Antibodies against human cluster of differentiation 3 protein and variants thereof

Abstract

The invention provides an antibody capable of specifically binding CD3 protein or an antigen binding fragment thereof, and a multispecific antibody containing the anti-CD3 antibody or the antigen binding fragment thereof. The present application also provides multispecific antibodies, pharmaceutical compositions, methods of making and using the antibodies or antigen binding fragments thereof, or uses of the multispecific antibodies in the manufacture of medicaments for the treatment of cancer.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to PCT International Application PCT / CN2023 / 084860, filed on March 29, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to antibodies or antigen-binding fragments thereof capable of specifically binding to the CD3 protein. This application also relates to bispecific antibodies comprising anti-CD3 antibodies or antigen-binding fragments thereof. Background Technology

[0004] The immune system is a host defense system comprised of a series of cells, tissues, and organs that work together to defend against attacks from "foreign" invaders or abnormal cells caused by mutations. Invaders are primarily organisms that cause infection, such as bacteria, viruses, parasites, and fungi. The immune system's ability to detect and destroy abnormal cells can prevent the development of many cancers and helps fight cancer. The immune system consists of central and peripheral immune organs that work together as a whole to fight infectious diseases. This ability to defend against millions of structurally diverse foreign threats demonstrates the complexity of the immune system. This complexity is achieved through a dynamic communication network of organs, tissues, cells, and molecules. These organs, tissues, cells, and molecules cooperate and maintain the balance of the immune system to fight foreign invasions while maintaining its own tolerance.

[0005] T cells are specialized lymphocytes that are an indispensable component of the immune system. During adaptive immune responses, T cells are typically classified as helper T cells, cytotoxic T cells, or regulatory T cells. These T cells can also be classified based on their activation status into naive, effector, and memory subsets; or they can be divided into two main subtypes: CD4+ T cells and CD8+ T cells. In addition to the conventional T cells mentioned above, there are innate-like T cells, including γδ T cells and natural killer T cells, which exhibit different immune responses. CD3 is a protein complex and T cell co-receptor involved in activating both cytotoxic T cells (CD8+ naive T cells) and helper T cells (CD4+ naive T cells). It consists of four distinct chains. In mammals, this complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and CD3-ζ (ζ-chain) to generate activation signals in T lymphocytes. The TCR, CD3-ζ, and other CD3 molecules together constitute the TCR complex.

[0006] Human tumors are the result of a combination of genetic and epigenetic alterations. When tumor cells form, some antigens on their surface may change. These so-called neoantigens are detected by the immune system and destroyed as foreign substances. Abnormal cells are eliminated before they progress to advanced stages of cancer. However, tumor cells develop a variety of resistance mechanisms to evade and suppress the immune system. One common mechanism used by tumors is to manipulate the immune checkpoint pathway by overexpressing inhibitory immune checkpoint regulatory molecules. Cancer immunotherapy utilizes the host's immune system to treat cancer. These mechanisms, ranging from activating effector cells to blocking inhibitory factors, strengthen the immune system and generate anti-tumor activity. Drugs that block inhibitory immune checkpoint pathways have demonstrated promising clinical activity in various solid tumors. T cell activation is a regulatory event in the adaptive immune response. T cell activation is initiated through the interaction of the TCR with other major histocompatibility complex (MHC) proteins on the surface of cells. CD3-bispecific antibodies (BsAbs) have emerged as a novel therapeutic modality in cancer immunotherapy. CD3-BsAbs can simultaneously bind to their antigens expressed on tumor cells and CD3 on T cells. By cross-linking T cells and tumor cells through CD3-BsAb, an immune synapse is formed, which leads to the activation of T cells and the secretion of inflammatory cytokines and cell-lysing molecules that kill tumor cells in the process. CD3-BsAb is a powerful modality that can bridge all available T cells in the field of immunotherapy. Summary of the Invention

[0007] In one aspect, an anti-CD3 antibody or antigen-binding fragment thereof is provided, the anti-CD3 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) containing: 1) HCDR1, the HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 13 and 23; 2) HCDR2, the HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 14 and 24; and 3) HCDR3, the HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 15 and 25, and a light chain variable domain (VL) containing: 1) LCDR1, the LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 18 and 28; 2) LCDR2, the LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 19 and 29; and 3) LCDR3, the LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 20 and 30.

[0008] In some embodiments, 1) VH comprises HCDR1, HCDR2, and HCDR3 sequences having amino acid sequences of SEQ ID NO:3, 4, and 5, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:8, 9, and 10, respectively; 2) VH comprises HCDR1, HCDR2, and HCDR3 sequences having amino acid sequences of SEQ ID NO:13, 14, and 15, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:18, 19, and 20, respectively; or 3) VH comprises HCDR1, HCDR2, and HCDR3 sequences having amino acid sequences of SEQ ID NO:23, 24, and 25, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:28, 29, and 30, respectively.

[0009] In some embodiments, respectively, VH comprises an amino acid sequence that is at least 90% identical to the sequence selected from the group consisting of SEQ ID NO:2, 12 and 22, and VL comprises an amino acid sequence that is at least 90% identical to the sequence selected from the group consisting of SEQ ID NO:7, 17 and 27.

[0010] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, 12 and 22 or a variant thereof, the variant comprising up to about 3 amino acid substitutions in VH; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, 17 and 27 or a variant thereof, the variant comprising up to about 3 amino acid substitutions in VL.

[0011] In some embodiments, 1) VH contains the amino acid sequence of SEQ ID NO:2 and VL contains the amino acid sequence of SEQ ID NO:7; 2) VH contains the amino acid sequence of SEQ ID NO:12 and VL contains the amino acid sequence of SEQ ID NO:17; or 3) VH contains the amino acid sequence of SEQ ID NO:22 and VL contains the amino acid sequence of SEQ ID NO:27.

[0012] In some embodiments, CD3 is human CD3 or cynomolgus monkey CD3.

[0013] In some embodiments, the anti-CD3 antibody is a mouse antibody.

[0014] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment is an activator of Jurkat T cells, with an EC50 value greater than 0.01 μg / mL as determined by IL-2 promoter activity; preferably at about 10-2 Between μg / mL and 1.0 μg / mL, such as between approximately 0.018 μg / mL and 0.82 μg / mL.

[0015] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment is an activator of Jurkat T cells, with an EC50 value determined by IL-2 promoter activity greater than that of the OKT3 antibody.

[0016] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment is an activator of primary human T cells, with an EC50 value greater than 0.1 μg / mL, determined by the concentration of IFN-γ in the supernatant of primary human T cells; preferably between about 0.1 μg / mL and 50.0 μg / mL, such as between about 0.50 μg / mL and 10.14 μg / mL.

[0017] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment is an activator of primary human T cells, and the EC50 value, determined by the IFN-γ concentration in the supernatant of primary human T cells, is greater than that of OKT3.

[0018] In some embodiments, the anti-CD3 antibody is a chimeric antibody comprising: 1) VH comprising the amino acid sequence of SEQ ID NO:22 and VL comprising the amino acid sequence of SEQ ID NO:34; 2) VH comprising the amino acid sequence of SEQ ID NO:36 and VL comprising the amino acid sequence of SEQ ID NO:38; or 3) VH comprising the amino acid sequence of SEQ ID NO:40 and VL comprising the amino acid sequence of SEQ ID NO:42, and optionally, VH is fused with a constant region of human IgG, preferably human IgG1.

[0019] In some embodiments, the EC50 value of the chimeric antibody or its antigen-binding fragment binding to Jurkat T cells is less than 1 μg / mL; preferably between about 0.1 μg / mL and 1.0 μg / mL, such as between about 0.20 μg / mL and 0.45 μg / mL.

[0020] In some embodiments, the EC50 value of the chimeric antibody or its antigen-binding fragment binding to Jurkat T cells is lower than that of the OKT3 antibody.

[0021] In some embodiments, the EC50 value of the chimeric antibody or its antigen-binding fragment binding to primary human T cells is less than 1 μg / mL; preferably between about 0.1 μg / mL and 1.0 μg / mL, such as between about 0.19 μg / mL and 0.30 μg / mL.

[0022] In some embodiments, the EC50 value of the chimeric antibody or its antigen-binding fragment binding to primary human T cells is lower than that of the OKT3 antibody.

[0023] In some embodiments, the EC50 value of the binding between the anti-CD3 antibody or its antigen-binding fragment and cynomolgus primary T cells is less than 10 μg / mL; preferably between about 0.1 μg / mL and 5.0 μg / mL, such as between about 0.14 μg / mL and 1.6 μg / mL.

[0024] In some embodiments, the chimeric antibody is an activator of Jurkat T cells with an EC50 value greater than 0.01 μg / mL as determined by IL-2 promoter activity; preferably between about 0.01 μg / mL and 10.0 μg / mL, such as between about 0.01 μg / mL and 5.0 μg / mL.

[0025] In some embodiments, the chimeric antibody is an activator of Jurkat T cells and has an EC50 value greater than that of the OKT3 antibody, as determined by IL-2 promoter activity.

[0026] In some embodiments, the chimeric antibody is an activator of primary human T cells, with an EC50 value greater than 0.1 μg / mL, determined by the concentration of IFN-γ in the supernatant of primary human T cells; preferably between about 0.1 μg / mL and 50.0 μg / mL, such as between about 0.20 μg / mL and 15.0 μg / mL.

[0027] In some embodiments, the chimeric antibody is an activator of primary human T cells, and the EC50 value, determined by the concentration of IFN-γ in the supernatant of primary human T cells, is greater than that of the OKT3 antibody.

[0028] In some embodiments, anti-CD3 antibodies or their antigen-binding fragments do not bind to NK cells, monocytes, or B cells.

[0029] In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), and sdAb.

[0030] In some embodiments, the multispecific antibody is in the form of BiTE.

[0031] In some embodiments, the multispecific antibody comprises the amino acid sequence of SEQ ID NO:45 or 46.

[0032] In some embodiments, anti-CD3 antibodies or their antigen-binding fragments exhibit a reduced tendency to induce cytokine release syndrome compared to OKT3 antibodies.

[0033] On the other hand, a multispecific antibody comprising a first binding portion and a second binding portion is provided, wherein the first binding portion comprises the anti-CD3 antibody described above or its antigen-binding fragment, and the second binding portion is capable of binding antigens other than CD3.

[0034] In some embodiments, a multispecific antibody is a bispecific antibody.

[0035] In some embodiments, the antigen is a cell surface antigen.

[0036] In some embodiments, the antigen is a tumor antigen.

[0037] In some embodiments, antigens other than CD3 are selected from the group consisting of CD19, CD20, EGFR, BCMA, GPRC5D, EpCAM, DLL3, and HER2.

[0038] In some embodiments, the first binding portion and / or the second binding portion are selected from the group consisting of: Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), and sdAb.

[0039] On the other hand, a pharmaceutical composition is provided comprising an anti-CD3 antibody or an antigen-binding fragment thereof, or a multispecific antibody; and a pharmaceutically acceptable carrier.

[0040] In some embodiments, the pharmaceutical composition further comprises one or more therapeutic agents other than an anti-CD3 antibody or its antigen-binding fragment or a multispecific antibody.

[0041] In some embodiments, the therapeutic agent is an antibody specific to CD39, CTLA-4, PD-L1, TIM-3, LAG-3, or A2aR.

[0042] On the other hand, methods for enhancing the immune function of subjects are provided, which include administering a therapeutically effective amount of an anti-CD3 antibody or its antigen-binding fragment, a multispecific antibody, or a pharmaceutical composition to subjects in need.

[0043] On the other hand, a method for treating cancer in a subject is provided, which includes administering a therapeutically effective amount of an anti-CD3 antibody or its antigen-binding fragment, a multispecific antibody, or a pharmaceutical composition to the subject in need.

[0044] On the other hand, the use of anti-CD3 antibodies or their antigen-binding fragments, or the multispecific antibody, in the manufacture of drugs for treating cancer is provided.

[0045] In some embodiments, an anti-CD3 antibody or its antigen-binding fragment, a multispecific antibody, or a pharmaceutical composition is used for the treatment of cancer in a subject in need.

[0046] In some embodiments, the cancer is multiple myeloma. Attached Figure Description

[0047] Figure 1 The binding of mouse hybridomas to human CD3 was demonstrated.

[0048] Figure 2 The binding of mouse anti-CD3 to CynoCD3 was demonstrated.

[0049] Figure 3 The activity of anti-CD3 mAb in activating reporter T cells was demonstrated.

[0050] Figure 4 The activity of anti-CD3 mAb in activating primary T cells was demonstrated.

[0051] Figure 5 The binding of the chimeric anti-CD3 antibody to CD3 on Jurkat cells was demonstrated.

[0052] Figure 6 The chimeric anti-CD3 antibody was shown to bind to human CD3 on human primary T cells.

[0053] Figure 7 The chimeric anti-CD3 antibody was shown to bind to CynoCD3 on primary T cells of cynomolgus monkeys.

[0054] Figure 8 The activity of the anti-CD3 chimeric antibody in activating reporter T cells was demonstrated.

[0055] Figure 9 The activity of the anti-CD3 chimeric antibody in activating primary T cells was demonstrated.

[0056] Figure 10 The binding activity of the anti-CD3 chimeric antibody to human NK cells was demonstrated.

[0057] Figure 11 The binding activity of the anti-CD3 chimeric antibody to human monocytes was demonstrated.

[0058] Figure 12 The binding activity of the anti-CD3 chimera to human B cells was demonstrated.

