Anti-CD3 antibodies and methods for using same

Abstract

The present invention relates to novel anti-CD3 antibodies (used as monoclonal antibodies or in other formats such as bispecific or multispecific formats) and compositions comprising such antibodies or cells activated by such antibodies for use in the treatment of CD3-related disorders, such as human cancer therapy. The present invention also includes compositions comprising one or more of these peptides / antibodies, or fragments thereof, and / or immune cells comprising and / or modified to be activated by one or more of these antibodies, or fragments thereof, for treating diseases or conditions such as cancer.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to novel anti-CD3 antibodies (used as monoclonal antibodies or in other formats such as bispecific or multispecific formats) and compositions comprising such antibodies or cells activated by such antibodies for use in the treatment of CD3-associated disorders, such as human cancer therapy. [Background technology]

[0002] Background of the Invention The body's immune system functions as a defense against various conditions, including injury, infection, and neoplasms, and is mediated by two separate but interrelated systems: the cellular immune system and the humoral immune system. Generally speaking, the humoral system is mediated by soluble products (antibodies or immunoglobulins) that have the ability to bind to and neutralize products recognized by the system as foreign to the body. In contrast, the cellular immune system is responsible for recruiting certain cells called T cells, which perform various therapeutic roles. T cells are lymphocytes derived from the thymus and circulate between tissues, the lymphatic system, and the circulatory system. They act against or in response to various foreign structures (antigens). Often, these foreign antigens are expressed on host cells as a result of neoplasms or infection. T cells do not secrete antibodies themselves; however, they are usually required for antibody secretion by a second class of lymphocyte, B cells (derived from the bone marrow). Importantly, T cells exhibit high immunological specificity, allowing them to distinguish one antigen from another.

[0003] Naive T cells, e.g., T cells that have not yet encountered their specific antigen, are activated when they first encounter a specific peptide:MHC complex on an antigen-presenting cell. The antigen-presenting cell can be a B cell, macrophage, or dendritic cell. When a naive T cell encounters a specific peptide:MHC complex on an antigen-presenting cell, a signal is delivered via the T cell receptor, which induces a conformational change in the T cell's lymphocyte function-associated antigen (LFA) molecules, increasing their affinity for intercellular adhesion molecules (ICAMs) present on the surface of the antigen-presenting cell. The signal generated by the interaction between the T cell and the antigen-presenting cell is necessary but not sufficient to activate naive T cells. A second costimulatory signal is required. Naive T cells can only be activated by antigen-presenting cells bearing both a specific peptide-MHC complex and a costimulatory molecule on their surface. Antigen recognition by naive T cells in the absence of costimulation renders the T cell anergic. The requirement for two signals to activate T and B cells to achieve an adaptive immune response may provide a mechanism to avoid responses to self-antigens that may be present on antigen-presenting cells in a system location where they can be recognized by T cells. If contact between a T cell and an antigen-presenting cell produces only one of the two necessary signals, the T cell will not be activated and an adaptive immune response will not occur.

[0004] The efficiency with which humans and other mammals generate immunological responses against pathogens and foreign substances depends on two attributes: the exquisite specificity of the immune response to antigen recognition, and immunological memory, which allows for a faster and more potent response upon reactivation by the same antigen (Portoles, P. et al. (2009) “The TCR / CD3 Complex: Opening the Gate to Successful Vaccination,” Current Pharmaceutical Design 15:3290-3300; Guy, C.S. et al. (2009) “Organization of Proximal Signal Initiation at the TCR:CD3 Complex,” Immunol Rev. 232(1):7-21). The specificity of T cell responses is mediated by recognition of antigens presented on antigen-presenting cells (APCs) by a molecular complex containing the T cell receptor ("TCR") and the cell surface receptor ligand, CD3. The TCR is a covalently linked heterodimer of an α chain and a β chain ("TCRαβ"). These chains are class I membrane polypeptides of 259 (α) and 296 (β) amino acids in length.The CD3 molecule is a complex containing three dimers (εγ, εδ, ζζ) and two CD3ε chains associated with the CD3γ chain, the CD3δ chain, and two CD3ε chains (Guy, CS et al. (2009) “Organization of Proximal Signal Initiation at the TCR: CD3 Complex,” Immunol Rev. 232(1):7-21; Call, ME et al. (2007) “Common Themes in the Assembly and Architecture of Activating Immune Receptors,” Nat. Rev. Immunol. 7:841-850; Weiss, A. (1993) “T Cell Antigen Receptor Signal Transduction: A Tale of Tails and Cytoplasmic Protein-Tyrosine Kinases,” Cell 73:209-212). The TCR and CD3 complex, together with the zeta chain of the CD3 zeta chain (also known as the T cell receptor T3 zeta chain or CD247), comprise the TCR complex (van der Merwe, PA et al. (epub Dec. 3, 2010) “Mechanisms For T Cell Receptor Triggering,” Nat. Rev. Immunol. 11:47-55; Wucherpfennig, KW et al. (2010) “Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling,” Cold Spring Harb. Perspect. Biol. 2:a005140). This complex is particularly important because it contains a large number (10) of immunoreceptor tyrosine-based activation motifs (ITAMs).

[0005] In mature T cells, activation of TCR / CD3 by foreign antigenic peptides associated with self-MHC molecules is a necessary initial step for the expansion of antigen-specific T cells and their differentiation into effector or memory T lymphocytes. These processes involve phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR complex. The TCR complex contains many such ITAMs (10 in total), which are arranged in tandem (through dimerization of the constituent chains). Upon TCR ligation, phosphorylation of the relevant tyrosine residues creates paired docking sites for proteins containing Src homology 2 (SH2) domains, such as 70-kDa zeta-chain-associated protein (ZAP-70), thereby initiating an amplifying signaling cascade leading to T cell activation and differentiation (Guy, C.S. et al. (2009) “Organization of Proximal Signal Initiation at the TCR:CD3 Complex,” Immunol Rev. 232(1):7-21).

[0006] The outcome of these processes is modulated by the strength and quality of antigen stimulation and the nature of the accompanying signals delivered by coreceptors and costimulatory surface molecules or by cytokine receptors (Portoles, P. et al. (2009) "The TCR / CD3 Complex: Opening the Gate to Successful Vaccination," Current Pharmaceutical Design 15:3290-3300; Riha, P. et al. (2010) "CD28 Co-Signaling In The Adaptive Immune Response," Self / Nonself 1(3):231-240). While TCR stimulation is a prerequisite for T cell activation, it is well recognized that engagement with costimulatory molecules such as CD28 is required for full T cell activation and differentiation (Guy, C.S. et al. (2009) "Organization of Proximal Signal Initiation at the TCR:CD3 Complex," Immunol Rev. 232(1):7-21). Due to the essential role of CD3 in initiating anti-antigen responses, monoclonal antibodies against this receptor can block or at least modulate immune processes and have therefore been proposed as drugs for the treatment of inflammatory and / or autoimmune diseases. In fact, anti-CD3 antibodies were the first antibodies approved for human therapy (St. Clair EW (2009) "Novel Targeted Therapies for Autoimmunity," Curr. Opin. Immunol. 21(6):648-657). Anti-CD3 antibodies (sold by Janssen-Cilag as ORTHOCLONE™ OKT3™) have been administered to alleviate acute rejection in patients with organ transplants and as a treatment for lymphoblastic leukemia (Cosimi, A.B. et al. (1981) “Use Of Monoclonal Antibodies To T-Cell Subsets For Immunologic Monitoring And Treatment In Recipients Of Renal Allografts,” N. Engl. J. Med. 305:308-314; Kung, P. et al. (1979) Monoclonal antibodies defining distinctive human T cell surface antigens,” Science 206:347-349; Vigeral, P. et al. (1986) “Prophylactic Use Of OKT3 Monoclonal Antibody In Cadaver Kidney Recipients. Utilization Of OKT3 As The Sole Immunosuppressive Agent,” Transplantation 41:730-733; Midtvedt, K. et al.(2003) “Individualized T Cell Monitored Administration Of ATG Versus OKT3 In Steroid-Resistant Kidney Graft Rejection,” Clin. Transplant. 17(1):69-74; Gramatzki, M. et al. (1995) “Therapy With OKT3 Monoclonal Antibody In Refractory T Cell Acute Lymphoblastic Leukemia Induces Interleukin-2 Responsiveness,” Leukemia 9(3):382-390; Herold, K. C. et al. (2002) “Anti-CD3 Monoclonal Antibody In New-Onset Type 1 Diabetes Mellitus,” N. Engl. J. Med. 346:1692-1698; Cole, M. S. et al. (1997) “Human IgG2 Variants Of Chimeric Anti-CD3 Are Nonmitogenic to T cells,” J. Immunol. 159(7):3613-3621; Cole, M. S. et al. (1999) “Hum291, A Humanized Anti-CD3 Antibody, Is Immunosuppressive To T Cells While Exhibiting Reduced Mitogenicity in vitro,” Transplantation 68:563-571; U.S. Patent Nos. 6,491,916; 5,585,097, and 6,706,265). However, such anti-CD3 treatment has proven insufficiently specific to avoid side effects (Ludvigsson, J. (2009) "The Role of Immunomodulation Therapy in Autoimmune Diabetes," J. Diabetes Sci. Technol. 3(2):320-330). Repeated daily administration of OKT3 results in significant immunosuppression and provides an effective treatment for rejection after kidney transplantation. In vivo administration of OKT3 results in both T cell activation and suppression of the immune response. However, the use of OKT3 has been hindered by an initial toxic dose-response syndrome associated with early T cell activation events and the release of cytokines prior to subsequent immunosuppression of the T cell response. Side effects reported after the first and sometimes second injections of this murine monoclonal antibody include a "flu-like" syndrome consisting of high fever, chills, headache, and gastrointestinal symptoms (vomiting and diarrhea); in severe cases, pulmonary edema has been documented within hours of treatment (Thistlethwaite, JR Jr. et al. (1988) "Complications and Monitoring of OKT3 Therapy," Am. J. Kidney Dis. 11:112-119).This syndrome is thought to reflect OKT3-mediated cross-linking of the TCR / CD3 complex on the surface of T cells and the resulting release of cytokines (e.g., tumor necrosis factor alpha (TNFα), interferon-γ, interleukins IL-2, IL-3, IL-4, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) (Masharani, U.B. et al. (2010) “Teplizumab Therapy For Type 1 Diabetes,” Expert Opin. Biol. Ther. 10(3):459-465; Abramowicz, D. et al. (1989) “Release Of Tumor Necrosis Factor, Interleukin-2, And Gamma-Interferon In Serum After Injection Of OKT3 Monoclonal Antibody In Kidney Transplant Recipients,” Transplantation 47:606-608; Ferran, C. et al. (1990) “Cytokine-Related Syndrome Following Injection Of Anti-CD3 Monoclonal Antibody: Further Evidence For Transient In Vivo T Cell Activation,” Eur. J. Immunol. 20:509-515; Hirsch, R. et al. (12989) “Effects Of In Vivo Administration Of Anti-CD3 Monoclonal Antibody On T Cell Function In Mice. II. In Vivo Activation Of T Cells,” J. Immunol. 142:737-743).The use of anti-CD3 antibodies is disclosed in U.S. Patent Nos. 7,883,703; 7,728,114; 7,635,472; 7,575,923; and 7,381,903, as well as U.S. Patent Publication Nos. 2010 / 0150918; 2010 / 0209437; 2010 / 0183554; 2010 / 0015142, 2008 / 0095766, 2007 / 0077246, and PCT Publication WO2008 / 119567. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 6,491,916 [Patent Document 2] U.S. Patent No. 5,585,097 [Patent Document 3] U.S. Patent No. 6,706,265 [Patent Document 4] U.S. Patent No. 7,883,703 [Patent Document 5] U.S. Patent No. 7,728,114 [Patent Document 6] U.S. Patent No. 7,635,472 [Patent Document 7] U.S. Patent No. 7,575,923 [Patent Document 8] U.S. Patent No. 7,381,903 [Patent Document 9] US Patent Application Publication No. 2010 / 0150918 [Patent Document 10] US Patent Application Publication No. 2010 / 0209437 [Patent Document 11] US Patent Application Publication No. 2010 / 0183554 [Patent Document 12] US Patent Application Publication No. 2010 / 0015142 [Patent Document 13] U.S. Patent and Trademark Publication No. 2008 / 0095766 [License 14] U.S. Patent and Trademark Publication No. 2007 / 0077246 [License 15] International Publication No. 2008 / 119567 [Non-licensed literature]

