Anti-CD3 antibodies

Abstract

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

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US63 / 426,626, filed November 18, 2022, the entire contents of which are incorporated by reference for all purposes. Sequence Listing

[0002] This application includes the sequence in the 66,000-byte XML file 603846SEQLST created on November 12, 2023, which is incorporated by reference. Background Art

[0003] The antigen-specific immune response of the adaptive immune system is a complex biological process controlled by multiple layers of positive and negative regulatory factors. Naive T cells are initially stimulated by recognition of their cognate peptide antigens presented by major histocompatibility complex (MHC; human proteins are also called HLA) molecules on antigen presenting cells (APCs) through the T cell receptor (TCR) complex (which contains TCRα and β (or γ and δ) chains and CD3 molecules). The initial interaction between TCR and MHC (or HLA) for T cell activation is called signal 1. Optimal T cell activation and proliferation requires a second signal, which is provided by the interaction of co-stimulatory molecules such as CD28 and ICOS of the CD28 superfamily expressed on T cells with their corresponding counter-receptors expressed on APCs (signal 2). The immune system is further positively regulated by other co-stimulatory molecules belonging to the TNF receptor superfamily, such as CD40, OX40, GITR, CD27, HVEM, and 4-1BB, and negatively regulated by checkpoint molecules such as PD-1, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD96, and CD112R. These co-stimulatory and checkpoint molecules are expressed in a cell type and developmental stage-dependent manner to finely control the immune response in the body. In addition, a variety of secretory proteins (such as cytokines and chemokines) participate in the regulation of immune responses by promoting the activation, differentiation, proliferation, maintenance, and suppression of certain subsets of immune cells. The effect of cytokines on T cells is often referred to as signal 3, which is the third mechanism required for T cell activation, differentiation, and proliferation. For review, see Curtsinger et al., Curr. Opin. Immunol. 22:333-340, 2010; Mahoney et al., Nat. Rev. Drug Discov. 14:561-584, 2015; Mercier et al., Front. Immunol. 6:418, 2015; Baumeister et al., Annu. Rev. Immunol. 34:539-573, 2016; Hurton et al., Proc. Natl. Acad. Sci. 113:E7788-E7797, 2016; Torphy et al., Int. J. Mol. Sci. 18:2642, 2017; Punt et al., Kuby Immunology, 8th ed. WH Freeman and Co., New York, 2018.

[0004] There are two main types of mature T cells: CD4+ helper T cells and CD8+ cytotoxic T cells. CD4+ helper T cells are further divided into T H 1. T H 2. T H 9. T H 17. TH 22. T FH CD4+ helper T cells and Treg cells, each with specific functions and unique cytokine expression patterns. The primary function of CD4+ helper T cells is to regulate other immune cells, such as B cells and CD8+ cytotoxic T cells, to enable appropriate and timely responses in the immune defense system. In contrast, the primary function of CD8+ cytotoxic T cells is to destroy cells infected or transformed by pathogens in an antigen-specific manner. Under activation by signals 1 and 2, CD8+ cytotoxic T cells secrete perforins and granzymes, which synergistically induce apoptosis of target cells. CD8+ cytotoxic T cells also play an important role in clearing 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 can bind to two different antigens. Recent studies have reported that bispecific antibodies that bind to CD3 expressed on T cells and cancer-associated surface molecules can bridge T cells to cancer cells and trigger T cell-mediated cytotoxicity against cancer cells. This type of bispecific antibody is called a T cell engager. Some T cell engagers have been approved for marketing as human therapeutics for the treatment of cancer, including leukemia. (belintozumab; anti-CD19 / CD3), for multiple myeloma (terituzumab; anti-BCMA / CD3) and for follicular lymphoma (Motuzumab; anti-CD20 / CD3). Many other T cell engagers are being evaluated in clinical studies for cancer treatment. For reviews, see Middelburg et al., Cancers, 13:287, 2021; Ma et al., Front. Immunol. 12:Article 6266116, 2021; Wang et al., EMBO Mol. Med. 13:e14291, 2021; Arvedson et al., Annu. Rev. Cancer Biol. 6:17–34, 2022. Summary of the Invention

[0006] The present invention provides an antibody that specifically binds to human CD3, comprising a mature heavy chain variable region and a mature light chain variable region, wherein the mature heavy chain variable region comprises CDRH1, CDRH2 and CDRH3 from SEQ ID NO: 1, and the mature light chain variable region comprises 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 of the humanized mature heavy chain variable region, as numbered by Kabat, 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 one 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 multispecific antibody comprising multiple pairs of mature heavy and light chain variable regions, wherein one pair comprises: 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, and another pair in the multiple pairs binds to the target antigen.

[0008] Optionally, the antibody is a bispecific antibody comprising two pairs of mature heavy and light chain variable regions, wherein one pair comprises: 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, and the other pair binds to the target antigen.

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

[0010] Optionally, one of the multiple pairs of mature heavy chain and light chain variable regions is an scFv, and the other pair of mature heavy chain and light chain variable regions 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, 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 are linked to form an scFv, and the other pair of mature heavy chain and light chain variable regions are linked to the heavy chain constant region and the light chain constant region, respectively.

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

[0012] The present invention also provides antibodies in the form of scFv or comprising scFv. The scFv comprises a heavy chain variable region fused to a light chain variable region via a linker, thereby forming a single chain. Optionally, the antibody has a sequence comprising any one of SEQ ID NO: 29, 31 or 33. Optionally, the antibody has a sequence consisting of or consisting essentially of any one of SEQ ID NO: 29, 31 or 33.

[0013] The present invention also provides a pharmaceutical composition comprising any of the antibodies described above or otherwise disclosed herein.

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

[0015] The present invention also provides a method of treating an immune disorder comprising administering to a patient suffering from the immune disorder an antibody as described above or otherwise disclosed herein.

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

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

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

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

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

[0021] Figure 5 AC: Schematic diagram of the structures of pHuSP34A (A), pHuSP34C (B), and pJB554 (C).

[0022] Figure 6 : Flow cytometric analysis of HuSP34A, HuSP34C, and HuSP34V binding to JurkatDual cells.

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

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

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

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

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

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

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

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

[0031] The antibodies of the present invention are typically provided in an isolated form. This means that the antibodies are typically at least 50% w / w pure from the interfering protein and other contaminants resulting from their production or purification, but does not exclude the possibility of combining the antibodies with excess pharmaceutically acceptable carriers or other vehicles designed to facilitate their use. Sometimes bispecific antibodies are at least 60, 70, 80, 90, 95, or 99% w / w pure from the interfering protein and contaminants resulting from their production or purification. Typically, the antibody is the primary macromolecular species remaining after purification.

[0032] For bispecific or multispecific antibodies, specific binding of an antibody to its target antigen(s) means that at least 10 6 , 10 7 , 10 8 , 10 9 or 10 10 M -1 Affinity. Antibodies that specifically bind to a target may also be referred to as antibodies against a target. The affinity for different targets may be different. The intensity of specific binding is higher in detection and can be distinguished from nonspecific binding that occurs to at least one unrelated target. Specific binding can be the result of a bond or a specific spatial fit (such as a lock and key type) between specific functional groups, while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that an antibody with the same two binding sites only binds to one target, or that a bispecific antibody with two different binding sites only binds to the targets of these two binding sites.

[0033] The basic antibody structural unit is a tetramer of subunits. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed in conjunction with a cleavable signal peptide. Variable regions without a signal peptide are sometimes referred to as mature variable regions. Thus, for example, a light chain mature variable region refers to a light chain variable region without a light chain signal peptide. However, reference to a variable region does not necessarily imply the presence of a signal sequence; in fact, the signal sequence is cleaved once the antibodies of the present invention are expressed and secreted. A pair of heavy and light chain variable regions defines the binding region of the antibody. The carboxyl-terminal portions of the light and heavy chains define the light and heavy chain constant regions, respectively. The heavy chain constant region is primarily responsible for effector function. In IgG antibodies, the heavy chain constant region is divided into the CH1, hinge, CH2, and CH3 regions. In IgA, the heavy chain constant region is divided into CH1, CH2, and CH3. The CH1 region is bound to the light chain constant region by disulfide bonds and non-covalent bonds. The hinge region provides flexibility between the antibody's binding and effector regions and also provides a site for intermolecular disulfide bonds between the two heavy chain constant regions in the tetrameric subunit. The CH2 and CH3 regions are the primary sites for effector function and FcRn binding.

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

[0035] The mature variable region of each light / heavy chain pair forms an antibody binding site. Therefore, a complete antibody has two binding sites, i.e., bivalent. In natural antibodies, the binding sites are identical. The binding sites within bispecific or multispecific antibodies can be identical or different from each other according to the form (see, for example, Songsivilai and Lachmann, Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148: 1547-53 (1992)). The variable region all exhibits the same general structure of a relatively conservative framework region (FR) connected by three hypervariable regions (also referred to as complementary determining regions or CDRs). The CDRs from each pair of two chains are aligned by the framework region, making it possible to bind to a specific epitope. From N-terminus to C-terminus, the light chain and the heavy chain all comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain are according to the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989), or other definitions of CDRs shown in Table 1 below. Table 1 Conventional definition of CDRs using Kabat numbering

[0036] *CDR-H1 by Chothia can end at H32, H33, or H34 (depending on the length of the loop). This is because the Kabat numbering scheme places the insertion of the extra residues at 35A and 35B, while Chothia numbering places them at 31A and 31B. If both H35A and H35B (Kabat numbering) are absent, 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 the corresponding residues between different heavy chain variable regions or different light chain variable regions are assigned the same numbering. Although Kabat numbering can be used for antibody heavy chain constant regions, more commonly used is the EU index (also referred to as EU numbering), as in the present application. When an antibody is considered to include CDRs by a certain definition of CDR (such as Kabat), the definition specifies the minimum number (i.e., Kabat CDR) of the CDR residues present in the antibody. It does not exclude the presence of other residues belonging to another conventional CDR definition but outside the specified definition. For example, the antibody comprising the CDR defined by Kabat, except for the following situation, also includes other possibilities, which are: CDR in the antibody includes Kabat CDR residues and does not include other CDR residues; and CDR H1 in the antibody is a composite Chothia-Kabat CDR H1 and other CDRs include Kabat CDR residues and do not include additional CDR residues based on other definitions.