[0059] Figure 13The binding of BiTE antibody to human CD3 on Jurkat cells is shown. Black: secondary antibody used for detection; red: 64F9G7 / BCMAT cell connector; green: SP34 / BCMA T cell connector; orange: 10A7C8 / BCMA T cell connector; blue: 39B12G6 / BCMAT cell connector.

[0060] Figure 14 The binding of BiTE antibody to human BCMA on RPMI 8226 cells is shown. Black: secondary antibody used for detection; red: 64F9G7 / BCMA T cell connector; green: SP34 / BCMAT cell connector; orange: 10A7C8 / BCMAT cell connector; blue: 39B12G6 / BCMAT cell connector. Figure 15 This study demonstrated BiTE-induced T-cell-specific killing of RPMI 8226 cells expressing human BCMA. Detailed Implementation

[0061] This disclosure provides information on anti-CD3 monoclonal antibodies and their applications. This disclosure relates to the heavy chain variable domain (V) of the following mouse anti-CD3 monoclonal antibody clones. H ) and light chain variable structural domain (V L The amino acid sequence of the disclosed clone is: 39B12G6, 10A7C8, and 64F9G7. This disclosure also provides a chimeric anti-CD3 monoclonal antibody by fusing the variable domains of the heavy and light chains of the disclosed clone with the constant region of human IgG (e.g., IgG1). This disclosure provides a mouse anti-CD3 monoclonal antibody clone with a heavy chain variable domain (V... H ) and light chain variable structural domain (V L Humanized forms of ) . In some instances, the CDR of a mouse anti-CD3 monoclonal antibody is transplanted onto a human IgG framework sequence, and the resulting humanized variable domain may or may not fuse with the constant region of human IgG.

[0062] I. Definition

[0063] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Any methods, apparatus, or materials similar to or equivalent to those described, used, or made herein may be used in the practice of this invention. The following definitions are provided for ease of understanding of certain terms used herein and are not intended to limit the scope of this disclosure.

[0064] The article “a / an” used in this article refers to one / a type or more than one / an of the grammatical objects of the article (i.e., at least one / a type). For example, “an element” means one element or more elements.

[0065] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps. When used herein, the term “comprise” may be replaced by the terms “containing” or “including”, or sometimes by the term “having”.

[0066] When used herein, "consisting of" excludes any element, step, or component not specified in the elements of the claims. When used herein, "consisting substantially of" does not exclude materials or steps that do not substantially affect the essential and novel features of the claims. Any of the foregoing terms "comprising," "containing," "including," and "having," whenever used herein in the context of aspects of or embodiments of this application, may be replaced with the terms "consisting of" or "substantially of" to change the scope of this disclosure.

[0067] As used herein, the connecting term “and / or” between multiple listed elements should be understood to encompass both individual and combined options. For example, when two elements are connected by “and / or”, the first option refers to the applicability of the first element in the absence of the second element, the second option refers to the applicability of the second element in the absence of the first element, and the third option refers to the applicability of the first and second elements together. Any one of these options should be understood to fall within this meaning and thus satisfy the requirement of the term “and / or” as used herein. The simultaneous applicability of more than one of these options should also be understood to fall within this meaning and thus satisfy the requirement of the term “and / or”.

[0068] Unless otherwise stated, any numerical value (such as concentrations or concentration ranges as described herein) should be understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include ±10% of the listed values. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1 mg / mL to 10 mg / mL includes 0.9 mg / mL to 11 mg / mL. As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0069] The terms “antibody,” “antibody moiety,” or “antibody construct” are used in their broadest sense and encompass a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and their antigen-binding fragments, provided they exhibit the desired antigen-binding activity.

[0070] A basic 4-chain antibody unit is a heterotetrameric protein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units along with an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2–5 basic 4-chain units that can polymerize to form multivalent assemblies combined with the J chain. In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to the H chain by a covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds depending on the H chain homotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (V) at its N-terminus. H ), followed by three constant structure domains C H (For each of the α and γ chains) and four C H Structural domains (for μ-isomorphism and ε-isomorphism). Each L-chain has a variable structural domain (V) at its N-terminus. L ), followed by a constant structural domain at its other end. V L With V H Align and C L With the first constant region of the heavy chain (C H 1) Alignment. Specific amino acid residues are believed to form interfaces between the variable regions of the light and heavy chains. V H and V L They pair together to form a single antigen-binding site. For information on the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. The L-chain from any vertebrate species can be designated as one of two distinct types based on the amino acid sequence of its constant structural domain, called κ and λ. According to the heavy chain constant region (C... H Immunoglobulins can be designated as different classes or isotypes based on their amino acid sequences. Five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated as α, δ, ε, γ, and μ, respectively. Based on C... HRelatively small differences in sequence and function further subdivide the γ and α classes into subclasses, such as the following subclasses expressed by humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0071] The term "heavy chain-only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in 4-chain antibodies. Cameloideas (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0072] The term "single-domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity-determining regions (CDRs). A single sdAb can bind to an antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camel HCAbs, and their heavy-chain variable domains are referred to herein as "V". H H (heavy chain variable domain of heavy chain antibody). Some V H H can also be called a nanobody. Cameloid sdAb is one of the smallest known antigen-binding antibody fragments (see, for example, Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). Basic V H H has the following structure from the N end to the C end: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to frame regions 1 to 4 respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3.

[0073] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains can be respectively referred to as "V" domains. H " and "V L These domains are typically the most variable parts of an antibody (relative to other antibodies of the same class) and contain antigen-binding sites. Camelidae species have heavy-chain-only antibodies with a single heavy-chain variable region, called the "V". H H”. Therefore, V H H is a special type of V H .

[0074] The term "variability" refers to the fact that certain segments of the variable domain in an antibody are widely different in sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire span of the variable domain. Instead, it is concentrated in three segments in the light and heavy chain variable domains called complementarity-determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FR regions, predominantly in a β-sheet configuration, linked by three CDRs to form loops that connect the β-sheet structure and, in some cases, form a portion of the β-sheet structure. The CDRs in each chain are tightly bound together by the FR regions and, together with CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD. (1991)). Constant domains are not directly involved in antibody-antigen binding, but they exhibit various effector functions, such as antibody involvement in antibody-dependent cytotoxicity.

[0075] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical, except for possibly small amounts of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Monoclonal antibodies are highly specific, targeting a single antigenic site. This is quite different from polyclonal antibody products, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized from hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used according to this application can be prepared by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, phage display technology, and techniques for producing human antibodies or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0076] The terms "full-length antibody," "intact antibody," or "complete antibody" are used interchangeably to refer to antibodies in essentially complete form, as opposed to antibody fragments. Specifically, full-length four-chain antibodies include those with both a heavy chain and a light chain, including the Fc region. Antibodies containing only a full-length heavy chain include the heavy chain (such as V...). HThe constant domain can be a natural sequence constant domain (e.g., a human natural sequence constant domain) or a variant of its amino acid sequence. In some cases, the intact antibody may have one or more effector functions.

[0077] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; double-chain antibodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; single-domain antibodies (such as V... H H) and multispecific antibodies formed from antibody fragments. Papain digests the antibody to produce two identical antigen-binding fragments (called the "Fab" fragment) and a residual "Fc" fragment, a name reflecting its tendency to crystallize. The Fab fragment consists of the entire L chain along with the variable region domain (V) of the H chain. H ) and the first constant structural domain of a heavy chain (C H 1) Composition. Each Fab fragment is monovalent in terms of antigen binding, meaning it has a single antigen-binding site. Treatment of the antibody with pepsin yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking the antigen. The difference between the Fab' fragment and the Fab fragment lies in the C... H The carboxyl terminus of the 1 domain has several additional residues, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name used herein for Fab', where the cysteine ​​residues of the constant domain are accompanied by free thiol groups. The F(ab')2 antibody fragments were initially generated as a Fab' fragment pair with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0078] The Fc fragment contains the carboxyl-terminal portions of two H chains linked together by a disulfide. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0079] The term "constant domain" or "constant region" refers to a portion of an immunoglobulin molecule that, relative to another portion of the immunoglobulin molecule, namely the variable domain, has a more conserved amino acid sequence containing antigen-binding sites. The constant domain contains the C16 heavy chain. H 1. C H 2 and C H The three structural domains (collectively referred to as CH) and the light chain CHL (or CL) structural domains.

[0080] The "light chain" of antibodies (immunoglobulins) in any mammalian species can be designated as one of two distinct types based on the amino acid sequence of their constant domains, referred to as kappa ("κ") and lambda ("λ").

[0081] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly, non-covalently associated heavy chain variable region domain and a light chain variable region domain. The folding of these two domains produces six hypervariable rings (three in the H chain and three in the L chain), which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three antigen-specific CDRs) can recognize and bind antigens, but with lower affinity than the entire binding site.

[0082] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is a V-shaped structure containing a single polypeptide chain. H and V L Antibody fragments containing antibody domains. Preferably, the sFv polypeptide further comprises V H and V L The polypeptide linker between the domains allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0083] The “functional fragment” of an antibody described herein comprises a portion of the complete antibody, typically including the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0084] The term "double-chain antibody" refers to a small antibody fragment prepared by constructing a V... H Domain and V L sFv fragments (see previous paragraph) have short linkers (approximately 5-10 residues) between their domains to enable interchain rather than intrachain pairing of the V domains, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific double-chain antibodies are heterodimers of two “crossed” sFv fragments, where the V domains of the two antibodies... H Domain and V LThe domains are located on different polypeptide chains. For example, biantibodies are described in more detail in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Nat'l. Acad. Sci. USA 90:6444-6448 (1993).

[0085] The monoclonal antibodies described herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851-6855 (1984)). “Humanized antibodies” are used as a subset of “chimeric antibodies”.

[0086] A “humanized” form of a nonhuman (e.g., llama or camel) antibody is an antibody containing a minimal sequence derived from a nonhuman immunoglobulin. In some embodiments, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient CDR (defined below) are replaced by residues from a CDR (donor antibody) from a nonhuman species (such as mouse, rat, rabbit, camel, llama, alpaca, or nonhuman primate) having the desired specificity, affinity, and / or capacity. In some cases, the framework (“FR”) residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity. Generally, humanized antibodies will contain substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulin sequences, and all or substantially all of the FR regions correspond to those of human immunoglobulin sequences. However, the FR regions may include one or more individual FR residue substitutions that improve antibody properties such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. Humanized antibodies will also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of human immunoglobulin. For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0087] When used herein, the terms “hypervariant region,” “HVR,” or “HV” refer to regions of antibody variable domains that are hypervariable in sequence and / or form structurally defined loops. Typically, single-domain antibodies contain three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) exhibits the greatest diversity among the three HVRs and is believed to play a unique role in conferring fine specificity to the antibody. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0088] The term “complementary determination region” or “CDR” is used to refer to a highly variable region as defined according to the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0089] Many HVRs are described and covered in this paper. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by the AbM antibody modeling software from Oxford Molecular. "Contact" HVRs are based on the analysis of available complex crystal structures. Single-domain antibodies (such as V...) HThe amino acid residues of VH are numbered according to the general numbering of the VH domain given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as in the paper by Riechmann and Muyldermans, J. Immunol. Methods, June 23, 2000; 240(1-2):185-195, applicable to VH from Camelidae. H H is a structural domain. Based on this number, V... H H's FR1 contains amino acid residues at positions 1-30, V H H's CDR1 is contained in amino acid residues at positions 31-35, V H H's FR2 is contained in amino acid residues at positions 36-49, V H H's CDR2 is contained in amino acid residues at positions 50-65, V H H's FR3 is contained in amino acid residues at positions 66-94, V H H's CDR3 is contained in amino acid residues at positions 95-102, and V H The FR4 of H consists of amino acid residues at positions 103-113. In this regard, it should be noted—as is discussed in the art regarding V… H Domain and for V H It is well known that the total number of amino acid residues in each CDR can vary and does not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number cannot be occupied in the actual sequence, or the actual sequence can contain more amino acid residues than the number allowed by the Kabat number).

[0090] “Frame” or “FR” residues are those variable domain residues other than HVR residues as defined in this paper.

[0091] As used herein, the terms “specific binding,” “specific recognition,” or “specific to” refer to a measurable and reproducible interaction, such as binding, between a target and an antigen-binding protein (such as a mAb), which determines the presence of the target in the presence of a population of heterogeneous molecules (including biomolecules). For example, an antigen-binding protein (such as a mAb) that specifically binds to a target (which may be an epitope) is an antigen-binding protein (such as a mAb) that binds to the target with greater affinity, stronger affinity, easier binding, and / or longer duration than it binds to other targets. In some embodiments, the antigen-binding protein (such as a mAb) binds to an unrelated target to a degree less than about 10% of the binding of the antigen-binding protein (such as a mAb) to the target. In some embodiments, the antigen-binding protein specifically binds to an epitope on a protein that is conserved across the protein from different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding.

[0092] The term "specificity" refers to the selective recognition of a specific epitope of an antigen by an antigen-binding protein (such as a mAb). For example, natural antibodies are monospecific. As used herein, the term "multispecific" refers to an antigen-binding protein that has multiple epitope specificity (i.e., the ability to bind specifically to two, three, or more different epitopes on a single biomolecule or the ability to bind specifically to epitopes on two, three, or more different biomolecules). As used herein, "bispecific" refers to an antigen-binding protein that has two different antigen-binding specificities. Unless otherwise indicated, the order in which listed bispecific antibodies bind to antigens is arbitrary. That is, for example, the terms "anti-CD3 / BCMA" and "anti-BCMA / CD3" are used interchangeably to refer to a bispecific antibody that specifically binds to both CD3 and BCMA. As used herein, the term "monospecific" refers to an antigen-binding protein (such as a mAb) having one or more binding sites, each binding to the same epitope of the same antigen.

