Anti-cd3 antibodies

Abstract

The invention provides antibodies specifically binding to human CD3. The antibodies can be monospecific (each binding site binds the same target, i.e., CD3), bispecific at least two binding sites for two targets including human CD3, or multi-specific (multiple binding sites for multiple targets including human CD3). The antibodies can be used in treatment of cancer, infectious disease and immune conditions among other disorders.

Description

ANTI-CD3 ANTIBODIES CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of US63 / 426,626 filed November 18, 2022, which is incorporated by reference in its entirety for all purposes. SEQUENCE LISTING

[0002] The application includes sequences in XML file 603846SEQLST of 66,000 bytes created November 12, 2023, which is incorporated by reference. BACKGROUND

[0003] Antigen-specific immune response by the adaptive immune system is a complex biological process that is controlled by multiple layers of positive and negative regulators. Naïve T cells are initially stimulated through the T cell receptor (TCR) complex, which comprises TCR α and β (or γ and δ) chains and CD3 molecules, by the recognition of their cognate peptide antigen presented by major histocompatibility complex (MHC; also called HLA for human proteins) molecules on antigen-presenting cells (APCs). The initial interaction between TCR and MHC (or HLA) for T cell activation is referred to as Signal 1. Optimal T cell activation and proliferation requires a second signal provided by 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 (Signal 2). The immune system is further regulated positively by other costimulatory molecules such as CD40, OX40, GITR, CD27, HVEM and 4-1BB that 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 in a cell type- and development stage-dependent manner to delicately control immune responses in the body. In addition, a variety of secreted proteins, such as cytokines and chemokines, are involved in regulation of immune responses by promoting activation, differentiation, proliferation, maintenance, and suppression of certain subsets of immune cells. The action of cytokines on T cells is often referred to as Signal 3, which is the third mechanism required for activation, differentiation, and proliferation of T cells. 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, 1 LEGAL02 / 43629467v12016; Torphy et al., Int. J. Mol. Sci.18:2642, 2017; Punt et al., Kuby Immunology, Eighth Edition. W.H. Freeman and Co., New York, 2018.

[0004] There are two major groups of mature T cells that are CD4+ helper T cells and CD8+ cytotoxic T cells. CD4+ helper T cells are further divided into TH1, TH2, TH9, TH17, TH22, TFH, and Treg cells, each of which has specific functions and distinctive cytokine expression pattern. The main function of CD4+ helper T cells is to regulate other immune cells, such as B cells and CD8+ cytotoxic T cells, for proper and timely responses in the immune defense system. In contrast, the main function of CD8+ cytotoxic T cells is to destroy cells infected or transformed by pathogens in an antigen-specific manner. On activation by Signals 1 and 2, CD8+ cytotoxic T cells secrete perfolin and granzymes, which synergistically induce apoptosis of target cells. CD8+ cytotoxic T cells also play an important role in 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 engineered monoclonal antibodies that are capable of binding to two distinct antigens. Recent studies have reported that bispecific antibodies binding to CD3 expressed on T cells and a cancer-associated surface molecule can bridge T cells to cancer cells and trigger T cell-mediated cytotoxicity against the cancer cells. This type of bispecific antibodies is called a T cell engager. Several T cell engagers have been approved for marketing as human therapeutics for treatment of cancer, including Blincyto® (blinatumomab; anti-CD19 / CD3) for treatment of 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 evaluated in clinical studies for 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. 2 LEGAL02 / 43629467v1SUMMARY OF THE CLAIMED INVENTION

[0006] The invention provides an antibody specifically binding to human CD3, comprising a mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 from SEQ ID NO:1 and a mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 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 mouse antibody. Optionally, the antibody is chimeric or veneered. Optionally, the antibody is a humanized antibody, comprising a humanized mature heavy chain variable region and a humanized mature light chain variable region. Optionally, positions 30, 49, 93 and 94 by 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 consisting essentially of SEQ ID NO:4 and the mature light chain variable region has an amino acid sequence consisting of or consisting essentially of SEQ ID NOS: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 multi-specific antibody comprising multiple pairs of mature heavy chain and light chain variable regions, one of which pairs comprises the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 from SEQ ID NO:1 and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 from SEQ ID NO:2, and another of which pairs binds to a target antigen.

[0008] Optionally, the antibody is a bispecific antibody comprising two pairs of mature heavy and light chain variable regions, one of which pairs comprises the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 from SEQ ID NO:1 and the mature 3 LEGAL02 / 43629467v1light chain variable region comprising CDRL1, CDRL2 and CDRL3 from SEQ ID NO:2, and the other of which pairs binds to a 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 pairs of mature heavy and light chain variable regions is an scFv and the other pair of mature heavy and 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 from SEQ ID NO:1 and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 from SEQ ID NO:2 are linked as an scFv and the mature heavy and light chain variable regions of the other pair are linked to a heavy chain constant region and a 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 consistent 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 mature heavy chain or light chain variable region of the other pair.

[0012] The invention further provides antibodies in the form of or comprising an scFv. An scFv comprises a heavy chain variable region fused via a linked to a light chain variable region as a single chain. Optionally, the antibody has a sequence comprising any of SEQ ID NO:29, 31 or 33. Optionally, the antibody has a sequence consisting of or consisting essentially of any of SEQ ID NO:29, 31 or 33.

[0013] The invention further provides a pharmaceutical composition comprising any antibody as described above or otherwise disclosed herein. 4 LEGAL02 / 43629467v1

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

[0015] The invention further provides a method of treating an immune condition comprising administering an antibody as described above or otherwise disclosed herein e to a patient having the immune condition.

[0016] The invention further provides a method of treating a pathogenic infection comprising administering the antibody as described above or otherwise disclosed herein to a patient infected with the pathogen, whether the target antigen is an antigen of the pathogen or a pathogen- infected cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Fig.1: Alignment of the mature SP34 VH, HuSP34 VH1, and the human acceptor M24236 VH amino acid sequences. 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 according to the definition of Kabat et al. (supra) are underlined in SP34 VH. The symbol “-“ indicates that no amino acid residue exists at the corresponding location.

[0018] Fig.2: Alignment of the mature SP34 VL, HuSP34 VL1, and the human acceptor Y14738 VL amino acid sequences. Residue numbers are assigned according to Kabat et al. (supra). CDR sequences according to the definition of Kabat et al. (supra) are underlined in SP34 VL. The symbol “-“ indicates that no amino acid residue exists at the corresponding location.

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

[0020] Fig.4: Nucleotide sequence of the HuSP34 VL1 gene flanked by NheI and EcoRI sites (underlined) (SEQ ID NO:59) along with the deduced amino acid sequence (SEQ ID NO:60). 5 LEGAL02 / 43629467v1

[0021] Figs.5A-C: Schematic structures of pHuSP34A (A), pHuSP34C (B) and pJB554 (C).

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

[0023] Fig.7: Alignment of the mature SP34 VL, HuSP34 VL3, HuSP34 VL4, and the human acceptor L37309 VL amino acid sequences. Residue numbers are assigned according to Kabat et al. (supra). CDR sequences according to the definition of Kabat et al. (supra) are underlined in SP34 VL. The symbol “-“ indicates that no amino acid residue exists at the corresponding location.

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

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

[0026] Fig.10: Schematic structure of JB554 and JB559 bispecific antibodies.

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

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

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

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

[0031] Antibodies of the invention are typically provided in isolated form. This means that an antibody is typically at least 50% w / w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the antibody is combined with an excess of pharmaceutical acceptable carrier(s) or other vehicle intended to 6 LEGAL02 / 43629467v1facilitate its use. Sometimes bispecific antibodies are at least 60, 70, 80, 90, 95 or 99% w / w pure of interfering proteins and contaminants from production or purification. Often an antibody is the predominant macromolecular species remaining after its purification.

[0032] Specific binding of an antibody to its target antigen or target antigens for a bispecific or multispecific antibody means an affinity of at least 106, 107, 108, 109, or 1010M-1. An antibody specifically binding to a target can also be referred to as an antibody against the target. Affinities can be different for the different targets. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody with the same two binding sites binds only one target or a bispecific antibody with two different binding sites binds only against targets for these two binding sites.

[0033] A basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without the light chain signal peptide. However, reference to a variable region does not mean that a signal sequence is necessarily present; and in fact signal sequences are cleaved once the antibodies of the invention have been expressed and secreted. A pair of heavy and light chain variable regions defines a binding region of an antibody. The carboxy-terminal portion of the light and heavy chains respectively defines light and heavy chain constant regions. 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. The CH1 region binds to the light chain constant region by disulfide and noncovalent bonding. The hinge region provides flexibility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide 7 LEGAL02 / 43629467v1bonding between the two heavy chain constant regions in a tetramer subunit. The CH2 and CH3 regions are the primary site of effector functions and FcRn binding.

[0034] Light chains are classified as either 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. Within light and heavy chains, the variable and constant regions are joined by a "J" segment of about 12 or more amino acids, with the heavy chain also including a "D" segment of about 10 or more amino acids. (See generally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 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 divalent. In natural antibodies, the binding sites are the same. Binding sites within bispecific or multi-specific antibodies can be the same or different from one another depending on the form (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). The variable regions all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with 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 definition of CDRs as indicated in Table 1 below. 8 LEGAL02 / 43629467v1Table 1 Conventional Definitions of CDRs Using Kabat Numbering Composite IMGT of Chothialoop). This is because the Kabat numbering scheme places insertions of extra residues at 35A and 35B, whereas Chothia numbering places them at 31A and 31B. If neither H35A nor H35B (Kabat numbering) is present, the Chothia CDR-H1 loop 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 convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. Although Kabat numbering can be used for antibody heavy chain constant regions, the EU index (also called EU numbering) is more commonly used, as is the case in this application. When an antibody is said to comprise CDRs by a certain definition of CDRs (e.g., Kabat) that definition specifies the minimum number of CDR residues present in the antibody (i.e., the Kabat CDRs). It does not exclude that other residues falling within another conventional CDR definition but outside the specified definition are also present. For example, an antibody comprising CDRs defined by Kabat includes among other possibilities, an antibody in which the CDRs contain Kabat CDR residues and no other CDR residues, and an antibody in which CDR H1 is a composite Chothia-Kabat 9 LEGAL02 / 43629467v1CDR H1 and other CDRs contain Kabat CDR residues and no additional CDR residues based on other definitions.

[0038] The term “antibody” includes intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, 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 a bispecific or multispecific antibody.

[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. An epitope 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 known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. Some antibodies bind to an end-specific epitope, meaning an antibody binds preferentially to a polypeptide with a free end relative to the same polypeptide fused to another polypeptide resulting in loss of the free end. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol.66, Glenn E. Morris, Ed. (1996).

[0040] The term "antigen" or "target antigen" indicates a target molecule bound by an antibody or one binding site of a bispecific antibody. An antigen may be a protein of any length (natural, synthetic or recombinantly expressed), a nucleic acid or carbohydrate among other molecules. Antigens include receptors, ligands, counter receptors, and coat proteins.

[0041] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined by X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or 10 LEGAL02 / 43629467v1eliminate binding of one antibody 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 reduce or eliminate binding of the other.

[0042] Competition between antibodies is determined by an assay in which an antibody under test inhibits 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 a test antibody (e.g., at least 2 times, 5 times, 10 times, 20 times or 100 times) inhibits binding of the reference antibody by at least 50% but preferably 75%, 90% or 99% as measured in a competitive binding assay. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.

[0043] The term "subject" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. Other mammalian subjects include animal models of a human condition (e.g., rodent, non-human primate) and veterinary subjects.

[0044] For purposes of classifying amino acids substitutions as conservative or nonconservative, 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 influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non- conservative substitutions constitute exchanging a member of one of these classes for a member of another.

[0045] Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention for a variable region or EU numbering for a constant region. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with 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 the same amino acid in both the subject and reference antibody region 11 LEGAL02 / 43629467v1divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.

[0046] Compositions or methods "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition that comprises an antibody may contain the antibody alone or in combination with other ingredients.

[0047] The term "antibody-dependent cellular cytotoxicity", or ADCC, is a mechanism for inducing cell death that depends upon the interaction of antibody-coated target cells (i.e., cells with bound antibody) with immune cells possessing lytic activity (also referred to as effector cells). Such effector cells include natural killer cells, monocytes / macrophages and neutrophils. ADCC is triggered by interactions between the Fc region of an antibody bound to a cell and Fcγ receptors, particularly FcγRI and FcγRIII, on immune effector cells such as neutrophils, macrophages and natural killer cells. The target cell is eliminated by phagocytosis or lysis, depending on the type of mediating effector cell. Death of the antibody-coated target cell 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, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils and dendritic cells) that bind to an immunoglobulin Fc region.

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

[0050] pH-dependent binding of an antibody to an FcRn receptor means that an antibody binds more strongly to such a receptor at pH 6.0 than at pH 7.5. Binding to FcRn at a low pH in endosomes after internalization by pinocytosis rescues IgG antibodies from catabolic degradation 12 LEGAL02 / 43629467v1in lysosomes. Rescued IgG antibodies are then released from FcRn at a neutral pH and recycled to the circulation. Such pH-dependent FcRn binding is the basis of the molecular mechanism for a long serum half-life of IgG antibodies (and bispecific antibodies of the invention) (Ghetie et al., Annu. Rev. Immunol.18:739-766, 2000). For example, human IgG antibodies bind to human neonatal Fc receptors (FcRn) at pH 6.0 while they bind only weakly to FcRn at pH 7.5. The FcRn binding site in IgG antibodies lies at the junction of the CH2 and CH3 domains. Because a mu heavy chain does not bind to FcRn at pH 6.0 or 7.5, natural IgM cannot take advantage of the FcRn-mediated pathway to rescue antibodies from degradation in lysosomes and therefore in general have shorter half-lives than natural IgG antibodies.

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

[0052] Protein G is a 65-kDa (G148 protein G) and a 58 kDa (C40 protein G) Streptococcal cell surface protein. It contains a serum albumin binding domain not needed for IgG binding, which is often deleted. Protein G specifically binds to all of the human IgG isotypes but not IgA or IgM. Protein G is also useful for antibody purification. DETAILED DESCRIPTION I. General

[0053] The invention provides antibodies specifically binding to human CD3. The antibodies can be monospecific (each binding site binds the same target, i.e., CD3), bispecific at least two binding sites for two targets, one of which is human CD3, or multi-specific (multiple binding sites for multiple targets, one of which is human CD3). The antibodies can be used in treatment of cancer, infectious disease and immune conditions among other disorders. II. Targets

[0054] Human CD3 is a complex including 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 13 LEGAL02 / 43629467v1includes the exemplified human forms and other known allelic variants in humans as indicated in the Swiss Prot database.

[0055] One class of protein that can serve as a second target in bispecific or multi-specific antibodies is a cancer-associated antigen. Such antigens are expressed by a cancer, typically at higher levels than control matched normal tissue (overexpressed). Some examples of cancer associated antigens include alpha-folate receptor (ovarian and epithelial cancers), CAIX (renal carcinoma), CD19 (B-cell malignancies, CLL, ALL), CD20 (B-cell malignancies, lymphomas), CD22 (B-cell malignancies), CD23 (CLL), CD24 (pancreatic CA), CD30 (lymphomas), CD33 (AML), CD38 (NHL), CD44v7 / 8 (cervical CA), CEA (colorectal CA), EGFRvIII (glioblastoma), EGP-2 (multiple malignancies), EGP-40 (colorectal CA), EphA2 (glioblastoma), Erb-B2 (breast, prostate, colon CA), FBP (ovarian CA), G.sub.D2 (neuroblastoma, melanoma), GD3 (melanoma), HER2 (pancreatic CA, ovarian CA, glioblastoma, osteosarcoma), HMW-MAA (melanoma), IL-11Rα (osteosarcoma), IL-13Rα2 (glioma, glioblastoma), KDR (tumor vasculature), kappa-light chain (B-cell malignancies), Lewis Y (various carcinomas), L1 (neuroblastoma), MAGE-A1 (melanoma), mesothelin (mesothelioma), MUC1 (breast and ovarian CA), MUC16 (ovarian CA), NKG2D (myeloma, ovarian CA), NY-ESO-1 (multiple myeloma), oncofetal antigen (various tumors), PSCA (prostate CA), PSMA (prostate CA), ROR1 (B-CLL), TAG-72 (adenocarcinomas), and VEGF-R2 (tumor neovasculature). (Sadelain et al., Cancer Discov 3:388-98, 2013). Other tumor-associated antigens that can be targeted include alpha-fetoprotein (AFP), alpha-actinin-4, A3, ART-4, B7, 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, CD55, 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, HER2 / neu, HMGB-1, hypoxia inducible factor 14 LEGAL02 / 43629467v1(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, placental 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 receptors, transferrin receptor, TNF-α, Tn antigen, 5T4,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl- 6, Kras, an oncogene marker or an oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006, 12:5023-32; 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 cancers of myeloid origin, such as acute myeloid leukemia. EGFR binds EGF and is overexpressed primarily in gastric, breast, endometrial, colorectal cancer, head and neck cancer, ovarian, cervical, bladder and esophageal cancers. PD-L1 binds PD1 and is overexpressed in cancers such as gastric cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer and bladder cancer.

[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 disease, such as autoimmune diseases. III. Antibodies against CD3

[0057] An exemplary antibody against CD3 is referred to as SP34. This antibody is characterized by a mature heavy chain variable region of SEQ ID NO:1 and a mature light chain variable region of SEQ ID NO:2. Kabat CDRs H1, H2 and H3 of the mature heavy chain variable region are provided by SEQ ID NOS:43-45 respectively and 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 agonist antibody that specifically binds to the extracellular domain of the epsilon chain of 15 LEGAL02 / 43629467v1human CD3. Cross-linking of CD3 by SP34 induces activation of T cells. SP34 specifically binds to human and a wide range of non-human primates (e.g., chimpanzee, cynomolgus, rhesus) CD3. By contrast other anti-CD3 antibodies widely used in T cell engagers (e.g., M291, OKT3) have been reported not to specifically bind to cynomolgus or rhesus. The ability to specifically bind CD3 from nonhuman primates is advantageous for preclinical work in animal models. 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 an antibody designated SP34 and / or compete with SP34 or other antibody sharing its mature variable regions for binding to human CD3. Other antibodies having such a binding specificity can be produced by immunizing mice with human CD3 or a portion thereof including the desired epitope, and screening resulting antibodies for binding to human CD3, optionally in competition with SP34. Antibodies can be screened against mutagenized forms of the human CD3 antigen to identify an antibody showing the same or similar binding profile to collection of mutational changes as SP34. The mutations can be systematic replacement substitution with alanine (or serine if an alanine is present already) one residue at a time, or more broadly spaced intervals, throughout the extracellular domain of human CD3 or through a section thereof in which an epitope is known to reside.

[0059] Antibodies having the binding specificity of SP34 can also be produced using a variant 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 the selected murine antibody is used as a starting material. If, for example, 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 murine starting material) and a different heavy chain variable region. The heavy chain variable regions can for example be obtained from a library of rearranged human heavy chain variable regions. A phage showing strong specific binding for human CD3 (e.g., at least 108and preferably at least 109M-1) is selected. The heavy chain variable region from this phage then serves as a starting material for constructing a further phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable regions can be obtained for example from a library of 16 LEGAL02 / 43629467v1rearranged human variable light chain regions. Again, phage showing strong specific binding for CD3 are selected. The resulting antibodies usually 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 an exemplary antibody, such as SP34. Monoclonal antibodies that are at least 90%, 95% or 99% identical to SP34 in amino acid sequence of the mature heavy and / or light chain variable regions and maintain its functional properties, and / or which differ from the respective antibody by a small number of functionally inconsequential amino acid substitutions (e.g., conservative substitutions), deletions, or insertions are also included in the invention. Monoclonal antibodies having at least one and preferably all six CDR(s) as defined by Kabat that are 90%, 95%, 99% or 100% identical to corresponding CDRs of SP34 are also included.

[0061] Antibodies of the invention can be provided in a conventional tetrameric format including two pairs of heavy and light chain variable regions, or in the form of or comprising fragments, such as scFv’s. Antibodies can comprise, consist of or consist essentially of any of the mature heavy chain and light chain variable regions disclosed and their combinations. A. Non-human antibodies

[0062] The production of other non-human monoclonal antibodies, e.g., murine, guinea pig, primate, rabbit or rat, against human CD3 can be accomplished by, for example, immunizing the animal with human CD3 or a fragment thereof, or cells bearing human CD3, optionally co- expressed with its co-receptor proteins as discussed above. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source, by peptide synthesis or by recombinant expression. Optionally, the immunogen can be administered fused or otherwise complexed with a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvant can be used as described below. Complete Freund’s adjuvant followed by incomplete adjuvant is preferred for immunization of laboratory animals. Rabbits or guinea pigs are typically used for making polyclonal antibodies. Mice are typically used for making monoclonal antibodies. Antibodies are screened for specific binding to human CD3. Optionally, antibodies are further screened for binding to a specific region of human CD3. Such screening 17 LEGAL02 / 43629467v1can be accomplished by determining binding of an antibody to a collection of deletion mutants of human CD3. Binding can be assessed, for example, by Western blot, FACS or ELISA. B. Humanized antibodies

[0063] A humanized antibody is a genetically engineered antibody in which the CDRs from a non-human “donor” antibody are grafted into human “acceptor” antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539, Carter, US 6,407,213, Adair, US 5,859,2056,881,557, Foote, US 6,881,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, 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 entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85, 90, 95 or 100% of corresponding residues defined by Kabat are identical.

[0064] Although humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5) CDRs from a mouse antibody (e.g., Pascalis et al., J. Immunol.169:3076, 2002; Vajdos 18 LEGAL02 / 43629467v1et 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 part of the CDRs, namely the subset of CDR residues required for binding, termed the SDRs, are needed to retain binding in a humanized antibody. CDR residues not contacting antigen and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from regions of Kabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol. Biol.196:901, 1987), by molecular modeling and / or empirically, or as described in Gonzales et al., Mol. Immunol.41: 863, 2004. In such humanized antibodies at positions in which one or more donor CDR residues is absent or in which an entire donor CR is omitted, the amino acid occupying the position can be an amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids in the CDRs to include reflects a balance of competing considerations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity. However, substitutions can also cause changes of affinity, and significant reductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.

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

[0067] Certain amino acids from the human variable region framework residues can be selected for substitution based on their possible influence on CDR conformation and / or binding to antigen. Investigation of such possible influences is by modeling, examination of the 19 LEGAL02 / 43629467v1characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.

[0068] For example, when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be substituted by the equivalent framework amino acid from the mouse antibody when it is reasonably expected that the amino acid: (1) noncovalently binds antigen directly, (2) is adjacent to a CDR region, (3) otherwise interacts with a CDR region (e.g. is within about 6 Å of a CDR region).

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

[0070] An exemplary humanized heavy chain variable region of SP34 is termed HuSP34 VH1 and assigned SEQ ID NO:4. At framework positions 30, 49, 93 and 94, by Kabat numbering, where the three-dimensional model of the SP34 variable regions indicated that amino acid residues could play significant roles in the formation of the antigen-binding site, amino acid residues of M24236 VH can be replaced by the corresponding residues of mouse SP34 VH, namely N, A, V and R. An exemplary humanized light chain variable region termed HuSP34 VL1 is assigned SEQ ID NO:6. This light chain variable region contains backmutations to mouse residues at positions 36, 46 and 49 by Kabat numbering, with these positions occupied by V, G and G respectively. Another exemplary humanized light chain variable region based on a kappa acceptor sequence is termed 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 by Kabat numbering with these positions occupied by V, G, G, V, L, I, D, K and A respectively. Another 20 LEGAL02 / 43629467v1humanized light chain variable region is designated HuSP34 VL4 and assigned SEQ ID NO:27. SEQ ID NO:27 is the same as SEQ ID NO:17 except it eliminates some backmutations not required for binding but which may increase immunogenicity. The remaining backmutations still present in SEQ ID NO:27 occur at positions 36, 46, 49, 66 and 71 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 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 NOS:6, 17 or 27, preferably SEQ ID NO:27. The invention also provides variants of the humanized anti-CD3 IgG1 / kappa antibody termed HuSP34V comprising HuSP34 VH1 (SEQ ID NO:4) and HuSP34 VL4 (SEQ ID NO:27). Such variants typically differ from the sequences of the variable regions of HuSP34V by a small number (e.g., typically no more than 1, 2, 3, 5, 7, 8, 9 or 10) of replacements, deletions or insertions. Such differences are usually in the framework but can also occur in the CDRs. For example, only a subset of substitutions at heavy chain positions 30, 49, 93 and 94 and light chain positions 36, 46, 49, 66 and 71 can be made. Many of the framework residues not in contact with the CDRs in the humanized mAb can accommodate substitutions of amino acids from the corresponding positions of the donor mouse mAb or other mouse or human antibodies, and even many potential CDR-contact residues are also amenable to substitution or even amino acids within the CDRs may be altered. One example of a CDR substitution is to substitute a residue in a CDR with the residue occupying the corresponding position of the human acceptor sequence used to supply variable region frameworks.

[0072] Often the replacements made in the variable regions of the variant HuSP34V sequences are conservative with respect to the replaced HuSP34V amino acids. Preferably, replacements in HuSP34V (whether or not conservative) have no substantial effect on the binding affinity or potency of the humanized mAb, that is, its ability to specifically bind to 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. Alternatively, other human 21 LEGAL02 / 43629467v1antibody acceptor sequences, particularly those with high sequence identity to the variable region framework sequences of HuSP34 are also suitable to provide the humanized antibody variable regions framework sequences.

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

[0074] The invention further provides chimeric and veneered forms of SP34.

[0075] A chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody (e.g., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence.

[0076] A veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T- cell epitopes, for example exposed residues (Padlan, Mol. Immunol.28:489, 1991) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions. Veneered forms of the SP34. D. Human Antibodies

[0077] Human antibodies against human CD3 are provided by a variety of techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter, above, or otherwise, to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the examples. Human antibodies can also be screened for a particular epitope specificity by using only a fragment of human CD3. 22 LEGAL02 / 43629467v1

[0078] Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No.4,634,664; and Engleman et al., US Patent 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al., WO93 / 12227 (1993); US 5,877,397; US 5,874,299; US 5,814,318; US 5,789,650; US 5,770,429; US 5,661,016; US 5,633,425; US 5,625,126; US 5,569,825; US 5,545,806; Neuberger, Nat. Biotechnol.14:826 (1996); and Kucherlapati, WO 91 / 10741 (1991)) 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 US 5,565,332); and methods described in WO 2008 / 081008 (e.g., immortalizing memory B cells isolated from humans, e.g., with EBV, screening for desired properties, and cloning and expressing recombinant forms). IV. Bispecific antibodies

[0079] Bispecific or multi-specific antibodies are formed from pairs of heavy and light chain variable regions from component antibodies. The component antibodies can be rodent, chimeric, veneered, humanized, primatized, primate or human among others. The component antibodies can be of the same or different types; for example, one can be humanized and the other human.

[0080] One of the component antibodies of a bispecific or multi-specific antibody is an antibody against human CD3 as described above. The other component antibody or antibodies bind an antigen or antigens other than human CD3. For example, the other component antibody or antibodies can bind to a target present on cells to be depleted. Such cells include cancer cells, pathogens, such as viruses, bacteria or fungi and cells infected with any of these, and immune cells associated with an immune disorder, particularly autoimmune disorders.

[0081] Methods for producing non-human, humanized, chimeric, veneered and human antibodies have been described for antibodies directed against human CD3. The same methods can be adapted to produce antibodies against other targets in which the relevant target is used in place of human CD3. Many antibodies approved for therapeutic use as monospecific antibodies can be incorporated into bispecific antibodies. Table 2 below provides examples of approved antibodies for treatment of cancer. 23 LEGAL02 / 43629467v1Table 2 Name Antigen Format Indications (Year of First Approval) Unconjugated Antibodies Atezolizumab PD-L1 Humanized IgG1 Bladder, Non-small cell lung (2016), and Triple- negative breast (2019) cancers (2019) Avelumab PD-L1 Human IgG1 Urothelial Carcinoma (2017) and Merkel Cell Carcinoma (2017) Bevacizumab VEGF Humanized IgG1 Colorectal (2004), Non-small cell lung (2006), Renal (2009), Glioblastoma (2009), and Ovarian (2018) Cancers Cemiplimab PD-1 Human IgG4 Cutaneous squamous-cell carcinoma (2018) Cetuximab EGFR Chimeric IgG1 Colorectal cancer (2004) and Head and neck squamous cell carcinoma (2006) Daratumumab CD38 Human IgG1 Multiple Myeloma (2015) Dinutuximab GD2 Chimeric IgG1 Neuroblastoma (2015) Durvalumab PD-L1 Human IgG1 Bladder Cancer (2017) Elotuzumab SLAMF7 Humanized IgG1 Multiple Myeloma (2015) Ipilimumab CTLA-4 Human IgG1 Melanoma (2011) and Renal cell carcinoma (2018) 24 LEGAL02 / 43629467v1Name Antigen Format Indications (Year of First Approval) Isatuximab CD38 Chimeric IgG1 Multiple Myeloma (2020) Mogamulizumab CCR4 Humanized IgG1 Cutaneous T-cell lymphoma (2018) Necitumumab EGFR Human IgG1 Non-small cell lung cancer (2015) Nivolumab PD-1 Human IgG4 Melanoma (2014), Lung (2015), and Renal (2018) cancers Obinutuzumab CD20 Humanized IgG2 Chronic lymphocytic leukemia (2013) Ofatumumab CD20 Human IgG1 Chronic lymphocytic leukemia (2014) Olaratumab PDGFRα Human IgG1 Sarcoma (2016) Panitumumab EGFR Human IgG2 Colorectal Cancer (2006) Pembrolizumab PD-1 Humanized IgG4 Melanoma (2014), Various (2015-) Pertuzumab HER2 Humanized IgG1 Breast cancer (2012) Ramucirumab VEGFR2 Human IgG1 Gastric cancer (2014) Rituximab CD20 Chimeric IgG1 B-Cell Lymphoma (1997) 25 LEGAL02 / 43629467v1Name Antigen Format Indications (Year of First Approval) Trastuzumab HER2 Humanized IgG1 Breast cancer (1998) Antibody–Drug Conjugates (ADCs) Gemtuzumab CD33 Humanized Acute myeloid leukemia (2000) ozogamicin ADC Brentuximab CD30 Chimeric ADC Hodgkin’s lymphoma and Anaplastic large-cell vedotin lymphoma (2011) Trastuzumab HER2 Humanized Breast cancer (2013) emtansine ADC Inotuzumab CD22 Humanized Acute lymphoblastic leukemia (2017) ozogamicin ADC Polatuzumab CD79B Humanized B-Cell Lymphoma (2019) vedotin ADC Enfortumab vedotin Nectin-4 Human ADC Bladder cancer (2019) Trastuzumab HER2 Humanized Breast cancer (2019) deruxtecan ADC Sacituzumab TROP2 Humanized Triple negative breast cancer (2020) govitecan ADC 26 LEGAL02 / 43629467v1Name Antigen Format Indications (Year of First Approval) Moxetumomab CD22 Mouse ADC Hairy-cell leukemia (2018) pasudotox Ibritumomab CD20 Mouse IgG1- Non-Hodgkin’s lymphoma (2002) tiuxetan Y90 or In111 Iodine (I131) CD20 Mouse IgG2- Non-Hodgkin’s lymphoma (2003) tositumomab I131 Blinatumomab CD19, Mouse BiTE Acute lymphoblastic leukemia (2014) CD3

[0082] Over 100 formats have been described for bispecific or multispecific antibodies (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 include at least one binding site for each of at least two targets. Some formats include two or more binding sites for each target.

[0083] Some formats have a similar tetrameric structure to a normal antibody 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 a normal antibody in that the two binding sites and pairs of heavy and light chains forming them are different. Thus, such antibodies require association of two different pairs of heavy and light chains.

[0084] The “knobs-into-holes” approach has been adopted to reduce formation of homodimers and mispairing of heavy chains by substituting a large amino acid for a small one in the CH3 domain (the “knob”) of one antibody and vice versa (the “hole”) of the other antibody (Ridgway et al., Protein Eng 9:617-21, 1996; Atwell et al., J Mol Biol 270:26-35, 1997; and U.S. Pat. No.7,695,936). Light chain mispairing in such formats can be reduced by a number of strategies. One strategy is to use a common light chain variable region for two different heavy 27 LEGAL02 / 43629467v1chain variable regions. But this is applicable only to some antibodies. Another approach has been to express the knob- and the hole-containing half-molecules separately in different bacteria. Another approach termed CrossMab swaps the CH1 domain of one of the heavy chain with the constant CL domain of the corresponding light chain to induce the right pairing between the engineered 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 has been to introduce additional mutations into VH-VL and CH1-CL interfaces (Lewis et al., Nat. Biotechnol., 32 (2014), pp.191-198). These mutations encourage a heavy chain to preferentially pair with a light chain. Another approach has been to introduce mutations promoting protein A binding into one of the Fc regions and select heterodimeric pairing having intermediate protein A binding from homodimers having higher or lower protein A binding by affinity chromatography (Tusdian et al, MAbs.2016 May-Jun;8(4):828-38).

[0085] Other bispecific antibodies avoid the problem of mispairing by combining multiple binding specificities in the same heavy and light chain pair. One approach for doing this, termed 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 an antibody can assemble as tetramer by association of two identical paired heavy and light chains. The assembled antibody includes two different binding sites for each target. Another approach is to link an scFv providing a binding site for one target to the N-terminus of the heavy chain variable region or light chain variable region of a heavy chain-light chain providing a binding site of a different target. Such a bispecific antibody can also assemble as a tetramer.

[0086] Another approach, which is followed in the examples of the present invention, is to incorporate a second binding specificity by linking a single-chain Fv (scFv) to the C-terminus of a heavy chain constant region, optionally omitting the C-terminal lysine residue. Such a bispecific antibody includes a first binding site formed by heavy and light chain variable regions attached to the N-termini of heavy and light chain constant regions as in a standard antibody. The C-terminus of the heavy chain is attached to an scFv providing the second binding site. The scFv is usually attached via a linker and a further linker connects the heavy and light chain variable regions in the scFv. The scFv can be attached either through its light chain variable 28 LEGAL02 / 43629467v1region or heavy chain variable region end via the linker to the Fc region. When assembled by complexing of two identical paired heavy and light chains, such a bispecific antibody includes two binding sites for each of two different specificities. Antigen-binding arms for CD3 and other target of such a bispecific antibody can be attached in either orientation. That is the antigen- binding arms for CD3 can be in the form of an scFv and the antigen-binding arms for the other target in a standard antibody format or vice versa. The arm to be attached to the N-termini of the heavy and light chain constant regions is provided as separate heavy and light chain variable regions, and that to be attached to the C-terminus is provided as an scFv fragment. An advantage of this format is that the two different binding spaces are separated by the entire heavy chain constant region, which may promote cell-to-cell bridging. Optionally a substitution to a cysteine residue can be introduced into each of the heavy and light chain variable regions in an scFv (e.g., at heavy chain variable region position 44 and at light chain variable region position 100 by Kabat numbering) for increased stability.

[0087] Another format links an scFv specifically binding to a first target to a heavy chain constant region and an scFv specifically binding to another target to a light chain constant region. Such an antibody assembles into a tetramer including 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 on a single chain without a constant region. For example, the BiTe format links two scFv fragments through a linker (see, e.g., Ross et al., PLoS ONE 12(8): e0183390, 2017). Such formats lack effector functions and tend to have a short half-life but may have advantages of accessibility and ease of manufacture due to their small size.

[0089] Many of the above formats include linker peptides between heavy and light variable regions or between variable regions and a constant region. Linkers are short peptide conferring flexibility often predominantly occupied by 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 of the combinations of heavy and light chain variable region described above for the SP34 antibody, and its chimeric, veneered and humanized forms or other CD3 antibodies described herein can be incorporated into an scFv in the above formats. Exemplary scFv’s have a sequence comprising, consisting of or consisting essentially any of SEQ ID NO:29, 31, or 33. 29 LEGAL02 / 43629467v1An exemplary bispecific antibody comprises an scFv linked to the heavy chain constant region of another antibody via the mature light chain variable region of the scFv in which the scFv has a sequence comprising SEQ ID NO:31 or 33. Another exemplary bispecific antibody comprises an scFv linked to the heavy chain constant region of another antibody via the mature heavy chain variable region of the scFv, in which the scFv has a sequence comprising, consisting of or consisting essentially of SEQ ID NO:29. Selection of Constant Region

[0091] Many of the formats for a monospecific antibody, bispecific or multi-specific antibodies include at least a portion of a human constant region. The choice of constant region depends, in part, whether antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis and / or complement dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not. Light chain constant regions can be lambda or kappa. Human IgG1 and IgG3 also induce stronger cell mediated effector functions than human IgG2 and IgG4. Here although ADCC, ADCP and CDC may be useful in providing an additional mechanism of action against cancer or infected cells bound by one arm of the bispecific antibodies, it is not useful for agonizing CD3 by the other arm to activate immune cells.

[0092] One or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Counter ions may or may not be present to form a pharmaceutically acceptable salt. Water or other solvent may or may not be associated with an antibody (e.g., as a hydrate or solvate). Amino acid substitutions can be made in the constant regions 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 prolong 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 in making the hybrid proteins of the present invention compete more strongly with 30 LEGAL02 / 43629467v1endogenous IgG for binding to FcRn. Also numerous mutations are known for reducing any of 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 amino acid residues at positions 234, 235, 236 and / or 237 reduce affinity for Fcγ receptors, particularly FcγRI receptor (see, e.g., US 6,624,821). Optionally, amino acid residues at positions 234, 236 and / or 237 in human IgG2 are substituted with Ala and at position 235 with Gln or Glu (See, e.g., US 5,624,821). Other substitutions reducing effector functions 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, Leu at position 309.

[0093] Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of one or more other isotypes. Expression of Recombinant Antibodies

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

[0095] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences. 31 LEGAL02 / 43629467v1

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

[0097] Mammalian cells can be used for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed, and include 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 can be nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev.89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol.148:1149 (1992).

[0098] Alternatively, antibody coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression 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 light and / or heavy chains operably linked with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.

[0099] The vectors containing the DNA segments of interest can be transferred into the host cell by methods depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, 32 LEGAL02 / 43629467v1protoplast fusion, liposomes, electroporation, and microinjection. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

[0100] Having introduced vector(s) encoding antibody heavy and light chains into cell culture, cell pools can be screened for productivity and quality of antibodies in serum-free media. Top-producing cell pools can then be subjected to FACS™-based single-cell cloning to generate monoclonal lines. Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g / L culture, can be used. Antibodies produced by single cell clones can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HP- SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or BIACORE™. A selected clone can then be banked in multiple vials and stored frozen for subsequent use.

[0101] Once expressed, bispecific antibodies can be purified according to standard procedures of the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0102] Methodology for commercial production of antibodies can be employed, including codon optimization, selection of promoters, selection of transcription elements, selection of terminators, serum-free single cell cloning, cell banking, use of selection markers for amplification of copy number, CHO terminator, or improvement of protein titers (see, e.g., US 5,786,464; US 6,114,148; US 6,063,598; US 7,569,339; W02004 / 050884; W02008 / 012142; W02008 / 012142; W02005 / 019442; W02008 / 107388; W02009 / 027471; and US 5,888,809). Nucleic Acids

[0103] The invention further provides nucleic acids encoding any of the heavy and light chains described above. Optionally, such nucleic acids further encode a signal peptide and can be expressed with the signal peptide linked to the constant region coding sequences of nucleic acids can be operably linked with regulatory sequences to ensure expression of the coding sequences, such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. The nucleic acids encoding heavy and light chains can occur in isolated form or can 33 LEGAL02 / 43629467v1be cloned into one or more vectors. The nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding heavy and light chains can be joined as one contiguous nucleic acid, e.g., within an expression vector, or can be separate, e.g., each cloned into its own expression vector. Methods of Treatment and Pharmaceutical Compositions

[0104] The bispecific or multi-specific antibodies of the invention can be used for treating cancers in which one arm of the bispecific or multi-specific antibody binds to a target expressed or overexpressed in the cancer, such as those disclosed above. The bispecific or multi-specific 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 skin (e.g., melanoma), ovarian, endometrial, kidney, liver, pancreas, bladder, breast, ovarian, prostate, rectum, colon, gastric, intestinal, pancreatic, lung, thymus, thyroid, kidney and brain.

[0105] Bispecific antibodies of the invention can also be used for treating pathogenic infections when the bispecific antibody has one arm specifically binding to an antigen present on a pathogen or expressed in a cell infected by a pathogen but not in matched uninfected cells. Such an antigen can be encoded by the pathogen or can be expressed by the cell in response to infection by the pathogen. Examples of such antigens expressed in infected cells are human immune deficiency virus (HIV) glycoproteins gp41 and gp120, human T-cell leukemia virus type 1 (HTLV-1) Env protein, herpes simplex virus (HSV) glycoproteins gB and gH, influenza hemagglutinin (HA) and neuraminidase (NA), and respiratory syncytial virus (RSV) F protein. Examples of pathogenic infections treatable with bispecific antibodies include viral, bacterial, protozoan or fungal infection. Some example of viral infections include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein Barr virus), adenovirus, XMRV, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, MLV-related virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus. Some examples of bacterial infections include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, 34 LEGAL02 / 43629467v1legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, Lymes disease bacteria, streptococci, or neisseria. Some examples of pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys. Examples of protozoa include Cryptosporidium, Giardia lamblia and plasmodium.

[0106] Bispecific antibodies can also be used for treating immune disorders as can monospecific antibodies against human CD3. Examples of immune disorders include autoimmune diseases such as type 1 diabetes, Crohn's disease, ulcerative colitis, multiple sclerosis, stiff man syndrome, rheumatoid arthritis, myasthenia gravis and lupus erythematosus. In these disease, the body develops a cellular and / or humoral immune response against one of its own antigens leading to destruction of that antigen, and potentially crippling and / or fatal consequences. Autoimmune diseases are treated by administering one of the monoclonal antibodies of the invention. Other immune disorders treatable by the monoclonal antibodies of the invention, include asthma, allergies, celiac disease, psoriasis, and uveitis. Celiac disease, psoriasis and uveitis are autoimmune diseases.

[0107] Monospecific, bispecific or multispecific antibodies are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and / or ameliorates at least one sign or symptom of a condition. If a subject is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the subject is at elevated risk of the condition relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual subject relative to historical controls or past experience in the same subject. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated subjects relative to a control population of untreated subjects.

[0108] Preferably a bispecific or multispecific antibody exhibits at least additive and more preferably synergistic activity against a cancer or infected cell compared with its component antibodies individually. Synergy is preferably assessed quantitatively such as discussed by Tallarida, Genes Cancer.2011 Nov; 2(11): 1003–1008. Preferably a bispecific antibody also exhibits increased activity compared with a mixture of its component antibodies, each at 35 LEGAL02 / 43629467v1equimolar concentration with the bispecific antibody. Such activity can be measured, for example, as cytotoxicity or cytostaticity against cancer cells, infected cells or immune cells expressing an antigen specifically bound by one arm of the bispecific antibody in the presence of an immune cell expressing CD3.

[0109] Exemplary dosages for a monospecific, bispecific or multispecific antibody 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 dosage. The dosage depends on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.

[0110] Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Administration into the systemic circulation by intravenous or subcutaneous administration is preferred. Intravenous administration can be, for example, by infusion over a period such as 30-90 min.

[0111] The frequency of administration depends on the half-life of the antibody in the circulation, the condition of the subject and the route of administration among other factors. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or progression of the disorder being treated. An exemplary frequency for intravenous administration is between weekly and quarterly over a continuous cause of treatment, although more or less frequent dosing is also possible. For subcutaneous administration, an exemplary dosing frequency is daily to monthly, although more or less frequent dosing is also possible.

[0112] The number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment. For acute disorders or acute exacerbations of chronic disorders, between 1 and 10 doses are often sufficient. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder. Treatment can be repeated for recurrence of an acute disorder or acute exacerbation. For chronic disorders, a bispecific antibody can be administered at regular intervals, e.g., weekly, fortnightly, monthly, quarterly, every six months for at least 1, 5 or 10 years, or the life of the subject. 36 LEGAL02 / 43629467v1

[0113] Pharmaceutical compositions are preferably suitable for parenteral administration to a human (e.g., according to the standard of the FDA). Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. Pharmaceutically acceptable means suitable for human administration, e.g., approved or approvable by the FDA. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively antibodies can be in lyophilized form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0114] Treatment with antibodies of the invention can be combined with other treatments effective against the disorder being treated. When used in treating cancer, the antibodies of the invention can be combined with chemotherapy, radiation, stem cell treatment, surgery or treatment with other biologics such as HerceptinTM(trastuzumab) against the HER2 antigen, AvastinTM(bevacizumab) against VEGF, or antibodies to the EGF receptor, such as (ErbituxTM, cetuximab), and VectibixTM(panitumumab) or other antibody indicated in Table 2. Chemotherapy agents include chlorambucil, cyclophosphamide or melphalan, carboplatinum, daunorubicin, doxorubicin, idarubicin, and mitoxantrone, methotrexate, fludarabine, and cytarabine, etoposide or topotecan, vincristine and vinblastine. For infections, treatment can be in combination with antibiotics, anti-virals, anti-fungal or anti-protozoan agents or the like. Other Methods

[0115] The antibodies of the invention also find use in diagnostic, prognostic and laboratory methods. They may be used to measure the level of an antigen expressed by a cancer or in the circulation of a patient with a cancer, to determine if the level is measurable or even elevated, and therefore to follow and guide treatment of the cancer, because cancers associated with measurable or elevated levels of an antigen are most susceptible to treatment with a bispecific antibody comprising an arm binding to the cancer. The antibodies can be used for an ELISA 37 LEGAL02 / 43629467v1assay, radioimmunoassay or immunohistochemistry among others. The antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes or radioisotopes, and may be provided in the form of a kit with all the necessary reagents to perform the assay.

[0116] All patent filings, websites, other publications, accession numbers and the like 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 so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although 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 may be practiced within the scope of the appended claims. Example 1: General procedures, methods, and materials

[0117] Gene cloning, mutagenesis, plasmid construction, protein expression and purification, cell culturing, ELISA, and flow cytometry were carried out following standard laboratory techniques such as those described by 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), and in vendors’ protocols. For the location 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. 38 LEGAL02 / 43629467v1

[0118] SP34 is a mouse IgG3 / lambda monoclonal agonist antibody that binds to human and cynomolgus CD3 epsilon protein. Cross-linking of CD3 epsilon proteins by SP34 on the surface of T cells induces activation of such T cells (Passano et al., EMBO J.4:337-344, 1985; Yang et al., J. Immunol.137:1097-1100, 1986; Salmerón 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). Amino acid sequence of the mature heavy chain variable region (VH) of SP34 is EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAY WGQGTLVTVSS (SEQ ID NO:1). Amino acid sequence of the mature light chain variable region (VL) of SP34 is QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPG VPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO:2). Example 2: Humanization of an anti-human CD3 antibody [I]

[0119] Humanized SP34 VH and VL amino acid sequences were designed according to the general procedure described in Tsurushita et al. (supra). Briefly, a three-dimensional molecular model of the mouse SP34 variable regions was first constructed with the aid of an appropriate software. Next, the framework amino acid residues important for the formation of the CDR structure were identified using the molecular model. In parallel, cDNA-derived human VH and VL amino acid sequences with high homology to SP34 VH and VL, respectively, were selected. Lastly, CDR sequences together with framework amino acid residues important for proper formation of the antigen-binding site were grafted from SP34 VH and VL into the corresponding selected human framework sequences.

[0120] For designing of 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 chosen as an 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 has 85.2% identity to that of SP34 VH. The CDR sequences of 39 LEGAL02 / 43629467v1SP34 VH were first transferred to the corresponding positions of M24236 VH. Next, at framework positions 30, 49, 93 and 94, where the three-dimensional model of the SP34 variable regions indicated that amino acid residues could play significant roles in the formation of the antigen-binding site, amino acid residues of M24236 VH were replaced by the corresponding residues of mouse SP34 VH. The amino acid sequence of the resulting mature humanized VH termed HuSP34 VH1 (SEQ ID NO:4) is shown in Fig.1 along with the mature SP34 and M24236 VH amino acid sequences.

[0121] For designing of 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 chosen as an acceptor for humanization. Y14738 VL, which belongs to the IGLV8-61 subgroups of human germline Vλ segments, has no somatic hypermutations in the amino acid sequence of the framework regions. The framework amino acid sequence of Y14738 VL has 67.1% identity to that of SP34 VL. The CDR sequences of SP34 VL were first transferred to the corresponding positions of Y14738 VL. Next, at framework positions 36, 46 and 49, where the three-dimensional model of the SP34 variable regions indicated that amino acid residues could play significant roles in the formation of the antigen-binding site, amino acid residues of Y14738 VL were replaced by the corresponding residues of mouse SP34 VL. The amino acid sequence of the resulting mature humanized VL termed HuSP34 VL1 (SEQ ID NO:6) is shown in Fig.2 along with the mature SP34 and Y14738 VL amino acid sequences.

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

[0123] Proceeding clockwise from the SalI site in Fig.5A, pHuSP34A contains the heavy chain transcription unit starting with the human cytomegalovirus (CMV) major immediate early 40 LEGAL02 / 43629467v1promoter and enhancer (CMV-P) to initiate transcription of the antibody heavy chain gene. The CMV promoter is followed by the VH exon encoding HuSP34 VH1, a genomic sequence containing the human gamma-1 heavy chain constant region including the CH1, hinge, CH2 and CH3 exons with the intervening introns, and the polyadenylation site following the CH3 exon. The CH2 region carries amino acid substitutions from leucine to alanine at positions 234 and 235 (Eu numbering; Kabat et al., supra) to eliminate effector functions (Hazareh et al., J. Virol. 75:12161-12168, 2001). After the heavy chain gene sequence, the light chain transcription unit begins with the CMV promoter, followed by the exon encoding HuSP34 VL1 and a genomic sequence containing the human lambda-2 chain constant region (Cλ2) with part of the intron preceding it, and the polyadenylation site following the Cλ2 exon. The light chain gene is then followed by the SV40 early promoter (SV40-P), the puromycin N-acetyl-transferase gene (puro) for resistance to puromycin, and a segment containing the SV40 polyadenylation site (SV40-A). Finally, the plasmid contains a part of the plasmid pUC19, comprising the bacterial origin of replication (pUC ori) and beta-lactamase gene (β lactamase). Locations of relevant restriction enzyme sites are shown in Fig.5A. Arrows indicate orientation of transcription.

[0124] Amino acid sequences of the CH1, hinge, CH2, and CH3 regions of human gamma-1 heavy chain encoded in pHuSP34A are ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:9), EPKSCDKTHTCPPCP (SEQ ID NO:10), APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO:11), and GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:12), respectively.

[0125] Amino acid sequences of the human lambda-2 constant region (Cλ2) encoded in pHuSP34A is GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:13) 41 LEGAL02 / 43629467v1

[0126] Amino acid sequence of a heavy chain encoded in pHuSP34A is MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVY YCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO:14). Mature HuSP34A heavy chain starts at a glutamate residue at position 20 of SEQ ID NO:14.

[0127] Amino acid sequence of a light chain encoded in pHuSP34A is MAWISLILSLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQ APRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGG GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:15). Mature HuSP34A light chain starts at a glutamine residue at position 17 of SEQ ID NO:15.

[0128] The expression vector pHuSP34A was introduced into the chromosome of a Chinese hamster ovary cell line CHO-K1. Stable transfection into CHO-K1 cells was carried out by electroporation. Before transfection, pHuSP34A was linearized using a restriction endonuclease FspI (Fig.5A). Approximately 2.5 x 106cells were transfected with 20 μg of linearized plasmid, suspended in SFM4CHO media (HyClone, Logan, UT), plated into several 96-well plates after appropriate dilutions of cells, and grown at 37°C in a 7.5% CO2 incubator. After 48 hrs, 10 μg / ml of puromycin was added for isolation of stable transfectants.

[0129] Approximately ten days after the initiation of selection, culture supernatants of CHO- K1 stable transfectants in 96-well plates were assayed for antibody production by sandwich ELISA. In typical experiments, wells of an ELISA plate were coated overnight at 4°C with goat anti-human IgG, Fcγ-specific, polyclonal antibody in PBS, washed with Washing Buffer (PBS containing 0.05% Tween 20), and blocked with ELISA Buffer (PBS containing 2% skim milk 42 LEGAL02 / 43629467v1and 0.05% Tween 20). After washing the wells with Washing Buffer, test antibodies appropriately diluted in ELISA Buffer were applied to the ELISA plate. An appropriate humanized IgG1 / lambda (or kappa as needed) antibody was used as a standard. After incubating the ELISA plate for 1 hr at room temperature and washing with Washing Buffer, bound antibodies were detected using HRP-conjugated goat anti-human lambda chain (or kappa chain as needed) polyclonal antibody. After incubating for 0.5 hr at room temperature and washing with Washing Buffer, color development was initiated with ABTS substrate (Sigma-Aldrich, St. Louis, MO) and stopped with 2% oxalic acid. Absorbance was read at 405 nm.

[0130] CHO-K1 stable transfectants producing HuSP34A (CHO-K1 / pHuSP34A) were grown in SFM4CHO in roller bottles until the cell viability became less than 50%. After centrifugation and filtration, culture supernatant was loaded onto a protein A column (HiTrap MabSelect SuRe, GE Healthcare, Piscataway, NJ). The column was washed with PBS before the antibody was eluted 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 buffer of eluted antibody was changed to PBS by dialysis. Antibody concentration was determined by measuring absorbance at 280 nm (1.4 OD = 1 mg / ml).

[0131] Binding of HuSP34A to human CD3 was analyzed by flow cytometry using the human T cell line Jurkat Dual (Invivogen, San Diego, CA). In typical experiments, HuSP34A was incubated at various concentrations with Jurkat Dual cells in FACS Buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.05% sodium azide) for 30 min. After washing with FACS Buffer, cells were incubated with PE-labeled goat anti-human IgG antibodies for 20 min. After washing with and suspending in FACS Buffer, cells were subjected to flow cytometry. As shown in Fig.6, HuSP34A bound to Jurkat Dual cells in a dose dependent manner. The EC50value of HuSP34A for binding to Jurkat Dual cells, which was calculated using the software Prism (GraphPad, San Diego, CA), was 92 ng / ml. Example 3: Humanization of an anti-human CD3 antibody [II]

[0132] A second humanized SP34 VL was designed in a different way. As an acceptor for humanization, 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. L37309 VL, which belongs to the IGKV3-11 subgroup of human germline Vκ segments, has no 43 LEGAL02 / 43629467v1somatic hypermutations in the amino acid sequence of the framework regions. The framework amino acid sequence of L37309 VL has 51.3% identity to that of SP34 VL. The CDR sequences of SP34 VL were first transferred to the corresponding positions of L37309 VL. Next, at framework positions 36, 46, 49, 58, 66, 67, 69, 70 and 71, where the three-dimensional model of the SP34 variable regions indicated that amino acid residues could play important roles in the formation of the antigen-binding site, amino acid residues of L37309 VL were replaced by the corresponding residues of mouse SP34 VL. The amino acid sequence of the resulting mature humanized VL termed HuSP34 VL3 (SEQ ID NO:17) is shown along with the SP34 and L37309 VL sequences in Fig.7.

[0133] A gene encoding HuSP34 VL3 was synthesized as an exon including a signal peptide (SEQ ID NO:18), a splice donor signal, a NheI site at the 5’ end, and an EcoRI site at the 3’ end (Fig.8). The HuSP34 VL3 gene was cloned between the NheI and EcoRI sites to replace HuSP34 VL1 in pHuSP34A. In addition, the human lambda-2 chain constant region (Cλ2) was substituted by the coding sequence of the human kappa chain constant region (Cκ). The resultant plasmid for expression of a humanized anti-CD3 IgG1 / kappa antibody comprising HuSP34 VH1 and VL3 (HuSP34C) was termed pHuSP34C. The schematic structure of pHuSP34C is shown in Fig.5B. Amino acid sequence of the human kappa chain constant region encoded in pHuSP34C is RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:19). Amino acid sequence of a heavy chain encoded in pHuSP34C is same as that encoded in pHuSP34A (SEQ ID NO:14). Amino acid sequence of a light chain encoded in pHuSP34C is MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQ QKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLW VFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:20). Mature HuSP34C light chain starts at a glutamate 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 stable transfectants expressing HuSP34C (CHO- 44 LEGAL02 / 43629467v1K1 / pHuSP34C) were expanded in SFM4CHO as described above. HuSP34C was purified from culture supernatants of CHO-K1 / pHuSP34C by protein A as described above. Binding of HuSP34C to human CD3 was measured by flow cytometry using Jurkat Dual cells as described above. The result is shown in Fig.6. HuSP34C bound to Jurkat Dual cells in a dose dependent manner. The EC50 value of HuSP34C for binding to Jurkat Dual cells was 209 ng / ml. Example 4: Mutagenesis of HuSP34 VL3

[0135] To reduce potential immunogenicity in humans, an attempt was carried out to lower the number of mouse-derived framework residues in HuSP34 VL3 (Fig.7). At each of positions 36, 58, 66, 67, 69 and 70 in the frameworks of HuSP34 VL3, a mouse-derived amino acid residue was substituted by the corresponding residue of the human L37309 VL acceptor by site- directed mutagenesis using the overlap-extension PCR method. These HuSP34 VL3 variants are substitutions of valine at position 36 to tyrosine (V36Y; SEQ ID NO:21), valine at position 58 to isoleucine (V58I; SEQ ID NO:22), leucine at position 66 to glycine (L66G; SEQ ID NO:23), isoleucine at position 67 to serine (I67S; SEQ ID NO:24), aspartate at position 69 to threonine (D69T; SEQ ID NO:25), or lysine at position 70 to aspartate (K70D; SEQ ID NO:26) in HuSP34 VL3.

[0136] Each of the six HuSP34 VL3 variants was combined with HuSP34 VH1 by replacing HuSP34VL3 in pHuSP34C for expression in the form of IgG1 / kappa antibody. When the HuSP34 VL3 L66G variant was combined with HuSP34 VH1, antibody expression severely decreased. A combination of HuSP34 VH1 with the HuSP34 VL3 V36Y variant resulted in drastic decrease of antigen binding. Each of the HuSP34 VL3 V58I, I67S, D69T and K70D variants in combination with HuSP34 VH1 showed antigen binding at the level similar to the combination of HuSP34 VH1 and VL3 (HuSP34C). The substitution of a mouse-derived hydrophobic isoleucine residue to a human-derived hydrophilic serine residue at position 67 (I67S) was accepted in HuSP34 VL3 without causing the loss of the antigen-binding affinity. Likewise, each of the substitutions of (i) a mouse-derived acidic aspartate residue to a human- derived neutral hydrophilic threonine residue at position 69 (D69T) and (ii) a mouse-derived basic lysine residue to a human-derived acidic aspartate residue at position 70 (K70D) did not cause the loss of the affinity. 45 LEGAL02 / 43629467v1

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

[0138] The HuSP34 VL4 gene was cloned between the NheI and EcoRI sites to replace HuSP34 VL3 in pHuSP34C. The resultant expression vector termed pHuSP34V expresses humanized SP34 IgG1 / kappa antibody comprising HuSP34 VH1 and VL4 (HuSP34V). Amino acid sequence of a heavy chain encoded in pHuSP34V is same as that encoded in pHuSP34A (SEQ ID NO:14). Amino acid sequence of a light chain encoded in pHuSP34V is MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQ QKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWV FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:28). Mature HuSP34V light chain starts at a glutamate 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 expanded in SFM4CHO as described above. HuSP34V was purified from culture supernatants of CHO-K1 / pHuSP34V by protein A as described above. Binding of HuSP34V to human CD3 was measured by flow cytometry using Jurkat Dual cells as described above. The result is shown in Fig.6. HuSP34V bound to Jurkat Dual cells in a dose dependent manner. The EC50value of HuSP34V for binding to Jurkat Dual cells was 234 ng / ml. Example 5: Generation and characterization of an anti-CD20 / CD3 bispecific antibody

[0140] The VH and VL regions of HuSP34A were converted to a single-chain Fv (scFv) form with an orientation of N’-VH-linker-VL-C’. In addition, two amino acid substitutions, one from glycine to cysteine at position 44 in HuSP34 VH1 (Fig.1) and another from glycine to cysteine at position 100 in HuSP34 VL1 (Fig.2), were introduced in such generated scFv (Brinkman et al., Proc. Natl. Acad. Sci.90:7538-7542, 1993). The resultant scFv form of HuSP34A (HuSP34A.scFv.HL.ds; SEQ ID NO:29) was then fused to the penultimate glycine 46 LEGAL02 / 43629467v1residue of the CH3 region with a flexible polypeptide linker between them (CH3- HuSP34A.scFv.HL.ds; SEQ ID NO:30) in an antibody expression vector which has the same structure as pHuSP34C (Fig.5B) except that the VH and VL exons were substituted by those of an anti-CD20 antibody. The resultant expression vector was named pJB509.

[0141] The VH and VL regions of HuSP34A were also converted to another scFv form which has an orientation of N’-VL-linker-VH-C’ with two amino acid substitutions, one from glycine to cysteine at position 44 in HuSP34 VH1 and another from glycine to cysteine at position 100 in HuSP34 VL1 (HuSP34A.scFv.LH.ds; SEQ ID NO:31). HuSP34A.scFv.LH.ds fused to the penultimate glycine residue of the CH3 region (CH3-HuSP34A.scFv.LH.ds; SEQ ID NO:32) was used to replace CH3-HuSP34A.scFv.HL.ds in pJB509. The resultant expression vector was named pJB510.

[0142] The expression vectors pJB509 and pJB510 were individually transfected into the human embryonic kidney cell line HEK293 using polyethylenimine (Durocher et al. Nucl. Acids Res.30: e9, 2002) for transient expression of recombinant antibodies. HEK293 cells were grown in DME medium containing 10% fetal bovine serum (FBS; Life Technologies, Grand Island, NY) at 37°C in a 7.5% CO2incubator. Expression of anti-CD20 / CD3 bispecific antibody in culture supernatants, which was determined by sandwich ELISA as described above, was very poor with both pJB509 and pJB510. When each of HuSP34A.scFv.HL and HuSP34A.scFv.LH was attached at the C-terminal area of CH3 of other chimeric or humanized IgG1 antibodies, antibody expression was again very poor.

[0143] An anti-CD3 scFv antibody was then formed using HuSP34 VH1 and VL4 with an orientation of N’-VL-linker-VH-C’. In addition, two amino acid substitutions, one from glycine to cysteine at position 44 in HuSP34 VH1 and another from glycine to cysteine at position 100 in HuSP34 VL4, were introduced in such generated scFv. The resulting scFv was named HuSP34V.scFv.ds. Amino acid sequence of HuSP34V.scFv.ds is EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPG IPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGS GGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIR SKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVS WFAYWGQGTLVTVSS (SEQ ID NO:33). 47 LEGAL02 / 43629467v1

[0144] HuSP34V.scFv.ds was fused to the penultimate glycine residue of CH3 with a flexible polypeptide linker separating them (CH3-HuSP34V.scFv.ds; SEQ ID NO:34) in an IgG1 / kappa antibody expression vector which has the same structure as pHuSP34C (Fig.5B) except that the VH and VL derived from a chimeric anti-CD20 antibody C2B8 (Reff et al. Blood 83:435-445, 1994; Maloney et al. Blood 84:2457-2466, 1994). The resultant expression vector was named pJB554. The schematic structure of pJB554 is shown in Fig.5C.

[0145] Amino acid sequence of mature C2B8 VH encoded in pJB554 is QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAG TTVTVSA (SEQ ID NO:35; https: / / go.drugbank.com / drugs / DB00073).

[0146] Amino acid sequence of mature C2B8 VL encoded in pJB554 is QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF SGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO:36; https: / / go.drugbank.com / drugs / DB00073).

[0147] Bispecific anti-CD20 / CD3 IgG1 / kappa antibody expressed from pJB554 was named JB554. The schematic structure of JB554 is shown in Fig.10. Amino acid sequence of a heavy chain encoded in pJB554 is MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVK QTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCA RSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPR GLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVE IKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQA PGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYC 48 LEGAL02 / 43629467v1VRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO:37). Mature JB554 heavy chain starts at a glutamine residue at position 20 of SEQ ID NO:37.

[0148] Amino acid sequence of a light chain encoded in pJB554 is MDFQVQIISFLLISASVIMSRGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPG SSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:38). Mature JB554 light chain starts at a glutamine residue at position 23 of SEQ ID NO:38.

[0149] The expression vector pJB554 was transfected into HEK293 cells as described above. Expression of anti-CD20 / CD3 bispecific antibody from pJB554 was improved when compared to the cases with pJB509 and pJB510. Transiently expressed JB554 was purified by protein A as described above. The activity of JB554, which is a bispecific antibody that can bind to CD20 and CD3, to induce T cell-mediated cytotoxicity against CD20-positive human Burkitt lymphoma Ramos cells was analyzed as follows. Purified Human CD3 Pan T Cells (Cat. # IQB- Hu1-T100, Lot # P19D0900, iQ Biosciences, Berkeley, CA) were incubated in RPMI 1640 media containing 10% FBS, 1 mM sodium pyruvate and 10 mM HEPES (RPMI 1640 complete media) in the presence of 50 ng / ml recombinant human IL-2 (Acro Biosystems, Newark, DE) for four days at 37°C in a 7.5% CO2 incubator (Activated T Cells). One hundred thousand Ramos cells labeled with a fluorescent dye (Calcein AM; BioLegend, San Diego, CA) were incubated in 200 μl of RPMI 1640 complete media for four hours with one million Activated T Cells in a well of a 96-well plate in the presence (or absence) of 150 ng / ml of JB554. To monitor the level of lysis of Ramos cells, fluorescence in culture media was measured according to the supplier’s instruction. As a 100% lysis control, fluorescence of culture supernatant of Calcein AM-labeled Ramos cells incubated alone as shown above and then treated with SDS for cell lysis was measured. As a background control, Calcein AM-labeled Ramos cells were incubated alone as shown above and fluorescence in culture supernatant was measured.

[0150] The result of the cytotoxicity test with Calcein AM-labeled Ramos cells is shown in Fig.11A. Relative fluorescence unit (RFU) values in 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 49 LEGAL02 / 43629467v1Cells and JB554. Anti-CD20 / CD3 bispecific antibody JB554 efficiently induced lysis of Ramos cells in the presence of Activated T Cells. Example 6: Generation and characterization of an anti-EGFR / CD3 bispecific antibody

[0151] To generate 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 substituted by those derived from a mouse anti-EGFR antibody 225 (Masui et al. Cancer Res.44:1002-1007, 1984; Gill et al. J. Biol. Chem.259:7755-7760, 1984). The resultant expression vector was named pJB559. The structure of pJB559 is same as pJB554 (Fig.5C) except for the VH and VL sequences.

[0152] Amino acid sequence of mature 225 VH encoded in pJB559 is QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVT VSA (SEQ ID NO:39; https: / / go.drugbank.com / drugs / DB00002).

[0153] Amino acid sequence of mature 225 VL encoded in pJB559 is DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO:40; https: / / go.drugbank.com / drugs / DB00002).

[0154] Bispecific anti-EGFR / CD3 IgG1 / kappa antibody expressed from pJB559 was named JB559. The schematic structure of JB559 is shown in Fig.10. Amino acid sequence of JB559 heavy chain is MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARAL TYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGG GGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIG GTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGG 50 LEGAL02 / 43629467v1GGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKC LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHG NFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO:41). The mature JB559 heavy chain starts at a glutamine residue at position 20 of SEQ ID NO:41.

[0155] Amino acid sequence of JB559 light chain is MRAPAQFLGFLLFWIPASRSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTN GSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTK LELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:42). The mature JB559 light chain starts at an aspartate 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 activity of JB559, which is a bispecific antibody that can bind to EGFR and CD3, to induce T cell-mediated cytotoxicity against EGFR-positive human colorectal adenocarcinoma HT-29 cells was analyzed as follows. HT-29 cells were first seeded in wells of a 96-well plate at a concentration of four hundred thousand cells / ml in RPMI 1640 complete media. After 48 hours, HT-29 cells were labeled with Calcein AM and then incubated with five million cells / ml of Activated T Cells in the presence (or absence) of a test antibody (JB554 or JB559) as shown above. As a 100% lysis control, fluorescence of culture supernatants of Calcein AM-labeled HT-29 cells incubated alone as shown above and then treated with SDS for cell lysis was measured. As a background control, Calcein AM-labeled HT-29 cells were incubated alone as shown above and fluorescence in culture supernatant was measured.

[0157] The result of the cytotoxicity test with Calcein AM-labeled HT-29 cells is shown in Fig.11B. RFU values in 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. Anti-EGFR / CD3 bispecific antibody JB559 induced lysis of HT-29 cells in the presence of Activated T Cells. Anti-CD20 / CD3 bispecific 51 LEGAL02 / 43629467v1antibody JB554, which does not bind to HT-29 cells, failed to induce lysis of HT-29 cells in the presence of Activated T Cells.

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

[0159] An expression vector pChC2B8, which expresses mouse-human chimeric anti-CD20 IgG1 / kappa antibody (ChC2B8), has the same structure as pJB554 (Fig.5C) except that (i) the CH3-HuSP34V.scFv.ds region (SEQ ID NO:34) is replaced by the wild type CH3 sequence (SEQ ID NO:12) and (ii) the CH2 region encodes the wild-type CH2 sequence of human gamma-1 heavy chain (SEQ ID NO:49). The light chain amino acid sequence encoded in pChC2B8 is the same as the light chain sequence encoded in pJB554 (SEQ ID NO:38). The heavy chain amino acid sequence encoded in pChC2B8 is MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVK QTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCA RSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:50). Mature ChC2B8 heavy chain starts at a glutamine residue at position 20 of SEQ ID NO:50.

[0160] An expression vector pCh225, which expresses 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 by the wild type CH3 sequence (SEQ ID NO:12) and (ii) the CH2 region encodes the wild-type CH2 sequence of human gamma-1 heavy chain (SEQ ID NO:49). The light chain amino acid sequence encoded in pCh225 is the same as the light chain sequence encoded in pJB559 (SEQ ID NO:42). The heavy chain amino acid sequence encoded in pCh225 is MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS 52 LEGAL02 / 43629467v1PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARAL TYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:51). The mature Ch225 heavy chain starts at a glutamine residue at position 20 of SEQ ID NO:51.

[0161] An expression vector pHuM195, which expresses humanized anti-CD33 IgG1 antibody HuM195 (Co et al., J. Immunol.148:1149-1154, 1992; US patent 5,693,761), has the same structure as pChC2B8 except that the VH and VL regions encode HuM195 VH (SEQ ID NO:52) and HuM195 VL (SEQ ID NO:53), respectively. Amino acid sequence of a heavy chain encoded in pHuM195 is MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVR QAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCA RGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:54). The mature HuM195 heavy chain starts at a glutamine residue at position 20 of SEQ ID NO:54. Amino acid sequence of a light chain encoded in pHuM195 is MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNW FQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVP WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO:55). The mature HuM195 light chain starts at an aspartate residue at position 21 of SEQ ID NO:55. 53 LEGAL02 / 43629467v1

[0162] Each of pChC2B8, pCh225 and pHuM195 was stably transfected into CHO-K1 as described above. ChC2B8, Ch225 and HuM195 IgG1 antibodies were purified from culture supernatants of respective CHO-K1 stable transfectants by protein A as described above. For each of ChC2B8, Ch225 and HuM195 antibodies, SDS-PAGE analysis under reducing conditions showed only two predominant bands of roughly 50 kDa heavy chains and 25 kDa light chains.

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

[0164] Generation of a CHO-K1 stable transfectant cell line producing JB554 (CHO- K1 / pJB554) was carried out by electroporation as described above. Expansion of CHO- K1 / pJB554 cells in SFM4CHO media and purification of JB554 by protein A were carried out as described above. SDS-PAGE analysis of purified JB554 under reducing conditions showed only two predominant bands of roughly 75 kDa heavy chains and 25 kDa light chains.

[0165] The biological activity of JB554 purified from CHO-K1 / pJB554 cells was examined with Jurkat Dual reporter cells (Invivogen, San Diego, CA). In Jurkat Dual cells, cross-linking of CD3 on the surface triggers activation of the intracellular NF-κB signaling pathway, which then leads to expression and secretion of recombinant Lucia luciferase. Approximately four hundred thousand Jurkat Dual cells were incubated in 200 μl of RPMI 1640 media containing 10% FBS in wells of a 96-well plate for one day at 37°C in a 7.5% CO2incubator in the presence (or absence) of two hundred thousand CD20-positive Ramos cells and test antibodies. Luciferase activity in culture supernatants was measured in triplicates with QUANTI-Luc reagents (Invivogen) according to the vendor’s protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek, Winooski, VT). The average 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 of ChC2B8 (anti-CD20 IgG1 antibody) and 1 μg / ml of HuSP34V (anti-CD3 IgG1 antibody), (iii) 827 for Jurkat Dual cells incubated with 1 μg / ml of JB554 (anti-CD20 / CD3 bispecific antibody), (iv) 459 for Jurkat Dual cells incubated with 1 μg / ml of JB559 (anti- EGFR / CD3 bispecific antibody), (v) 256 for Jurkat Dual cells incubated with Ramos cells, (vi) 54 LEGAL02 / 43629467v12,212 for Jurkat Dual cells incubated with 1 μg / ml of ChC2B8, 1 μg / ml of HuSP34V, and Ramos cells, (vii) 14,730 for Jurkat Dual cells incubated with 1 μg / ml of JB554 and Ramos cells, and (viii) 691 for Jurkat Duals cells incubated with 1 μg / ml of JB559 and Ramos cells. Fig.12A shows the RLU values of the Jurkat Dual assay with standard deviation error bars. Only when Jurkat Dual cells were incubated with JB554 and Ramos cells, a high level of luciferase activity was observed in culture supernatants.

[0166] JB554 purified from CHO-K / pJB554 cells was further analyzed for the activity to induce 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 derived from Donor 3820 of Human CD3 Pan T Cells (Cat. # IQB-Hu1-T100, iQ Biosciences, Berkeley, CA). Relative fluorescence unit (RFU) values in 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, and (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 (Fig.12B). Anti-CD20 / CD3 bispecific antibody JB554, but neither anti-EGFR / CD3 bispecific antibody JB559 nor a combination of anti-CD20 antibody ChC2B8 and anti-CD3 antibody HuSP34V, efficiently induced lysis of Ramos cells in the presence of Activated T Cells.

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

[0168] Generation of a CHO-K1 stable transfectant cell line producing JB559 (CHO- K1 / pJB559) was carried out by electroporation as described above. Expansion of CHO- K1 / pJB559 cells in SFM4CHO media and purification of JB559 by protein A were carried out as described above. SDS-PAGE analysis of purified JB559 under reducing conditions showed only two predominant bands of roughly 75 kDa heavy chains and 25 kDa light chains.

[0169] JB559 purified from CHO-K1 / pJB559 cells was analyzed for the activity to induce T cell-mediated cytotoxicity against HT-29 cells labeled with Calcein AM as described above. Each test antibody was used at 1 μg / ml. Activated T Cells used in the first cytotoxicity 55 LEGAL02 / 43629467v1experiment were derived from Donor 3820 of Human CD3 Pan T Cells (Cat. # IQB-Hu1-T100, iQ Biosciences, Berkeley, CA). In the second experiment, Activated T Cells derived from Donor 3661 of Human CD3 Pan T Cells were used. Relative fluorescence unit (RFU) values in culture supernatants of 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) (Fig.13A). In the second experiment, the RFU values in 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 (Fig.13B). In both experiments with HT-29 cells, anti- EGFR / CD3 bispecific antibody JB559, but neither anti-CD20 / CD3 bispecific antibody JB554 nor a combination of Ch225 and HuSP34V, efficiently induced lysis of HT-29 cells in the presence of Activated T Cells.

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

[0171] To examine if circulating T cells are activated by JB559 in an EGFR-independent manner, 105human peripheral blood mononuclear cells (PBMC) were incubated in 200 μl of RPMI 1640 media containing 10% FBS with (i) no additional reagents, (ii) 1 μg / ml of JB559, or (iii) 8 x 104Human CD3 / CD28 T Cell Activation Beads (Anti-CD3 / CD28 Beads; BioLegend, CA) in wells of a 96-well plate for 3 days at 37°C in a 7.5% CO2incubator. The assay was carried out in duplicate wells. JB559 is a bispecific antibody that binds to EGFR and CD3. EGFR is reported to be not expressed, or only weakly if any, in PBMC (http: / / proteinatlas.org).

[0172] The expression level of each of IFN-γ and IL-2 in culture supernatants was measured using ELISA MAX Standard Set Human IFN-γ and ELISA MAX Standard Set Human IL-2 kits 56 LEGAL02 / 43629467v1(BioLegend), respectively. The average IFN-γ expression levels of duplicate samples were (a) 23.3 pg / ml when no additional reagents were added, (b) 11.6 pg / ml in the presence of JB559, and (c) 2,670 pg / ml in the presence of Anti-CD3 / CD28 Beads. The average IL-2 expression levels of duplicate samples were (d) 33.8 pg / ml when no additional reagents were added, (e) 31.9 pg / ml in the presence of JB559, and (f) 11,500 pg / ml in the presence of Anti-CD3 / CD28 Beads. No sign of T cell activation was observed when PBMC was incubated with JB559 for 3 days in the absence of cells expressing EGFR.

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

[0174] To generate an expression vector for a bispecific antibody that binds to CD33 and CD3, the VH and VL genes of pJB554 were substituted by those encoding HuM195 VH (SEQ ID NO:52) and HuM195 VL (SEQ ID NO:53), respectively. The resultant plasmid was named pJB564. The structure of pJB564 is the same as pJB554 (Fig.5C) except for the VH and VL sequences. Anti-CD33 / CD3 bispecific antibody expressed from pJB564 was named JB564. The schematic structure of JB564 is shown in Fig.10. Amino acid sequence of a heavy chain encoded in pJB564 is MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVR QAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCA RGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGG GSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGG GSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHG NFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO:56). The mature JB564 heavy chain starts 57 LEGAL02 / 43629467v1at a glutamine residue at position 20 of SEQ ID NO:56. Amino acid sequence of a light chain encoded in pJB564 is the same as the light chain sequence encoded in pHuM195 (SEQ ID NO:55).

[0175] Generation of a CHO-K1 stable transfectant cell line producing JB564 (CHO- K1 / pJB564) was carried out by electroporation as described above. Expansion of CHO- K1 / pJB564 cells in SFM4CHO media and purification of JB564 by protein A were carried out as described above. SDS-PAGE analysis under reducing conditions showed that JB564 is composed of two polypeptides of roughly 75 kDa heavy chains and 25 kDa light chains.

[0176] JB564 was analyzed for the activity to induce T cell-mediated cytotoxicity against human CD33-positive HL-60 cells (Cat. # CCL-240, American Type Culture Collection, Manassas, VA) labeled with Calcein AM in the presence (or absence) of Activated T Cells and 1 μg / ml test antibody as described above. Relative fluorescence unit (RFU) values in 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, and (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) (Fig.14). Anti-CD33 / CD3 bispecific antibody JB564, but neither anti-EGFR / CD3 bispecific antibody JB559 nor a combination of HuM195 and HuSP34V, efficiently induced lysis of HL-60 cells in the presence of Activated T Cells. Sequence listing

[0177] SEQ ID NO:1

[0178] Amino acid sequence of mature SP34 VH

[0179] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYV SWFAYWGQGTLVTVSS

[0180] SEQ ID NO:2

[0181] Amino acid sequence of mature SP34 VL 58 LEGAL02 / 43629467v1

[0182] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGG TNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL

[0183] SEQ ID NO:3

[0184] Amino acid sequence of mature M24236 VH

[0185] EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRI KSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDSLPPHRVW GQGTLVTVSS

[0186] SEQ ID NO:4

[0187] Amino acid sequence of mature HuSP34 VH1

[0188] EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSY VSWFAYWGQGTLVTVSS

[0189] SEQ ID NO:5

[0190] Amino acid sequence of mature Y14738 VL

[0191] QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYQQTPGQAPRTLIYTT NTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCVLYMGGVWVFGGGTKLTVL

[0192] SEQ ID NO:6

[0193] Amino acid sequence of mature HuSP34 VL1

[0194] QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGG TNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL

[0195] SEQ ID NO:7

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

[0197] MLLGLKWVFFVVFYQGVHC

[0198] SEQ ID NO:8

[0199] Amino acid sequence of the signal peptide in HuSP34 VL1 59 LEGAL02 / 43629467v1

[0200] MAWISLILSLLALSSG

[0201] SEQ ID NO:9

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

[0203] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0204] SEQ ID NO:10

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

[0206] EPKSCDKTHTCPPCP

[0207] SEQ ID NO:11

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

[0209] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0210] SEQ ID NO:12

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

[0212] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0213] SEQ ID NO:13

[0214] Amino acid sequences of the human lambda-2 constant region encoded in pHuSP34A

[0215] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:13) 60 LEGAL02 / 43629467v1

[0216] SEQ ID NO:14

[0217] Amino acid sequence of a heavy chain encoded in pHuSP34A

[0218] MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAASGFTFNTY AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLK TEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

[0219] SEQ ID NO:15

[0220] Amino acid sequence of a light chain encoded in HuSP34A

[0221] MAWISLILSLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANW VQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSN LWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC S

[0222] SEQ ID NO:16

[0223] Amino acid sequence of mature L37309 VL

[0224] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK

[0225] SEQ ID NO:17

[0226] Amino acid sequence of mature HuSP34 VL3

[0227] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0228] SEQ ID NO:18 61 LEGAL02 / 43629467v1

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

[0230] MEAPAQLLFLLLLWLPDTTG

[0231] SEQ ID NO:19

[0232] Amino acid sequence of the human kappa chain constant region encoded in pHuSP34C and pHuSP34V

[0233] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0234] SEQ ID NO:20

[0235] Amino acid sequence of a light chain encoded in pHuSP34C

[0236] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSN YANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCAL WYSNLWVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

[0237] SEQ ID NO:21

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

[0239] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWYQQKPGQAPRGLIGG TNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0240] SEQ ID NO:22

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

[0242] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGIPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0243] SEQ ID NO:23

[0244] Amino acid sequence of mature HuSP34 VL3 L66G variant 62 LEGAL02 / 43629467v1

[0245] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGVPARFSGSGIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0246] SEQ ID NO:24

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

[0248] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGVPARFSGSLSGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0249] SEQ ID NO:25

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

[0251] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGVPARFSGSLIGTKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0252] SEQ ID NO:26

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

[0254] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGVPARFSGSLIGDDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0255] SEQ ID NO:27

[0256] Amino acid sequence of mature HuSP34 VL4

[0257] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0258] SEQ ID NO:28

[0259] Amino acid sequence of a light chain encoded in pHuSP34V

[0260] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSN YANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCAL WYSNLWVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 63 LEGAL02 / 43629467v1

[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] EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARI RSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSY VSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSFSVSPGGTVTLTCRSST GAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADD ESDYYCALWYSNLWVFGCGTKLTVL

[0264] SEQ ID NO:30

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

[0266] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGG GSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR IRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSY VSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSFSVSPGGTVTLTCRSST GAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADD ESDYYCALWYSNLWVFGCGTKLTVL

[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] QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGG TNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVLG GGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGK CLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRH GNFGNSYVSWFAYWGQGTLVTVSS

[0270] SEQ ID NO:32 64 LEGAL02 / 43629467v1

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

[0272] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGG GSGGGGSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGG TNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVLG GGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGK CLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRH GNFGNSYVSWFAYWGQGTLVTVSS

[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] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGG GSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHG NFGNSYVSWFAYWGQGTLVTVSS

[0276] SEQ ID NO:34

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

[0278] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGG GSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGG GSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHG NFGNSYVSWFAYWGQGTLVTVSS

[0279] SEQ ID NO:35 65 LEGAL02 / 43629467v1

[0280] Amino acid sequence of mature C2B8 VH

[0281] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGA IYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYF NVWGAGTTVTVSA

[0282] SEQ ID NO:36

[0283] Amino acid sequence of mature C2B8 VL

[0284] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLA SGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK

[0285] SEQ ID NO:37

[0286] Amino acid sequence of a heavy chain encoded in pJB554

[0287] MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSED SAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQ QKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWV FGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYA MNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKT EDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0288] SEQ ID NO:38

[0289] Amino acid sequence of a light chain encoded in pJB554

[0290] MDFQVQIISFLLISASVIMSRGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIH WFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTS 66 LEGAL02 / 43629467v1NPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

[0291] SEQ ID NO:39

[0292] Amino acid sequence of mature 225 VH

[0293] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIW SGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWG QGTLVTVSA

[0294] SEQ ID NO:40

[0295] Amino acid sequence of mature 225 VL

[0296] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK

[0297] SEQ ID NO:41

[0298] Amino acid sequence of a heavy chain encoded in pJB559

[0299] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGV HWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAI YYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPG QAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCG TKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNW VRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTA VYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 67 LEGAL02 / 43629467v1

[0300] SEQ ID NO:42

[0301] Amino acid sequence of a light chain encoded in pJB559

[0302] MRAPAQFLGFLLFWIPASRSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT FGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[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 in pChC2B8

[0311] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0312]

[0313] SEQ ID NO:50

[0314] Amino acid sequence of a heavy chain encoded in pChC2B8

[0315] MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSY NMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSED SAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 68 LEGAL02 / 43629467v1

[0316]

[0317] SEQ ID NO:51

[0318] Amino acid sequence of a heavy chain encoded in pCh225

[0319] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGV HWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAI YYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

[0320]

[0321] SEQ ID NO:52

[0322] Amino acid sequence of HuM195 VH

[0323] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDY NMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSED TAVYYCARGRPAMDYWGQGTLVTVSS

[0324]

[0325] SEQ ID NO:53

[0326] Amino acid sequence of HuM195 VL

[0327] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASESVDNY GISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYC QQSKEVPWTFGQGTKVEIK

[0328]

[0329] SEQ ID NO:54

[0330] Amino acid sequence of a heavy chain encoded in pHuM195

[0331] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDY NMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSED TAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT 69 LEGAL02 / 43629467v1KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

[0332]

[0333] SEQ ID NO:55

[0334] Amino acid sequence of a light chain encoded in pHuM195

[0335] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASESVDNY GISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYC QQSKEVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

[0336]

[0337] SEQ ID NO:56

[0338] Amino acid sequence of a heavy chain encoded in pJB564

[0339] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDY NMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSED TAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQ APRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGT KVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWV RQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAV YYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0340] SEQ ID NO:57

[0341] Nucleotide sequence of the HuSP34 VH1 gene flanked by SpeI and HindIII sites 70 LEGAL02 / 43629467v1

[0342] ACTAGTACCACCATGCTGTTGGGGCTGAAGTGGGTTTTCTTTGTTGTTTTT TATCAAGGAGTGCATTGTGAAGTGCAGCTTGTGGAAAGTGGCGGAGGACTGGTGAA GCCAGGCGGATCACTGAGACTGTCCTGCGCAGCTAGTGGCTTCACCTTTAACACATA CGCTATGAATTGGGTCCGACAGGCACCTGGCAAGGGCCTGGAGTGGGTGGCAAGGA TCAGGTCCAAGTACAACAATTATGCAACCTACTATGCCGACTCTGTGAAGGATAGAT TCACAATCAGTCGCGACGATTCCAAGAACACTCTGTATCTGCAGATGAACAGTCTGA AAACTGAAGACACCGCCGTGTACTATTGTGTGCGGCACGGAAACTTCGGCAATTCTT ACGTCTCTTGGTTTGCTTATTGGGGACAGGGGACACTGGTCACTGTGTCTTCAGGTG AGTCCTAACTTCTCCCATTCTAAGCTT

[0343] SEQ ID NO:58

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

[0345] MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAASGFTFNTY AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLK TEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0346] SEQ ID NO:59

[0347] Nucleotide sequence of the HuSP34 VL1 gene flanked by NheI and EcoRI sites GCTAGCACCACCATGGCCTGGATTTCACTTATCCTCTCTCTCCTGGCTCTCAGCTCAG GGCAGACTGTCGTGACACAGGAACCCTCATTTTCCGTCAGCCCTGGCGGAACAGTG ACCCTGACCTGCAGATCTAGCACAGGCGCAGTGACCACAAGCAACTACGCCAACTG GGTCCAGCAAACTCCAGGCCAAGCTCCCAGAGGCCTGATCGGCGGCACCAACAAAA GGGCTCCAGGCGTGCCAGACAGATTCAGCGGCAGCATCCTTGGCAATAAGGCTGCC CTGACAATCACTGGAGCCCAGGCCGACGACGAGTCCGACTACTATTGCGCCCTGTG GTACAGCAACCTGTGGGTCTTCGGCGGAGGCACCAAGCTGACAGTGCTAGGTGAGT CCTTCCTCCTTTGTTATTGAATTC

[0348] SEQ ID NO:60

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

[0350] MAWISLILSLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANW VQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSN LWVFGGGTKLTVL

[0351] SEQ ID NO:61

[0352] Nucleotide sequence of the HuSP34 VL3 gene flanked by NheI and EcoRI sites 71 LEGAL02 / 43629467v1

[0353] GCTAGCGCCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTTCTC TGGCTCCCAGATACCACTGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGATCTAGCACAGGAGCCGTGAC CACAAGCAACTATGCCAACTGGGTCCAACAGAAACCTGGCCAGGCTCCCAGGGGAC TCATCGGAGGCACCAACAAAAGGGCTCCAGGAGTCCCAGCCAGGTTCAGTGGCAGT CTGATTGGGGATAAAGCTACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCA GTGTATTACTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGAGGAGGCACCAAA GTCGAAATCAAACGTAAGTAGAATCCAAAGTGAATTC

[0354] SEQ ID NO:62

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

[0356] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSN YANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCAL WYSNLWVFGGGTKVEIK

[0357] SEQ ID NO:63

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

[0359] GCTAGCGCCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTTCTC TGGCTCCCAGATACCACTGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGATCTAGCACAGGAGCCGTGAC CACAAGCAACTATGCCAACTGGGTCCAACAGAAACCTGGCCAGGCTCCCAGGGGAC TCATCGGAGGCACCAACAAAAGGGCTCCAGGAATCCCAGCCAGGTTCAGTGGCAGT CTGAGCGGGACTGATGCTACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCA GTGTATTACTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGAGGAGGCACCAAA GTCGAAATCAAACGTAAGTAGAATCCAAAGTGAATTC

[0360]

[0361] SEQ ID NO:64

[0362] Amino acid sequence of HuSP34 VL4 including signal peptide

[0363] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSN YANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCAL WYSNLWVFGGGTKVEIK 72 LEGAL02 / 43629467v1

Claims

What is claimed is:

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

2.

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

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

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

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

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

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. 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. 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. 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. The antibody of any one of claims 1-10, which is a multi-specific antibody comprising multiple pairs of mature heavy chain and light chain variable regions, one of which pairs comprises the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 from SEQ ID NO:1 and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 from SEQ ID NO:2, and another of which pairs binds to a target antigen. 73 LEGAL02 / 43629467v112. The antibody of any one of claims 1-10, which is a bispecific antibody comprising two pairs of mature heavy and light chain variable regions, one of which pairs comprises the mature heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 from SEQ ID NO:1 and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 from SEQ ID NO:2, and the other of which pairs binds to a target antigen.

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 pathogen-infected cell.

14. The antibody of claim 12 or 13, wherein one of the pairs of mature heavy and light chain variable regions is an scFv and the other pair of mature heavy and 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 from SEQ ID NO:1 and the mature light chain variable region comprising CDRL1, CDRL2 and CDRL3 from SEQ ID NO:2 are linked as an scFv and the mature heavy and light chain variable regions of the other 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. 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 mature heavy chain or light chain variable region of the other pair.

22. The antibody of claim 1 in the form of or comprising an scFv, the scFv comprising the mature heavy chain variable region fused via a linker to the mature light chain variable region. 74 LEGAL02 / 43629467v123. The antibody of claim 1, having a sequence comprising any of SEQ ID NO:29, 31 or 33.

24. A pharmaceutical composition comprising the antibody of any preceding claim.

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

26. A method of treating an immune condition comprising administering the antibody of any one of claims 1-23 to a patient having the immune condition.

27. A method of treating a pathogenic infection comprising administering the antibody of any one of claims 11-23 to a patient infected with the pathogen, whether the target antigen is an antigen of the pathogen or a pathogen-infected cell. 75 LEGAL02 / 43629467v1

Images