[0008] [Non-licensed Document 1] Portoles, P. et al. (2009) “The TCR / CD3 Complex: Opening the Gate to Successful Vaccination,” Current Pharmaceutical Design 15:3290-3300 [Non-licensed Document 2] Guy, CS et al. (2009) “Organization of Proximal Signal Initiation at the TCR: CD3 Complex,” Immunol Rev. 232(1):7-21 [Non-licensed Document 3] Call, ME et al. (2007) “Common Themes In The Assembly And Architecture Of Activating Immune Receptors,” Nat. Rev. Immunol. 7:841-850 [Non-licensed Document 4] Weiss, A. (1993) “T Cell Antigen Receptor Signal Transduction: A Tale Of Tails And Cytoplasmic Protein-Tyrosine Kinases,” Cell 73:209-212 [Non-licensed Document 5] van der Merwe, PA etc. (epub Dec. 3, 2010) “Mechanisms For T Cell Receptor Triggering,” Nat. Rev. Immunol. 11:47-55 [Non-patent document 6] Wucherpfennig, KW et al. (2010) “Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling,” Cold Spring Harb. Perspect. Biol. 2:a005140 [Non-Patent Document 7] Riha, P. et al. (2010) “CD28 Co-Signaling In The Adaptive Immune Response,” Self / Nonself 1(3):231-240 [Non-patent document 8] St. Clair EW (2009) “Novel Targeted Therapies for Autoimmunity,” Curr. Opin. Immunol. 21(6):648-657 [Non-Patent Document 9] Cosimi, AB et al. (1981) “Use Of Monoclonal Antibodies To T-Cell Subsets For Immunologic Monitoring And Treatment In Recipients Of Renal Allografts,” N. Engl. J. Med. 305:308-314 [Non-Patent Document 10] Kung, P. et al. (1979) Monoclonal antibodies defining distinctive human T cell surface antigens,” Science 206:347-349 [Non-Patent Document 11] Vigeral, P. et al. (1986) “Prophylactic Use Of OKT3 Monoclonal Antibody In Cadaver Kidney Recipients. Utilization Of OKT3 As The Sole Immunosuppressive Agent,” Transplantation 41:730-733 [Non-Patent Document 12] Midtvedt, K. et al. (2003) “Individualized T Cell Monitored Administration Of ATG Versus OKT3 In Steroid-Resistant Kidney Graft Rejection,” Clin. Transplant. 17(1):69-74 [Non-Patent Document 13] Gramatzki, M. et al. (1995) “Therapy With OKT3 Monoclonal Antibody In Refractory T Cell Acute Lymphoblastic Leukemia Induces Interleukin-2 Responsiveness,” Leukemia 9(3):382-390 [Non-Patent Document 14] Herold, KC et al. (2002) “Anti-CD3 Monoclonal Antibody In New-Onset Type 1 Diabetes Mellitus,” N. Engl. J. Med. 346:1692-1698 [Non-Patent Document 15] Cole, MS et al. (1997) “Human IgG2 Variants Of Chimeric Anti-CD3 Are Nonmitogenic to T cells,” J. Immunol. 159(7):3613-3621 [Non-Patent Document 16] Cole, MS et al. (1999) “Hum291, A Humanized Anti-CD3 Antibody, Is Immunosuppressive To T Cells While Exhibiting Reduced Mitogenicity in vitro,” Transplantation 68:563-571 [Non-Patent Document 17] Ludvigsson, J. (2009) “The Role of Immunomodulation Therapy in Autoimmune Diabetes,” J. Diabetes Sci. Technol. 3(2):320-330 [Non-Patent Document 18] Thistlethwaite, JR Jr. et al. (1988) "Complications and Monitoring of OKT3 Therapy," Am. J. Kidney Dis. 11:112-119 [Non-Patent Document 19] Masharani, UB et al. (2010) “Teplizumab Therapy For Type 1 Diabetes,” Expert Opin. Biol. Ther. 10(3):459-465 [Non-Patent Document 20] Abramowicz, D. et al. (1989) “Release Of Tumor Necrosis Factor, Interleukin-2, And Gamma-Interferon In Serum After Injection Of OKT3 Monoclonal Antibody In Kidney Transplant Recipients,” Transplantation 47:606-608 [Non-Patent Document 21] Ferran, C. et al. (1990) “Cytokine-Related Syndrome Following Injection Of Anti-CD3 Monoclonal Antibody: Further Evidence For Transient In Vivo T Cell Activation,” Eur. J. Immunol. 20:509-515 [Non-Patent Document 22] Hirsch, R. et al. (12989) “Effects Of In Vivo Administration Of Anti-CD3 Monoclonal Antibody On T Cell Function In Mice. II. In Vivo Activation Of T Cells,” J. Immunol. 142:737-743 Summary of the Invention [Means for solving the problem]

[0009] Brief summary of the invention The present invention relates to novel anti-CD3 antibodies (used as monoclonal antibodies or in other formats, such as bispecific or multispecific formats) and compositions comprising such antibodies or cells activated by such antibodies for use in treating CD3-related disorders, such as human cancer therapy. The present invention also includes compositions comprising one or more of these peptides / antibodies, or fragments thereof, and / or immune cells comprising and / or modified to be activated by one or more of these antibodies, or fragments thereof, for treating diseases or conditions, such as cancer. The antibodies of the present invention may similarly find use as the targeting arm of bispecific or multispecific formats.

[0010] In one aspect, the invention provides an anti-CD3 antibody or antibody fragment comprising a heavy chain CDR3 sequence (VH) comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59. Additionally or alternatively, the anti-CD3 antibody or antibody fragment of the invention may comprise a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117.

[0011] Additionally or alternatively, the present invention provides an anti-CD3 antibody or antibody fragment comprising a heavy chain CDR3 sequence (VH) comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59, and a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117.

[0012] Additionally or alternatively, an anti-CD3 antibody or antibody fragment of the invention may comprise a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175 and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175. Additionally or alternatively, an anti-CD3 antibody or antibody fragment of the invention may comprise a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233 and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

[0013] Additionally or alternatively, an anti-CD3 antibody or antibody fragment of the invention may comprise a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175 and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175, and a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233 and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

[0014] In certain aspects, the present invention provides methods for treating disorders such as cancer using one or more antibodies of the invention, as described above, by at least administering such antibodies to a subject, such as a human subject.

[0015] In certain embodiments, the present invention provides immunoconjugates and / or compositions comprising such immunoconjugates, wherein the immunoconjugate comprises an anti-CD3 antibody of the present invention conjugated to another therapeutic agent, such as an anti-cancer agent. The present invention further provides immunoconjugates comprising two or more different anti-CD3 antibodies or fragments thereof, wherein each different anti-CD3 antibody or fragment targets a different CD3 fragment or epitope.

[0016] Further aspects of the present invention pertain to nucleic acid molecules having a nucleotide sequence encoding an anti-CD3 antibody or fragment thereof, as disclosed herein, as well as expression vectors comprising such polynucleotides, and host cells transfected with such expression vectors.

[0017] Aspects of the present invention also provide methods for making the anti-CD3 antibodies, fragments thereof, and compositions of the present invention.

[0018] The invention also provides methods for treating disease in a human or animal subject, particularly cancer in humans, by administering to said subject an anti-CD3 antibody or composition of the invention. The invention also includes the use of one or more anti-CD3 antibodies of the invention for the preparation of a medicament for use in treating disease in humans or animals, particularly cancer in humans.

[0019] In some embodiments, the antibody herein is a full-length antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the anti-CD3 antibody is an IgG antibody (e.g., an IgG1, IgG2, or IgG3 antibody).

[0020] In some embodiments, the antibody herein is an antibody fragment. In some embodiments, the antibody is an Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a Fab'-SH fragment, an scFv (sFv) fragment, or an scFv-Fc fragment. In some embodiments, the bispecific antibody is an scFv fragment. In some embodiments, the antibody herein is monoclonal, human, humanized, or chimeric.

[0021] In some embodiments, the antibody further comprises an Fc region. In some embodiments, the antibody comprises one or more heavy chain constant domains, wherein the one or more heavy chain constant domains are selected from a first CH1 domain, a first CH2 domain, a first CH3 domain, a second CH1 domain, a second CH2 domain, and a second CH3 domain. In some embodiments, the one or more heavy constant chain domains are paired with another heavy chain constant domain.