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

[0039] The term "epitope" refers to a site on an antigen to which an antibody or bispecific or multispecific antibody arm binds. An epitope can be formed by continuous amino acids or by non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also referred to as linear epitopes) are typically retained when exposed to denaturing solvents, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost when treated with denaturing solvents. Some antibodies bind to terminal-specific epitopes, meaning that antibodies preferentially bind to polypeptides with free ends compared to the same polypeptide fused to other polypeptides (which can cause the free end to be lost). An epitope typically includes at least 3, more commonly at least 5 or 8-10 amino acids with a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, Methods in Molecular Biology, Vol. 66, Glenn E. Morris, ed. (1996).

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

[0041] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that shows the ability of one antibody to compete with another for binding to the target antigen. Antibody epitopes can also be defined by X-ray crystallography of antibodies bound to their antigens to identify contact residues. Alternatively, if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody, then the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody, then the two antibodies have overlapping epitopes.

[0042] Competition between antibodies is determined by an assay in which a test antibody 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 the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits binding of the reference antibody by at least 50%, but preferably 75%, 90%, or 99%, as measured in a competitive binding assay. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, as well as antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to cause steric hindrance.

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

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

[0045] The percentage of sequence identity is determined using the antibody sequences that are maximally aligned using the Kabat numbering convention (for variable regions) or EU numbering (for constant regions). After alignment, if a test antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the test and reference antibody regions is the number of positions occupied by the same amino acid in the test and reference antibody regions divided by the total number of aligned positions in the two regions (excluding gaps), multiplied by 100 to convert to a percentage.

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

[0047] The term "antibody-dependent cellular cytotoxicity" or ADCC is a mechanism of inducing cell death that depends on the interaction of antibody-coated target cells (i.e., cells with bound antibodies) with immune cells with lytic activity (also referred to as effector cells). Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. ADCC is initiated by the interaction between the Fc region of the cell-bound antibody and Fcγ receptors (particularly FcγRI and FcγRIII) on immune effector cells (e.g., neutrophils, macrophages, and natural killer cells). Target cells are cleared by phagocytosis or lysis, depending on the type of mediating effector cell. The death of the antibody-coated target cell is 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 fully or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind the Fc region of immunoglobulins.

[0049] The term "complement-dependent cytotoxicity" or CDC (also known as CMC) refers to a mechanism of cell death induction in which the Fc effector domain of a target-binding antibody activates a series of enzymatic reactions that ultimately form pores in the target cell membrane. Typically, antigen-antibody complexes (such as those on antibody-coated target cells) bind and activate the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation may also result in the deposition of complement components on the target cell surface, promoting ADCC by binding to complement receptors (such as CR3) on leukocytes.

[0050] The pH-dependent binding of antibodies to FcRn receptors means that antibodies bind to this receptor more strongly at pH 6.0 than at pH 7.5. After internalization by pinocytosis, IgG antibodies bind to FcRn at low pH in the endosome, thereby saving the IgG antibodies and avoiding their catabolism in the lysosome. The rescued IgG antibodies are then released from FcRn at neutral pH and recycled into the circulation. This pH-dependent FcRn binding is the basis of the molecular mechanism of the long serum half-life of IgG antibodies (and the bispecific antibodies of the present invention) (Ghetie et al., Annu. Rev. Immunol. 18: 739-766, 2000). For example, human IgG antibodies bind to human neonatal Fc receptors (FcRn) at pH 6.0, and only weakly bind to FcRn at pH 7.5. The FcRn binding site in IgG antibodies is located at the junction of the CH2 and CH3 domains. Because the μ heavy chain does not bind to FcRn at pH 6.0 or 7.5, native IgM cannot utilize the FcRn-mediated pathway to rescue the antibody from lysosomal degradation and thus typically has a shorter half-life than native IgG antibodies.

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

[0052] Protein G is a 65 kDa (G148 Protein G) and 58 kDa (C40 Protein G) streptococcal cell surface protein. It contains a serum albumin binding domain that is not required for IgG binding and is often deleted. Protein G specifically binds to all human IgG isotypes but does not bind IgA or IgM. Protein G is also used for antibody purification. DETAILED DESCRIPTION I. General Principles

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

[0054] Human CD3 is a complex comprising CD3δ (e.g., Swiss Prot P04234), CD3γ (e.g., Swiss Prot PO9693), two CD3ε molecules (e.g., Swiss Prot P07766), and two CD3ζ molecules (e.g., Swiss Prot P20963). References to human CD3 and its subunits include exemplary human forms as shown in the Swiss Prot database and other known allelic variants in humans.

[0055] One class of proteins that can serve as secondary targets in bispecific or multispecific antibodies is cancer-associated antigens. Such antigens are expressed by cancers, often at higher levels (overexpressed) than in control matched normal tissue. Some examples of cancer-associated antigens include α-folate receptor (ovarian and epithelial cancers), CAIX (kidney cancer), CD19 (B-cell malignancies, CLL, ALL), CD20 (B-cell malignancies, lymphomas), CD22 (B-cell malignancies), CD23 (CLL), CD24 (pancreatic cancer), CD30 (lymphomas), CD33 (AML), CD38 (NHL), CD44v7 / 8 (cervical cancer), CEA (colorectal cancer), EGFRvIII (glioblastoma), EGP-2 (multiple malignancies), EGP-40 (colorectal cancer), EphA2 (glioblastoma), Erb-B2 (breast cancer, prostate cancer, colon cancer), FBP (ovarian cancer), G.sub.D2 (neuroblastoma, melanoma), GD3 (melanoma), HER2 (pancreatic cancer, ovarian cancer, glioblastoma, osteosarcoma), HMW-MAA (melanoma), IL-11Rα (osteosarcoma), IL-13Rα2 (glioma, glioblastoma), KDR (tumor vasculature), kappa light chain (B-cell malignancies), Lewis Y (various carcinomas), L1 (neuroblastoma), MAGE-A1 (melanoma), mesothelin (mesothelioma), MUC1 (breast and ovarian cancers), MUC16 (ovarian cancer), NKG2D (myeloma, ovarian cancer), NY-ESO-1 (multiple myeloma), carcinoembryonic antigen (various tumors), PSCA (prostate cancer), PSMA (prostate cancer), ROR1 (B-CLL), TAG-72 (adenocarcinoma), and VEGF-R2 (tumor neovascularization) (Sadelain et al., Cancer Discov 3:388-98, 2013). Other tumor-associated antigens that can be targeted include alpha-fetoprotein (AFP), alpha-actin-4, A3, ART-4, B7, Ba 733, BAGE, BCMA, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, 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-β, GUCY2C, HLA-DR, HLA-A*02:01:gp100, HM1.24. Human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, IGF-1R, IFN-γ, IFN-, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, Insulin-like growth factor-1 (IGF-1), integrin β4, 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 receptor, transferrin receptor, TNF-α, Tn antigen, 5T4, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factor C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, Kras, oncogene markers or oncogene products (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 primarily overexpressed in bone marrow-derived cancers, such as acute myeloid leukemia. EGFR binds EGF and is primarily overexpressed in gastric, breast, endometrial, colorectal, head and neck, ovarian, cervical, bladder, and esophageal cancers. PD-L1 binds PD1 and is overexpressed in cancers such as gastric, hepatocellular, renal, esophageal, pancreatic, ovarian, and bladder cancers.

[0056] Another class of proteins are antigens expressed on the surface of pathogens or on the surface of cells infected by pathogens.Another class of proteins are antigens expressed on immune cells associated with diseases (eg, autoimmune diseases). III. anti-CD3 antibodies

[0057] An exemplary anti-CD3 antibody is designated 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 human CD3. Crosslinking of CD3 by SP34 induces T cell activation. SP34 specifically binds to CD3 from humans and various non-human primates (e.g., chimpanzees, cynomolgus monkeys, and rhesus monkeys). In contrast, other anti-CD3 antibodies widely used in T cell engagers (e.g., M291 and OKT3) have been reported to not specifically bind to cynomolgus monkeys or rhesus monkeys. The ability to specifically bind to non-human primate CD3 facilitates preclinical work in animal models. Other antibodies of the present invention preferably possess this and other properties of SP34.

[0058] Some antibodies of the present invention bind to the same or overlapping epitopes on human CD3 as the antibody designated SP34 and / or compete for binding to human CD3 with SP34 or other antibodies sharing its mature variable region. Other antibodies with such binding specificity can be generated by immunizing mice with human CD3 or a portion thereof containing the desired epitope and screening the 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 antibodies that exhibit the same or similar binding characteristics as SP34 to a collection of mutational changes. Mutations can be systematic substitutions, replacing one residue at a time with alanine (or serine, if alanine is already present) or more widely spaced substitutions throughout the extracellular domain of human CD3 or a portion thereof where the epitope is known to be present.

[0059] Antibodies with SP34 binding specificity 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, the heavy chain or light chain variable region of a selected murine antibody is used as the starting material. For example, if the light chain variable region is selected as the starting material, a phage library is constructed in which the members display the same light chain variable region (i.e., the murine starting material) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. Phage that show strong specific binding to human CD3 are selected (e.g., at least 10 8 M -1 , preferably at least 10 9 M -1 ). The heavy chain variable region from this phage is then used as the starting material for constructing another 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 region can be obtained, for example, from a library of rearranged human variable light chain regions. Again, phage that show strong specific binding to CD3 are selected. The resulting antibodies generally have the same or similar epitope specificity as the murine starting material.

[0060] Other antibodies can be obtained by mutagenizing cDNAs encoding the heavy and light chains of exemplary antibodies (e.g., SP34). The present invention also includes monoclonal antibodies that are at least 90%, 95%, or 99% identical to SP34 in the amino acid sequence of the mature heavy and / or light chain variable regions and retain their functional properties, and / or monoclonal antibodies that differ from the corresponding antibody by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Also included are monoclonal antibodies that have at least one, and preferably all six, Kabat-defined CDRs that are 90%, 95%, 99%, or 100% identical to the corresponding CDRs of SP34.