[0093] "Antibody effector functions" refer to those biological activities attributable to the Fc region of an antibody (either the native Fc region or the Fc region of an amino acid sequence variant) and that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0094] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. Binding affinity can be determined by KD, K... off or K on Instructions. As used herein, the term "K" off "This refers to the dissociation rate constant of the antibody (or antigen-binding domain) from the antibody / antigen complex, as determined by kinetic selection settings, expressed in seconds." -1 Indicated. As used in this article, the term "K" is used to refer to... on "This refers to the association rate constant of an antibody (or antigen-binding domain) forming an antibody / antigen complex, expressed in units of M." -1 s -1 As used herein, the term equilibrium dissociation constant “KD” refers to the dissociation constant of a particular antibody-antigen interaction and describes the antigen concentration required to occupy half of all antibody-binding domains present in the antibody molecule solution at equilibrium, and is equal to K. off / K on The value is expressed in units of M. The measurement of KD presupposes that all binding agents are in solution. When the antigen is tethered to the cell membrane, the corresponding equilibrium rate constant is expressed as EC. 50 A good approximation of KD is given.

[0095] The binding specificity of an antibody or antigen-binding fragment can be determined experimentally using methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIAcore assay, peptide scanning, or FACS.

[0096] EC 50 "or "EC" 50 The "value" indicates the concentration required to achieve 50% of the maximum effect. EC 50 It can be measured by bioassays, such as FACS analysis, cell-based cytokine release assays, or amplified luminescent proximity homogeneous assays (AlphaLISA).

[0097] The "percentage of amino acid sequence identity (%)" and "homology" for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after sequence alignment and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in various ways available in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN. TM (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms that require maximum alignment across the full length of the sequences being compared.

[0098] The “isolated” nucleic acid molecule encoding the constructs, antibodies, or antigen-binding fragments thereof described herein is a nucleic acid molecule identified and isolated from at least one contaminant nucleic acid molecule typically associated with it in its production environment. Preferably, the isolated nucleic acid does not associate with any components related to the production environment. The isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is in a form different from its naturally occurring form or background. Therefore, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that are naturally present in cells. The isolated nucleic acid includes the nucleic acid molecule typically contained in cells, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0099] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell (in which the vector has been introduced). Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0100] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced using exogenous nucleic acids. These cells include primary target cells and their progeny.

[0101] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are typically composed of chemically active surface groups of a molecule, such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes lies in their loss of binding to the former, rather than the latter, in the presence of denaturing solvents.

[0102] As used herein, “treatment” is a method for achieving beneficial or desired outcomes (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relieving one or more symptoms caused by a disease; reducing the severity of the disease; stabilizing the disease (e.g., preventing or delaying the worsening of the disease); preventing or delaying the spread of the disease (e.g., metastasis); preventing or delaying the recurrence of the disease; delaying or slowing the progression of the disease; improving the disease state; providing partial or complete remission of the disease; reducing the dosage of one or more other medications required to treat the disease; delaying the progression of the disease; increasing quality of life and / or prolonging survival. “Treatment” also encompasses reducing the pathological outcomes of the disease. The methods of this invention are contemplated in relation to any one or more of these treatment aspects.

[0103] As used herein, the term "effective amount" refers to an amount of agent or combination of agents sufficient to treat a specified condition, symptom, or disease, such as improving, alleviating, reducing, and / or delaying one or more of its symptoms. In the context of cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce its growth rate (e.g., inhibit tumor growth) or prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more doses. An effective amount of a drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) to some extent inhibit, delay, slow, and preferably terminate the infiltration of cancer cells into surrounding organs; (iv) inhibit (i.e., to some extent slow and preferably terminate) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) to some extent alleviate one or more symptoms associated with cancer.

[0104] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include “transformers” and “transformed cells,” encompassing primary transformed cells and their derived progeny, regardless of passage number. The nucleic acid content of progeny cells may not be entirely identical to that of the parent cells and may contain mutations. Mutant progeny cells possessing the same function or biological activity as those screened or selected in the original transformed cells are included herein.

[0105] The term "pharmaceutical composition" in the context of "pharmaceutical formulation" refers to an article whose form allows for the effective bioactivity of the active ingredient and contains no other components that would have unacceptable toxicity to a subject to be administered the formulation. Such formulations are sterile. "Sterile" formulations are sterilized or free of live microorganisms and their spores.

[0106] It should be understood that the embodiments of the invention described herein include "consisting of the embodiments" and / or "consisting substantially of the embodiments".

[0107] References to “about” values ​​or parameters in this document include (and describe) variations of that value or parameter itself. For example, a description involving “about X” includes a description of “X”.

[0108] The term “about XY” used in this article has the same meaning as “about X to about Y”.

[0109] II. Mouse anti-CD3 antibody

[0110] The isolated mouse anti-CD3 construct described herein comprises a monoclonal antibody (mAb) moiety that specifically recognizes or binds to human CD3 (or "anti-CD3 mAb"). In some embodiments of the invention, the isolated mouse anti-CD3 antibody is full-length IgG. In some embodiments, the disclosed mouse anti-CD3 mAb may cross-react with CD3 from species other than humans or with other proteins structurally related to human CD3. In some embodiments, the anti-CD3 mAb of this application is capable of specifically recognizing or binding to cynomolgus monkey CD3 (or cyno CD3).

[0111] In some embodiments, a mouse anti-CD3 mAb is provided, comprising: a heavy chain variable domain (VH) having: CDR1, the CDR1 comprising an amino acid sequence or a variant thereof of any one of SEQ ID NO:3 (TYAMN), SEQ ID NO:13 (SQYLH), and SEQ ID NO:23 (TSYIH), the variant comprising up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3 amino acid substitutions); a heavy chain CDR2, the heavy chain CDR2 comprising an amino acid sequence or a variant thereof of any one of SEQ ID NO:4 (RIRSKIYNYATFYDDSVKD), SEQ ID NO:14 (WINPGDDTTKYNEKFKV), and SEQ ID NO:24 (WISPGDVNTKYSEKFKG), the variant comprising up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3 amino acid substitutions); and a heavy chain CDR3, the heavy chain CDR3 comprising SEQ ID NO:3 (TYAMN), SEQ ID NO:13 (SQYLH), and SEQ ID NO:23 (TSYIH), the amino acid sequence or a variant thereof, the variant comprising up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3 amino acid substitutions); and a heavy chain CDR3, the heavy chain CDR3 comprising an amino acid sequence of any one of SEQ ID NO:3 (TYAMN), SEQ ID NO:13 (SQYLH), and SEQ ID NO:23 (TSYIH), the amino acid sequence of any one of SEQ ID NO:13 (TYAMN), SEQ ID NO:23 (SQYL ... The amino acid sequence of any one of SEQ ID NO:5 (YYGNDWIAK), SEQ ID NO:15 (DYGYYFDY), and SEQ ID NO:25 (DYGYYFDY), or a variant thereof, the variant comprising up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3 amino acid substitutions); and a light chain variable domain (VL) having: a light chain CDR1 comprising the amino acid sequence of any one of SEQ ID NO:8 (RSSTGAVTTSNYAN), SEQ ID NO:18 (KSSQSLLNSRTRKNYLA), and SEQ ID NO:28 (KSSQSLLNSRTRKNYLA), or a variant thereof, the variant comprising up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3 amino acid substitutions); and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:9 (GTSNRAP), SEQ ID NO:19 (WASTRES), and SEQ ID NO:25 (DYGYYFDY). The amino acid sequence of any one of NO:29 (WASTRES) or a variant thereof, the variant comprising up to about 3 amino acid substitutions (such as any one of about 1, 2 or 3 amino acid substitutions); and the light chain CDR3 comprising the amino acid sequence of any one of SEQ ID NO:10 (ALWYSTHYV), SEQ ID NO:20 (KQSFILRT) and SEQ ID NO:30 (KQSFILRT) or a variant thereof, the variant comprising up to about 3 amino acid substitutions (such as any one of about 1, 2 or 3 amino acid substitutions).

[0112] In some embodiments, a mouse anti-CD3 mAb is provided, comprising: a heavy chain variable domain (VH) having: CDR1 comprising an amino acid sequence of any one of SEQ ID NO: 3, 13, and 23; CDR2 comprising an amino acid sequence of any one of SEQ ID NO: 4, 14, and 24; and CDR3 comprising an amino acid sequence of any one of SEQ ID NO: 5, 15, and 25; and a light chain variable domain (VL) having: CDR1 comprising an amino acid sequence of any one of SEQ ID NO: 8, 18, and 28; CDR2 comprising an amino acid sequence of any one of SEQ ID NO: 9, 19, and 29; and CDR3 comprising an amino acid sequence of any one of SEQ ID NO: 10, 20, and 30.

[0113] In some embodiments, a mouse anti-CD3 mAb is provided, comprising: 1) a VH comprising heavy chain CDR1, CDR2, and CDR3 sequences having amino acid sequences of SEQ ID NO: 3, 4, and 5, respectively; and a VL comprising light chain CDR1, CDR2, and CDR3 sequences having amino acid sequences of SEQ ID NO: 8, 9, and 10, respectively; 2) a VH comprising heavy chain CDR1, CDR2, and CDR3 sequences having amino acid sequences of SEQ ID NO: 13, 14, and 15, respectively; and a VL comprising light chain CDR1, CDR2, and CDR3 sequences having amino acid sequences of SEQ ID NO: 18, 19, and 20, respectively; or 3) a VH comprising heavy chain CDR1, CDR2, and CDR3 sequences having amino acid sequences of SEQ ID NO: 23, 24, and 25, respectively; and a VL comprising light chain CDR1, CDR2, and CDR3 sequences having amino acid sequences of SEQ ID NO: 3, 4, and 5, respectively. The light chains CDR1, CDR2, and CDR3 of amino acid sequences NO:28, 29, and 30.

[0114] In some embodiments, a mouse anti-CD3 mAb is provided, comprising: 1) a VH comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:2; and a VL comprising the amino acid sequence of SEQ ID NO:7 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:7; 2) a VH comprising the amino acid sequence of SEQ ID NO:12 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:7; 2) a VH comprising the amino acid sequence of SEQ ID NO:12 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:7; NO:12 has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL, which contains the amino acid sequence of SEQ ID NO:17 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:17; or 3) VH, which contains the amino acid sequence of SEQ ID NO:22 or a variant thereof, the variant having at least about 80% (such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:17; NO:22 has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL, which contains the amino acid sequence of SEQ ID NO:27 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:27.

[0115] In some embodiments, a mouse anti-CD3 mAb is provided, comprising: 1) a heavy chain comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:1; and a light chain comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:6; 2) a heavy chain comprising the amino acid sequence of SEQ ID NO:11 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:6; NO:11 has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and a light chain comprising the amino acid sequence of SEQ ID NO:16 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:16; or 3) a heavy chain comprising the amino acid sequence of SEQ ID NO:21 or a variant thereof, the variant having at least about 80% (such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:16; NO:21 has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and a light chain comprising the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:26.

[0116] In some embodiments, an anti-CD3 antibody, such as a mAb (hereinafter referred to as "competitive anti-CD3 antibody" or "competitive anti-CD3 mAb"), which competitively binds to CD3 specifically with any of the anti-CD3 mAbs described herein, or an antigen-binding fragment thereof, is provided. In some embodiments, competitive binding can be determined using an ELISA assay. In some embodiments, the competitive anti-CD3 antibody and the aforementioned anti-CD3 antibody bind to the same epitope on CD3.

[0117] In some embodiments, the binding specificity of the disclosed mouse anti-CD3 mAb to CD3 is detected by the binding of the mouse anti-CD3 mAb to CD3-expressing cells (such as Jurkat T cells). In some embodiments, the binding of the disclosed mouse anti-CD3 mAb to CD3-expressing cells is detected by FACS.

[0118] Upon binding to CD3 on T cells, the mouse anti-CD3 mAb disclosed herein is able to induce T cell activation. In some embodiments, T cell activation is determined by IL-2 or IFN-γ secretion. In other embodiments, T cell activation is determined by the expression of an IL-2 promoter-driven reporter gene (e.g., luciferase).

[0119] In some embodiments, as determined by a reporter gene driven by the IL-2 promoter, the mouse anti-CD3mAb disclosed herein activates EC2 cells (e.g., Jurkat T cells). 50 Greater than 0.01 μg / mL, preferably at about 10 -2 Between μg / mL and 1.0 μg / mL, such as between approximately 0.018 μg / mL and 0.82 μg / mL.

[0120] In some embodiments, such as those identified via a reporter gene driven by the IL-2 promoter, the mouse anti-CD3mAb T cell activation EC of this disclosure 50 Higher than OKT3.

[0121] In some embodiments, such as those determined by IFN-γ secretion, the mouse anti-CD3mAb T cell activation EC of this disclosure 50 Greater than 0.1 μg / mL, preferably between about 0.1 μg / mL and 50.0 μg / mL, such as between about 0.50 μg / mL and 10.14 μg / mL.

[0122] In some embodiments, such as those determined by IFN-γ secretion, the mouse anti-CD3mAb T cell activation EC of this disclosure 50 Higher than OKT3.

[0123] III chimeric anti-CD3 antibody

[0124] In some embodiments, the anti-CD3 antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a rodent species such as a mouse) and a human constant region. Chimeric antibodies include their antigen-binding fragment.

[0125] In some embodiments, the chimeric anti-CD3 antibody provided herein comprises a variable region of the mouse anti-CD3 antibody disclosed herein and a constant region of human IgG (e.g., IgG1) comprising the amino acid sequence of SEQ ID NO:43.

[0126] In some embodiments, the chimeric anti-CD3 antibody provided herein is a humanized antibody comprising the CDR, human FR, and constant region of human IgG (e.g., IgG1) containing the amino acid sequence of SEQ ID NO:43 of the mouse anti-CD3 antibody disclosed herein.

[0127] In some embodiments, a chimeric anti-CD3 mAb is provided, comprising: 1) VH, the VH comprising the amino acid sequence of SEQ ID NO:32 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:32; and VL, the VL comprising the amino acid sequence of SEQ ID NO:34 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:34; 2) VH, the VH comprising the amino acid sequence of SEQ ID NO:36 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:34; NO:36 has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL, which contains the amino acid sequence of SEQ ID NO:38 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:38; or 3) VH, which contains the amino acid sequence of SEQ ID NO:40 or a variant thereof, the variant having at least about 80% (such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:38; NO:40 has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL, which contains the amino acid sequence of SEQ ID NO:42 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:42.

[0128] In some embodiments, the chimeric anti-CD3 antibody provided herein comprises: 1) a heavy chain comprising the amino acid sequence of SEQ ID NO:31 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:31; and a light chain comprising the amino acid sequence of SEQ ID NO:33 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:33; 2) a heavy chain comprising the amino acid sequence of SEQ ID NO:35 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:33; NO:35 has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and a light chain comprising the amino acid sequence of SEQ ID NO:37 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:37; or 3) a heavy chain comprising the amino acid sequence of SEQ ID NO:39 or a variant thereof, the variant having at least about 80% (such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:37; NO:39 has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and a light chain comprising the amino acid sequence of SEQ ID NO:41 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:41.

[0129] In some embodiments, the ECG binding between the chimeric anti-CD3 antibody or its antigen-binding fragment disclosed herein and Jurkat T cells 50 The value is below 1 μg / mL; preferably between about 0.1 μg / mL and 1.0 μg / mL, such as between about 0.20 μg / mL and 0.45 μg / mL.

[0130] In some embodiments, the chimeric antibody or its antigen-binding fragment disclosed herein binds to ECs of Jurkat T cells. 50 The value is lower than that of OKT3 antibody.

[0131] In some embodiments, the EC50 value of the chimeric antibody or its antigen-binding fragment thereof bound to primary human T cells is less than 1 μg / mL; preferably between about 0.1 μg / mL and 1.0 μg / mL, such as between about 0.19 μg / mL and 0.30 μg / mL.

[0132] In some embodiments, the EC50 value of the chimeric antibody or its antigen-binding fragment disclosed herein binding to primary human T cells is lower than that of the OKT3 antibody.

[0133] In some embodiments, the EC50 value of the chimeric antibody or its antigen-binding fragment thereof bound to primary T cells of cynomolgus monkeys is less than 10 μg / mL; preferably between about 0.1 μg / mL and 5.0 μg / mL, such as between about 0.14 μg / mL and 1.6 μg / mL.

[0134] Upon binding to CD3 on T cells, the chimeric anti-CD3 mAb disclosed herein can induce T cell activation. In some embodiments, T cell activation is determined by IL-2 or IFN-γ secretion. In other embodiments, T cell activation is determined by the expression of an IL-2 promoter-driven reporter gene (e.g., luciferase).

[0135] In some embodiments, as determined by a reporter gene driven by the IL-2 promoter, the chimeric anti-CD3mAb disclosed herein activates EC2 cells (e.g., Jurkat T cells). 50 Greater than 0.01 μg / mL, preferably at about 10 -2 Between μg / mL and 10 μg / mL, such as between approximately 0.01 μg / mL and 5.0 μg / mL.

[0136] In some embodiments, such as those identified via an IL-2 promoter-driven reporter gene, the chimeric anti-CD3mAb of this disclosure activates ECGs in T cells. 50 Higher than OKT3.

[0137] In some embodiments, such as those determined by IFN-γ secretion, the chimeric anti-CD3mAb T-cell activation EC of this disclosure 50 Greater than 0.1 μg / mL, preferably between about 0.1 μg / mL and 50.0 μg / mL, such as between about 0.20 μg / mL and 15.0 μg / mL.

[0138] In some embodiments, such as those determined by IFN-γ secretion, the chimeric anti-CD3mAb T-cell activation EC of this disclosure 50 Higher than OKT3.

[0139] In some embodiments, the chimeric antibody is a humanized antibody. Typically, nonhuman antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parental nonhuman antibody. Typically, humanized antibodies contain one or more variable domains, wherein the HVR, such as the CDR, (or a portion thereof) is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the nonhuman antibody (e.g., antibodies derived from HVR residues) to restore or improve antibody specificity or affinity, for example.

[0140] Humanized antibodies and their manufacturing methods are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, the following references: Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes SDR(a-CDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “reworked surfaces”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes the “guided selection” approach to FR reorganization).

[0141] In some embodiments, mAbs are modified, such as through humanization, without diminishing the natural affinity of the domains for the antigen and simultaneously reducing their immunogenicity relative to a foreign species. For example, amino acid residues of the variable domains (VH and VL) of the antibody heavy and light chains can be determined, and one or more mouse amino acids, for example, in the frame region, can be replaced by human controls of these amino acids as found in human common sequences, without the peptide losing its characteristic features; that is, humanization does not significantly affect the antigen-binding capacity of the resulting peptide. Humanization of mouse monoclonal antibodies requires the introduction and mutagenesis of a limited number of amino acids in both chains (light and heavy chains) and the maintenance of the assembly of the two chains.

[0142] IV contains a construct against CD3 mAb.

[0143] The anti-CD3 construct containing anti-CD3 mAb can be in any possible form.

[0144] In some embodiments, an anti-CD3 construct comprising an anti-CD3 mAb may further include additional polypeptide sequences, such as one or more antibody moieties. Such additional polypeptide sequences may or may not alter or otherwise affect the (biological) properties of the anti-CD3 mAb, and may or may not add additional functionality to the anti-CD3 mAb described herein. In some embodiments, the additional polypeptide sequences confer one or more desired properties or functions to the anti-CD3 mAb of this application.

[0145] In some embodiments, the additional polypeptide sequence may be a second antibody moiety (such as sdAb, scFv) that specifically recognizes a second antigen. In some embodiments, the second antigen is not CD3. In some embodiments, the second antibody moiety specifically recognizes the same epitope on CD3 as the anti-CD3 mAb described herein. In some embodiments, the second antibody moiety specifically recognizes a different epitope on CD3 than the anti-CD3 mAb described herein.

[0146] In some embodiments, the addition of a polypeptide sequence may increase the molecular stability, solubility, or absorption of the anti-CD3 construct of the present invention, reduce immunogenicity or toxicity, eliminate or mitigate undesirable side effects, and / or impart other advantageous properties and / or reduce undesirable properties to the anti-CD3 construct of the present invention compared to the anti-CD3 mAb itself described herein.

[0147] In some embodiments, anti-CD3 mAb is full-length IgG. In some embodiments, anti-CD3 mAb comprises a constant region of IgG (such as any one of IgG1, IgG2, IgG3, or IgG4). In some embodiments, this constant region is a human constant region. In some embodiments, this constant region is a human IgG1 constant region.

[0148] In some embodiments, the full-length anti-CD3 IgG is rodent-derived, chimeric, human, partially humanized, or fully humanized.

[0149] In some embodiments, anti-CD3 mAb is not a full-length anti-CD3 IgG. In some embodiments, anti-CD3 mAb does not contain an Fc domain and does not have the ability to induce ADCC in vivo.

[0150] In some embodiments, the anti-CD3 construct comprises an anti-CD3 mAb as described herein fused with one or more other antibody moieties, such as an antibody moieties that specifically recognize another antigen. These one or more other antibody moieties can be in the form of any antibody or antibody fragment, such as sdAb, full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), scFv-scFv, microantibody, or double-chain antibody. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); also see WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an extended in vivo half-life, see U.S. Patent No. 5,869,046. For reviews of multispecific antibodies, see Weidle et al., Cancer Genomics Proteomics, 10(1):1-18, 2013; Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015; Stamova et al., Antibodies, 1(2):172-198, 2012. Double-chain antibodies are antibody fragments with two antigen-binding sites and can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Nat'l. Acad. Sci. USA 90:6444-6448 (1993). Triple-chain and quadruple-chain antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or bacteriophages), as described herein. In some embodiments, the one or more other antibody portions are antibody mimics, which are small engineered proteins containing antigen-binding domains reminiscent of antibodies (Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and contain a single polypeptide.Exemplary antibody mimics that may be included in the anti-CD3 constructs described herein may be, but are not limited to, designed ankyrin repeat proteins (DARPin; fully synthetic ankyrin repeat sequences containing 3-5 flanked by N-terminal and C-terminal Cap domains), affinity multimers (avimer; high-affinity proteins containing multiple A domains, each with low affinity for the target), or anticalin (a lipid-carrying protein scaffold with four accessible loops, each of which may be randomized).

[0151] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, Traunecker et al., EMBO J.10:3655 (1991)) and engineered "knob-in-hole" structures (see, for example, US Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineered electrostatic redirection effects (WO) for the preparation of antibody Fc-heterodimeric molecules. 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, US Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); generating bispecific antibodies using leucine zippers (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using “double-chain antibody” technology for preparing bispecific antibody fragments (see, for example, Hollinger et al., Proc. Nat'l. Acad. Sci. USA). ,90:6444-6448(1993)); and the use of single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368(1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147:60(1991); and the generation of peptides comprising tandem single-domain antibodies (see, for example, U.S. Patent Application No. 20110028695; and Conrath et al., J. Biol. Chem., 2001; 276(10):7346-50). This document also includes engineered antibodies having three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US2006 / 0025576A1).

[0152] In some embodiments, two or more antibody moieties within an anti-CD3 construct may optionally be linked by peptide linkers. The length, flexibility, and / or other characteristics of the peptide linkers used in the anti-CD3 construct may have some influence on the properties, including but not limited to affinity, specificity, or affinity for one or more specific antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not interfere with each other spatially. In some embodiments, the peptide linkers contain flexible residues (such as glycine and serine) that allow adjacent domains to move freely relative to each other. For example, a glycine-serine duplex may be a suitable peptide linker.

[0153] In some embodiments, the isolated antibody or antigen-binding fragment of this application is a bispecific or multispecific antibody comprising a second antibody partially fused with the anti-CD3 IgG described herein, wherein the second antibody partially specifically binds to another antigen, preferably a tumor-associated antigen or a tumor-specific antigen, such as CD19, CD20, EGFR, BCMA, GPRC5D, EpCAM, DLL3, and HER2.

[0154] In some embodiments, the bispecific antibody takes the form of a bispecific T-cell adaptor (BiTE). “BiTE” generally refers to a single polypeptide chain molecule having two antigen-binding domains, one of which binds to an immune effector cell antigen (e.g., CD3), and the second antigen-binding domain binds to an antigen present on the surface of a target cell (e.g., tumor cell). In some embodiments, the BiTE is a fusion protein consisting of two single-chain variable fragments (scFvs) linked by an adaptor. In some embodiments, one antigen-binding domain (first scFv) is specific to immune cells (such as T-cell antigens, such as the CD3 receptor expressed on the surface of T cells). In some embodiments, the second antigen-binding domain (second scFv) binds to tumor cells via a tumor-specific molecule. Thus, the BiTE is able to form a connection between T cells and tumor cells due to its specificity for antigens on T cells and antigens on tumor cells. This leads to T-cell activation and can trigger T cells to exert their cytotoxic effects on tumor cells independently of MHC I or co-stimulatory molecules. In some embodiments, the second antigen is selected from the group consisting of: CD19, EpCAM, CD20, CD123, BCMA, B7-H3, and PSMA.

[0155] In some embodiments, BiTE comprises a first scFv from an anti-CD3 antibody of the present invention and a second scFv from an anti-BCMA antibody (such as the anti-human BCMA antibody described in WO 2014 / 140248 A1). In some embodiments, BiTE comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, BiTE comprises the amino acid sequence of SEQ ID NO:45 without a C-terminal His tag. In some embodiments, BiTE comprises the amino acid sequence of SEQ ID NO:46. In some embodiments, BiTE comprises the amino acid sequence of SEQ ID NO:46 without a C-terminal His tag. In some embodiments, BiTE is capable of activating T cells and / or promoting T cell killing of target cells expressing the BCMA antigen. In some embodiments, the target cells are tumor cells of multiple myeloma (MM).

[0156] Human monoclonal antibodies specific to CD3 or their antigen-binding fragments can be conjugated to pharmaceutical agents, such as effector molecules or detectable markers, in a variety of ways known to those skilled in the art. Covalent and non-covalent linkages can be used. Conjugates include, but are not limited to, molecules in which the effector molecule or detectable marker is covalently linked to an antibody or antigen-binding fragment that specifically binds to CD3. Those skilled in the art will understand that a variety of effector molecules and detectable markers can be used, including (but not limited to) chemotherapeutic agents, anti-angiogenic agents, toxins, and radiation agents (such as...). 125 I, 32 P, 14 C 3 H and 35 S) and other tags, target moieties, and ligands, etc. The selection of specific effector molecules or detectable markers depends on the specific target molecule or target cell and the desired biological effect.

[0157] Effector molecules and detectable markers can be linked to target antibody or antigen-binding fragments in any manner known to those skilled in the art. Covalent and non-covalent linkages can be used. The procedure for attaching effector molecules or detectable markers to antibody or antigen-binding fragments varies depending on the chemical structure of the effector. Peptides typically contain multiple functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to induce binding of the effector molecule or detectable marker.

[0158] The antibody or antigen-binding fragments disclosed herein can be derivatized, for example, by crosslinking two or more antibodies (antibodies of the same or different types, such as to generate bispecific antibodies). Suitable crosslinking agents include heterobifunctional crosslinking agents having two distinct reactive groups separated by an appropriate spacer (such as m-maleimide benzoyl-N-hydroxysuccinimide ester) or homobifunctional crosslinking agents (such as disuccinimide octanoate). Such linkers are commercially available.

[0159] Given the numerous methods already reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, tags (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies, those skilled in the art will be able to determine suitable methods for attaching a given agent to an antibody or antigen-binding fragment or other peptide. For example, an antibody or antigen-binding fragment can be conjugated with a low molecular weight drug such as monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), maytansine, maytansine derivatives (including maytansine derivatives called DM1, also known as metansine)), or other chemotherapeutic agents to prepare an antibody-drug conjugate (ADC). In several embodiments, the various chemotherapeutic agents described herein can be conjugated with the provided antibody to generate a conjugate.

[0160] V antiCD3 antibody variant

[0161] In some embodiments, amino acid sequence variants of the antibodies provided herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0162] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. Conserved substitutions are shown in Table 1, titled “Preferred Substitutions.” More substantial variations are provided in Table 1, titled “Exemplary Substitutions,” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into target antibodies, and products can be screened for desired activities (e.g., preserved / improved antigen binding or reduced immunogenicity).

[0163] Table 1. Amino acid substitutions

[0164]

[0165]

[0166] Amino acids can be grouped according to their common side chain characteristics:

[0167] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0168] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0169] (3) Acidity: Asp, Glu;

[0170] (4) Alkaline: His, Lys, Arg;

[0171] (5) Residues that affect chain orientation: Gly, Pro;

[0172] (6) Aromatics: Trp, Tyr, Phe.

[0173] Non-conservative replacement would require swapping members of one of these categories with members of another category.

[0174] One type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further research will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody that can be readily generated, for example using phage display-based affinity maturation techniques (such as those described herein). In short, one or more HVR residues are mutated and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

[0175] Alterations (e.g., substitutions) can be made in the HVR, for example, to improve antibody affinity. Such alterations can be made in HVR “hotspots,” which are residues encoded by codons that undergo frequent mutations during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or SDR (a-CDR), where the binding affinity of the resulting variant VH or VL is tested. For example, Hoogenboom et al. have described affinity maturation by constructing and reselecting from a secondary library in Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variant gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variant with the desired affinity. Another approach to introducing diversity involves HVR-directed methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

[0176] In some embodiments, substitution, insertion, or deletion can occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not significantly reduce binding affinity (e.g., conserved substitutions as provided herein) can be made in the HVR. Such changes can occur outside of the HVR "hotspot" or CDR. In the variant V provided above... H In some embodiments of the H sequence, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0177] In some embodiments, the anti-CD3 constructs provided herein can be modified to increase or decrease the degree of glycosylation in the construct. Adding or deleting glycosylation sites in an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0178] When an anti-CD3 construct includes an Fc region, the carbohydrates to which they are attached can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region via an N-bond. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the anti-CD3 construct of this application can be modified to produce antibody variants with certain improved properties.

[0179] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the anti-CD3 construct provided herein to generate an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0180] In some embodiments, the anti-CD3 constructs provided herein may be further modified to contain additional non-protein moieties known in the art and readily available. Suitable antibody-derived moieties include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used in a defined therapeutic condition.

[0181] VI. Pharmaceutical Composition

[0182] This application further provides pharmaceutical compositions comprising any anti-CD3 construct (such as anti-CD3 IgG, anti-CD3 fragment, full-length anti-CD3 IgG, or anti-CD3 bispecific antibody) and optionally a pharmaceutically acceptable carrier. The pharmaceutical compositions can be prepared by mixing the anti-CD3 construct described herein with the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the form of a lyophilized formulation or an aqueous solution.

[0183] The pharmaceutical composition is preferably stable, and the anti-CD3 construct containing the anti-CD3 mAb described herein retains substantially its physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability are available in the art and have been reviewed in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at selected temperatures for selected time periods. For rapid screening, the formulation can be kept at 40°C for 2 weeks to 1 month, during which stability is measured. When the formulation is to be stored at 2°C–8°C, it should generally be stable at 30°C or 40°C for at least 1 month, and / or stable at 2°C–8°C for at least 2 years. When the formulation is to be stored at 30°C, it should generally be stable at 30°C for at least 2 years, and / or stable at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. In some embodiments, a stabilizing formulation of the antiCD3 construct described herein may contain less than about 10% (preferably less than about 5%) of the antiCD3 construct present as aggregates in the formulation.

[0184] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium metabisulfite); preservatives, isotonic agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents (such as EDTA) and / or nonionic surfactants.

[0185] Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; and proteins. Examples of active ingredients include: serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium ions; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN. TM Polyethylene glycol (PEG) and PLURONICS TM Or polyethylene glycol (PEG).

[0186] Buffers are used to control the pH within a range that optimizes therapeutic efficacy, especially if stability is pH-dependent. Buffers are preferably present at a concentration of about 50 mM to about 250 mM. Suitable buffers for use in this application include organic and inorganic acids and their salts. Examples include citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, and acetates. Additionally, buffers may contain histidine salts and trimethylamine salts, such as Tris.

[0187] Preservatives are added to slow microbial growth, and these preservatives are typically present in the range of 0.2%–1.0% (w / v). The addition of preservatives can, for example, facilitate the production of multi-use (multi-dose) formulations. Suitable preservatives for use in this application include octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium halides (e.g., benzalkonium chloride, benzalkonium bromide, benzalkonium iodide), benzyl chloride; thimerosal, phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0188] Tensile agents (sometimes called "stabilizers") are present to regulate or maintain the surface tension of liquids in a composition. They are often referred to as "stabilizers" when used with large, charged biomolecules such as proteins and antibodies because they can interact with charged groups on the side chains of amino acids, thereby reducing the potential for intermolecular and intramolecular interactions. Tensile agents can be present in any amount from 0.1% to 25% by weight, preferably from 1% to 5% by weight, taking into account the relative amounts of other components. Preferred tensile agents include polyhydroxy sugar alcohols, preferably trihydroxy or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol.

[0189] Other excipients include agents that can be used as one or more of the following: (1) fillers, (2) solubilizers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include: polyhydroxy sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbitol, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclic polyols ( Examples include inositol and polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0190] For pharmaceutical compositions to be used in vivo, they must be sterile. Sterility can be achieved through filtration using a sterile filter membrane. The pharmaceutical compositions described herein are typically placed in containers with sterile inlets, such as intravenous solution bags or vials with stoppers that can be punctured by a subcutaneous needle.

[0191] The route of administration is based on known and accepted methods, such as by single or multiple bolus injections or by infusion over a prolonged period of time in a suitable manner, for example by injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intra-articular routes, local application, inhalation, or by sustained or prolonged release. In some embodiments, the pharmaceutical composition is applied locally, such as intratumoral application.

[0192] Sustained-release products can be prepared. Suitable examples of sustained-release products include semi-permeable matrices of solid hydrophobic polymers containing antagonists, which are in the form of molded articles, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid esters, non-degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

[0193] The pharmaceutical compositions described herein may also contain one or more active compounds necessary for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or additionally, the composition may contain cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, or growth inhibitors. Such molecules are suitably present in a combination of amounts effective for the intended purpose. In some embodiments, the therapeutic agent or immunosuppressant other than the antibodies provided herein is an antibody or antigen-binding fragment thereof specific to CD39, CTLA-4, PD-L1, TIM-3, LAG-3, or A2aR.

[0194] Active ingredients can also be encapsulated in microcapsules, such as those prepared by coagulation techniques or interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition.

[0195] In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a reusable vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is frozen.

[0196] VII. Method of Use or Application

[0197] Anti-CD3 constructs and their compositions (such as pharmaceutical compositions) containing mAbs that specifically recognize CD3 as described herein (e.g., anti-CD3 IgG, anti-CD3 fragment, full-length anti-CD3 IgG, anti-CD3 bispecific antibody) can be used in a variety of applications, such as in diagnostics, molecular assays and therapies.

[0198] One aspect of the invention provides a method for treating CD3-related diseases or conditions in an individual in need, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising the anti-CD3 construct described herein. Another aspect of the invention provides a method for treating diseases or conditions associated with the expression of a specific antigen in an individual in need, wherein T cells can be cross-linked with target cells via a CD3-BsAb comprising the anti-CD3 antibody of the present invention and an antibody targeting the specific antigen. In some embodiments, the disease or condition is a tumor or cancer. In some embodiments, the tumor or cancer is a solid tumor or cancer.

[0199] This application partially considers protein constructs (such as full-length anti-CD3 IgG, anti-CD3 bispecific antibodies), nucleic acid molecules and / or carriers encoding protein constructs, and host cells containing nucleic acid molecules and / or carriers encoding protein constructs, which can be administered alone or in combination with another therapy, and in at least some respects, together with pharmaceutically acceptable carriers or excipients. In some embodiments, they can be combined with suitable pharmaceutical carriers and excipients well known in the art prior to administration of the anti-CD3 construct. Compositions prepared according to this disclosure can be used for the treatment of cancer or for the delay of its progression.

[0200] In some embodiments, a method of treating cancer is provided, the method comprising administering to an individual an effective amount of a pharmaceutical composition comprising an isolated anti-CD3 construct comprising a mAb that specifically recognizes CD3 (such as full-length anti-CD3 IgG, anti-CD3 bispecific antibody). In some embodiments, the cancer is a solid tumor (such as lung cancer). In some embodiments, the pharmaceutical composition is administered systemically (such as intravenously). In some embodiments, the pharmaceutical composition is administered locally (such as within a tumor). In some embodiments, the method further comprises administering to the individual additional cancer therapy (such as surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof). In some embodiments, the individual is a person. In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting cancer cell proliferation; (3) inducing an immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual with cancer; (6) inhibiting tumor metastasis; (7) prolonging survival; (8) prolonging the time of cancer progression; and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher percentage. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (not infected by oncolytic cells VV) death rate of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher percentage. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce the tumor size by at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including, for example, any one of at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of metastasis. In some embodiments, the method of prolonging the survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolong the survival of the individual by at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging the time of cancer progression mediated by the pharmaceutical composition described herein can prolong the time of cancer progression by at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0201] The methods described herein are applicable to the treatment of a variety of cancers, including both solid and liquid-filled cancers. These methods are applicable to all stages of cancer, including early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The methods described herein can be used in adjunctive or neoadjunctive settings as a first-line, second-line, third-line therapy, or in combination with other types of cancer therapies known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (e.g., T-cell therapy), bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc. (i.e., the method can be performed prior to a primary / deterministic therapy). In some embodiments, the method is used to treat individuals who have previously been treated. In some embodiments, the cancer is refractory to previous therapies. In some embodiments, the method is used to treat individuals who have not previously been treated.

[0202] In some embodiments, the method is suitable for treating tumors or cancers with aberrant CD3 expression, activity, and / or signaling. In some embodiments, the method is suitable for treating tumors or cancers associated with the expression of tumor-associated antigens or tumor-specific antigens such as CD19, CD20, EGFR, BCMA, GPRC5D, EpCAM, DLL3, and HER2. In some embodiments, the tumors or cancers include, by way of non-limiting examples, bladder cancer, cervical cancer, colon cancer, colorectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, breast cancer, head and neck cancer, skin cancer, sarcoma, brain tumors, brain and spinal cord cancers, adrenal cancer, uterine cancer, neuroblastoma, small round cell tumors, peripheral nerve sheath tumors, bone cancer, rhabdoid tumors, lymphoma, multiple myeloma, leukemia, neuroendocrine tumors, and melanoma.

[0203] In some embodiments, the pharmaceutical composition is administered systemically (e.g., intravenously). In some embodiments, the pharmaceutical composition is administered locally (e.g., intratumorally). In some embodiments, the method further includes administering additional cancer therapy (e.g., surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or combinations thereof) to the individual. In some embodiments, the individual is a person. In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting cancer cell proliferation; (3) inducing an immune response in the tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual with cancer; (6) inhibiting tumor metastasis; (7) prolonging survival; (8) prolonging the time of cancer progression; and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell mortality rate of at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher percentages. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a mortality rate of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher percentage of bystander tumor cells (not infected with oncolytic cells VV). In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce tumor size by at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of metastasis. In some embodiments, the method of prolonging the survival of an individual (such as a human) mediated by the pharmaceutical composition described herein may prolong the survival of the individual by at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging the time of cancer progression mediated by the pharmaceutical composition described herein may prolong the time of cancer progression by at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0204] The dosage and desired drug concentration of the pharmaceutical composition of this application may vary depending on the intended specific use. Determining the appropriate dosage or route of administration is entirely within the skill of a person skilled in the art. Animal studies have provided reliable guidance for determining effective dosages for human treatment. Interspecies analogies for effective dosages can be made according to the principles established by Mordenti, J. and Chappell, W., “The Use of Interspecies Scaling in Toxicokinetics”, Toxicokinetics and New Drug Development, Yacobi et al., Pergamon Press, New York, 1989, pp. 42-46.

[0205] When administered in vivo using the anti-CD3 construct containing anti-CD3 mAb as described herein, the normal dose can vary from about 10 ng / kg per day to about 100 mg / kg of mammalian body weight or more, preferably from about 1 mg / kg / day to 10 mg / kg / day, such as about 1-3 mg / kg / day, about 2-4 mg / kg / day, about 3-5 mg / kg / day, about 4-6 mg / kg / day, about 5-7 mg / kg / day, about 6-8 mg / kg / day, about 6-6.5 mg / kg / day, about 6.5-7 mg / kg / day, about 7-9 mg / kg / day, or about 8-10 mg / kg / day, depending on the route of administration. Within the scope of this application, different formulations will be effective for different treatments and different conditions, and administration intended to treat a specific organ or tissue may require delivery in a manner different from delivery to another organ or tissue. Moreover, the dose can be administered by single or multiple separate administrations or by continuous infusion. For repeated administration over several days or longer, treatment should continue until the desired suppression of disease symptoms is achieved, depending on the condition. However, other dosage regimens may also be useful. Progression of this therapy can be easily monitored using routine techniques and assays.

[0206] In some embodiments, the pharmaceutical composition is administered once (e.g., by bolus injection). In some embodiments, the pharmaceutical composition is administered multiple times (such as any number of 2, 3, 4, 5, 6, or more times). If administered multiple times, they can be done via the same or different routes and can be done at the same site or at alternative sites. The pharmaceutical composition can be administered twice a week, three times a week, four times a week, five times a week, once daily, once daily without interruption, once a week, once a week, once a week without interruption, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once a year. The interval between administrations can be any of about 24 hours to 48 hours, 2 days to 3 days, 3 days to 5 days, 5 days to 1 week, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 months to 3 months, 3 months to 6 months, or 6 months to 1 year. The interval can also be irregular (e.g., after tumor progression). In some embodiments, the dosing schedule is not interrupted. In some embodiments, the pharmaceutical composition is administered every 4 days for a total of 4 times. The optimal dosage and treatment regimen for a particular patient can be easily determined by a medical professional by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0207] VIII. Preparation Method

[0208] The anti-CD3 constructs described herein (such as anti-CD3 monoclonal antibodies) can be prepared using any method known in the art or as described herein.

[0209] Rodent monoclonal antibodies can be obtained using methods known in the art, such as by immunizing a rodent species (such as a mouse or rat) and obtaining a hybridoma from it, or by cloning a Fab fragment or a single-chain Fc (scFv) library using molecular biology techniques known in the art and subsequently selecting the library by ELISA or FACS with a single clone of the unselected library or by using phage display.

[0210] To recombinantly generate monoclonal antibodies, the nucleic acid encoding the monoclonal antibody is isolated or synthesized and inserted into a reproducible vector for further cloning (DNA amplification) or for expression. The DNA encoding the monoclonal antibody can be easily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody. Many vectors are available. The choice of vector depends in part on the host cell to be used. Typically, the preferred host cell is of prokaryotic or eukaryotic (usually mammalian) origin.

[0211] Developing anti-human CD3 antibodies is crucial in the field of immunotherapy. Anti-human CD3 antibodies that only bind to CD3 are insufficient. They must further activate primary T cells. The human CD3 antigen consists of four distinct chains: CD3γ, CD3δ, two CD3ε, and CD3ζ. It is impossible to express and purify all four distinct human CD3 chains in vitro. Primary human T cells expressing endogenous membrane CD3 are isolated from peripheral blood mononuclear cells (PBMCs). They are a good choice for immunogens to obtain anti-human CD3 antibodies in animals. However, using primary human T cells as immunogens is less effective than using purified human CD3 protein. Upon exposure to immunized animals, the amount of all proteins on the membrane of primary human T cells, except for human CD3, induces an adaptive immune response in B cells. The B cells of the immunized animals will then generate various antibodies targeting these proteins. Distinguishing anti-human CD3 antibodies from an antibody library is not an easy task. Therefore, obtaining anti-human CD3 antibodies will be very difficult. In general, the number of anti-human CD3 antibodies is limited. The most well-known anti-human CD3 antibody, OKT3, does not bind to non-human primate CD3 proteins. Due to its high T cell activation activity, OKT3 induces cytokine release syndrome, thus raising safety concerns. Our anti-human CD3 not only binds to human CD3 proteins but also induces T cell activation upon binding to CD3 proteins on T cells. Compared to OKT3, our anti-human CD3 has a higher binding affinity for CD3 but lower T cell activation, providing unique characteristics for its potential applications in immunotherapy. Compared to OKT3, it enables T cells to maintain a high affinity binding to CD3 expressed on T cells, while its lower T cell activation activity makes T cells extremely effective in natural killing tumor cells with fewer side effects. It can establish an appropriate therapeutic window while balancing its efficacy and safety. Furthermore, it exhibits cross-reactivity against non-human primate CD3 proteins.

[0212] Example 1. Preparation of mouse anti-human CD3 monoclonal antibody

[0213] For animal immunity, the immunogen was primary T cells isolated from human PBMCs. Five female Balb / c mice were injected intraperitoneally with 5 × 10⁵ cells. 6 Primary human T cells. Then, mice were given 5 × 10⁸ cells every two weeks. 6 One primary human T-cell intraperitoneal booster immunization may be administered once, or at most twice.

[0214] Four mice were selected for hybridoma fusion and screening. The isolated spleens were prepared into homogenized single-cell suspensions, and single-cell suspensions of myeloma cells (SP 2 / 0 cells) were also prepared. 8.9 × 10⁸ cells were used. 74.1 × 10⁻⁶ spleen cells and 4.1 × 10⁻⁶ 7 SP2 / 0 cells were fused using an electrofusion method. Fusion cells from each hybridoma fusion were resuspended in 100 ml of DMEM / 10% FBS medium containing thymidine, hypoxanthine, and aminopterin hybridoma selectivity reagent. These cell suspensions were aliquoted into fifty 96-well plates at 100 μl per well. The 96-well plates were incubated at 37°C and 6% CO2 for 7 days. The hybridoma supernatant was then tested by FACS binding assay to detect the binding of anti-human CD3 antibody to CD3 expressed in Jurkat cells. Stable human CD3 binding was confirmed in hybridoma supernatants from 39B12G6, 10A7C8, and 64F9G7 cells along with the positive control commercial anti-human CD3 antibody (clone UCHT1) using a single dose. Figure 1 Negative controls (PBS) or mouse IgG isotype controls showed no binding activity. Mouse anti-human CD3 monoclonal antibodies 39B12G6, 10A7C8, and 64F9G7 were purified from hybridoma supernatant. In a single-dose FACS study, the binding of mouse anti-human CD3 monoclonal antibodies 39B12G6, 10A7C8, and 64F9G7 to cynomolgus monkey CD3 was confirmed using primary T cells isolated from cynomolgus monkeys. Figure 2 Negative controls PBS or mouse IgG isotype controls showed no binding activity against primary T cells isolated from cynomolgus monkeys.

[0215] Example 2. Activation of Jurkat cells using mouse anti-human CD3 monoclonal antibody

[0216] Jurkat cells are immortalized cell lines of human T lymphocytes, widely used to study signal transduction in T cell activation. Once activated, T cells secrete interleukin-2 (IL-2). T-activated reporter gene cell lines are generated by constructing an IL-2 promoter-driven luciferase reporter gene into Jurkat cells (Jurkat / IL 2 promoter-luciferase). Measuring T cell activation by luciferase intensity is a highly reliable method for assessing T cell activation. 39B12G6, 10A7C8, and 64F9G7 cells were tested in T-activated reporter gene studies. 8,000 CHO-K1 cells overexpressing human CD80 (CHO-K1 / CD80) were seeded into each well of a 384-well plate. After overnight incubation, 40,000 Jurkat / IL 2 promoter-luciferase cells were added to the wells along with the antibody sample to be tested. After 5 hours, the luciferase intensity in each well was read using a microplate reader. All mouse anti-human CD3 monoclonal antibodies induced concentration-dependent luciferase signaling in T-activated reporter cell lines. Figure 3The EC50 values ​​for reporter gene T cell activation ranged from 0.018 μg / ml to 0.82 μg / ml (Table 2).

[0217] Table 2. Effects of anti-human CD3 hybridoma on reporter T cell activation.

[0218]

[0219] Example 3. Activation of primary T cells using mouse anti-human CD3 monoclonal antibody

[0220] 39B12G6, 10A7C8, 64F9G7, and OKT3 were plated in 96-well plates and incubated overnight at 4°C. Two × 10⁵ primary human pan-T cells derived from peripheral blood mononuclear cells were added to the plated wells using a human pan T cell isolation kit (Miltenyi: catalog number: 130-096-535) and incubated for 72 hours at 37°C in a humid atmosphere with 5% CO₂. IFN-γ levels in the supernatant of each well were measured using a human IFN-γ kit from Cisbio. 39B12G6, 10A7C8, 64F9G7, and OKT3 induced concentration-dependent IFN-γ release in primary T cells. Figure 4 EC21 activated by primary T cells. 50 The values ​​ranged from 0.50 μg / ml to 10.14 μg / ml (Table 3).

[0221] Table 3. Effects of anti-human CD3 hybridoma on primary T cell activation.

[0222]

[0223] Example 4. Isotype Analysis

[0224] Monoclonal antibody isotypes were identified using a mouse isotype rapid detection ELISA kit (Clonotyping System-HRP, SouthernBiotech). Then, TRIzol (Ambion) was used from 3 × 10⁻⁶ ppm. 6 -5×10 6Total RNA was extracted from single clones of hybridoma cells. RNA was reverse transcribed into cDNA using isotype-specific primers and universal primers (PrimeScript™ 1st Strand cDNA Synthesis Kit, Takara). RACE PCR (GenScript) was then used to amplify the variable regions of the antibody heavy and light chains, and the PCR products were subcloned into the pMD18-T vector system (Takara). Vector-specific primers were used to verify the inserted regions, and they were sequenced. Finally, the variable region DNA / protein sequences of 39B12G6, 10A7C8, and 64F9G7 were obtained. 39B12G6 is the mouse IgG2bκ isotype. 10A7C8 is the mouse IgG1κ isotype. 64F9G7 is the mouse IgG1λ isotype. Chimeric anti-human CD3 monoclonal antibodies (39B12G6, 10A7C8, or 64F9G7 chimeras) were prepared by fusing the variable domains of the heavy and light chains of 39B12G6, 10A7C8, or 64F9G7 with the constant region of human IgG1.

[0225] Example 5. Binding affinity of chimeric anti-human CD3 monoclonal antibodies

[0226] The binding affinity of chimeric anti-human CD3 monoclonal antibodies to human or cynomolgus CD3 was determined using a fluorescence-activated cell sorting (FACS) assay. Chimeric anti-human CD3 monoclonal antibodies and OKT3 (starting at 7.5 μg / ml, serially diluted 2-fold, 10 concentrations) were prepared as primary antibodies for FACS analysis. Jurkat cells were detached from adherent culture flasks. Human primary T cells were purified from human PBMCs using a human PanT cell isolation kit (Miltenyi: catalog number: 130-096-535). Decoupled Jurkat cells, human primary T cells, or cynomolgus PBMCs were mixed with different concentrations of mouse anti-human CD3 monoclonal antibody (all in 96-well plates). The mixtures were equilibrated at room temperature for 30 minutes and washed three times with FACS buffer (PBS containing 1% BSA). Add F(ab')2 goat anti-human IgG Fc PE (Thermo: catalog number: H10104) or goat anti-mouse IgG PE (Biolegend: catalog number: 405307) as secondary antibodies against chimeric anti-human CD3 monoclonal antibodies, and incubate at room temperature for 45 min. Finally, wash the cells three times with PBS and read the signal using FACS BD calibur. Analyze the data using PRISMTM (GraphPad Software, San Diego, CA) with nonlinear regression and calculate the EC50 value. Figure 5 As shown in Table 4, FACS studies have demonstrated that chimeric anti-human CD3 monoclonal antibodies bind to human CD3 expressed in Jurkat cells, EC...50 The values ​​ranged from 0.20 μg / ml to 0.44 μg / ml. Compared with OKT3, 64F9G7 had a higher binding affinity for CD3 expressed on Jurkat cells.

[0227] Table 4. Binding of anti-human CD3 monoclonal antibodies on Jurkat cells.

[0228]

[0229] like Figure 6 As shown in Table 5, FACS studies have demonstrated that chimeric anti-human CD3 monoclonal antibodies bind to human CD3 expressed in human primary T cells, where EC... 50 The values ​​ranged from 0.19 μg / ml to 0.30 μg / ml. Compared with OKT3, all chimeric anti-human CD3 monoclonal antibodies showed higher binding affinity to CD3 expressed on human primary T cells.

[0230] Table 5. Binding of anti-human CD3 monoclonal antibodies to human primary T cells.

[0231]

[0232] Chimeric anti-human CD3 monoclonal antibodies also bind to cynomolgus monkey CD3. For example... Figure 7 As shown in Table 6, the FACS study demonstrated that the chimeric anti-human CD3 monoclonal antibody binds to cynomolgus monkey PBMCs, among which EC... 50 The values ​​ranged from 0.14 μg / ml to 1.6 μg / ml. However, OKT3 did not bind to cynomolgus monkey CD3.

[0233] Table 6. Binding of anti-human CD3 monoclonal antibodies to cynomolgus monkey PBMCs.

[0234]

[0235] Example 6. Activation of Jurkat cells with chimeric anti-human CD3 monoclonal antibody

[0236] Chimeric anti-human CD3 monoclonal antibodies were tested in a T-activated reporter gene study. 8,000 CHO-K1 (CHO-K1 / CD80) cells overexpressing human CD80 were seeded into each well of a 384-well plate. After overnight incubation, 40,000 Jurkat / IL-2 promoter-luciferase cells were added to the wells along with the antibody sample to be tested. After 5 hours, luciferase intensity was read in each well using a microplate reader. All chimeric anti-human CD3 monoclonal antibodies elicited concentration-dependent luciferase signaling in the T-activated reporter gene cell line. Figure 8 ). Reporter gene T cell activation of EC 50Values ​​ranged from 0.047 μg / ml to 3.64 μg / ml (Table 7). Since 39B12G6, 10A7C8, and 64F9G7 belong to either mouse IgG1 (mIgG1) or mouse IgG2b (mIgG2b), unrelated mouse IgG1 and mouse IgG2b were used as negative controls in this study. Antibodies against mouse IgG1 and mouse IgG2b did not induce concentration-dependent luciferase signaling in T-activated reporter cell lines.

[0237] Table 7. Effects of anti-human CD3 chimerism on reporter T cell activation.

[0238]

[0239] Example 7. Activation of primary T cells using chimeric anti-human CD3 monoclonal antibody

[0240] Chimeric anti-human CD3 monoclonal antibody and OKT3 were plated on 96-well plates and incubated overnight at 4°C. 2 × 10⁶ human peripheral blood mononuclear cells were isolated using a human Pan T cell isolation kit (Miltenyi: catalog number: 130-096-535). 5 Primary human pan-T cells were added to the coated wells and incubated at 37°C in a humid atmosphere with 5% CO2 for 72 hours. IFN-γ in the supernatant of each well was measured using a human IFN-γ kit from Cisbio. Chimeric anti-human CD3 monoclonal antibody and OKT3 induced concentration-dependent IFN-γ release in primary T cells. Figure 9 EC21 activated by primary T cells. 50 Values ​​ranged from 0.20 μg / ml to 13.73 μg / ml (Table 8). Primary T-cell activation with 64F9G7 was less than with OKT3. It is expected to produce fewer unnecessary side effects due to excessive T-cell activation in potential immunotherapies.

[0241] Table 8. Effects of anti-human CD3 chimerism on primary T cell activation.

[0242]

[0243]

[0244] Example 8. Target Specificity

[0245] The target specificity of the chimeric anti-human CD3 monoclonal antibody was tested on immune cells isolated from human peripheral blood mononuclear cells. A 5 μg / ml chimeric anti-human CD3 monoclonal antibody and OKT3 were prepared as primary antibodies for FACS analysis. 2 μg / ml rat anti-human IgG μg PE (Biolegend: catalog number: 410708) was used as the secondary antibody for the chimeric anti-human CD3 monoclonal antibody. The target specificity of the chimeric anti-human CD3 monoclonal antibody was tested on 7-ADD (Biolegend: catalog number: 420403) negative, CD45 (Biolegend: catalog number: 368540) positive, and CD16 (Biolegend: catalog number: 302016) / CD56 (Biolegend: catalog number: 362504) positive NK cells. Figure 10 No significant binding was detected in the sample. A 5 μg / ml chimeric anti-human CD3 monoclonal antibody and OKT3 were prepared as primary antibodies for FACS analysis. 2 μg / ml F(ab')2 goat anti-human IgG μg PE (Thermo: catalog number: H10104) was used as the secondary antibody for the chimeric anti-human CD3 monoclonal antibody. The assay was performed on 7-ADD (Biolegend: catalog number: 420403) negative, CD45 (Biolegend: catalog number: 368540) positive, and CD14 (Biolegend: catalog number: 325620) positive monocytes. Figure 11 No significant binding was detected in ). 5 μg / ml of chimeric anti-human CD3 monoclonal antibody directly labeled with iFluor 647 and OKT3 directly labeled with APC were prepared as primary antibodies for FACS analysis. In 7-ADD (Biolegend: catalog number: 420403) negative, CD45 (Biolegend: catalog number: 368540) positive, and CD19 (Biolegend: catalog number: 302238) positive B cells (… Figure 12 No obvious binding was detected in ).

[0246] Example 9. Bispecific T-cell adaptor (BiTE)

[0247] Bispecific T-cell adaptors (BiTEs) were expressed to evaluate the potency of anti-human CD3 antibodies in T-cell-mediated cytotoxicity against tumor cells by simultaneously binding to the target antigen and CD3. Two single-chain variable fragments (scFvs) from anti-human BCMA and anti-CD3 monoclonal antibodies formed the classic BiTE molecule, linked tandemly by short GS adapters, respectively. The scFv sequence for anti-human BCMA is derived from patent (WO 2014 / 140248 A1). The scFv sequence for anti-human CD3 SP34 was also used for BiTE expression. A total of three BiTEs were expressed for the study: anti-human BCMA-SP34, anti-human BCMA-64F9G7, and anti-human BCMA-10A7C8. The sequences of the three BiTEs are shown below.

[0248] Sequence of anti-human BCMA-SP34:

[0249] (SEQ ID NO:44)

[0250] The sequence of anti-human BCMA-64F9G7:

[0251] AATMGWSWILLFLLSVTAGVHSDIQMTQSPSSLSASVGDRVTITCRANQGISNNLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFTSLPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGGGDTYYADSVKGRFTISRDNAKSTLYLQMDSLRSEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKIYNYATFYDDSVKDRFTISRDDSQTMLYLQMNNLKTEDTAMYYCVTYYGNDWIAKWGQGTRVTVSAGGGGSGGGGSGGGGSQAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHYVFGGGTKVTVLHHHHHH(SEQ ID NO:45)

[0252] Sequence of anti-human BCMA-10A7C8:

[0253] AATMGWSWILLFLLSVTAGVHSDIQMTQSPSSSLSASVGDRVTITCRANQGISNNLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFTSLPYTFGQGTKLEIKGG GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGGGDTYYADSVKGRFTISRDNAKSTLYLQMDSLRSEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTV SSSGGGGSQVQLQQSGPELVKPGASVKMSCKAAGYTFTSQYLHWMKQRPGQGLEWIGWINPGDDTTKYNEKFKVRTTLTADKSSSTVYMLLSSLTSDDSAIYFCARDYGYYFDYWGQGTTLTVSSGGGGSGG GGSGGGGSDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCKQSFILRTFGGGTRLEIKHHHHHH(SEQID NO:46)

[0254] The binding affinity of three purified BiTEs for human CD3 was determined using a fluorescence-activated cell sorting (FACS) assay. Purified BiTE was prepared at 45 μg / ml as the primary antibody for FACS analysis. Jurkat cells harvested from culture flasks were mixed with the three purified BiTEs. The mixture was equilibrated at room temperature for 30 min and washed three times with FACS buffer (PBS containing 1% BSA). Anti-His antibody (GenScript, catalog number: A01802-100) was added as the secondary antibody, and the cells were incubated at room temperature for 45 min. Finally, the cells were washed three times with PBS, and the signal was read out using a FACS BD calibur. Figure 13 The results showed that FACS studies demonstrated that three purified BiTEs bound to human CD3 expressed on Jurkat cells.

[0255] The binding affinity of three purified BiTEs for human BCMA was determined using a fluorescence-activated cell sorting (FACS) assay. Purified BiTE was prepared at 15 μg / ml as the primary antibody for FACS analysis. RPMI 8226 cells, a human BCMA-expressing cell line harvested from culture flasks, were mixed with the three purified BiTEs. The mixture was equilibrated at room temperature for 30 min and washed three times with FACS buffer (PBS containing 1% BSA). Anti-His antibody (GenScript, catalog number: A01802-100) was added as a secondary antibody and incubated at room temperature for 45 min. Finally, the cells were washed three times with PBS, and the signal was read out using a FACS BD calibur. Figure 14 The results showed that FACS studies demonstrated that three purified BiTEs bound to human BCMA expressed on RPMI 8226 cells.

[0256] These three purified BiTEs were used in a study of T cell-mediated cytotoxicity in the BCMA-expressing tumor cell line RPMI 8226. The RPMI 8226-Luc cell line was generated through the stable integration of constitutive firefly luciferase. Luciferase activity in RPMI 8226-Luc cells could be measured using a luciferase assay kit, allowing for the detection of surviving RPMI 8226-Luc cells in the co-culture system, thus indirectly reflecting BiTE-induced T cell-mediated cytotoxic activity. RPMI 8226-Luc cells were the target cells in this study. Primary CD8+ T cells derived from peripheral blood mononuclear cells were used as effector cells in the study using a human CD8+ T cell isolation kit (Miltenyi catalog number: 130-097-057). RPMI 8226-Luc cells were gently mixed and added to the wells of a 96-well plate. After adding RPMI 8226-Luc cells, the 96-well plates were covered with cells and placed in a horizontal centrifuge. The plates were centrifuged at 300g for 1 minute at room temperature to allow liquid on the well walls to flow to the bottom. The wells containing RPMI 8226-Luc cells were labeled with three groups: the sampling group (added with effector cells and purified BiTE), the maximum survival group (Max) (added with only complete culture medium), and the minimum survival group (Min) (added with 2% Triton X-100 to kill all RPMI 8226-Luc cells). 0.1 μg / ml of anti-human BCMA-SP34, anti-human BCMA-64F9G7, anti-human BCMA-10A7C8, 64F9G7 chimera, and 10A7C8 chimera were added to the wells of the sampling group, respectively. The sample wells were incubated in a cell culture incubator at room temperature for 30 min, and then effector cells were added. The effector cell to target cell ratio was 5:1. After 24 hours of incubation, Fire-lumi (Fire-lumi luciferase assay kit, GenScript, catalog number: L00877C-1000) was added to the wells and luciferase intensity was measured. Target cell killing percentage = [1 - (X - Min) / (Max - Min)] * 100% (X: luciferase intensity of the sampling group; Max: luciferase intensity of Max; Min: luciferase intensity of Min). BiTE-induced killing of primary CD8+ T cells of RMI 8226 was shown in... Figure 15 In this study, both anti-human BCMA-SP34 and anti-human BCMA-64F9G7 induced significant T-cell-specific killing of RPMI 8226 cells expressing human BCMA.

[0257] The following lists some of the amino acid and nucleic acid sequences mentioned in this article. CDR sequences are based on Kabat numbering.

[0258] Hybridoma

[0259] 64F9G7 (Mouse IgG1λ)

[0260] Heavy chain

[0261] MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKIYNYATFYDDSVKDRFTISRDDSQTMLYLQMNNLKTEDTAMYYCVTYYGNDWIAKWGQGTRVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:1)

[0262] VH:

[0263] MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKIYNYATFYDDSVKDRFTISRDDSQTMLYLQMNNLKTEDTAMYYCVTYYGNDWIAKWGQGTRVTVSA (SEQID NO:2)

[0264] CDR1: TYAMN (SEQ ID NO:3)

[0265] CDR2: RIRSKIYNYATFYDDSVKD (SEQ ID NO:4)

[0266] CDR3: YYGNDWIAK (SEQ ID NO:5)

[0267] Light chain

[0268] MAWTSLILSLLALCSGASSQAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHYVFGGGTKVTVLQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS(SEQ ID NO:6)

[0269] VL:

[0270] MAWTSLILSLLALCSGASSQAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHYVFGGGTKVTVL(SEQ ID NO:7)

[0271] CDR1: RSSTGAVTTSNYAN(SEQ ID NO:8)

[0272] CDR2: GTSNRAP(SEQ ID NO:9)

[0273] CDR3: ALWYSTHYV(SEQ ID NO:10)

[0274] 10A7C8 (mouse IgG1κ)

[0275] Heavy chain

[0276] MRWSWFFLFLLSITAGVHCQVQLQQSGPELVKPGASVKMSCKAAGYTFTSQYLHWMKQRPGQGLEWIGWINPGDDTTKYNEKFKVRTTLTADKSSSTVYMLLSSLTSDDSAIYFCARDYGYYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:11)

[0277] VH:

[0278] MRWSWFFLFLLSITAGVHCQVQLQQSGPELVKPGASVKMSCKAAGYTFTSQYLHWMKQRPGQGLEWIGWINPGDDTTKYNEKFKVRTTLTADKSSSTVYMLLSSLTSDDSAIYFCARDYGYYFDYWGQGTTLTVSS

[0279] (SEQ ID NO:12)

[0280] CDR1: SQYLH(SEQ ID NO:13)

[0281] CDR2: WINPGDDTTKYNEKFKV(SEQ ID NO:14)

[0282] CDR3: DYGYYFDY(SEQ ID NO:15)

[0283] Light chain

[0284] MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCKQSFILRTFGGGTRLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC(SEQ ID NO:16)

[0285] VL:

[0286] MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCKQSFILRTFGGGTRLEIK(SEQ ID NO:17)

[0287] CDR1: KSSQSLLNSRTRKNYLA(SEQ ID NO:18)

[0288] CDR2: WASTRES(SEQ ID NO:19)

[0289] CDR3: KQSFILRT(SEQ ID NO:20)

[0290] 39B12G6 (mouse IgG2bκ)

[0291] Heavy chain

[0292] MGWSRIFLFLLSIIAGVHCQVQLQQSGPELVKPGASVRISCKASGYTFTTSYIHFVRQRPGQGLEWIGWISPGDVNTKYSEKFKGRATLTADKSSTTAYMQLSSLTSEDSAVYFCTRDYGYYFDYWGQGTTLTVSSKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTIS(SEQ ID NO:21)

[0293] VH:

[0294] MGWSRIFLFLLSIIAGVHCQVQLQQSGPELVKPGASVRISCKASGYTFTTSYIHFVRQRPGQGLEWIGWISPGDVNTKYSEKFKGRATLTADKSSTTAYMQLSSLTSEDSAVYFCTRDYGYYFDYWGQGTTLTVSS(SEQ IDNO:22)

[0295] CDR1: TSYIH(SEQ ID NO:23)

[0296] CDR2: WISPGDVNTKYSEKFKG(SEQ ID NO:24)

[0297] CDR3: DYGYYFDY(SEQ ID NO:25)

[0298] Light chain

[0299] MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQKKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSFILRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC(SEQ ID NO:26)

[0300] VL:

[0301] MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQKKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSFILRTFGGGTKLEIK(SEQ ID NO:27)

[0302] CDR1: KSSQSLLNSRTRKNYLA(SEQ ID NO:28)

[0303] CDR2: WASTRES(SEQ ID NO:29)

[0304] CDR3: KQSFILRT(SEQ ID NO:30)

[0305] Chimeric antibody

[0306] 64F9G7

[0307] Heavy chain

[0308] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKIYNYATFYDDSVKDRFTISRDDSQTMLYLQMNNLKTEDTAMYYCVTYYGNDWIAKWGQGTRVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:31)

[0309] VH:

[0310] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKIYNYATFYDDSVKDRFTISRDDSQTMLYLQMNNLKTEDTAMYYCVTYYGNDWIAKWGQGTRVTVSA(SEQ ID NO:32) Light chain

[0311] QAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHYVFGGGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQID NO:33) VL:

[0312] QAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHYVFGGGTKVTVL(SEQ ID NO:34)

[0313] 10A7C8

[0314] Heavy chain

[0315] QVQLQQSGPELVKPGASVKMSCKAAGYTFTSQYLHWMKQRPGQGLEWIGWINPGDDTTKYNEKFKVRTTLTADKSSSTVYMLLSSLTSDDSAIYFCARDYGYYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:35)

[0316] VH:

[0317] QVQLQQSGPELVKPGASVKMSCKAAGYTFTSQYLHWMKQRPGQGLEWIGWINPGDDTTKYNEKFKVRTTLTADKSSSTVYMLLSSLTSDDSAIYFCARDYGYYFDYWGQGTTLTVSS(SEQ ID NO:36)

[0318] Light chain

[0319] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCKQSFILRTFGGGTRLEIKAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:37)VL:

[0320] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCKQSFILRTFGGGTRLEIK(SEQ ID NO:38)

[0321] 39B12G6

[0322] Heavy chain

[0323] QVQLQQSGPELVKPGASVRISCKASGYTFTTSYIHFVRQRPGQGLEWIGWISPGDVNTKYSEKFKGRATLTADKSSTTAYMQLSSLTSEDSAVYFCTRDYGYYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39)

[0324] VH:

[0325] QVQLQQSGPELVKPGASVRISCKASGYTFTTSYIHFVRQRPGQGLEWIGWISPGDVNTKYSEKFKGRATLTADKSSTTAYMQLSSLTSEDSAVYFCTRDYGYYFDYWGQGTTLTVSS(SEQ ID NO:40)

[0326] Light chain

[0327] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQKKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSFILRTFGGGTKLEIKAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:41)VL:

[0328] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQKKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSFILRTFGGGTKLEIK(SEQ ID NO:42)

[0329] Constant region of human IgG1

[0330] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:43)

Claims

1. An anti-CD3 antibody or an antigen-binding fragment thereof, said anti-CD3 antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) containing: 1) HCDR1, said HCDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 13 and 23; 2) HCDR2, said HCDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 14 and 24; and 3) HCDR3, said HCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 15 and 25, and a light chain variable domain (VL) containing: 1) LCDR1, said LCDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 18 and 28; 2) LCDR2, said LCDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 19 and 29; and 3) LCDR3, said LCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 20 and 30.

2. The anti-CD3 antibody or its antigen-binding fragment as described in claim 1, wherein... 1) The VH comprises HCDR1, HCDR2 and HCDR3 sequences having amino acid sequences of SEQ ID NO:3, 4 and 5 respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:8, 9 and 10 respectively; 2) The VH comprises HCDR1, HCDR2, and HCDR3 sequences having amino acid sequences of SEQ ID NO:13, 14, and 15, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 sequences having amino acid sequences of SEQ ID NO:18, 19, and 20, respectively; or 3) The VH comprises HCDR1, HCDR2 and HCDR3 sequences having amino acid sequences of SEQ ID NO:23, 24 and 25 respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:28, 29 and 30 respectively.

3. The anti-CD3 antibody or antigen-binding fragment thereof as claimed in any one of claims 1 or 2, wherein, respectively, the VH comprises an amino acid sequence that is at least 90% identical to the sequence selected from the group consisting of SEQ ID NO: 2, 12 and 22, and the VL comprises an amino acid sequence that is at least 90% identical to the sequence selected from the group consisting of SEQ ID NO: 7, 17 and 27.

4. The anti-CD3 antibody or antigen-binding fragment thereof as claimed in any one of claims 1-3, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, 12 and 22 or a variant thereof, the variant comprising up to about 3 amino acid substitutions in the VH; and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, 17 and 27 or a variant thereof, the variant comprising up to about 3 amino acid substitutions in the VL.

5. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-4, wherein... 1) The VH contains the amino acid sequence of SEQ ID NO:2, and the VL contains the amino acid sequence of SEQ ID NO:7; 2) The VH contains the amino acid sequence of SEQ ID NO:12, and the VL contains the amino acid sequence of SEQ ID NO:17; or 3) The VH contains the amino acid sequence of SEQ ID NO:22, and the VL contains the amino acid sequence of SEQ ID NO:

27.

6. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-5, wherein the CD3 is human CD3 or cynomolgus monkey CD3.

7. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-6, wherein the anti-CD3 antibody is a mouse antibody.

8. The anti-CD3 antibody or antigen-binding fragment thereof as described in any one of claims 1-7, wherein the anti-CD3 antibody or antigen-binding fragment thereof is an activator of Jurkat T cells, and the EC50 value determined by IL-2 promoter activity is greater than 0.01 μg / mL; preferably at about 10 -2 Between μg / mL and 1.0 μg / mL, such as between approximately 0.018 μg / mL and 0.82 μg / mL.

9. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-8, wherein the anti-CD3 antibody or its antigen-binding fragment is an activator of Jurkat T cells and has an EC50 value determined by IL-2 promoter activity greater than that of the OKT3 antibody.

10. The anti-CD3 antibody or antigen-binding fragment thereof as claimed in any one of claims 1-9, wherein the anti-CD3 antibody or antigen-binding fragment thereof is an activator of primary human T cells, and the EC50 value determined by the IFN-γ concentration in the supernatant of the primary human T cells is greater than 0.1 μg / mL; preferably between about 0.1 μg / mL and 50.0 μg / mL, such as between about 0.50 μg / mL and 10.14 μg / mL.

11. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-10, wherein the anti-CD3 antibody or its antigen-binding fragment is an activator of primary human T cells, and the EC50 value determined by the IFN-γ concentration in the supernatant of the primary human T cells is greater than OKT3.

12. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-11, wherein the anti-CD3 antibody is a chimeric antibody comprising: 1) VH contains the amino acid sequence of SEQ ID NO:32, and VL contains the amino acid sequence of SEQ ID NO:34; 2) VH contains the amino acid sequence of SEQ ID NO:36, and VL contains the amino acid sequence of SEQ ID NO:38; or 3) VH contains the amino acid sequence of SEQ ID NO:40, and VL contains the amino acid sequence of SEQ ID NO:

42. Optionally, the VH is fused with the constant region of human IgG, preferably human IgG1.

13. The anti-CD3 antibody or antigen-binding fragment thereof as claimed in any one of claims 1-12, wherein the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and Jurkat T cells is less than 1 μg / mL; preferably between about 0.1 μg / mL and 1.0 μg / mL, such as between about 0.20 μg / mL and 0.45 μg / mL.

14. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-13, wherein the EC50 value of the chimeric antibody or its antigen-binding fragment binding to Jurkat T cells is lower than that of the OKT3 antibody.

15. The anti-CD3 antibody or antigen-binding fragment thereof as claimed in any one of claims 1-14, wherein the EC50 value of the binding between the chimeric antibody or antigen-binding fragment thereof and primary human T cells is less than 1 μg / mL; preferably between about 0.1 μg / mL and 1.0 μg / mL, such as between about 0.19 μg / mL and 0.30 μg / mL.

16. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-15, wherein the EC50 value of the chimeric antibody or its antigen-binding fragment binding to primary human T cells is lower than that of the OKT3 antibody.

17. The anti-CD3 antibody or antigen-binding fragment thereof as claimed in any one of claims 1-16, wherein the EC50 value of the binding between the anti-CD3 antibody or antigen-binding fragment thereof and primary T cells of cynomolgus monkeys is less than 10 μg / mL; preferably between about 0.1 μg / mL and 5.0 μg / mL, such as between about 0.14 μg / mL and 1.6 μg / mL.

18. The anti-CD3 antibody or antigen-binding fragment thereof as claimed in any one of claims 1-17, wherein the chimeric antibody is an activator of Jurkat T cells and has an EC50 value greater than 0.01 μg / mL as determined by IL-2 promoter activity; preferably between about 0.01 μg / mL and 10.0 μg / mL, such as between about 0.01 μg / mL and 5.0 μg / mL.

19. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-18, wherein the chimeric antibody is an activator of Jurkat T cells and has an EC50 value determined by IL-2 promoter activity greater than that of the OKT3 antibody.

20. The anti-CD3 antibody or antigen-binding fragment thereof as claimed in any one of claims 1-19, wherein the chimeric antibody is an activator of primary human T cells, and the EC50 value determined by the concentration of IFN-γ in the supernatant of the primary human T cells is greater than 0.1 μg / mL; preferably between about 0.1 μg / mL and 50.0 μg / mL, such as between about 0.20 μg / mL and 15.0 μg / mL.

21. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-20, wherein the chimeric antibody is an activator of primary human T cells, and the EC50 value determined by the IFN-γ concentration in the supernatant of the primary human T cells is greater than that of the OKT3 antibody.

22. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-21, wherein the anti-CD3 antibody or its antigen-binding fragment does not bind to NK cells, monocytes or B cells.

23. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-22, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), and sdAb.

24. The antiCD3 antibody or antigen-binding fragment thereof as described in any one of claims 1-23, wherein the antiCD3 antibody or antigen-binding fragment thereof exhibits a reduced tendency to induce cytokine release syndrome compared to the OKT3 antibody.

25. A multispecific antibody comprising a first binding portion and a second binding portion, wherein the first binding portion comprises an anti-CD3 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-24, and the second binding portion is capable of binding antigens other than CD3.

26. The multispecific antibody of claim 25, wherein the multispecific antibody is a bispecific antibody.

27. The multispecific antibody of claim 25 or 26, wherein the antigen is a cell surface antigen.

28. The multispecific antibody according to any one of claims 25-27, wherein the antigen is a tumor antigen.

29. The multispecific antibody according to any one of claims 25-28, wherein the antigen other than CD3 is selected from the group consisting of: CD19, CD20, EGFR, BCMA, GPRC5D, EpCAM, DLL3 and HER2.

30. The multispecific antibody of any one of claims 25-29, wherein the first binding portion and / or the second binding portion is selected from the group consisting of: Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), and sdAb.

31. The multispecific antibody according to any one of claims 25-29, wherein the multispecific antibody is in the form of BiTE.

32. The multispecific antibody according to any one of claims 25-30, wherein the multispecific antibody comprises the amino acid sequence of SEQ ID NO: 45 or 46.

33. A pharmaceutical composition comprising an anti-CD3 antibody or an antigen-binding fragment thereof as described in any one of claims 1-24, or a multispecific antibody as described in any one of claims 25-32; and a pharmaceutically acceptable carrier.

34. The pharmaceutical composition of claim 33, wherein the pharmaceutical composition further comprises one or more therapeutic agents other than the anti-CD3 antibody or its antigen-binding fragment or the multispecific antibody.

35. The pharmaceutical composition of claim 34, wherein the therapeutic agent is an antibody specific to CD39, CTLA-4, PD-L1, TIM-3, LAG-3, or A2aR.

36. A method for enhancing the immune function of a subject, the method comprising administering to a subject in need a therapeutically effective amount of an antiCD3 antibody or an antigen-binding fragment thereof as described in any one of claims 1-24, a multispecific antibody as described in any one of claims 25-32, or a pharmaceutical composition as described in any one of claims 33-35.

37. A method of treating a subject with cancer, the method comprising administering to a subject in need a therapeutically effective amount of an antiCD3 antibody or an antigen-binding fragment thereof as described in any one of claims 1-24, a multispecific antibody as described in any one of claims 25-32, or a pharmaceutical composition as described in any one of claims 33-35.

38. The method of claim 37, wherein the cancer is multiple myeloma.

39. Use of the antiCD3 antibody or its antigen-binding fragment as described in any one of claims 1-24, or the multispecific antibody as described in any one of claims 25-32, in the manufacture of a medicament for treating cancer.

40. The method of claim 39, wherein the cancer is multiple myeloma.

41. The anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1-24, the multispecific antibody as described in any one of claims 25-32, or the pharmaceutical composition as described in any one of claims 33-35, for use in treating cancer in a subject in need.

42. The method of claim 41, wherein the cancer is multiple myeloma.

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