[0022] In some embodiments, the antibody further comprises a glycosylation site mutation. In some embodiments, the mutation reduces effector function. In some embodiments, the mutation is a substitution mutation. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention The T cell receptor (TCR) binds to antigens (Ag) presented by the major histocompatibility complex (MHC) and plays a critical role in T cell function. However, the TCR does not have its own intracellular signaling pathway. Instead, the TCR noncovalently associates with the cluster of differentiation 3 (CD3) complex and triggers intracellular signaling via the CD3 immunoreceptor tyrosine-based activation motif (ITAM). The CD3 T cell coreceptor serves to activate both cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It consists of a protein complex composed of four distinct chains. In mammals, the complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and the CD3ζ chain (zeta chain) to generate activation signals in T lymphocytes. The TCR, ζ chain, and CD3γ, δ, and ε chains together comprise the TCR complex. The CD3 four-chain complex then forms CD3εγ, CD3εδ, and δδ dimers with a 1:1:1 stoichiometry.

[0024] CD3 is first expressed in the cytoplasm of prothymocytes, the stem cells from which T cells arise in the thymus. Prothymocytes differentiate into common thymocytes and then medullary thymocytes, and at this latter stage the CD3 antigen begins to move to the cell membrane. The antigen is found membrane-associated on all mature T cells and is rarely found on other cell types, but appears to be present in small amounts on Purkinje cells.

[0025] This high specificity, coupled with the presence of CD3 at all stages of T cell development, makes CD3 a useful immunohistochemical marker for T cells in tissue sections. The antigen remains present in nearly all T cell lymphomas and leukemias and can therefore be used to distinguish them from superficially similar B cell and myeloid neoplasms. Some antibodies against the CD3ε chain have been shown to activate the TCR-CD3 complex, likely by clustering the CD3 complex on T cells. In addition, bispecific antibodies targeting both CD3 and tumor-specific antigens are being investigated for redirected tumor eradication by T cells. Because CD3 is required for T cell activation, drugs (often monoclonal antibodies) targeting it are being investigated as immunosuppressive therapies (e.g., otelixizumab) for type 1 diabetes and other autoimmune diseases.

[0026] The present invention is directed to novel peptides (e.g., antibodies and antibody fragments) that bind to CD3. The present invention also includes compositions comprising one or more of these peptides / antibodies, or fragments thereof, and / or immune cells that contain and / or have been modified to be activated by one or more of these antibodies, or fragments thereof, for treating diseases or conditions such as cancer.

[0027] The antibodies disclosed herein can provide a much more effective treatment than current CD3 treatments. The anti-CD3 antibodies disclosed herein of the invention can be included as part of a treatment regimen that can include, for example, providing two or more such antibodies and / or in combination with other treatments, such as chemotherapy. definition

[0028] The term "antibody" or "antibody molecule" refers to a functional component of serum and is often referred to as a group of molecules (antibodies or immunoglobulins) or as a single molecule (antibody molecule or immunoglobulin molecule). Antibodies are capable of binding to or reacting with a specific antigenic determinant (antigen or antigenic epitope), which in turn can result in the induction of immunological effector mechanisms. Antibodies are generally considered to be monospecific, and compositions of antibodies may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., multiple distinct antibodies capable of reacting with the same or different epitopes on the same antigen or on separate / different antigens). Antibodies have a unique structure that enables them to specifically bind to their corresponding antigens, and all naturally occurring antibodies have the same overall basic structure of two identical light chains and two identical heavy chains.

[0029] As used herein, "antibody" or "antibodies" can include chimeric and single chain antibodies, binding fragments of antibodies such as Fab, Fv fragments, or single-chain Fv (scFv) fragments, and multimeric forms such as dimeric IgA molecules or pentavalent IgM. Antibodies of the invention can be of human or non-human origin, e.g., antibodies derived from mice or other rodents, or chimeric, humanized, or reshaped antibodies based on, e.g., mouse antibodies.

[0030] The heavy chain of an antibody typically comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region usually comprises three domains designated CH1, CH2, and CH3. The light chain of an antibody comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises a single domain designated CL. The VH and VL regions are subdivided into regions of hypervariability ("hypervariable regions") that may be hypervariable in sequence and / or in loop structure. These regions are also referred to as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Amino acid residues in variable regions are often numbered using a standard numbering method known as the Kabat numbering scheme (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., USA).

[0031] Antibody identifiers found in the tables of this application, for example, "70701_06A03A," refer to specific antibodies.

[0032] As used herein, an antibody or a fragment "derived from" or "based on" an antibody means that the "derived" antibody comprises, depending on the particular context, one of the following: the heavy chain CDR3 sequence of said specified antibody; the heavy chain CDR3 sequence and the light chain CDR3 sequence of said specified antibody; the heavy chain CDR1, CDR2, and CDR3 sequence and the light chain CDR1, CDR2, and CDR3 sequence of said specified antibody; or the heavy chain and light chain variable region sequences of said specified antibody, or humanized variants of said heavy chain variable region sequence and / or light chain variable region sequence, or heavy and / or light chain variable region sequences having at least 80%, 85%, 90%, or 95% sequence identity, for example at least 96%, 97%, 98%, or 99% sequence identity, to the heavy and light chain variable region sequences, respectively.

[0033] Antibodies derived from or based on the identified antibodies described herein generally bind to the same CD3 epitope as the identified antibody and preferably exhibit substantially the same activity as the identified antibody.

[0034] The specificity of antibody interaction with target antigens is primarily driven by amino acid residues located in the six CDRs of the heavy and light chains. The amino acid sequences within the CDRs are more variable between individual antibodies than the sequences outside the CDRs. Because the CDR sequences are responsible for most of the antibody-antigen interaction, antibodies that mimic the characteristics of a particular naturally occurring antibody, or any specific antibody with a given amino acid sequence, can be expressed by constructing an expression vector that expresses the CDR sequences from a particular antibody grafted onto framework sequences from a different antibody. This allows for the "humanization" of non-human antibodies, which still substantially maintain the binding specificity and affinity of the original antibody. Nevertheless, in a preferred embodiment, the anti-CD3 antibody is a human antibody.

[0035] "Chimeric antibody" refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more different antibodies. "Chimeric antibodies" are typically antibodies that are partially human and partially non-human in origin. Chimeric antibodies may be preferred over non-human antibodies because they have been shown to reduce the risk of human anti-antibody responses. Chimeric antibodies may include antibodies in which the variable region sequences are mouse sequences derived from mouse immunization, while the constant region sequences are human. In the case of chimeric antibodies, the non-human portions, including the framework regions of the variable region sequences, may often be further modified to humanize the antibody.

[0036] In a preferred embodiment, the antibodies disclosed herein of the present invention are derived from transgenic mice containing human antibody gene segments, such that the antibodies are human antibodies produced by hybridoma technology from the transgenic mice.

[0037] The terms "heavy chain variable region sequence" and "light chain variable region sequence," and similar terms, when used herein with reference to any particular amino acid sequence, encompass not only that particular sequence but also any recombinant antibody, human antibody, including those derived from transgenic mice containing human antibody gene segments, and thus the antibody is a human antibody produced by hybridoma technology, and / or a humanized variant thereof.

[0038] As used herein, a reference to a heavy or light chain variable region sequence that has a certain minimum level of sequence identity compared to a specified heavy or light chain variable region sequence.

[0039] A "recombinant antibody" is an antibody that is expressed from a cell or cell line that has been transfected with an expression vector (or optionally more than one expression vector, typically two expression vectors) that contains an antibody coding sequence that is not naturally associated with the cell.

[0040] A "vector" is a nucleic acid molecule into which a nucleic acid sequence can be inserted for transport between different genetic environments and / or expression in a host cell. A vector carrying regulatory elements (at least a suitable promoter) for transcription of a nucleic acid sequence is called an "expression vector." The terms "plasmid" and "vector" can be used interchangeably. The expression vector used in the context of the present invention can be of any suitable type known in the art, such as a viral vector or a plasmid.

[0041] It is well known in the art that antibodies exist as different isotypes, such as the human isotypes IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, or the mouse isotypes IgG1, IgG2a, IgG2b, IgG3, and IgA. The antibodies of the present invention may be of either isotype.

[0042] In certain embodiments, the compositions of the invention include antibody compositions comprising multiple individual anti-CD3 antibodies, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more different CD3 antibodies.

[0043] "CDR" or "complementarity determining region" refers to the "hypervariable" region found in the variable domain of an antibody that is primarily responsible for determining the binding specificity of the antibody. Each of the heavy and light chains of an antibody contains three CDR regions, designated CDR1, CDR2, and CDR3, of which CDR3 shows the most variability.

[0044] "Epitope" refers to a portion of a larger molecule (e.g., an antigen or antigenic site) that has antigenic or immunogenic activity in an animal. An epitope with immunogenic activity is a portion of a larger molecule that elicits an antibody response. An epitope with antigenic activity is a portion of a larger molecule to which an antibody immunospecifically binds. An antigenic epitope is not necessarily immunogenic. An antigen is a substance, such as a toxin, virus, bacterium, protein, or DNA, to which an antibody or antibody fragment specifically binds. An antigen or antigenic site may have more than one epitope and may be capable of stimulating an immune response.

[0045] Epitopes can be linear or conformational. Linear epitopes generally consist of approximately 6-10 contiguous amino acids on a protein molecule that are recognized by antibodies. In contrast, conformational epitopes consist of amino acids that are not arranged contiguously, but antibodies recognize specific three-dimensional structures. When a protein molecule folds into a three-dimensional structure, the amino acids that form the epitope are juxtaposed, allowing antibodies to recognize conformational epitopes. In denatured proteins, only linear epitopes are recognized. Conformational epitopes, by definition, must reside on the outside of the folded protein.

[0046] The term "distinct epitopes" refers to the fact that when two different antibodies of the invention bind to distinct epitopes, there is less than 100% competition for antigen binding, preferably less than 80% competition for antigen binding, more preferably less than 50% competition for antigen binding, and most preferably as low competition as possible, e.g., less than about 25% competition for antigen binding.

[0047] Antibodies that may compete with each other for binding to the same antigen may bind to the same or overlapping epitopes, or may have binding sites that are close to each other, so competition is mainly caused by steric hindrance.The analysis of the "distinct epitopes" of antibody pairs can be carried out by methods known in the art, for example, by binding experiments under saturating antibody conditions using FACS (fluorescence-activated cell sorting) or other flow cytometry analysis on cells that express CD3 and individual fluorescently labeled antibodies, or by surface plasmon resonance (SPR) using CD3 antigen bound to the surface of a flow cell.

[0048] Antibodies that bind to different epitopes on the same antigen can have different effects on the activity of the antigen they bind to, depending on the location of the epitope.An antibody that binds to an epitope in the active site of an antigen can completely block the function of the antigen, while another antibody that binds to a different epitope can have no or little effect on the activity of the antigen alone.However, such an antibody can still activate complement, thereby causing the elimination of the antigen, and when combined with one or more antibodies that bind to different epitopes on the same antigen, it can produce a synergistic effect.

[0049] "Immunoglobulin" is a general term for the mixture of antibodies found in blood or serum, but is also sometimes used to designate mixtures of antibodies from other sources.

[0050] "Homogenous V H and V L The term "coding pair" refers to Vs contained within or derived from the same antibody-producing cell. H and V L represents the original pair of coding sequences. Therefore, the cognate V H and V L The pair is the V originally present in the donor from which such cells are derived. H and V L represents the involution of "V H and V LThe term "antibody" refers to an antibody or antibody fragment expressed from a coding pair, such as a V H and V L It indicates that the antibody is produced from a vector, plasmid, or other polynucleotide containing the coding sequence. H and V L When the coding pairs are expressed, they retain the binding affinity and specificity of the antibody originally expressed from the cell from which they were derived. Libraries of cognate pairs are also called repertoires or collections of cognate pairs, and may be maintained individually or pooled.

[0051] By "protein" or "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification. Proteins can exist as monomers or multimers, including two or more assembled polypeptide chains, fragments of proteins, polypeptides, oligopeptides, or peptides.

[0052] The term "head-to-head promoter" refers to a pair of promoters positioned in close proximity so that transcription of the two gene segments driven by the promoters occurs in opposite directions. Head-to-head promoters are also known as bidirectional promoters.

[0053] The term "transfection" is used herein broadly as a term for introducing foreign DNA into a cell. The term is also intended to include other functionally equivalent methods for introducing foreign DNA into a cell, such as, for example, transformation, infection, transduction, or fusion of a donor cell with an acceptor cell.

[0054] As used herein, CD3 is intended to include variants, isoforms, and species homologs of CD3. Preferably, binding of the antibodies of the present invention to CD3 inhibits the growth of cells expressing CD3. In certain embodiments, this inhabitation is caused by inhibiting the formation of heterologous complexes between CD3 and other ErbB family members.

[0055] As used herein, the term "inhibit growth" (e.g., when referring to cells) is intended to include any measurable decrease in proliferation (increase in cell number) or metabolism of cells when contacted with an anti-CD3 antibody compared to the growth of the same cells in the absence of the anti-CD3 antibody, e.g., an inhibition of growth of a cell culture by at least about 10%, more preferably, e.g., at least about 20% or 30%, more preferably at least about 40% or 50%, e.g., at least about 60%, 70%, 80%, 90%, 99%, or even 100%.

[0056] As used herein, the term "treatment" refers to the administration of an anti-CD3 antibody, antibody composition, or composition of immune cells expressing or activated by a CD3 antibody or fragment thereof of the invention in an amount sufficient to alleviate, relieve, ameliorate, or eradicate (cure) the symptoms or disease state.

[0057] The percent identity between two sequences, for example, variable region sequences, refers to the number of identical positions shared by the sequences (calculated as the number of identical positions / total number of positions x 100), taking into account gaps that must be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be achieved using readily available software. Suitable software programs are available from various sources, both for online use and download, and for alignment of both protein and nucleotide sequences. One suitable program is ClustalW (Thompson et al. (1994) Nucleic Acids Res. 11; 22(22):4673-80), available at www.clustal.org.

[0058] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0059] "Affinity" refers to the strength of the sum total 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 (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0060] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions compared to a parent antibody that does not possess such alterations, which alterations result in an improvement in the affinity of the antibody for antigen.

[0061] The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody that is capable of binding to CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD3. In one embodiment, the extent of binding of the anti-CD3 antibody to an unrelated, non-CD3 protein is less than about 10% of the binding of the antibody to CD3, as measured, for example, by radioimmunoassay (RIA).

[0062] In certain embodiments, an antibody that binds to CD3 has a concentration of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -6 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13The anti-CD3 antibodies have a dissociation constant (Kd) of 100-150 nM (Kd = 0.01 M). In preferred embodiments, this affinity range represents the "optimal affinity range" that retains anti-tumor activity but reduces toxicity due to reduced cytokine release. In certain preferred embodiments, the anti-CD3 antibodies have an affinity in the range of 30-40 nM, as measured by alanine scanning of the HC CDR3 of the antibody.

[0063] In certain embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species.

[0064] As used herein, the term "cluster of differentiation 3" or "CD3," unless otherwise indicated, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including, for example, the CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses "full-length," unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 obtained from intracellular processing. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein, which is 207 amino acids long (NCBI Reference SEQ ID NO: NP-000724), and the human CD3γ protein, which is 182 amino acids long (NCBI Reference SEQ ID NO: NP-000064).

[0065] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0066] "Effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on the 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.

[0067] An "effective amount" of a compound, e.g., an anti-CD3 antibody of the invention, or composition thereof (e.g., pharmaceutical composition), is at least the minimum amount necessary to achieve a desired therapeutic or prophylactic result, such as measurable improvement or prevention of a particular disorder (e.g., a cell proliferative disorder, e.g., cancer). The effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. In the case of prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes manifest during disease development. In the case of therapeutic use, beneficial or desired results include clinical results such as a reduction in one or more symptoms caused by the disease, an improvement in the quality of life of those suffering from the disease, a reduction in the dose of other medications required to treat the disease, an enhancement of the effect of another medication by targeting or the like, a delay in disease progression, and / or an increase in survival time. In the case of cancer or tumors, an effective amount of a drug may be effective in reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., delaying to some extent or desirably stopping) cancer cell invasion into peripheral organs; inhibiting (i.e., delaying to some extent and desirably stopping) tumor metastasis; inhibiting to some extent tumor growth; and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in relation to administering one or more therapeutic agents, and in combination with one or more other agents, where a desired result may be or is achieved, a single agent may be considered to be administered in an effective amount.

[0068] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0069] "Framework" or "FR" refers to variable domain residues other than the hypervariable region residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the VH (or VL) in the following order: FR1-H1(L)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0070] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure, or an antibody having a heavy chain containing an Fc region as defined herein.

[0071] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or to the amino acid sequence of an antibody derived from a non-human source utilizing the human antibody repertoire, or to the sequence encoding another human antibody. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Human monoclonal antibodies can also be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomice, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies generated by human B-cell hybridoma technology.

[0072] For example, in preferred embodiments, the anti-CD3 antibodies described herein are generated using technology such as AlivaMab mouse technology. Generally, AlivaMab mouse technology is used to generate a panel of monoclonal antibodies (mAbs) directed against a human antigen of interest, such as a human antigen expressed by or associated with tumor cells, e.g., a variant of HER2.

[0073] AlivaMab mice are transgenic mice that produce chimeric human-mouse monoclonal antibodies containing fully human Fab and upper hinge regions and mouse middle hinge and Fc regions. Optimized constant domains facilitate the generation and identification of antibodies that retain structure-function characteristics. Antibodies generated using AlivaMab mouse technology have predictable and biophysical properties comparable to those of their fully human antibody counterparts.

[0074] The antibodies produced by AlivaMab Kappa mice contain a chimeric immunoglobulin heavy (IgH) chain and a human immunoglobulin kappa (IgK) light chain. The antibodies produced by AlivaMab Lambda mice contain a chimeric IgH chain and a human immunoglobulin lambda (IgK) light chain. The chimeric IgH chain of the AlivaMab mouse antibody contains a human variable region including a human variable heavy (VH) domain, a human diversity heavy (DH) domain, and a human joining heavy (JH) domain, a human constant heavy 1 (CHI) domain, a human upper hinge region (except for Oμ, which naturally lacks an upper hinge region), a mouse middle hinge region, a mouse CH2 domain, and a mouse CH3 domain.

[0075] Once a lead candidate antibody is identified, the human heavy chain variable region is readily added to a fully human constant region while maintaining the antigen-binding characteristics of the parent chimeric antibody developed in vivo in the AlivaMab mouse. In one embodiment, the human heavy chain variable region, CHI, and optionally the upper hinge region of the chimeric antibody are added to a human hinge, a human CH2 domain, and a human CH3 domain to create a fully human antibody.

[0076] Portions of the variable regions derived from antibodies generated using AlivaMab mouse technology may contain all or a combination of VH and / or VL complementarity-determining regions (CDRs). The variable regions may be configured in a standard antibody structure (two heavy chains and two light chains) with either native constant regions or constant regions engineered for various desired effector functions. The variable regions may also be configured as multispecific antibodies, e.g., bispecific antibodies that bind to two different epitopes or two different antigens. The variable regions may also be configured as antibody fragments, e.g., single-domain antibodies containing a single VH or VL, Fab, or Fab'2. Antibodies may also be used as antibody-drug conjugates or may carry other appendages, such as small molecule toxins, biological toxins, cytokines, oligopeptides, or RNA, to improve therapeutic modalities and / or safety.

[0077] Methods for producing anti-CDR3 antibodies of the present invention using AlivaMab mouse technology can include immunizing AlivaMab kappa and AlivaMab lambda mice with an antigen of interest. Generally, mice are sacrificed within two weeks and terminal material is harvested. Spleens and lymph nodes can be prepared and fused with myeloma cells (such as CRL-2016 cells) to establish hybridomas, generally using PEG-based methods as described in "Antibodies: A Laboratory Manual" (Harlow and Lane, 1988, CSH Press).

[0078] Hybridomas can be grown in 384-well tissue culture plates, and supernatants from individual wells are screened by ELISA for the production of antibodies that recognize the antigen of interest. Positive wells are then transferred and expanded to 48-well plates, and supernatants are collected for antigen binding confirmation by ELISA. Positive supernatants can also be counterscreened against unrelated histidine-tagged proteins. Hybridoma lines derived from AlivaMab kappa mice and AlivaMab lambda mice are confirmed to specifically bind to the antigen by ELISA, randomly picked, and single-cell cloned into 96-well plates. They are grown into colonies, and supernatants from these individual colonies are screened by ELISA to reconfirm the presence of monoclonal antibodies that bind to the antigen of interest. These supernatants are then screened by FACS to confirm binding to the native antigen expressed on the cells.

[0079] AlivaMab mouse technology and methods for generating antibodies using such technology can be found in WO2010 / 039900 and WO2011 / 123708, which are incorporated herein in their entireties.

[0080] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, Md. (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0081] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to HVRs of a non-human antibody and all or substantially all of the FRs correspond to FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0082] "Humanizing" an antibody means that an antibody of fully or partially non-human origin, such as a murine antibody obtained by immunizing a mouse with an antigen of interest, or a chimeric antibody based on such a murine antibody, can avoid or minimize an immune response in humans by replacing amino acids, particularly in the framework regions and constant domains of the heavy and light chains. All antibodies are known to have the potential to elicit a human anti-antibody response, which to some extent correlates with the degree of "humanity" of the antibody.

[0083] Non-human antibodies tend to be more immunogenic than human antibodies. Chimeric antibodies, in which foreign (usually rodent) constant regions are replaced with sequences of human origin, have been shown to be less immunogenic than antibodies of completely foreign origin, and most therapeutic antibody development efforts tend toward the use of humanized or fully human antibodies. Preferably, chimeric antibodies or other antibodies of non-human origin are humanized to reduce the risk of human anti-antibody responses. In the case of chimeric antibodies, humanization may include, for example, modification of the framework regions of the variable region sequences. While amino acid residues in CDRs often cannot be changed during humanization, in certain cases, it may be desirable to modify individual CDR amino acid residues, for example, to remove glycosylation sites, deamidation sites, or undesired cysteine ​​residues.

[0084] Numerous methods for humanizing antibody sequences are known in the art. A commonly used method is CDR grafting, which may involve identifying human germline gene counterparts for mouse variable region genes and grafting mouse CDR sequences onto this framework. Because CDR grafting reduces the binding specificity and affinity, as well as the likelihood of biological activity, of the CDR-grafted non-human antibody, back mutations are often introduced at selected positions of the CDR-grafted antibody to preserve binding specificity and affinity. Amino acid residues for back mutations may include amino acid residues located on the surface of the antibody molecule. Another humanization technique for CDR grafting and back mutation is resurfacing, in which surface residues are changed to human variants while non-surface-exposed residues of non-human origin are retained.

[0085] Humanized antibodies and methods for making them are reviewed, e.g., by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0086] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (2008)). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0087] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity-determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact antigen ("antigen contacts"). Generally, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0088] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0089] A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.

[0090] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0091] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0092] An "isolated nucleic acid encoding an anti-CD3 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), and includes such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present in one or more locations within a host cell.

[0093] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or that arise during the production of the monoclonal antibody preparation; such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0094] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (K) or lambda (A).

[0095] The term "package insert" is used to refer to instructions customarily included in commercial packaging for a therapeutic product, including information regarding the indications, uses, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0096] As used herein, the term "protein," unless otherwise indicated, refers to any naturally occurring protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed proteins and any form of protein resulting from processing within a cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0097] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain from an antibody that binds the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0098] As used herein, "administering" refers to a method of providing a subject with a dosage of a compound (e.g., an anti-CD3 antibody of the invention or a nucleic acid encoding an anti-CD3 antibody of the invention) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an anti-CD3 antibody of the invention). Compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, transcutaneously, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion to directly bathe target cells, by catheter, by lavage, as a cream, or as a lipid composition. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0099] As used herein, "somatic hypermutation" or "SHM" ​​refers to mutations in a polynucleotide sequence initiated by or associated with the action of activation-induced cytidine deaminase (AID), functional AID mutants, uracil glycosylase, and / or error-prone polymerases on a polynucleotide sequence. As used herein, the term includes mutagenesis that occurs as a result of error-prone repair, including mutagenesis mediated by mismatch repair mechanisms and related enzymes.

[0100] SHM is typically initiated by targeting AID to the rearranged V(D)J and switch regions of Ig genes. The mutation rate of this programmed mutagenesis is one million-fold higher than that of AID-untargeted genomes in B cells. AID is a processive enzyme that binds to single-stranded DNA and deaminates cytosines in the DNA. Deamination of cytosine generates the highly mutagenic deoxyuracil (U) in the DNA of Ig loci. Mutagenic processing of uracil via the DNA damage response generates the full spectrum of base substitutions that characterize SHM at and around the initial U lesion. At least five identified mutagenic DNA damage response pathways are known to generate a well-defined spectrum of SHM around this initial lesion: C / G transitions, C / G transversions, and A / T mutations. These pathways include (1) replication opposite the template U, resulting in a C / G transition; (2) UNG2-dependent translesion synthesis (TLS), resulting in a C / G transversion; (3) a hybrid pathway involving noncanonical mismatch repair (ncMMR) and UNG2-dependent TLS, resulting in a C / G transversion; (4) ncMMR, resulting in an A / T mutation; and (5) UNG2- and PCNA ubiquitination (PCNA-Ub)-dependent mutation at A / T. Specific strand bias in the SHM spectrum arises as a result of biased AID targeting, ncMMR, and antimutagenic repriming. By elucidating the amino acid and / or nucleotide sequences of one of the CDR3 variable regions and / or the CDR3 heavy chain (HC) and light chain (LC or λ) variable regions of the anti-CD3 antibodies disclosed herein, the inventors identified a series of "clusters" or "motifs" within these sequences. These clusters represent convergent somatic hypermutation (SHM) in variable region sequences. Clustering can provide insight into functionally related sequences and the diversity of the entire population of antibodies and their variable region sequences. Sequences that descend from the same parent B cell or that have convergently evolved within the same cluster should be more closely related functionally than sequences belonging to other clusters.Convergent SHM can be functionally related mutations, e.g., they share a specific affinity for CD3. These SHM can inform the development of recombinant anti-CD3 antibodies with improved properties, such as specific binding to CD3 fragments.

[0101] Selected Embodiments One aspect of the present invention relates to various novel anti-CD3 antibodies and fragments thereof.

[0102] The antibodies disclosed herein can provide a much more effective treatment than current therapies. The CD3 antibodies disclosed herein of the present invention can be included as part of a treatment regimen, which can include, for example, providing two or more such antibodies and / or in combination with other treatments, such as chemotherapy.

[0103] In one embodiment, the present invention relates to novel CD3 antigen-binding peptides, which may be antibodies and / or fragments thereof. In certain embodiments, the antibodies and / or fragments thereof bind to a CD3 fragment having the amino acid sequence of SEQ ID NO: 1 and / or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In certain embodiments, the antibodies and / or fragments thereof bind to a CD3 fragment having the amino acid sequence of SEQ ID NO: 234 and / or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 234. In certain embodiments, the antibodies and / or fragments thereof bind to a CD3 fragment having the amino acid sequence of SEQ ID NO: 235 and / or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 235.

[0104] In certain embodiments, the present invention provides anti-CD3 antibodies or antibody fragments comprising a heavy chain CDR3 sequence (VH) comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59. Additionally or alternatively, the anti-CD3 antibodies or antibody fragments of the present invention may comprise a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117.

[0105] Additionally or alternatively, the present invention provides an anti-CD3 antibody or antibody fragment comprising a heavy chain CDR3 sequence (VH) comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59, and a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117.

[0106] Additionally or alternatively, an anti-CD3 antibody or antibody fragment of the invention may comprise a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175 and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175. Additionally or alternatively, an anti-CD3 antibody or antibody fragment of the invention may comprise a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233 and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

[0107] Additionally or alternatively, an anti-CD3 antibody or antibody fragment of the invention may comprise a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175 and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175, and a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233 and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

[0108] Additionally or alternatively, an anti-CD3 antibody or antibody of the present invention may be selected from the clusters listed in Tables 3-4: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; Clust 3.10; Clust 3.11; Clust 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; Clust 4.2; Clust 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 5.8; Clust 5.9; Clust 6.1; Clust 6.10; Clust 6.11; Clust 6.12; Clust 6.13; Clust 6.14; Clust 6.15; Clust 6.16; Clust 6.17; Clust 6.2; Clust 6.3; Clust 6.4; Clust 6.5; Clust 6.6; Clust 6.7; Clust 6.8; Clust 6.9; Clust 7.1; Clust 7.2; Clust 8.1 and / or Clust 8.2, and including heavy chain variable region and / or light chain variable region sequences containing somatic hypermutation (SMH).

[0109] In certain embodiments, the present invention provides compositions, including therapeutic compositions, comprising an anti-CD3 antibody or antibody fragment described herein. In certain embodiments, the present invention provides therapeutic compositions, compositions, comprising two or more of the CD3 antibodies disclosed herein. Certain compositions of the present invention comprise multiple different CD3 antibodies disclosed herein, each of the different antibodies binding to a distinct CD3 epitope or fragment.

[0110] In certain embodiments, the present invention provides methods for treating breast cancer using compositions comprising one or more CD3 antibodies described herein. In certain embodiments, administration of such compositions results in reduced CD3 and / or HER2 expression, internalization of CD3 and / or HER2 receptors, and / or ligand-induced HER3 phosphorylation.

[0111] In certain embodiments, the present invention provides immunoconjugates and / or compositions comprising such immunoconjugates, wherein the immunoconjugate comprises a CD3 antibody of the present invention conjugated to another therapeutic agent, such as an anti-cancer agent. The present invention further provides immunoconjugates comprising two or more different CD3 antibodies or fragments thereof, wherein each different CD3 antibody or fragment targets a different CD3 fragment or epitope.

[0112] Further aspects of the present invention pertain to nucleic acid molecules having nucleotide sequences encoding the CD3 antibodies or fragments thereof disclosed herein, as well as expression vectors comprising such polynucleotides, and host cells transfected with such expression vectors.

[0113] Embodiments of the invention also provide methods for producing the CD3 antibodies, fragments thereof, and compositions of the invention.

[0114] The invention also provides methods for treating disease in a human or animal subject, particularly treating cancer in humans, by administering to said subject an anti-CD3 antibody or composition of the invention. The invention also includes the use of one or more anti-CD3 antibodies of the invention for the preparation of a medicament for use in treating disease in humans or animals, particularly treating cancer in humans.

[0115] Another embodiment of this aspect of the invention relates to an antibody composition comprising at least a first and a second anti-CD3 antibody, wherein the first and second antibodies bind to distinct epitopes of CD3, and wherein the first and second antibodies independently comprise a heavy chain CDR3 sequence (VH) comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59 and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59. Additionally or alternatively, the first and second anti-CD3 antibodies comprise a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117 and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117.

[0116] Another aspect of the present invention relates to a nucleic acid molecule comprising a sequence having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 2-233, and / or a nucleotide sequence encoding the antibody, VL variable region sequence, VH variable region sequence, VL CDR3 sequence, and / or VH CDR3 sequence described herein.

[0117] A further aspect of the present invention relates to an expression vector comprising the nucleic acid molecule as defined above. As described above, expression vectors for use in the context of the present invention may be of any suitable type known in the art, such as a plasmid or a viral vector.

[0118] A still further aspect of the present invention relates to a host cell comprising a nucleic acid molecule as defined above, said host cell being capable of expressing an anti-CD3 antibody encoded by said nucleic acid molecule.

[0119] In some embodiments, the antibodies provided herein have a cytotoxicity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -6 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 The anti-CD3 antibodies have a dissociation constant (Kd) of 100-150 nM (Kd = 0.01 M). In preferred embodiments, this affinity range represents the "optimal affinity range" that retains anti-tumor activity but reduces toxicity due to reduced cytokine release. In certain preferred embodiments, the anti-CD3 antibodies have an affinity in the range of 30-40 nM, as measured by alanine scanning of the HC CDR3 of the antibody.

[0120] In some embodiments, Kd is measured by radiolabeled antigen binding assay (RIA). In some embodiments, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of a Fab to an antigen is measured by equilibrating the Fab with a minimum concentration of (I)-labeled antigen in the presence of a titration series of unlabeled antigen, and then capturing the bound antigen with a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).

[0121] In some embodiments, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using an immobilized antigen CM5 chip of approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 g / ml (approximately 0.2 M) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to reach approximately 10 response units (RU) of binding protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at a flow rate of approximately 25 μl / min at 25°C. Association rates (k) and dissociation rates (k) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K) is calculated as the ratio k / k. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).If the on-rate by the surface plasmon resonance assay described above exceeds 10 M s, the on-rate may be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations as measured in a spectrometer such as a spectrophotometer equipped with stop-flow (Aviv Instruments) or an 8000 Series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0122] In a preferred embodiment, the modality for estimating affinity is the generation of monovalent anti-CD3 antibodies, followed by titration on live CD3-expressing cells and determination of the MFI by flow cytometry to determine the EC50 value. Advantageously, this can represent the exact context (monovalent and CD3 on cells) in which the anti-CD3 antibody-based therapy of the present invention will be used.

[0123] In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. 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, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0124] Diabodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific.See, for example, EP404,097; WO1993 / 01161; Hudson et al. Nat. Med. 9:129-134 (2003); and Hollinger et al. Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003).

[0125] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516 B1).

[0126] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage).

[0127] In some embodiments, the antibody provided herein is a chimeric antibody.Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984).In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region.In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody.A chimeric antibody includes its antigen-binding fragment.

[0128] In a preferred embodiment, the antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0129] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). (See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Patent Application Publication No. US2007 / 0061900, which describes VELOCIMOUSE® technology.) The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0130] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. LISA. 103; 3557-3562 (2006). Additional methods include those described in, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0131] For example, in preferred embodiments, the anti-CD3 antibodies described herein are generated using technology such as AlivaMab mouse technology, which is used to generate a panel of monoclonal antibodies (mAbs) against CD3.

[0132] AlivaMab mice are transgenic mice that produce chimeric human-mouse monoclonal antibodies containing fully human Fab and upper hinge regions and mouse middle hinge and Fc regions. Optimized constant domains facilitate the generation and identification of antibodies that retain structure-function characteristics. Antibodies of the present invention generated using AlivaMab mouse technology have predictable biophysical properties comparable to those of their fully human antibody counterparts.

[0133] The antibodies of the present invention can be produced by AlivaMab Kappa mice and can comprise a chimeric immunoglobulin heavy (IgH) chain and a human immunoglobulin kappa (IgK) light chain. The antibodies of the present invention produced by AlivaMab Lambda mice can comprise a chimeric IgH chain and a human immunoglobulin lambda (IgK) light chain. The chimeric IgH chain of the AlivaMab mouse anti-CD3 antibody can comprise a human variable region including a human variable heavy (VH) domain, a human diversity heavy (DH) domain, and a human joining heavy (JH) domain, a human constant heavy 1 (CHI) domain, a human upper hinge region (except for Oμ, which naturally lacks an upper hinge region), a mouse middle hinge region, a mouse CH2 domain, and a mouse CH3 domain.

[0134] Once an anti-CD3 antibody is discovered, a human heavy chain variable region is readily added to a fully human constant region while maintaining the antigen-binding properties of the parent chimeric antibody developed in vivo in the AlivaMab mouse. In one embodiment, the human heavy chain variable region, CHI, and optionally the upper hinge region of the chimeric antibody are added to a human hinge, a human CH2 domain, and a human CH3 domain to create the fully human anti-CD3 antibody disclosed herein.

[0135] Portions of the variable regions derived from antibodies generated using AlivaMab mouse technology may contain all or a combination of VH and / or VL complementarity-determining regions (CDRs). The variable regions may be configured in a standard antibody structure (two heavy chains and two light chains) with either native constant regions or constant regions engineered for various desired effector functions. The variable regions may also be configured as multispecific antibodies, e.g., bispecific antibodies that bind to two different epitopes or two different antigens. The variable regions may also be configured as antibody fragments, e.g., single-domain antibodies containing a single VH or VL, Fab, or Fab'2. Antibodies may also be used as antibody-drug conjugates or may carry other appendages, such as small molecule toxins, biological toxins, cytokines, oligopeptides, or RNA, to improve therapeutic modalities and / or safety.

[0136] Methods for producing anti-CDR3 antibodies of the present invention using AlivaMab mouse technology can include immunizing AlivaMab kappa and AlivaMab lambda mice with an antigen. Generally, within two weeks, the mice are sacrificed and terminal material is harvested. Spleens and lymph nodes can be prepared and fused with myeloma cells (such as CRL-2016 cells) to establish hybridomas, generally using PEG-based methods as described in "Antibodies: A Laboratory Manual" (Harlow and Lane, 1988, CSH Press).

[0137] Hybridomas can be grown in 384-well tissue culture plates, and supernatants from individual wells are screened by ELISA for the production of antibodies that recognize the antigen of interest. Positive wells are then transferred and expanded to 48-well plates, and supernatants are collected for antigen binding confirmation by ELISA. Positive supernatants can also be counterscreened against unrelated histidine-tagged proteins. Hybridoma lines derived from AlivaMab kappa mice and AlivaMab lambda mice are confirmed to specifically bind to the antigen by ELISA, randomly picked, and single-cell cloned into 96-well plates. They are grown into colonies, and supernatants from these individual colonies are screened by ELISA to reconfirm the presence of monoclonal antibodies that bind to the antigen of interest. These supernatants are then screened by FACS to confirm binding to the native antigen expressed on the cells.

[0138] AlivaMab mouse technology and methods for generating antibodies using such technology can be found in WO2010 / 039900 and WO2011 / 123708, which are incorporated herein in their entireties.

[0139] In one particular method for designing and / or generating anti-CD3 antibodies of the present invention, for example, by elucidating the amino acid and / or nucleotide sequence of one of the CDR3 variable regions and / or CDR3 heavy chain (HC) and light chain (LC or λ) variable regions of an anti-CD3 antibody generated using AlivaMab mouse technology, a series of "clusters" or "motifs" are identified within the sequence. These clusters represent convergent somatic hypermutation (SHM) in the variable region sequences. Clustering can provide functionally related sequences and insight into the diversity of the entire population of antibodies and their variable region sequences. Sequences that descend from the same parent B cell or that have convergently evolved within the same cluster should be more functionally related than sequences belonging to other clusters. Convergent SHM can be functionally related mutations, e.g., they share a specific affinity for CD3. These SHM can inform the development of recombinant anti-CD3 antibodies with improved properties, such as specific binding to CD3 fragments.

[0140] Specific SHMs in the variable regions of the anti-CD3 antibodies of the antibodies disclosed herein are found in Tables 1-4, with the amino acids representing the SHMs shown in bold.

[0141] Human antibodies can also be produced by isolating the variable domain sequences of Fv clones selected from human-derived phage display libraries, which can then be combined with the desired human constant domains.

[0142] Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0143] In one particular phage display method, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self-antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, encoding highly variable CDR3 regions, and using PCR primers containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0144] Alternatively, in certain aspects, the invention includes humanized variants of the antibodies described herein, or humanized antibodies comprising one or more of SEQ ID NOs: 2-233, or fragments thereof. Methods for humanizing antibodies are well known in the art.

[0145] In certain embodiments, the anti-CD3 antibodies of the invention are, or form part of, multispecific antibodies.

[0146] A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different sites. In some embodiments, a bispecific antibody can bind to two different epitopes of CD3 (e.g., CD3ε or CD3γ). In some embodiments, one of the binding specificities is for CD3 (e.g., CD3ε or CD3γ), and the other is for any other antigen (e.g., a second biological molecule, e.g., a cell surface antigen, e.g., a tumor antigen). Thus, a bispecific anti-CD3 antibody can have binding specificities for CD3 and a second biological molecule, such as a second biological molecule (e.g., a tumor antigen).

[0147] Techniques for generating multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). "Knob-in-hole" engineering of multispecific antibodies can be used to generate a first arm containing a knob and a second arm containing a hole to which the knob of the first arm can bind. The knob of the multispecific antibody of the present invention can, in one embodiment, be an anti-CD3 arm. Alternatively, the knob of the multispecific antibody of the present invention can, in one embodiment, be an anti-target / antigen arm. The hole of the multispecific antibody of the present invention can, in one embodiment, be an anti-CD3 arm. Alternatively, the hole of the multispecific antibody of the present invention may in one embodiment be an anti-target / antigen arm.

[0148] Other means of generating multispecific antibodies exist. For example, multispecific antibodies can be engineered using immunoglobulin cross-linking (also known as Fab domain swapping or CrossMab format) technology (see, e.g., WO2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al., J. ImmunoL, 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. ImmunoL, 152:5368 (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. ImmunoL 147: 60 (1991).

[0149] In certain embodiments, amino acid sequence variants of the anti-CD3 antibodies of the invention (e.g., bispecific anti-CD3 antibodies of the invention that bind to CD3 and a second biological molecule, e.g., a cell surface antigen, e.g., a tumor antigen, e.g., a particular member and / or fragment of the epidermal growth factor receptor (HER / EGFR / ERBB) family, and / or a different CD3 epitope) are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be used to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0150] In certain aspects, the anti-CD3 antibodies of the present invention are multispecific antibodies. In certain aspects, the multispecific antibodies are bispecific, trispecific, and / or more multispecific antibodies that exhibit specificity for CD3 and for another molecule and / or another epitope on CD3. For example, such antibodies can bind to both CD3 and an antigen important for targeting the antibody to a particular cell type or tissue (e.g., an antigen associated with the cancer antigen of the tumor being treated). In some embodiments, the multispecific antibodies of the present invention bind to a molecule (receptor or ligand) involved in an immunoregulatory pathway, such as CTLA4, TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, CD27 / CD70, ICOS, B7-H4, LIGHT, PD-1, or LAG3, thereby controlling or modulating the therapeutic effect of the multispecific antibody. Furthermore, multispecific antibodies can bind to effector molecules such as cytokines (e.g., IL-7, IL-15, IL-12, IL-4 TGF-beta, IL-10, IL-17, IFNg, Flt3, BLys) and / or chemokines (e.g., CCL21). Methods for producing bispecific antibodies are known in the art.

[0151] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table A under the heading of "Preferred Substitutions." More substantial changes are provided in Table A under the heading of "Exemplary Substitutions," and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0152] [Table A-1] [Table A-2] Amino acids can be classified according to common side chain properties: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0153] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing have modified (e.g., improved) certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or substantially retain certain biological properties of the parent antibody. Exemplary substitutional variants are affinity-matured antibodies, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques. Briefly, one or more HVR residues are mutated, and variant antibodies displayed on phage are screened for a particular biological activity (e.g., binding affinity).

[0154] Alterations (e.g., substitutions) can be made in HVRs to, for example, improve antibody affinity. Such alterations may be made in HVR "hot spots," i.e., residues encoded by codons that frequently undergo mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or antigen-contacting residues, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, 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 scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0155] In certain embodiments, substitutions, insertions, or deletions may 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, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may, for example, be outside the antigen contact residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0156] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain desired properties.

[0157] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion of an enzyme or a polypeptide to the N- or C-terminus of the antibody to increase the serum half-life of the antibody.

[0158] In certain embodiments, anti-CD3 antibodies of the invention can be altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an anti-CD3 antibody of the invention can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0159] If an antibody contains an Fc region, the carbohydrate attached thereto may be altered. Typically, natural antibodies produced by mammalian cells contain branched, biantennary oligosaccharides that are usually N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications in the antibodies of the present invention may be performed to generate antibody variants with improved specific properties.

[0160] In one embodiment, an anti-CD3 antibody variant is provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the total amount of all glycan structures attached to Asn297 (e.g., complex structures, hybrid structures, and high-mannose structures) measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Fc region residues in EU numbering) within the Fc region; however, due to slight sequence variations in antibodies, Asn297 may also be located approximately three amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 01321 40; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108 A1, Presta, L; and WO2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0161] Further provided are anti-CD3 antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0162] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an anti-CD3 antibody of the present invention, thereby generating an Fc region variant (see, e.g., US2012 / 0251531). The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions. In certain embodiments, the present invention contemplates anti-CD3 antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc., Mountain View, Calif.); and CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0163] Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al. J. ImmunoL Methods 202:163 (1996); Cragg, MS et al. Blood. 101:1045-1052 (2003); and Cragg, MS and MJ Glennie Blood. 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al. Int'l. ImmunoL 18(12):1759-1769 (2006)).

[0164] Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent Nos. 6,737,056 and 8,219,149). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0165] In certain embodiments, the proline at position 329 of the wild-type human Fc region in the antibody is substituted with glycine or arginine or an amino acid residue large enough to disrupt the proline sandwich in the Fc / Fcγ receptor interface formed between proline 329 of the Fc and tryptophan residues Trp87 and Trp110 of FcgRIII (Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In certain embodiments, the antibody comprises at least one additional amino acid substitution. In one embodiment, the additional amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S; in yet another embodiment, the at least one additional amino acid substitution is L234A and L235A in the human IgG1 Fc region, or S228P and L235E in the human IgG4 Fc region (see, e.g., US2012 / 0251531); and in yet another embodiment, the at least one additional amino acid substitution is L234A and L235A and P329G in the human IgG1 Fc region.

[0166] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0167] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering residues) of the Fc region.

[0168] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0169] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260; 5,624,821; and WO 94 / 29351.

[0170] In some aspects, the bispecific antibody comprises an Fc region comprising an N297G mutation. In some embodiments, the bispecific antibody comprising the N297G mutation comprises one or more heavy chain constant domains, wherein the one or more heavy chain constant domains are selected from a first CH1 domain, a first CH2 domain, a first CH3 domain, a second CH1 domain, a second CH2 domain, and a second CH3 domain.

[0171] In certain embodiments, it may be desirable to create a cysteine-engineered antibody in which one or more residues of the antibody are replaced with cysteine ​​residues. In some embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be produced, for example, as described in WO2016 / 040856, the entire contents of which are incorporated herein by reference, including any drawings.

[0172] In certain embodiments, the bispecific antibodies provided herein can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of 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. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0173] In another embodiment, a conjugate of an antibody and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, wavelengths that are not harmful to normal cells but heat the nonproteinaceous moiety to temperatures that kill cells proximal to the antibody-nonproteinaceous moiety.

[0174] Bispecific antibodies of the present invention can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CD3 antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, for example, a Chinese hamster ovary (CHO) cell or a lymphocytic cell (e.g., a Y0, NS0, or Sp20 cell). In one embodiment, a method of making a bispecific antibody is provided, comprising culturing a host cell containing nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0175] For recombinant production of an antibody, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0176] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0177] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0178] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified and can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0179] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0180] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0181] The antibodies of the present invention may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0182] In one embodiment, the antibodies of the present invention are tested for their antigen-binding activity by known methods, such as ELISA or Western blot. In another embodiment, a competitive assay can be used to identify antibodies that compete with the anti-CD3 antibodies of the present invention for binding to CD3. In an exemplary competitive assay, immobilized CD3 is incubated in a solution containing a first labeled antibody that binds to CD3 and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to CD3. The second antibody may be present in hybridoma supernatant. As a control, immobilized CD3 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to CD3, excess unbound antibody is removed and the amount of label associated with immobilized CD3 is measured. If the amount of label associated with immobilized CD3 is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to CD3. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0183] In one aspect, an assay is provided for identifying antibodies with biological activity. Biological activity can include, for example, binding to CD3 (e.g., CD3 on the surface of T cells), or a peptide fragment thereof, either in vivo, in vitro, or ex vivo. In the case of bispecific antibodies of the present invention, biological activity can also include, for example, effector cell activation (e.g., activation of T cells (e.g., CD8+ and / or CD4+ T cells)), expansion of effector cell populations (i.e., increase in T cell count), reduction of target cell populations (i.e., reduction of the population of cells expressing a second biological molecule on their cell surface), and / or target cell killing. In some embodiments, activity includes the ability to support B cell killing and / or activation of cytotoxic T cells.

[0184] In certain embodiments, any of the antibodies of the present invention can be used to detect the presence of CD3 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In certain embodiments, the method includes contacting the biological sample with an anti-CD3 antibody described herein under conditions that allow binding of the bispecific antibody to CD3 and another antigen, and detecting whether a complex is formed between the bispecific antibody and CD3. Such methods can be in vitro or in vivo methods.

[0185] In certain embodiments, labeled antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (such as fluorescent labels, chromophoric labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and indirectly detectable moieties, such as enzymes or ligands, for example, by enzymatic reaction or molecular interaction. Exemplary labels include radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (see, for example, U.S. Patent No. 4,737,456, the entire contents of which, including any drawings, are incorporated herein by reference), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), These include, but are not limited to, alkaline phosphatase, O-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors using hydrogen peroxide, such as heterocyclic oxidases coupled with HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0186] Production of anti-CD3 antibodies and antibody compositions A further aspect of the invention relates to methods for producing the anti-CD3 antibodies and compositions of the invention. One embodiment of this aspect of the invention relates to a method for producing an anti-CD3 antibody as defined herein, comprising providing a host cell capable of expressing the anti-CD3 antibody, culturing the host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody.

[0187] The antibodies or antibody compositions of the present invention can be produced by methods generally known in the art for producing recombinant monoclonal or polyclonal antibodies. Therefore, for the production of a single antibody of the present invention, any method known in the art for producing recombinant monoclonal antibodies can be used. For the production of an antibody composition containing two or more anti-CD3 antibodies of the present invention, the individual antibodies can be produced separately, i.e., each antibody can be produced in a separate bioreactor, or the individual antibodies can be produced together in a single bioreactor. When the number of different antibodies in the composition is greater than, for example, two or three, it is generally preferable to produce the antibodies together in a single bioreactor for cost-effective reasons. On the other hand, when a composition contains only a small number of different antibodies, for example, two, three, or even four different antibodies, the decision of whether to produce them separately in different bioreactors or together in a single bioreactor must be made based on the individual circumstances. When an antibody composition is produced in more than one bioreactor, a purified anti-CD3 antibody composition can be obtained by pooling antibodies obtained from the supernatants purified individually from each bioreactor. A variety of techniques are known in the art for producing polyclonal antibody compositions in multiple bioreactors, where cell lines or antibody preparations are combined at a later point upstream or before or during downstream processing.

[0188] When producing two or more individual antibodies in a single bioreactor, this can be done, for example, based on site-specific integration of antibody coding sequences into the genome of individual host cells, V H and V LThis ensures that the protein chains are maintained in their original pairing during production. Furthermore, site-specific integration minimizes position effects, so that the growth and expression characteristics of individual cells within a polyclonal cell line are expected to be very similar. In general, this method involves: i) a host cell containing one or more recombinase recognition sites; ii) an expression vector containing at least one recombinase recognition site that is compatible with the recombinase recognition site of the host cell; iii) transferring the selected V from the screening vector to an expression vector so that a full-length antibody or antibody fragment can be expressed from the vector. H and V L generating a group of expression vectors by transferring the coding pairs (such transfer may not be necessary if the screening vector is identical to the expression vector); iv) transfecting host cells with the group of expression vectors and a vector encoding a recombinase that can bind recombinase recognition sites in the vector with recombinase recognition sites in the genome of the host cells; v) obtaining / generating a polyclonal cell line from the transfected host cells; and vi) expressing and harvesting an antibody composition from the polyclonal cell line.

[0189] An alternative approach is to produce two or more different antibodies in a single bioreactor. This method involves generating a polyclonal cell line capable of expressing a polyclonal antibody or other polyclonal protein comprising two or more distinct members by: a) providing a set of expression vectors, each of which contains at least one copy of a distinct nucleic acid encoding a distinct member of a polyclonal protein; separately transfecting each of the expression vectors into a host cell under conditions that avoid site-specific integration of the expression vector into the genome of the cell, thereby obtaining two or more cell compositions, each expressing one distinct member of the polyclonal protein; and c) combining the at least two cell compositions to obtain the polyclonal cell line.

[0190] The antibodies of the present invention can be produced in a variety of cell types, including mammalian cells, and non-mammalian eukaryotic or prokaryotic cells such as plant cells, insect cells, yeast cells, fungi, E. coli, etc. However, the antibodies are preferably produced in mammalian cells, such as CHO cells, COS cells, BHK cells, myeloma cells (e.g., Sp2 / 0 or NS0 cells), fibroblasts such as NIH 3T3, or immortalized human cells such as HeLa cells or HEK 293 cells.

[0191] Methods for transfecting a nucleic acid sequence into a host cell are well known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Edition, 2001). For site-specific integration, a suitable host cell contains one or more recombinase recognition sites in its genome. In this case, a suitable expression vector contains a recombinase recognition site(s) that matches the recombinase recognition site(s) of the host cell.

[0192] Thus, one embodiment of the present invention is a polyclonal cell line capable of expressing two or more anti-CD3 antibodies of the present invention. A further embodiment is a polyclonal cell line in which each individual cell expresses a single V H and V L The polyclonal cell line is capable of expressing a group of V H and V L A pair can be expressed, and V H and V L Each of the pairs is a polyclonal cell line encoding an anti-CD3 antibody.

[0193] therapeutic composition Another aspect of the present invention is a pharmaceutical composition comprising, as an active ingredient, at least one anti-CD3 antibody, or anti-CD3 Fab or other anti-CD3 antibody fragment composition of the present invention. Such compositions are intended for the amelioration, prevention, and / or treatment of cancer. The pharmaceutical composition can be administered to humans or domestic animals.

[0194] In addition to at least one antibody or fragment thereof of the present invention, the pharmaceutical composition further comprises at least one pharmaceutically acceptable diluent, carrier, or excipient. These may include, for example, preservatives, stabilizers, surfactants / wetting agents, emulsifiers, solubilizers, salts for adjusting osmotic pressure, and / or buffers. The solution or suspension may further comprise a viscosity-increasing substance, such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatin. The pH value suitable for pharmaceutical compositions is generally in the range of about 5.5 to 8.5, e.g., about 6 to 8, e.g., about 7, and is maintained appropriately using a buffer.

[0195] Conventional pharmaceutical practice can be used to provide suitable formulations or compositions for administration to, for example, cancer patients.Typically, administration is therapeutic, that is, administered after cancer condition is diagnosed.Any suitable administration route can be adopted, for example, parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intranasal, aerosol, suppository or oral administration.The pharmaceutical composition of the present invention is typically administered in the form of liquid solution or suspension, more typically in the form of aqueous solution or suspension, particularly isotonic aqueous solution or suspension.

[0196] As an alternative to liquid formulations, compositions of the invention can be prepared in lyophilized form comprising at least one antibody, alone or together with a carrier, e.g., mannitol, in which case the composition is reconstituted with a liquid, such as sterile water, prior to use.

[0197] The pharmaceutical composition contains approximately 1% to approximately 95%, preferably approximately 20% to approximately 90%, of the active ingredient. The pharmaceutical composition according to the present invention can be prepared in a unit dosage form, such as an ampule, vial, suppository, tablet, or capsule. The formulation can be administered to a human individual in a therapeutically or prophylactically effective amount (e.g., an amount that prevents, eliminates, or reduces a pathological condition) to provide treatment for cancerous diseases or other conditions. The preferred dosage of the administered therapeutic agent may depend on variables such as the severity of the cancer, the overall health of the particular patient, the formulation of the excipients, and its administration route.

[0198] Therapeutic Uses of Antibodies and Compositions According to the Invention The anti-CD3 antibodies and pharmaceutical compositions according to the invention can be used for the treatment or amelioration of diseases in mammals, particularly for the treatment of cancer in humans.

[0199] Some embodiments provide a method of treating or delaying the progression of a cell proliferative disorder or an autoimmune disorder in a subject in need thereof, comprising administering to the subject an effective amount of any one of the antibodies (in a monospecific, bispecific, or multispecific format) described herein. In another aspect, the invention features a method of enhancing or decreasing immune function in a subject with a cell proliferative disorder or an autoimmune disorder, comprising administering to the subject any one of the antibodies (in a monospecific, bispecific, or multispecific format) described herein.

[0200] In any of the uses or methods described herein, the cell proliferative disorder can be cancer. In some embodiments, the cancer is breast cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or marginal zone lymphoma. (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenström's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassifiable splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, Waldenström's macroglobulinemia, heavy chain disease, a heavy chain disease, gamma heavy chain disease, heavy chain disease, Plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T-cell / histiocytocyte-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, leg type, age-related EBV-positive DLBCL, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma The lymphoma is selected from the group consisting of diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma: unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.In some embodiments, the preferred cancer is germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), or Burkitt's lymphoma (BL).

[0201] In some embodiments, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjögeren's syndrome, glomerulonephritis, neuromyelitis optica (NMO), and IgG neuropathy.

[0202] In some embodiments, the antibody is in a kit comprising (a) a composition comprising any one of the antibodies described herein (in a monospecific, bispecific, or multispecific format), and (b) a package insert containing instructions for administering the composition to a subject for treating or delaying the progression of a cell proliferative disorder. In some embodiments, the antibody in the kit is lyophilized.

[0203] In any of the foregoing uses or methods, the subject may be a human.

[0204] Dosage and route of administration The antibodies and compositions of the present invention are administered in an amount effective to treat the condition in question, i.e., at a dosage and for a period of time necessary to achieve the desired result. A therapeutically effective amount may vary depending on factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether the anti-CD3 antibody is administered as the sole treatment or in combination with one or more additional anti-cancer treatments.

[0205] The effective amount for tumor treatment can be measured by its ability to stabilize the progression of disease and / or improve symptoms in patients, preferably by reversing the progression of disease, for example, by reducing tumor size.The ability of the antibody or composition of the present invention to suppress cancer can be evaluated by in vitro assays, for example, as described in the Examples, and also in appropriate animal models that predict efficacy in human tumors.Appropriate dosage regimens can be selected to provide optimal therapeutic responses in each specific situation, for example, by administering a single bolus or continuous infusion, and dosage adjustments can be made according to the exigencies of each case.

[0206] In some embodiments, the antibody is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg. In some embodiments, the antibody is administered to the subject at a dose of about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the antibody is administered to the subject at a dose of about 1 mg / kg. In some embodiments, the antibody is administered subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the antibody is administered subcutaneously. In some embodiments, the bispecific antibody is administered intravenously.

[0207] Pharmaceutical preparations Pharmaceutical formulations of the antibodies of the present invention can be prepared in the form of a lyophilized formulation or aqueous solution by mixing such antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers, e.g., phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) soluble or unsaturated fatty acids; (of which the term "polypeptide" is used herein) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0208] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations containing a histidine acetate buffer.

[0209] The formulations herein may also contain two or more active ingredients as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an additional therapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, and / or an antihormonal agent, such as those listed hereinabove). Such active ingredients are suitably present in a combination in an amount effective for the intended purpose. The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation technology or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0210] Sustained-release preparations can be prepared.Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing bispecific antibodies, which matrices are in the form of shaped articles, for example, films or microcapsules.The preparations used for in vivo administration are generally sterile.Sterilization can be easily achieved, for example, by filtration through sterile filtration membranes. [Example]

[0211] Example 1 This example provides the results of a hybridoma-based method used to generate multiple different anti-CD3 antibodies of the present invention.

[0212] Briefly, we used hybridoma technology to produce multiple different anti-CD3 antibodies. While we have produced numerous anti-CD3 antibodies using this method, this example presents 58 CD3-specific antibodies we discovered. By elucidating the amino acid and / or nucleotide sequences of the CDR3 heavy chain (HC) and light chain (LC or λ) variable regions of these antibodies, we identified a series of "clusters" or "motifs" within the sequences. These clusters represent convergent somatic hypermutation (SHM) in the variable region sequences. Clustering provides insight into functionally related sequences as well as the diversity of the entire population of antibodies and their variable region sequences. Sequences descended from the same parent B cell or that have convergently evolved within the same cluster should be more closely related functionally than sequences belonging to other clusters.

[0213] In this example, the variable chain region sequences were provided by different antibodies. Therefore, any clustering is most likely due to convergent SHM, which is likely to be functionally related mutations. For example, they share specific affinity for CD3. These SHMs can inform the development of recombinant anti-CD3 antibodies with improved properties, such as specific binding to CD3 fragments.

[0214] Hybridoma Hybridomas were produced, each expressing a different anti-CD3 antibody. Art-known methods for producing antibodies and / or hybridoma cells are known in the art.

[0215] For example, different anti-CD3 antibodies are obtained from different populations of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that arise during the production of the monoclonal antibody, such variants being generally present in minor amounts.

[0216] By way of example, in one particular hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized with the CD3 antigen to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing protein. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells.

[0217] The hybridoma cells thus prepared are preferably seeded and grown in an appropriate culture medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells are deficient in the hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT) enzyme, the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells.

[0218] In some embodiments, the myeloma cells fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In some embodiments, the myeloma cell line is a mouse myeloma line, such as one derived from a mouse tumor. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.

[0219] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against CD3. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

[0220] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution and grown by standard methods. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.

[0221] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0222] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). In some embodiments, hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be introduced into an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or in other cases, myeloma cells that do not produce immunoglobulin proteins, resulting in the synthesis of monoclonal antibodies in the recombinant host cells.

[0223] In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0224] The DNA can also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains for the mouse homologous sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0225] Sequencing Briefly, a cDNA library was generated from anti-CD3 antibody mRNA obtained from hybridoma cultures of cells expressing different anti-CD3 antibodies.

[0226] The sequences were aligned to identify all unique anti-CD3 antibody sequences. Sequence alignment revealed the uniqueness of each particular sequence and its corresponding antibody. The antibody variable sequences of each different antibody were analyzed using custom AlivaAlign sequencing software.

[0227] The identified CDR3 HC and VL sequences are provided in Table 1 as SEQ ID NOs: 2-59 and 60-117. The corresponding heavy and light chain variable region sequences are provided in Table 2 as SEQ ID NOs: 118-175 and 176-233, respectively. Paired heavy and light chain variable sequences were identified for specific antibodies, as shown in Table 2.

[0228] Clustering HC and VL chain variable region sequences were clustered using an algorithm that assigned sequences to clusters if they shared identical hV, hJ, IV, and IJ genes, had identical HCDR3 lengths, and were at least 90% identical (Hamming distance) to their HCDR3s within the cluster.

[0229] