acid sequence encoded by pHuSP34A

[0218] MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCV RHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0219] SEQ ID NO: 15

[0220] Light chain amino acid sequence encoded by pHuSP34A

[0221] MAWISLILSLLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWV FGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0222] SEQ ID NO: 16

[0223] Amino acid sequence of mature L37309 VL

[0224] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK

[0225] SEQ ID NO: 17

[0226] Amino acid sequence of mature HuSP34 VL3

[0227] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0228] SEQ ID NO: 18

[0229] Amino acid sequence of the signal peptide of HuSP34 VL3

[0230] MEAPAQLLFLLLLWLPDTTG

[0231] SEQ ID NO: 19

[0232] Amino acid sequences of human kappa constant regions encoded by pHuSP34C and pHuSP34V

[0233] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0234] SEQ ID NO: 20

[0235] Light chain amino acid sequence encoded by pHuSP34C

[0236] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNL WVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0237] SEQ ID NO: 21

[0238] Amino acid sequence of the mature HuSP34 VL3 V36Y variant

[0239] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWYQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0240] SEQ ID NO: 22

[0241] Amino acid sequence of the mature HuSP34 VL3 V58I variant

[0242] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0243] SEQ ID NO: 23

[0244] Amino acid sequence of the mature HuSP34 VL3 L66G variant

[0245] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSGIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0246] SEQ ID NO: 24

[0247] Amino acid sequence of the mature HuSP34 VL3 I67S variant

[0248] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLSGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0249] SEQ ID NO: 25

[0250] Amino acid sequence of the mature HuSP34 VL3 D69T variant

[0251] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGTKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0252] SEQ ID NO: 26

[0253] Amino acid sequence of the mature HuSP34 VL3 K70D variant

[0254] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0255] SEQ ID NO: 27

[0256] Amino acid sequence of mature HuSP34 VL4

[0257] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0258] SEQ ID NO: 28

[0259] Light chain amino acid sequence encoded by pHuSP34V

[0260] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNL WVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0261] SEQ ID NO: 29

[0262] Amino acid sequence of the scFv form of HuSP34A in the N'-VH-linker-VL-C' orientation (HuSP34A.scFv.HL.ds)

[0263] EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVL

[0264] SEQ ID NO: 30

[0265] Amino acid sequence of the CH3 region fused to HuSP34A.scFv.HL (CH3-HuSP34A.scFv.HL.ds)

[0266] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSV KDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVL

[0267] SEQ ID NO: 31

[0268] Amino acid sequence of the scFv form of HuSP34A in the N'-VL-linker-VH-C' orientation (HuSP34A.scFv.LH.ds)

[0269] QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVLGGGGSGGGGSGGGGS EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0270] SEQ ID NO: 32

[0271] Amino acid sequence of the CH3 region fused to HuSP34A.scFv.LH.ds (CH3-HuSP34A.scFv.LH.ds)

[0272] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSG SILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0273] SEQ ID NO: 33

[0274] Amino acid sequence of the scFv form of HuSP34V in the N'-VL-linker-VH-C' orientation (HuSP34V.scFv.ds)

[0275] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGS EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0276] SEQ ID NO: 34

[0277] Amino acid sequence of the CH3 region fused to HuSP34V.scFv.ds (CH3-HuSP34V.scFv.ds)

[0278] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFS GSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0279] SEQ ID NO: 35

[0280] Mature C2B8 VH amino acid sequence

[0281] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA

[0282] SEQ ID NO: 36

[0283] Amino acid sequence of mature C2B8 VL

[0284] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK

[0285] SEQ ID NO: 37

[0286] Heavy chain amino acid sequence encoded by pJB554

[0287] MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSG SLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0288] SEQ ID NO: 38

[0289] Light chain amino acid sequence encoded by pJB554

[0290] MDFQVQIISFLLISASVIMSRGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPP TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0291] SEQ ID NO: 39

[0292] Amino acid sequence of mature 225 VH

[0293] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA

[0294] SEQ ID NO: 40

[0295] Amino acid sequence of mature 225 VL

[0296] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK

[0297] SEQ ID NO: 41

[0298] Heavy chain amino acid sequence encoded by pJB559

[0299] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGS LSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0300] SEQ ID NO: 42

[0301] Light chain amino acid sequence encoded by pJB559

[0302] MRAPAQFLGFLLFWIPASRSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT FGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0303] SEQ ID NO: 43 CDRH1 TYAMN

[0304] SEQ ID NO: 44: CDRH2 RIRSKYNNYATYYADSVKD

[0305] SEQ ID NO: 45 CDRH3 HGNFGNSYVSWFAY

[0306] SEQ ID NO: 46 CDRL1 RSSTGAVTTSNYAN

[0307] SEQ ID NO: 47 CDRL2 GTNKRAP

[0308] SEQ ID NO: 48 CDRL3 ALWYSNLWV

[0309] SEQ ID NO: 49

[0310] Amino acid sequence of the CH2 region encoded by pChC2B8

[0311] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0312]

[0313] SEQ ID NO:50

[0314] Heavy chain amino acid sequence encoded by pChC2B8

[0315] MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCAR STYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0316]

[0317] SEQ ID NO:51

[0318] Heavy chain amino acid sequence encoded by pCh225

[0319] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARA LTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0320]

[0321] SEQ ID NO:52

[0322] HuM195 VH amino acid sequence

[0323] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS

[0324]

[0325] SEQ ID NO:53

[0326] Amino acid sequence of HuM195 VL

[0327] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK

[0328]

[0329] SEQ ID NO:54

[0330] Heavy chain amino acid sequence encoded by pHuM195

[0331] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCA RGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0332]

[0333] SEQ ID NO: 55

[0334] Light chain amino acid sequence encoded by pHuM195

[0335] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVP WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0336]

[0337] SEQ ID NO:56

[0338] Heavy chain amino acid sequence encoded by pJB564

[0339] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGS LSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0340] SEQ ID NO:57

[0341] Nucleotide sequence of the HuSP34 VH1 gene flanked by SpeI and HindIII sites

[0342] ACTAGTACCACCATGCTGTTGGGGCTGAAGTGGGTTTTCTTTGTTGTTTTTTATCAAGGAGTGCATTGTGAAGTGCAGCTTGTGGAAAGTGGCGGAGGACTGGTGAAGCCAGGCGGATCACTGAGACTGTCCTGCGCAGCTAGTGGCTTCACCTTTAACACATACGCTATGAATTGGGTCCGACAGGCACCTGGCAAGGGCCTGGAGTGGGTGGCAAGGATCAGGTCCAAGTACAAC AATTATGCAACCTACTATGCCGACTCTGTGAAGGATAGATTCACAATCAGTCGCGACGATTCCAAGAACACTCTGTATCTGCAGATGAACAGTCTGAAAACTGAAGACACCGCCGTGTACTATTGTGTGCGGCACGGAAACTTCGGCAATTCTTACGTCTCTTGGTTTGCTTATTGGGGACAGGGGACACTGGTCACTGTGTCTTCAGGTGAGTCCTAACTTCTCCCATTCTAAGCTT

[0343] SEQ ID NO:58

[0344] Amino acid sequence of HuSP34 VH1 including signal peptide

[0345] MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0346] SEQ ID NO:59

[0347] Nucleotide sequence of the HuSP34 VL1 gene flanked by NheI and EcoRI sites GCTAGCACCACCATGGCCTGGATTTCACTTATCCTCTCTCTCCTGGCTCTCAGCTCAGGGCAGACTGTCGTGACACAGGAACCCTCATTTTCCGTCAGCCCTGGCGGAACAGTGACCCTGACCTGCAGATCTAGCACAGGCGCAGTGACCACAAGCAACTACGCCAACTGGGTCCAGCAAACTCCAGGCCAAGCTCCCAGAGGGCCTGAT CGGCGGCACCAACAAAAGGGCTCCAGGCGTGCCAGACAGATTCAGCGGCAGCATCCTTGGCAATAAGGCTGCCCTGACAATCACTGGAGCCCAGGCCGACGACGAGTCCGACTACTATTGCGCCCTGTGGTACAGCAACCTGTGGGTCTTCGGCGGAGGCACCAAGCTGACAGTGCTAGGTGAGTCCTTCCTCCTTTGTTATTGAATTC

[0348] SEQ ID NO: 60

[0349] Amino acid sequence of HuSP34 VL1 including the signal peptide

[0350] MAWISLILSLLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL

[0351] SEQ ID NO: 61

[0352] Nucleotide sequence of the HuSP34 VL3 gene flanked by NheI and EcoRI sites

[0353] GCTAGCGCCACCATGGAAGCCCAGCTCAGCTTCTCTTCCTCCTGCTTCTCTGGCTCCCAGATACCACTGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGATCTAGCACAGGAGCCGTGACCACAAGCAACTATGCCAACTGGGTCCAACAGAAACCTGGCCAGGCTCCC AGGGGACTCATCGGAGGCACCAACAAAAGGGCTCCAGGAGTCCCAGCCAGGTTCAGTGGCAGTCTGATTGGGGATAAAGCTACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGAGGAGGCACCAAAGTCGAAATCAAACGTAAGTAGAATCCAAAGTGAATTC

[0354] SEQ ID NO: 62

[0355] Amino acid sequence of HuSP34 VL3 including signal peptide

[0356] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0357] SEQ ID NO: 63

[0358] Nucleotide sequence of the HuSP34 VL4 gene flanked by NheI and EcoRI sites

[0359] GCTAGCGCCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTTCTCTGCTCCAGATACCACTGGAGAAATTGTGTTGACACAGTCTCCAGCCCTGTCTTTGTCTCCAGGGAAAGAGCCACCCTCTCCTGCAGATCTAGCACAGGAGCCGTGACCACAAGCAACTATGCCAACTGGGTCCAACAGAAACCTGGCCAGGCTCCC AGGGGACTCATCGGAGGCACACAAAAGGGCTCCAGGAATCCCAGCCAGGTTCAGTGGCAGTCTGAGCGGGACTGATGCTACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGAGGAGCACCAAAGTCGAAATCAAACGTAAGTAGAATCCAAAGTGAATTC

[0360]

[0361] sequence no.64

[0362] ションドフムテッチションHuSP34 VL4のaminoacids

[0363] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

Claims

1. An antibody that specifically binds to human CD3, comprising a mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 derived from SEQ ID NO: 1, and a mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 derived from SEQ ID NO:

2.

2. The antibody of claim 1, wherein CDRH1, CDRH2 and CDRH3 comprise SEQ ID NOs: 43 to 45, respectively, and CDRL1, CDRL2 and CDRL3 comprise SEQ ID NOs: 46 to 48, respectively.

3. The antibody of claim 1, which is a mouse antibody.

4. The antibody of claim 1, which is chimerized or veneered.

5. The antibody of claim 1, which is a humanized antibody and comprises a humanized mature heavy chain variable region and a humanized mature light chain variable region.

6. 6. The antibody of claim 5, wherein positions 30, 49, 93, and 94 according to the Kabat numbering of the humanized mature heavy chain variable region are occupied by N, A, V, and R, respectively.

7. 7. The antibody of claim 6, wherein positions 36, 46, 49, 66 and 71 of the humanized mature light chain variable region are occupied by V, G, G, L and A, respectively.

8. 2. The antibody of claim 1, wherein the mature heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 1, and the mature light chain variable region has an amino acid sequence comprising SEQ ID NO:

2.

9. 2. The antibody of claim 1, wherein the mature heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 4, and the mature light chain variable region has an amino acid sequence comprising any of SEQ ID NOs: 6, 17, or 27.

10. 10. The antibody of any preceding claim, further comprising a heavy chain constant region fused to the mature heavy chain variable region and a light chain constant region fused to the mature light chain variable region.

11. 11. The antibody of any one of claims 1 to 10, comprising multiple pairs of mature heavy chain variable regions and mature light chain variable regions, wherein one of the pairs comprises the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 derived from SEQ ID NO: 1, and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 derived from SEQ ID NO: 2, and the other of the pair binds to a target antigen.

12. 11. A bispecific antibody comprising two pairs of mature heavy chain variable regions and mature light chain variable regions, one of which comprises the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 derived from SEQ ID NO: 1, and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 derived from SEQ ID NO: 2, and the other of which binds to a target antigen, the antibody described in any one of claims 1 to 10.

13. 13. The antibody of claim 11 or 12, wherein the target antigen is a cancer-associated antigen, an immune cell antigen, or an antigen on a pathogen or a pathogen-infected cell.

14. The antibody of claim 12 or 13, wherein one of the pair of mature heavy chain variable region and mature light chain variable region is an scFv, and the other of the pair of mature heavy chain variable region and mature light chain variable region further comprises a heavy chain constant region linked to the mature heavy chain variable region and a light chain constant region linked to the mature light chain variable region.

15. The antibody of claim 12 or 13, wherein the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 derived from SEQ ID NO: 1 and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 derived from SEQ ID NO: 2 are linked as an scFv, and the other mature heavy chain variable region and mature light chain variable region of the pair are linked to a heavy chain constant region and a light chain constant region, respectively.

16. The antibody of claim 15, wherein the scFv is linked to the heavy chain constant region.

17. The antibody of claim 16, wherein the scFv is linked to the heavy chain constant region via the mature light chain variable region of the scFv.

18. The antibody of claim 17, wherein the scFv has a sequence comprising SEQ ID NO: 31 or 33.

19. The antibody of claim 16, wherein the scFv is linked to the heavy chain constant region via the mature heavy chain variable region of the scFv.

20. 20. The antibody of claim 19, wherein the scFv has a sequence comprising SEQ ID NO:

29.

21. The antibody of claim 15, wherein the scFv is linked to the N-terminus of the other mature heavy chain variable region or mature light chain variable region of the pair.

22. 2. The antibody of claim 1, having the form of or comprising an scFv, wherein the scFv comprises the mature heavy chain variable region fused to the mature light chain variable region via a linker.

23. 2. The antibody of claim 1, having an array comprising any one of SEQ ID NOs: 29, 31 or 33.

24. A pharmaceutical composition comprising an antibody according to any of the preceding claims.

25. 24. A method for treating cancer, comprising administering to a patient having cancer the antibody of any one of claims 11 to 23, wherein the target antigen is a cancer-associated antigen.

26. A method for treating an immune disease, comprising administering to a patient having said immune disease an antibody according to any one of claims 1 to 23.

27. 24. A method for treating a pathogen infection, comprising administering to a patient infected with said pathogen an antibody according to any one of claims 11 to 23, wherein the target antigen is an antigen of said pathogen or a pathogen-infected cell.

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