[0061] The antibodies of the present invention can be provided in the form of traditional tetramers, including two pairs of heavy and light chain variable regions, or in the form of fragments or including fragments, such as scFv. The antibodies can comprise, consist of, or consist essentially of any of the disclosed mature heavy and light chain variable regions and combinations thereof. A. Non-human antibodies

[0062] The production of other non-human monoclonal antibodies (e.g., mice, guinea pigs, primates, rabbits or rats) against human CD3 can be achieved by, for example, immunizing animals with human CD3 or a fragment thereof or cells carrying human CD3 (optionally co-expressed with its co-receptor protein), as described above. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such immunogens can be obtained from natural sources, by peptide synthesis or by recombinant expression. Optionally, the immunogen can be fused to a carrier protein or administered after complexing in other ways. Optionally, the immunogen can be administered together with an adjuvant. As described below, several types of adjuvants can be used. Complete Freund's adjuvant followed by incomplete adjuvant is preferred for immunization of laboratory animals. Rabbits or guinea pigs are typically used to prepare polyclonal antibodies. Mice are typically used to prepare monoclonal antibodies. The antibodies are screened for specific binding to human CD3. Optionally, the antibodies are further screened for binding to specific regions of human CD3. This screening can be achieved by determining the binding of the antibodies 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 transplanted into a human "acceptor" antibody sequence (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 sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody in which some or all of the CDRs are derived entirely or substantially from a donor antibody, and the variable region framework sequences and constant region (if present) are derived entirely or substantially from human antibody sequences. In some embodiments, the humanized heavy chain comprises at least one, two and usually all three CDRs from the heavy chain of the donor antibody, and at least one, two and usually all three CDRs from the heavy chain of the donor antibody, and at least one, two and usually all three CDRs from the heavy chain of the human heavy chain variable region framework and constant region sequence (if present). Similarly, the humanized light chain comprises at least one, two and usually all three CDRs from the light chain of the donor antibody, and at least one, two and usually all three CDRs from the light chain of the human light chain variable region framework and constant region sequence (if present). In addition to nano antibodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. When at least 85%, 90%, 95% or 100% of the corresponding residues (as defined by Kabat) are identical between the corresponding CDRs, the CDRs in the humanized antibodies are substantially from the corresponding CDRs in the non-human antibody. The variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially derived from a human variable region framework sequence or a human constant region, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues (as defined by Kabat) are identical.

[0064] Although humanized antibodies typically incorporate all six CDRs from a mouse antibody (preferably as defined by Kabat), they can also be made with fewer than all (e.g., at least 3, 4, or 5) CDRs from a mouse antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0065] In some antibodies, only part of the CDR is needed, that is, the subset of the CDR residues required for binding, referred to as SDR, to maintain the binding of humanized antibodies. The CDR residues that do not contact the antigen and are not in the SDR can be based on previous studies (for example, the residues H60-H65 in CDR H2 are usually not needed), from the Kabat CDR regions (Chothia, J.Mol.Biol.196:901,1987) outside the Chothia hypervariable ring, by molecular modeling and / or experience, or as described in Gonzales et al., Mol.Immunol.41:863,2004, identified. In this humanized antibody, at one or more donor CDR residues, there is no or the position of the entire donor CDR is omitted, and the amino acid occupying this position can be the amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. The number of this replacement of the acceptor to be included in the CDR to the donor amino acid reflects the balance considered in competition. This replacement has a potential advantage in reducing the number of mouse amino acids in the humanized antibody, thereby reducing potential immunogenicity. However, substitutions can also result in changes in affinity, and it is best to avoid a significant decrease in affinity. The position of substitution within the CDR and the amino acid to be substituted can also be selected empirically.

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

[0067] Based on their possible effects on CDR conformation and / or antigen binding, certain amino acids from human variable region framework residues can be selected for substitution. Investigation of such possible effects can be performed by modeling, examining the characteristics of amino acids at specific positions, or empirical observation of the effects of substitution or mutagenesis of specific amino acids.

[0068] For example, when the amino acid between a murine variable region framework residue and a selected human variable region framework residue differs, the human framework amino acid may be substituted with the equivalent framework amino acid from a mouse antibody when it is reasonable to expect that the amino acid meets the following requirements: (1) Non-covalent direct binding to antigens, (2) adjacent to the CDR region, (3) Otherwise interact with the CDR region (e.g., Inside).

[0069] Other replacement candidates are acceptor human framework amino acids, which are uncommon for human immunoglobulins at this position. These amino acids can be replaced by amino acids from the equivalent positions of mouse donor antibodies or from the equivalent positions of more typical human immunoglobulins. Other replacement candidates are acceptor human framework amino acids, which are uncommon for human immunoglobulins at this position. The VH sequence encoded by people M24236 cDNA is an exemplary acceptor sequence for heavy chain humanization. The people Vλ region encoded by Y14738 cDNA or the people mature Vκ region encoded by L37309 cDNA is an exemplary acceptor sequence for light chain humanization.

[0070] An exemplary humanized heavy chain variable region of SP34 is designated HuSP34 VH1 and is assigned to SEQ ID NO: 4. At framework positions 30, 49, 93, and 94 (by Kabat numbering), where the three-dimensional model of the SP34 variable region suggests that amino acid residues may play an important role in the formation of the antigen-binding site, the amino acid residues of M24236 VH can be replaced by the corresponding residues (i.e., N, A, V, and R) of mouse SP34 VH. An exemplary humanized light chain variable region designated HuSP34 VL1 is assigned to SEQ ID NO: 6. This light chain variable region contains backmutations to mouse residues at positions 36, 46, and 49 (by Kabat numbering), where these positions are occupied by V, G, and G, respectively. Another exemplary humanized light chain variable region based on the kappa receptor sequence is designated HuSP34 VL3 and is assigned to 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), where these positions are occupied by V, G, G, V, L, I, D, K, and A, respectively. Another humanized light chain variable region was designated HuSP34VL4 and assigned to SEQ ID NO: 27. SEQ ID NO: 27 is identical to SEQ ID NO: 17, except that some back mutations that are not required for binding but may increase immunogenicity have been eliminated. The remaining back mutations still present in SEQ ID NO: 27 occur at positions 36, 46, 49, 66, and 71, which are occupied by V, G, G, L, and A, respectively.

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

[0072] Typically, substitutions made in the variable regions of the variant HuSP34V sequence are conservative relative to the HuSP34V amino acids being replaced. Preferably, the substitutions in HuSP34V, whether conservative or not, do not substantially affect the binding affinity or potency of the humanized mAb, i.e., its ability to specifically bind to and stimulate human CD3. Preferably, the mature light and heavy chain variable region sequences are at least 90%, more preferably at least 95%, and most preferably at least 98% identical to the corresponding HuSP34V mature light and heavy chain variable regions. Alternatively, other human antibody receptor sequences, particularly those with high sequence identity to the HuSP34 variable region framework sequences, are also suitable for providing humanized antibody variable region framework sequences.

[0073] In some variants of HuSP34V, at least 1, 2, 3, 4, 5, 7, or all 8 of the mentioned acceptor-to-donor substitution positions with respect to the exemplary antibodies, 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 residues occupying the corresponding positions in the SP34 heavy or light chain variable region. C. Chimeric and veneered antibodies

[0074] The present invention also provides SP34 in mosaic and veneered forms.

[0075] A chimeric antibody is an antibody in which the mature variable regions of the light and heavy chains of a non-human antibody (such as a mouse) are combined with the constant regions of the light and heavy chains of a human. This antibody substantially or completely retains the binding specificity of the mouse antibody and is approximately two-thirds human sequence.

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

[0077] Anti-human CD3 human antibodies are provided by various techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter (supra), or other methods to have the same epitope specificity as a particular mouse antibody (e.g., one of the mouse monoclonal antibodies described in the Examples). It is also possible to screen for specific epitope specificity of human antibodies by using only fragments of human CD3.

[0078] Methods for producing human antibodies include the trioma approach of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, using transgenic mice that include human immunoglobulin genes (see, e.g., Lonberg et al., WO 93 / 12227 (1993); U.S. Pat. Nos. 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126; 5,569,825; 5,633,425). 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; U.S. Pat. No. 5,877,218; U.S. Pat. No. 5,871,907; U.S. Pat. No. 5,858,657; U.S. Pat. No. 5,837,242; U.S. Pat. No. 5,733,743; and U.S. Pat. No. 5,565,332); and the methods described in WO 2008 / 081008 (e.g., immortalizing memory B cells isolated from humans with EBV, screening for desired properties, and cloning and expressing recombinant forms). IV. Bispecific Antibodies

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

[0080] One of the component antibodies of the bispecific or multispecific antibody is an anti-human CD3 antibody as described above. The other one or more component antibodies bind to one or more antigens other than human CD3. For example, the other one or more component antibodies can bind to a target present on the cell to be depleted. These cells include cancer cells, pathogens (such as viruses, bacteria or fungi) and cells infected by any of these, as well as immune cells associated with immune diseases (particularly autoimmune diseases).

[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 used to produce antibodies against other targets, using related targets instead of human CD3. Many antibodies approved for therapeutic use as monospecific antibodies can be incorporated into bispecific antibodies. Table 2 below provides examples of antibodies approved for the treatment of cancer. Table 2

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

[0083] Some forms have a tetrameric structure similar to normal antibodies, with two binding regions, one for each target. Each binding region is formed by a pair of heavy and light chain variable regions, which are connected to heavy and light chain constant regions, respectively. This bispecific antibody differs from a normal antibody in that the two binding sites and the heavy and light chain pairs that form them are different. Therefore, this antibody requires the binding of two different pairs of heavy and light chains.

[0084] The "knob-in-hole" approach has been used to reduce homodimer formation and heavy chain mispairing by replacing small amino acids with large amino acids in the CH3 domain of one antibody (the "knob") and vice versa in the other antibody (the "hole"). (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 this format can be reduced by a variety of strategies. One strategy is to use a common light chain variable region for two different heavy chain variable regions. However, this only works for certain antibodies. Another approach is to express the knob- and hole-containing half-molecules separately in different bacteria. Another approach, called CrossMab, swaps the CH1 domain of one of the heavy chains with the constant CL domain of the corresponding light chain to induce the correct 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 is to introduce additional mutations into the VH-VL and CH1-CL interfaces (Lewis et al., Nat. Biotechnol., 32 (2014), pp. 191-198). These mutations encourage the heavy chain to preferentially pair with the light chain. Another approach is to introduce mutations that promote protein A binding into one of the Fc regions and select heterodimer pairings with intermediate protein A binding from homodimers with higher or lower protein A binding by affinity chromatography (Tusdian et al., MAbs. 2016 May-Jun;8(4):828-38).

[0085] Other bispecific antibodies avoid the mispairing problem by combining multiple binding specificities in the same heavy and light chain pair. One approach to doing this, called a dual variable domain, is to link two different heavy chain variable regions in series to a heavy chain constant region, and two different light chain variable regions in series to a light chain constant region (Correia et al., MAbs. 2013 May 1;5(3):364–372). Such antibodies can be assembled into tetramers by combining two identical pairs of heavy and light chains. The assembled antibody includes two different binding sites for each target. Another approach is to link an scFv that provides 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 that provides a binding site for a different target. Such bispecific antibodies can also be assembled into tetramers.

[0086] Another approach followed in the examples of the present invention is to introduce a second binding specificity by connecting a single-chain Fv (scFv) to the C-terminus of the heavy chain constant region, optionally omitting the C-terminal lysine residue. Like standard antibodies, this bispecific antibody includes a first binding site formed by the heavy chain and light chain variable regions attached to the N-terminus of the heavy and light chain constant regions. The C-terminus of the heavy chain is attached to the scFv, which provides a second binding site. scFv is usually attached through a linker, with another linker connecting the heavy and light chain variable regions in the scFv. The scFv can be attached to the Fc region through a linker via its light chain variable region or the end of the heavy chain variable region. When assembled by a complex of two identical paired heavy and light chains, this bispecific antibody includes two binding sites for each of the two different specificities. The antigen-binding arms of this bispecific antibody for CD3 and the other target can be attached in any direction. That is, the antigen-binding arm for CD3 can be in the form of an scFv, while the antigen-binding arm for the other target can be in the form of a standard antibody, and vice versa. The arms attached to the N-terminal of the heavy and light chain constant regions are provided as separate heavy chains and heavy chain variable regions, and the arms attached to the C-terminal are provided as scFv fragments. The advantage of this format is that the two different binding spaces are separated by the entire heavy chain constant region, which may promote intercellular bridging. Optionally, substitutions to cysteine ​​residues can be introduced into each of the heavy and light chain variable regions in the scFv (e.g., by Kabat numbering, at heavy chain variable region position 44 and light chain variable region position 100) to improve stability.

[0087] Another format connects a scFv that specifically binds to the first target to the heavy chain constant region, and connects a scFv that specifically binds to the other target to the light chain constant region. This antibody assembles into a tetramer that includes two copies of each binding site (Bs(scFv)4-IgG) (Zuo et al., Protein Eng 13:361-367, 2000).

[0088] Other formats connect the scFv binding region to a single chain without a constant region. For example, the BiTe format connects two scFv fragments via a linker (see, for example, Ross et al., PLoS ONE 12(8):e0183390, 2017). This format lacks effector function and has a short half-life, but may have advantages in accessibility and ease of manufacture due to its small size.

[0089] Many of the above forms include linker peptides between the heavy chain variable region and the light chain variable region or between the variable region and the constant region. The linker is a short peptide that confers flexibility and is usually mainly 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 combination of heavy and light chain variable regions 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-described formats. The sequence of an exemplary scFv comprises, consists of, or consists essentially of any one of SEQ ID NOs: 29, 31, or 33. An 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, wherein 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, wherein the scFv has a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 29. Selection of constant regions

[0091] Many forms of monospecific antibodies, bispecific or multispecific antibodies include at least a portion of a human constant region. The selection of constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and / or complement-dependent cytotoxicity are needed. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. The light chain constant region can be λ or κ. Human IgG1 and IgG3 also induce cell-mediated effector functions that are stronger than human IgG2 and IgG4. Here, although ADCC, ADCP and CDC can be used to provide additional mechanisms of action for cancer cells or infected cells bound by one arm of the bispecific antibody, it is useless for exciting CD3 to activate immune cells by the other arm.

[0092] One or more amino acids at the amino or carboxyl termini of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in part or all of the molecule. Counterions may be present or absent to form pharmaceutically acceptable salts. Water or other solvents may or may not be bound to the antibody (e.g., as a hydrate or solvate). Amino acid substitutions may be made in the constant region to reduce or increase effector function, such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend human half-life (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). For example, there are many known mutations in IgG Fc to 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 allowing the hybrid protein of the present invention to compete more strongly with endogenous IgG for FcRn binding. In addition, many mutations are known to reduce any one of ADCC, ADCP, or CDC. (See, for example, Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006). For example, substitution of any amino acid residue at positions 234, 235, 236 and / or 237 reduces affinity for Fcγ receptors, particularly FcγRI receptors (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 position 235 is substituted with Gln or Glu (see, e.g., US 5,624,821). Other substitutions that reduce effector function include Ala at position 268, Gly or Ala at position 297, Leu at position 309, Ala at position 322, Gly at position 327, Ser at position 330, Ser at position 331, Ser at position 238, Ala at position 268, and Leu at position 309.

[0093] Human constant regions exhibit both allotypic and idiotypic variation between individuals, meaning that the constant regions may differ from individual to individual at one or more polymorphic positions. Idiotypic allotypes differ from allotypes in that serum that recognizes the idiotypic allotype binds to non-polymorphic regions of one or more other isotypes. Expression of recombinant antibodies

[0094] Monospecific, bispecific antibodies or multispecific antibodies are usually produced by recombinant expression. Depending on the form, one, two or more antibody chains may need to be expressed. If multiple chains are expressed, they can be expressed from the same or different vectors. The recombinant polynucleotide construct usually includes an expression control sequence operably linked to the coding sequence of the antibody chain, including natural binding or heterologous expression control elements, such as promoters. The expression control sequence can be a promoter system in a vector capable of transforming or transfecting a eukaryotic or prokaryotic host cell. Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of the bispecific antibody.

[0095] These expression vectors can usually replicate in the host organism as episomes or as an integral part of the host chromosomal DNA. Typically, the expression vector contains a selection marker, such as ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the desired DNA sequence.

[0096] Escherichia coli is a prokaryotic host that can be used to express antibodies, particularly antibody fragments. Microorganisms such as yeast can also be used for expression. Yeast is a yeast host using a suitable vector having the desired expression control sequences, replication origin, termination sequences, and desired similar elements. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, and other promoters.

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

[0098] Alternatively, the antibody coding sequence can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequently expressed in the milk of the transgenic animal (see, e.g., U.S. Patent No. 5,741,957; U.S. Patent No. 5,304,489; and U.S. Patent No. 5,849,992). Suitable transgenes include coding sequences for light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes (e.g., casein or beta lactoglobulin).

[0099] The vector containing the DNA fragment of interest can be transferred into the host cell by methods determined by the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, bio-bombing or virus-based transfection can be used for other cell hosts. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection. In order to produce transgenic animals, the transgene can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of these cells are transferred into enucleated oocytes.

[0100] After the vectors encoding the antibody heavy and light chains are introduced into the cell culture, the cell pool can be screened for antibody productivity and quality in serum-free medium. TMSingle cell clones can be used to generate monoclonal cell lines. Specific productivity of 50 pg or 100 pg per cell per day or more can be used, corresponding to product titers greater than 7.5 g / L of culture. Antibodies produced by single cell clones can also be subjected to turbidity, filterability, PAGE, IEF, UV scanning, HP-SEC, carbohydrate-oligosaccharide profiling, mass spectrometry, and binding assays such as ELISA or BIACORE. TM Selected clones can then be stored in multiple vials and cryopreserved for subsequent use.

[0101] Once expressed, the bispecific antibodies can be purified according to standard procedures in 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] Methods for commercial production of antibodies can be employed including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single-cell cloning, cell banking, use of selectable markers to amplify copy number, CHO terminators, or increasing protein titer (see, e.g., U.S. Pat. No. 5,786,464; U.S. Pat. No. 6,114,148; U.S. Pat. No. 6,063,598; U.S. Pat. No. 7,569,339; WO2004 / 050884; WO2008 / 012142; WO2008 / 012142; WO2005 / 019442; WO2008 / 107388; WO2009 / 027471; and U.S. Pat. No. 5,888,809). Nucleic Acids

[0103] The present invention also provides nucleic acids encoding any one of the above-mentioned heavy and light chains. Optionally, such nucleic acids further encode a signal peptide, and can be expressed with a signal peptide of the constant region coding sequence connected to the nucleic acid, and can be operably connected to a regulatory sequence to ensure the expression of the coding sequence, such as a promoter, enhancer, ribosome binding site, transcription termination signal, etc. The nucleic acids encoding the heavy and light chains can exist in an isolated form, or can be cloned into one or more vectors. The nucleic acids can be synthesized by, for example, solid-state synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains can be connected into a continuous nucleic acid, such as in an expression vector, or can be separate, such as each cloned into its own expression vector. Methods of treatment and pharmaceutical compositions

[0104] The bispecific or multispecific antibodies of the present invention can be used to treat cancer, wherein one arm of the bispecific or multispecific antibody binds to a target expressed or overexpressed in the cancer, such as those disclosed above. Bispecific or multispecific antibodies can be used to treat solid tumors and hematologic malignancies. Hematologic malignancies include leukemias (e.g., T-cell large granular lymphocytic leukemia), lymphomas (Hodgkin or non-Hodgkin), or multiple myeloma. Solid tumors include skin (e.g., melanoma), ovary, endometrium, kidney, liver, pancreas, bladder, breast, ovary, prostate, rectum, colon, stomach, intestine, pancreas, lung, thymus, thyroid, kidney, and brain.

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

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

[0107] Monospecific, bispecific or multispecific antibodies are administered in an effective regimen, which means that the dosage, route of administration and frequency of administration delay onset, reduce severity, inhibit further deterioration and / or improve at least one sign or symptom of the disease. If the subject is already ill, the regimen may be referred to as a therapeutically effective regimen. If the subject's risk of illness is higher than the general population, but symptoms have not yet appeared, the regimen may be referred to as a prophylactic effective regimen. In some cases, treatment or prophylactic efficacy may be observed in individual subjects relative to historical controls or past experience of the same subject. In other cases, treatment or prophylactic efficacy may be demonstrated in a treatment subject population in preclinical or clinical trials relative to a control population of untreated subjects.

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

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

[0110] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Preferably, the drug is administered intravenously or subcutaneously to the systemic circulation. Intravenous administration can be, for example, by infusion over a period of, for example, 30-90 minutes.

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

[0112] The number of doses administered depends on whether the disease is acute or chronic, and the response of the disease to treatment. For acute diseases or acute exacerbations of chronic diseases, 1 to 10 doses are generally sufficient. Sometimes, for acute diseases or acute exacerbations of chronic diseases, a single bolus dose (optionally in the form of fractions) is sufficient. For acute disease recurrence or acute exacerbations, treatment can be repeated. For chronic diseases, bispecific antibodies can be administered regularly, for example weekly, every two weeks, monthly, quarterly, every six months, for at least 1, 5 or 10 years, or for the lifetime of the subject.

[0113] The pharmaceutical composition is preferably suitable for parenteral administration to humans (e.g., according to FDA standards). Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a dose for a single administration). The pharmaceutical composition can be formulated using one or more pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Pharmaceutically acceptable means suitable for human administration, for example, approved or approvable by the FDA. The formulation depends on the selected route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain a preparaton, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, the antibody can be in lyophilized form and reconstituted with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.

[0114] Treatment with the antibodies of the present invention can be combined with other treatments that are effective against the disease being treated. When used to treat cancer, the antibodies of the present invention can be combined with chemotherapy, radiation, stem cell therapy, surgery, or treatment with other biological products, such as Herceptin, which targets the HER2 antigen. TM (trastuzumab), Avastin targeting VEGF TM (bevacizumab) or antibodies against EGF receptors, such as (Erbitux TM , cetuximab) and Vectibix TM (panitumumab) or other antibodies shown in Table 2. Chemotherapeutic agents include chlorambucil, cyclophosphamide or melphalan, carboplatin, daunorubicin, doxorubicin, idarubicin, and mitoxantrone, methotrexate, fludarabine, and cytarabine, etoposide or topotecan, vincristine, and vinblastine. For infections, treatment can be combined with antibiotics, antivirals, antifungals, or antiprotozoal agents. Other methods

[0115] The antibodies of the present invention can also be used for diagnostic, prognostic and laboratory methods. They can be used to measure the level of antigen expressed in the circulation of cancer or cancer patients to determine whether the level is measurable or even elevated, and thus track and guide the treatment of cancer, because cancers associated with measurable or elevated levels of antigen are most suitable for treatment with bispecific antibodies comprising arms that bind to the cancer. The antibodies can be used for ELISA assays, radioimmunoassays or immunohistochemistry, etc. The antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes or radioisotopes and can be provided in the form of kits, which contain all the reagents required for the assay.

[0116] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually pointed out to be incorporated by reference. If different versions of a sequence are associated with accession numbers at different times, it refers to the version associated with the accession number of the effective filing date of this application. The effective filing date refers to the earlier of the actual filing date or the filing date of the priority application (if applicable) that mentions the accession number. Similarly, if different versions of a publication, website, etc. are published at different times, unless otherwise stated, it should refer to the version most recently published on the effective filing date of the application. Unless otherwise specifically stated, any feature, step, element, embodiment or aspect of the present invention can be used in combination with any other. Although the present invention has been described in some detail by way of illustration and example for the purpose of clarity and understanding, it is apparent that certain changes and modifications can be implemented 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 culture, ELISA, and flow cytometry were performed according to standard laboratory techniques, such as those described in Green and Sambrook (Molecular Cloning, A Laboratory Manual, 4th ed., 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Greenfield (Antibodies, A Laboratory Manual, 2nd ed., 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Kostelny et al. (Int. J. Cancer 93:556-565, 2001), Cole et al. (J. Immunol. 159:3613-3621, 1997), and Tsurushita et al. (Methods 36:69-83, 2005), as well as the suppliers' instructions. For the positions of amino acid residues in chimeric, humanized, and human IgG1 / κ (or λ) antibodies, the numbering system of Kabat et al. (Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991) is used.

[0118] SP34 is a mouse IgG3 / λ monoclonal agonist antibody that binds to human and cynomolgus monkey CD3ε protein. SP34 cross-links CD3ε protein on the surface of T cells, inducing activation of these T cells (Passano et al., EMBO J. 4:337-344, 1985; Yang et al., J. Immunol. 137:1097-1100, 1986). et al., J. Immunol. 147:3047-3052, 1991; Perez-Aciego et al., J. Exp. Med. 174:319-326, 1991; Conrad et al., Cytom. A71A:925-933, 2007; U.S. Patents 8,236,308 and 10,066,015). The amino acid sequence of the mature heavy chain variable region (VH) of SP34 is EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 1). The amino acid sequence of the mature light chain variable region (VL) of SP34 is QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLI GGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWV FGGGTKLTVL (SEQ ID NO: 2). Example 2: Humanization of 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. (above). In short, a three-dimensional molecular model of the mouse SP34 variable region was first constructed using appropriate software. Next, molecular modeling was used to identify framework amino acid residues important for CDR structure formation. Simultaneously, human VH and VL amino acid sequences derived from cDNAs with a high degree of homology to SP34 VH and VL were selected. Finally, the CDR sequences, along with framework amino acid residues important for the correct formation of the antigen-binding site, were transplanted from SP34 VH and VL into the corresponding selected human framework sequences.

[0120] To design the humanized SP34 VH, the human VH sequence encoded by the M24236 cDNA (GenBank accession number; Sanz et al., J. Immunol. 142:883-887, 1989) (M24236 VH; SEQ ID NO: 3) was selected as the humanized receptor. M24236 VH belongs to the IGHV3-15 subgroup of human germline VH fragments and has no somatic hypermutations in the amino acid sequence of the framework region. The framework amino acid sequence of M24236 VH is 85.2% identical to SP34 VH. First, the CDR sequences of SP34 VH were 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 region suggests that amino acid residues may play an important role in the formation of the antigen binding site, the amino acid residues of M24236 VH were replaced with the corresponding residues of mouse SP34 VH. The amino acid sequence of the resulting mature humanized VH, designated HuSP34 VH1 (SEQ ID NO: 4), is identical to the amino acid sequences of mature SP34 and M24236 VH. Figure 1 shown.

[0121] In order to design the humanized SP34 VL, the human Vλ region encoded by the Y14738 cDNA (GenBank accession number; Paterson et al., Immunotechnol. 4: 37-47, 1998) (Y14738 VL; SEQ ID NO: 5) was selected as the humanized receptor. Y14738 VL belongs to the IGLV8-61 subgroup of the human germline Vλ segment and has no somatic hypermutations in the amino acid sequence of the framework region. The framework amino acid sequence of Y14738 VL has 67.1% identity with SP34 VL. First, the CDR sequence of SP34 VL was 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 region indicates that the amino acid residues may play an important role in the formation of the antigen binding site, the 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, designated HuSP34 VL1 (SEQ ID NO: 6), is identical to the amino acid sequences of the mature SP34 and Y14738 VLs as shown in FIG. Figure 2 shown.

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

[0123] from Figure 5 Starting clockwise from the SalI site in A, pHuSP34A contains the heavy chain transcription unit, which starts with the human cytomegalovirus (CMV) major immediate early promoter 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, which is a genomic sequence containing the human gamma-1 heavy chain constant region, including CH1, hinge, CH2 and CH3 exons and the introns between them, as well as a polyadenylation site after the CH3 exon. The CH2 region carries an amino acid substitution from leucine to alanine at positions 234 and 235 (Eu numbering; Kabat et al., supra) to eliminate effector function (Hazareh et al., J. Virol. 75: 12161-12168, 2001). After the heavy chain gene sequence, the light chain transcription unit starts with the CMV promoter, followed by the exons encoding HuSP34 VL1 and the genomic sequence containing the human lambda-2 chain constant region (Cλ2), which is preceded by a portion of the introns and followed by the polyadenylation site of the Cλ2 exon. The light chain gene is followed by the SV40 early promoter (SV40-P), the puromycin N-acetyltransferase gene (puro) that confers resistance to puromycin, and a fragment containing the SV40 polyadenylation site (SV40-A). Finally, the plasmid contains a portion of the plasmid pUC19, which contains the bacterial origin of replication (pUC ori) and the beta-lactamase gene (β-lactamase). The locations of the relevant restriction enzyme sites are shown in Figure 2. Figure 5 As shown in A. Arrows indicate the direction of transcription.

[0124] The amino acid sequences of the CH1, hinge, CH2, and CH3 regions of the human gamma-1 heavy chain encoded in pHuSP34A are ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:9), EPKSCDKTHTCPPCP (SEQ ID NO:10), APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAK (SEQ ID NO:11), and GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN, respectively. NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12).

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

[0126] The amino acid sequence of the heavy chain encoded in pHuSP34A is MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14). The mature HuSP34A heavy chain starts at the glutamic acid residue at position 20 of SEQ ID NO: 14.

[0127] The amino acid sequence of the light chain encoded in pHusp34A is MAWISLILSLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 15). The 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 the Chinese hamster ovary cell line CHO-K1. Stable transfection into CHO-K1 cells was performed by electroporation. Before transfection, pHuSP34A was linearized using the restriction endonuclease FspI ( Figure 5 A) Approximately 2.5 x 10 cells were transfected with 20 μg of linearized plasmid. 6 Cells were suspended in SFM4 CHO medium (HyClone, Logan, UT) and, after appropriate dilution, plated in multiple 96-well plates and grown at 37°C in a 7.5% CO2 incubator. After 48 hours, 10 μg / ml puromycin was added to isolate stable transfectants.

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

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

[0131] The binding of HuSP34A to human CD3 was analyzed by flow cytometry using the human T cell line Jurkat Dual (Invivogen, San Diego, CA). In a typical experiment, different concentrations of HuSP34A were incubated with Jurkat Dual cells in FACS buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.05% sodium azide) for 30 minutes. After washing with FACS buffer, the cells were incubated with PE-labeled goat anti-human IgG antibody for 20 minutes. After washing with FACS buffer and resuspending in it, the cells were subjected to flow cytometry. Figure 6 As shown, HuSP34A bound to Jurkat Dual cells in a dose-dependent manner. The EC values ​​for HuSP34A binding to Jurkat Dual cells were calculated using Prism software (GraphPad, San Diego, CA). 50 The value is 92ng / ml. Example 3: Humanization of anti-human CD3 antibody [II]

[0132] The second humanized SP34 VL was designed in a different manner. As the humanized receptor, 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 belongs to the IGKV3-11 subgroup of human germline Vκ segments and lacks somatic hypermutations in the amino acid sequence of the framework regions. The framework amino acid sequence of L37309 VL shares 51.3% identity with 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 region suggests that the amino acid residues may play an important role in the formation of the antigen binding site, the amino acid residues of L37309 VL were replaced with the corresponding residues of mouse SP34 VL. The amino acid sequence of the resulting mature humanized VL, designated HuSP34 VL3 (SEQ ID NO: 17), is identical to the SP34 and L37309 VL sequences as shown in FIG. Figure 7 shown.

[0133] The gene encoding HuSP34 VL3 was synthesized as an exon, which includes a signal peptide (SEQ ID NO: 18), a splice donor signal, an NheI site at the 5' end, and an EcoRI site at the 3' end ( Figure 8The HuSP34 VL3 gene was cloned between the NheI and EcoRI sites to replace the HuSP34 VL1 in pHuSP34A. In addition, the human λ-2 chain constant region (Cλ2) was replaced by the coding sequence of the human κ chain constant region (Cκ). The resulting plasmid for expressing a humanized anti-CD3 IgG1 / κ antibody (HuSP34C) containing HuSP34 VH1 and VL3 was designated pHuSP34C. The schematic diagram of the structure of pHuSP34C is shown in FIG. Figure 5 B. The amino acid sequence of the human kappa chain constant region encoded in pHuSP34C is RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19). The amino acid sequence of the heavy chain encoded in pHuSP34C is identical to that encoded in pHuSP34A (SEQ ID NO: 14). The amino acid sequence of the light chain encoded in pHusp34C is MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20). The mature HuSP34C light chain starts at the glutamic acid residue at position 21 of SEQ ID NO: 20.

[0134] The expression vector pHuSP34C was introduced into CHO-K1 cells by electroporation as described above. CHO-K1 stable transfectants expressing HuSP34C (CHO-K1 / pHuSP34C) were amplified in SFM4CHO as described above. HuSP34C was purified from the culture supernatant 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 results are shown in Figure 4. Figure 6 HuSP34C binds to JurkatDual cells in a dose-dependent manner. 50The value is 209ng / ml. Example 4: Mutagenesis of HuSP34 VL3

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

[0136] By replacing HuSP34 VL3 in pHuSP34C, each of the six HuSP34 VL3 variants was combined with HuSP34 VH1 for expression as an IgG1 / κ antibody. When the HuSP34 VL3 L66G variant was combined with HuSP34 VH1, antibody expression was severely reduced. The combination of HuSP34 VH1 with the HuSP34 VL3 V36Y variant resulted in a dramatic reduction in antigen binding. Each of the HuSP34 VL3 V58I, I67S, D69T, and K70D variants, in combination with HuSP34 VH1, exhibited similar levels of antigen binding as the HuSP34 VH1 and VL3 combination (HuSP34C). The mouse-derived hydrophobic isoleucine residue at position 67 in HuSP34 VL3 was replaced with a human-derived hydrophilic serine residue (I67S) without loss of antigen binding affinity. Likewise, each of the substitution of (i) the mouse-derived acidic aspartic acid residue at position 69 to a human-derived neutral hydrophilic threonine residue (D69T) and (ii) the mouse-derived basic lysine residue at position 70 to a human-derived acidic aspartic acid residue (K70D) did not result in a loss of affinity.

[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. Figure 7 The gene encoding HuSP34 VL4 was generated by site-directed mutagenesis of HuSP34 VL3. The nucleotide sequence and deduced amino acid sequence of the HuSP34 VL4 gene are shown in Figure 9 shown.

[0138] The HuSP34 VL4 gene was cloned between the NheI and EcoRI sites to replace the HuSP34 VL3 in pHuSP34C. The resulting expression vector, designated pHuSP34V, expressed a humanized SP34 IgG1 / κ antibody (HuSP34V) comprising HuSP34 VH1 and VL4. The amino acid sequence of the heavy chain encoded in pHuSP34V was identical to that encoded in pHuSP34A (SEQ ID NO: 14). The amino acid sequence of the light chain encoded in pHusp34v is MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28). The mature HuSP34V light chain starts at the glutamic acid residue at position 21 of SEQ ID NO: 28.

[0139] The expression vector pHuSP34V was introduced into CHO-K1 cells by electroporation as described above. CHO-K1 stable transfectants expressing HuSP34V (CHO-K1 / pHuSP34V) were selected and amplified in SFM4CHO as described above. HuSP34V was purified from the culture supernatant 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 results are shown in Figure 4. Figure 6 HuSP34V binds to JurkatDual cells in a dose-dependent manner. 50 The value is 234ng / ml. Example 5: Generation and characterization of anti-CD20 / CD3 bispecific antibodies

[0140] The VH and VL regions of HuSP34A were converted into single-chain Fv (scFv) formats with an N'-VH-linker-VL-C' orientation. In addition, two amino acid substitutions were introduced into the resulting scFv, one of which was a substitution of glycine to cysteine ​​at position 44 in HuSP34 VH1 ( Figure 1 ), and another is the substitution of glycine to cysteine ​​at position 100 in HuSP34 VL1 ( Figure 2 (Brinkman et al., Proc. Natl. Acad. Sci. 90:7538-7542, 1993). The resulting HuSP34A scFv form (HuSP34A.scFv.HL.ds; SEQ ID NO:29) was then fused to the penultimate glycine residue of the CH3 region in an antibody expression vector having the same structure as pHuSP34C using a flexible polypeptide linker (CH3-HuSP34A.scFv.HL.ds; SEQ ID NO:30). Figure 5 B), except that the VH and VL exons were replaced by exons from an anti-CD20 antibody. The resulting expression vector was named pJB509.

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

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

[0143] HuSP34 VH1 and VL4 were then used to generate an anti-CD3 scFv antibody with an N'-VL-linker-VH-C' orientation. Furthermore, two amino acid substitutions were introduced into this generated scFv: a glycine to cysteine ​​substitution at position 44 in HuSP34 VH1 and a glycine to cysteine ​​substitution at position 100 in HuSP34 VL4. The resulting scFv was designated HuSP34V.scFv.ds. The amino acid sequence of HuSP34V.scFv.ds is EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSG GGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS(SEQ ID NO:33).

[0144] In the IgG1 / κ antibody expression vector, HuSP34V.scFv.ds was fused to the penultimate glycine residue of CH3 using a flexible polypeptide linker (CH3-HuSP34V.scFv.ds; SEQ ID NO: 34) that separated them. The structure of this antibody expression vector was the same as that of pHuSP34C ( Figure 5B), except that VH and VL were derived from the chimeric anti-CD20 antibody C2B8 (Reff et al. Blood 83:435-445, 1994; Maloney et al. Blood 84:2457-2466, 1994). The resulting expression vector was named pJB554. The schematic diagram of the structure of pJB554 is shown in FIG. Figure 5 As shown in C.

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

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

[0147] The bispecific anti-CD20 / CD3 IgG1 / κ antibody expressed from pJB554 was named JB554. The schematic diagram of the structure of JB554 is shown in Figure 10As shown. The amino acid sequence of the heavy chain encoded in pJB554 is MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO:37). The mature JB554 heavy chain starts at the glutamine residue at position 20 of SEQ ID NO:37.

[0148] The amino acid sequence of the light chain encoded in pJB554 is MDFQVQIISFLLISASVIMSRGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 38). The mature JB554 light chain starts at a glutamine residue at position 23 of SEQ ID NO: 38.

[0149] As described above, the expression vector pJB554 was transfected into HEK293 cells. Expression of the anti-CD20 / CD3 bispecific antibody from pJB554 was improved compared to that using pJB509 and pJB510. Transiently expressed JB554 was purified using Protein A as described above. The activity of JB554 (a bispecific antibody that binds to both CD20 and CD3) in inducing T cell-mediated cytotoxicity against CD20-positive human Burkitt's lymphoma Ramos cells was analyzed as follows. Purified human CD3 pan T cells (catalog number IQB-Hu1-T100, batch number P19D0900, iQ Biosciences, Berkeley, California) were cultured in RPMI 1640 medium (RPMI 1640 complete medium) containing 10% FBS, 1mM sodium pyruvate, and 10mM HEPES at 37°C for four days in a 7.5% CO2 incubator (activated T cells) in the presence (or absence) of 150ng / ml JB554. 100,000 Ramos cells labeled with a fluorescent dye (calcein AM; BioLegend, San Diego, California) were incubated in 200μl RPMI 1640 complete medium with 1 million activated T cells in a well of a 96-well plate for 4 hours. To monitor the level of lysis of Ramos cells, fluorescence in the culture medium was measured according to the supplier's instructions. As a 100% lysis control, fluorescence was measured in the culture supernatant of Calcein AM-labeled Ramos cells incubated alone as above and then lysed by SDS treatment. As a background control, fluorescence was measured in the culture supernatant of Calcein AM-labeled Ramos cells incubated alone as above.

[0150] The results of the cytotoxicity test of Ramos cells labeled with Calcein AM are as follows Figure 11 As shown in Figure A. The relative fluorescence unit (RFU) values ​​in the culture supernatant were 23,911 for (i) Ramos cells alone, 74,000 for (ii) SDS-treated Ramos cells, 33,668 for (iii) Ramos cells incubated with activated T cells, and 82,104 for (iv) Ramos cells incubated with activated T cells and JB554. The anti-CD20 / CD3 bispecific antibody JB554 effectively induced Ramos cell lysis in the presence of activated T cells. Example 6: Generation and characterization of anti-EGFR / CD3 bispecific antibodies

[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 replaced with genes derived from the 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 resulting expression vector was named pJB559. The structure of pJB559 is identical to that of pJB554 ( Figure 5 C), except for VH and VL sequences.

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

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

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

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

[0156] As described above, the expression vector pJB559 was transfected into HEK293 cells. As described above, the transiently expressed JB559 was purified by protein A. The activity analysis of JB559 (a bispecific antibody that can bind to EGFR and CD3) inducing T cell-mediated cytotoxicity against EGFR-positive human colorectal adenocarcinoma HT-29 cells is as follows. First, HT-29 cells were seeded in the wells of a 96-well plate at a concentration of 400,000 cells / ml in RPMI1640 complete medium. After 48 hours, HT-29 cells were labeled with calcein AM and then incubated with 5 million cells / ml of activated T cells in the presence (or absence) of the test antibody (JB554 or JB559), as shown above. As a 100% lysis control, the fluorescence of the culture supernatant of HT-29 cells labeled with calcein AM that were 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 indicated above, and fluorescence in the culture supernatant was measured.

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

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

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

[0160] The expression vector pCh225 for expressing a mouse-human chimeric anti-EGFR IgG1 / κ 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 the human gamma-1 heavy chain (SEQ ID NO: 49). The light chain amino acid sequence encoded in pCh225 is identical to the light chain sequence encoded in pJB559 (SEQ ID NO: 42). The amino acid sequence of the heavy chain encoded in pCh225 is MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51). The mature Ch225 heavy chain starts at a glutamine residue at position 20 of SEQ ID NO: 51.

[0161] The expression vector pHuM195 (Co et al., J. Immunol. 148: 1149-1154, 1992; U.S. Patent 5,693,761) expressing the humanized anti-CD33 IgG1 antibody HuM195 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. The amino acid sequence of the heavy chain encoded in pHuM195 is MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54). The mature HuM195 heavy chain starts at a glutamine residue at position 20 of SEQ ID NO: 54. The amino acid sequence of the light chain encoded in pHuM195 is MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 55).The mature HuM195 light chain starts at the aspartic acid residue at position 21 of SEQ ID NO:55.

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

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

[0164] As described above, a CHO-K1 stable transfectant cell line (CHO-K1 / pJB554) producing JB554 was generated by electroporation. As described above, CHO-K1 / pJB554 cells were expanded in SFM4 CHO medium and JB554 was purified by protein A. SDS-PAGE analysis of the purified JB554 under reducing conditions showed only two major bands of approximately 75 kDa heavy chain and 25 kDa light chain.

[0165] The biological activity of JB554 purified from CHO-K1 / pJB554 cells was examined using Jurkat Dual reporter cells (Invivogen, San Diego, California). In Jurkat Dual cells, cross-linking with CD3 on the surface triggers activation of the intracellular NF-κB signaling pathway, resulting in the expression and secretion of recombinant Lucia luciferase. Approximately 400,000 Jurkat Dual cells were incubated in 200 μl of RPMI 1640 medium containing 10% FBS in wells of a 96-well plate at 37°C in a 7.5% CO2 incubator for one day in the presence (or absence) of 200,000 CD20-positive Ramos cells and test antibodies. Luciferase activity in the culture supernatant was measured in triplicate using QUANTI-Luc reagent (Invivogen) according to the supplier's protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek, Winooski, Vermont). The mean relative luminescence unit (RLU) values ​​were 539 for (i) JurkatDual cells alone, 1,940 for (ii) Jurkat Dual cells incubated with 1 μg / ml ChC2B8 (anti-CD20 IgG1 antibody) and 1 μg / ml HuSP34V (anti-CD3 IgG1 antibody), 827 for (iii) JurkatDual cells incubated with 1 μg / ml JB554 (anti-CD20 / CD3 bispecific antibody), 459 for (iv) JurkatDual cells incubated with 1 μg / ml JB559 (anti-EGFR / CD3 bispecific antibody), 256 for (v) JurkatDual cells incubated with Ramos cells, and 257 for (vi) JurkatDual cells incubated with 1 μg / ml ChC2B8, 1 μg / ml The number of JurkatDual cells incubated with HuSP34V and Ramos cells was 2,212, (vii) the number of JurkatDual cells incubated with 1 μg / ml JB554 and Ramos cells was 14,730, and (viii) the number of JurkatDuals cells incubated with 1 μg / ml JB559 and Ramos cells was 691. Figure 12 A shows the RLU values ​​and standard deviation error bars for the Jurkat Dual assay. High levels of luciferase activity were observed in the culture supernatant only when Jurkat Dual cells were incubated with JB554 and Ramos cells.

[0166] JB554 purified from CHO-K / pJB554 cells was further analyzed for its ability 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. The activated T cells used in this experiment were derived from donor 3820 of human CD3 pan T cells (Cat. No. IQB-Hu1-T100, iQ Biosciences, Berkeley, CA). The relative fluorescence unit (RFU) values ​​in the culture supernatants were 18,474 for (i) Ramos cells alone, 79,395 for (ii) SDS-treated Ramos cells, 18,940 for (iii) Ramos cells incubated with activated T cells, 19,220 for (iv) Ramos cells incubated with activated T cells, ChC2B8, and HuSP34V, 79,395 for (v) Ramos cells incubated with activated T cells and JB554, and 20,663 for (vi) Ramos cells incubated with activated T cells and JB559. Figure 12 B) The anti-CD20 / CD3 bispecific antibody JB554 effectively induced Ramos cell lysis in the presence of activated T cells, but the anti-EGFR / CD3 bispecific antibody JB559 did not have this activity, nor did the combination of the anti-CD20 antibody ChC2B8 and the anti-CD3 antibody HuSP34V.

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

[0168] As described above, a CHO-K1 stable transfectant cell line (CHO-K1 / pJB559) producing JB559 was generated by electroporation. As described above, CHO-K1 / pJB559 cells were expanded in SFM4 CHO medium and JB559 was purified by protein A. SDS-PAGE analysis of the purified JB559 under reducing conditions showed only two major bands of approximately 75 kDa heavy chain and 25 kDa light chain.

[0169] The activity of JB559 induced T cell-mediated cytotoxicity against calcein AM-labeled HT-29 cells purified from CHO-K1 / pJB559 cells was analyzed, as described above. Each test antibody was used at 1 μg / ml. The activated T cells used in the first cytotoxicity experiment were derived from donor 3820 (Cat. No. IQB-Hu1-T100, iQBiosciences, Berkeley, California) of human CD3 pan T cells. In the second experiment, activated T cells derived from donor 3661 of human CD3 pan T cells were used. The relative fluorescence unit (RFU) values ​​in the culture supernatant of the first experiment were 6,112 for (i) HT-29 cells alone, 75,173 for (ii) SDS-treated HT-29 cells, 6,482 for (iii) HT-29 cells incubated with activated T cells, 5,968 for (iv) HT-29 cells incubated with activated T cells, Ch225 (anti-EGFR IgG1 antibody) and HuSP34V (anti-CD3 IgG1 antibody), 6,350 for (v) HT-29 cells incubated with activated T cells and JB554 (anti-CD20 / CD3 bispecific antibody), and 56,634 for (vi) HT-29 cells incubated with activated T cells and JB559 (anti-EGFR / CD3 bispecific antibody). Figure 13 A). In the second experiment, the RFU values ​​in the culture supernatant were 5,046 for (vii) HT-29 cells alone, 72,990 for (viii) SDS-treated HT-29 cells, 6,114 for (ix) HT-29 cells incubated with activated T cells, 5,391 for (x) HT-29 cells incubated with activated T cells, Ch225, and HuSP34V, 5,662 for (xi) HT-29 cells incubated with activated T cells and JB554, and 35,443 for (xii) HT-29 cells incubated with activated T cells and JB559. Figure 13 B) In two experiments using HT-29 cells, the anti-EGFR / CD3 bispecific antibody JB559 effectively induced HT-29 cell lysis in the presence of activated T cells, but the anti-CD20 / CD3 bispecific antibody JB554 had no such activity, nor did the combination of Ch225 and HuSP34V.

[0170] Example 10: Anti-EGFR / CD3 bispecific antibody JB559 does not induce EGFR-independent activation of T cells

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

[0172] The expression levels of IFN-γ and IL-2 in culture supernatants were measured using the ELISA MAX Standard Human IFN-γ and ELISA MAX Standard Human IL-2 kits (BioLegend), respectively. The average IFN-γ expression levels of replicate samples were (a) 23.3 pg / ml without additional reagents, (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 replicate samples were (d) 33.8 pg / ml without additional reagents, (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 signs of T cell activation were observed after PBMCs were incubated with JB559 for 3 days in the absence of cells expressing EGFR.

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

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

[0175] As described above, a CHO-K1 stable transfectant cell line (CHO-K1 / pJB564) producing JB564 was generated by electroporation. As described above, CHO-K1 / pJB564 cells were expanded in SFM4 CHO medium and JB564 was purified by protein A. SDS-PAGE analysis under reducing conditions showed that JB564 consists of two polypeptides of approximately 75 kDa heavy chain and 25 kDa light chain.

[0176] JB564 was analyzed for its activity in inducing T cell-mediated cytotoxicity against Calcein AM-labeled human CD33-positive HL-60 cells (Cat. No. CCL-240, American Type Culture Collection, Manassas, VA) in the presence (or absence) of activated T cells and 1 μg / ml of test antibody, as described above. The relative fluorescence unit (RFU) values ​​in the culture supernatant were 25,364 for (i) HL-60 cells alone, 75,253 for (ii) SDS-treated HL-60 cells, 22,895 for (iii) HL-60 cells incubated with activated T cells, and 25,756 for (iv) HL-60 cells incubated with activated T cells, HuM195 (anti-CD33 IgG1 antibody), and HuSP34V (anti-CD3 IgG1 antibody), (v) 21,928 for HL-60 cells incubated with activated T cells and JB559 (anti-EGFR / CD3 bispecific antibody), and (vi) 68,226 for HL-60 cells incubated with activated T cells and JB564 (anti-CD33 / CD3 bispecific antibody). Figure 14 The anti-CD33 / CD3 bispecific antibody JB564 effectively induced HL-60 cell lysis in the presence of activated T cells, but the anti-EGFR / CD3 bispecific antibody JB559 did not have this activity, nor did the combination of HuM195 and HuSP34V. Sequence Listing

[0177] SEQ ID NO: 1

[0178] Amino acid sequence of mature SP34 VH

[0179] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0180] SEQ ID NO:2

[0181] Amino acid sequence of mature SP34 VL

[0182] QAVVTQESALTSPGETVTTLTCRSSTGAVTTSNYANWVQEKPDHL FTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYS NLWVFGGGTKLTVL

[0183] SEQ ID NO:3

[0184] Amino acid sequence of mature M24236 VH

[0185] EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKG LEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDT AVYYCTTDSLPPHRVWGQGTLVTVSS

[0186] SEQ ID NO:4

[0187] Amino acid sequence of mature HuSP34 VH1

[0188] EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGK GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTED TAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0189] SEQ ID NO:5

[0190] Amino acid sequence of mature Y14738 VL

[0191] QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYQQTPGQAP RTLIYTTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCVLYMGG VWVFGGGTKLTVL

[0192] SEQ ID NO:6

[0193] Amino acid sequence of mature HuSP34 VL1

[0194] QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQA PRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWY SNLWVFGGGTKLTVL

[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

[0200] MAWISLILSLLALSSG

[0201] SEQ ID NO:9

[0202] Amino acid sequence of the CH1 region of the human gamma-1 heavy chain encoded in pHusp34A

[0203] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0204] SEQ ID NO: 10

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

[0206] EPKSCDKTHTCPPCP

[0207] SEQ ID NO:11

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

[0209] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAK

[0210] SEQ ID NO:12

[0211] Amino acid sequence of the CH3 region of the human gamma-1 heavy chain encoded in pHusp34A

[0212] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK

[0213] SEQ ID NO:13

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

[0215] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST VEKTVAPTECS(SEQ ID NO:13)

[0216] SEQ ID NO:14

[0217] Amino acid sequence of the heavy chain encoded in pHusP34A

[0218] MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAAS GFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0219] SEQ ID NO:15

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

[0221] MAWISLILSLLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTS NYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0222] SEQ ID NO:16

[0223] Amino acid sequence of mature L37309 VL

[0224] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK

[0225] SEQ ID NO: 17

[0226] Amino acid sequence of mature HuSP34 VL3

[0227] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0228] SEQ ID NO: 18

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

[0230] MEAPAQLLFLLLLWLPDTTG

[0231] SEQ ID NO: 19

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

[0233] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0234] SEQ ID NO:20

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

[0236] MEAPAQLLFLLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNL WVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0237] SEQ ID NO:21

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

[0239] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWYQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0240] SEQ ID NO:22

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

[0242] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLIGDKATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0243] SEQ ID NO:23

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

[0245] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQA PRGLIGGTNKRAPGVPARFSGSGIGDKATLTISSLEPEDFAVYYCALWYSN LWVFGGGTKVEIK

[0246] SEQ ID NO:24

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

[0248] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQA PRGLIGGTNKRAPGVPARFSGSLSGDKATLTISSLEPEDFAVYYCALWYSN LWVFGGGTKVEIK

[0249] SEQ ID NO:25

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

[0251] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQA PRGLIGGTNKRAPGVPARFSGSLIGTKATLTISSLEPEDFAVYYCALWYSN LWVFGGGTKVEIK

[0252] SEQ ID NO:26

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

[0254] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQA PRGLIGGTNKRAPGVPARFSGSLIGDDATLTISSLEPEDFAVYYCALWYSN LWVFGGGTKVEIK

[0255] SEQ ID NO:27

[0256] Amino acid sequence of mature HuSP34 VL4

[0257] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQA PRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSN LWVFGGGTKVEIK

[0258] SEQ ID NO:28

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

[0260] MEAPAQLLFLLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSST GAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIKRTVAAPSVFIFPPSDEQSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0261] SEQ ID NO:29

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

[0263] EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKC LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVL

[0264] SEQ ID NO:30

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

[0266] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVL

[0267] SEQ ID NO:31

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

[0269] QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQA PRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0270] SEQ ID NO:32

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

[0272] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0273] SEQ ID NO:33

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

[0275] EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQA PRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0276] SEQ ID NO:34

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

[0278] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0279] SEQ ID NO:35

[0280] Amino acid sequence of mature C2B8 VH

[0281] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGR GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAV YYCARSTYYGGDWYFNVWGAGTTVTVSA

[0282] SEQ ID NO:36

[0283] Amino acid sequence of mature C2B8 VL

[0284] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWI YATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFG GGTKLEIK

[0285] SEQ ID NO:37

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

[0287] MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0288] SEQ ID NO:38

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

[0290] MDFQVQIISFLLISASVIMSRGQIVLSQSPAILSASPGEKVTMTCRASS SVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0291] SEQ ID NO:39

[0292] Amino acid sequence of mature 225 VH

[0293] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGL EWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAYWGQGTLVTVSA

[0294] SEQ ID NO:40

[0295] Amino acid sequence of mature 225VL

[0296] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLI KYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA GTKLELK

[0297] SEQ ID NO:41

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

[0299] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFS LTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0300] SEQ ID NO:42

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

[0302] MRAPAQFLGFLLFWIPASRSDILLTQSPVILSVSPGERVSFSCRASQSI GTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0303] SEQ ID NO:43CDRH1 TYAMN

[0304] SEQ ID NO:44:CDRH2 RIRSKYNNYATYYADSVKD

[0305] SEQ ID NO:45CDRH3HGNFGNSYVSWFAY

[0306] SEQ ID NO:46CDRL1 RSSTGAVTTSNYAN

[0307] SEQ ID NO:47CDRL2 GTNKRAP

[0308] SEQ ID NO:48CDRL3 ALWYSNLWV

[0309] SEQ ID NO:49

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

[0311] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAK

[0312] SEQ ID NO:50

[0313] Amino acid sequence of the heavy chain encoded in pChC2B8

[0314] MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0315] SEQ ID NO:51

[0316] Amino acid sequence of the heavy chain encoded in pCh225

[0317] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFS LTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0318] SEQ ID NO:52

[0319] Amino acid sequence of HuM195 VH

[0320] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASG YTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITAD ESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS

[0321] SEQ ID NO:53

[0322] Amino acid sequence of HuM195 VL

[0323] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRAS ESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFT LTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK

[0324] SEQ ID NO:54

[0325] Amino acid sequence of the heavy chain encoded in pHuM195

[0326] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASG YTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0327] SEQ ID NO:55

[0328] Amino acid sequence of the light chain encoded in pHuM195

[0329] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSSLSASVGDRVTITCRAS ESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0330] SEQ ID NO:56

[0331] Amino acid sequence of the heavy chain encoded in pJB564

[0332] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASG YTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGCGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0333] SEQ ID NO:57

[0334] Nucleotide sequence of HuSP34 VH1 gene flanked by SpeI and HindIII sites

[0335] ACTAGTACCACCATGCTGTTGGGGCTGAAGTGGGTTTTCTTTG TTGTTTTTTATCAAGGAGTGCATTGTGAAGTGCAGCTTGTGGAAAGTGGCGGAGGACTGGTGAAGCCAGGCGGATCACTGAGACTGTCCTGCGCAGCTAGTGGCTTCACCTTTAACACATACGCTATGAATTGGGTCCGACAGGCACCTGGCAAGGGCCTGGAGTGGGTGGCAAGGATCAGGTCCAAGTACAACAATTATGCAACCTACTATGCCGACTCTGTGAAGGATAGATTCACAATCAGTCGCGACGATTCCAAGAACACTCTGTATCTGCAGATGAACAGTCTGAAAACTGAAGACACCGCCGTGTACTATTGTGTGCGGCACGGAAACTTCGGCAATTCTTACGTCTCTTGGTTTGCTTATTGGGGACAGGGGACACTGGTCACTGTGTCTTCAGGTGAGTCCTAACTTCTCCCATTCTAAGCTT

[0336] SEQ ID NO:58

[0337] Amino acid sequence of HuSP34 VH1 including signal peptide

[0338] MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVKPGGSLRLSCAAS GFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0339] SEQ ID NO:59

[0340] HuSP34 flanked by Nhel and EcoRI sites Nucleotide sequence of the VL1 gene GCTAGCACCACCATGGCCTGGATTTCACTTATCCTCTCTCCTGGCTCTCAGCTCAGGGCAGACTGTCGTGACACAGGAACCCTCATTTTCCGTCAGCCCTGGCGGAACAGTGACCCTGACCTGCAGATCTAGCACAGGCGCAGTGACCACAAGCAACTACGCCAACTGGGTCCAGCAAACTCCAGGCCAAGCTCCCAGAGG CCTGATCGGCGGCACCAACAAAAGGGCTCCAGGCGTGCCAGACAGATTCAGCGGCAGCATCCTTGGCAATAAGGCTGCCCTGACAATCACTGGAGCCCAGGCCGACGACGAGTCCGACTACTATTGCGCCCTGTGGTACAGCAACCTGTGGGTCTTCGGCGGAGGCACCAAGCTGACAGTGCTAGGTGAGTCCTTCCTCCTTTGTTATTGAATTC

[0341] SEQ ID NO:60

[0342] Amino acid sequence of HuSP34 VL1 including signal peptide

[0343] MAWISLILSLLALSSGQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTS NYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGA QADDESDYYCALWYSNLWVFGGGTKLTVL

[0344] SEQ ID NO:61

[0345] Nucleotide sequence of the HuSP34 VL3 gene flanked by NheI and EcoRI sites

[0346] GCTAGCGCCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCT GCTTCTCTGGCTCCCAGATACCACTGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGATCTAGCACAGGAGCCGTGACCACAAGCAACTATGCCAACTGGGTCCAACAGAAACCTGGCCAGGCTCCCAGGGGACTCATCGGAGGCACCA ACAAAAGGGCTCCAGGAGTCCCAGCCAGGTTCAGTGGCAGTCTGATTGGGGATAAAGCTACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGAGGAGGCACCAAAGTCGAAATCAAACGTAAGTAGAATCCAAAGTGAATTC

[0347] SEQ ID NO:62

[0348] Amino acid sequence of HuSP34 VL3 including signal peptide

[0349] MEAPAQLLFLLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSST GAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLIGDKAT LTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

[0350] SEQ ID NO:63

[0351] Nucleotide sequence of the HuSP34 VL4 gene flanked by NheI and EcoRI sites

[0352] GCTAGCGCCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCT GCTTCTCTGGCTCCCAGATACCACTGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGATCTAGCACAGGAGCCGTGACCACAAGCAACTATGCCAACTGGGTCCAACAGAAACCTGGCCAGGCTCCCAGGGGACTCATCGGAGGCACCAACAAAAGGGCTCCAGGAATCCCAGCCAGGTTCAGTGGCAGTCTGAGCGGGACTGATGCTACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGAGGAGGCACCAAAGTCGAAATCAAACGTAAGTAGAATCCAAAGTGAATTC

[0353] SEQ ID NO:64

[0354] Amino acid sequence of HuSP34 VL4 including a signal peptide

[0355] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGTDATLTISSLEPEDFAVYYCALWYSNLWVFGGGTKVEIK

Claims

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

2.

2. The antibody of claim 1, wherein the CDRH1, CDRH2, and CDRH3 comprise SEQ ID NOs: 43-45, respectively, and the CDRL1, CDRL2, and CDRL3 comprise SEQ ID NOs: 46-48, respectively. The antibody according to claim 1 , which is a mouse antibody.

4. The antibody of claim 1, which is chimeric or veneered. The antibody according to 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 of the humanized mature heavy chain variable region by Kabat numbering 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 one of SEQ ID NO: 6, 17 or 27.

10. The antibody of any one of the preceding claims, 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 multispecific antibody comprising a plurality of pairs of mature heavy and light chain variable regions, wherein one pair comprises: 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, and another pair of the plurality of pairs binds a target antigen.

12. The antibody according to any one of claims 1 to 10, which is a bispecific antibody comprising two pairs of mature heavy and light chain variable regions, wherein one pair comprises: 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, and the other pair binds to a target antigen.

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

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

22. The antibody of claim 1 which is in the form of or comprises an scFv comprising the mature heavy chain variable region fused to the mature light chain variable region via a linker.

23. The antibody of claim 1, having a sequence comprising any one of SEQ ID NO: 29, 31 or 33.

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

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

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

27. A method of treating infection by a pathogen, comprising administering the antibody according to any one of claims 11 to 23 to a patient infected with the pathogen, regardless of whether the target antigen is an antigen of the pathogen or an antigen of a cell infected by the pathogen.

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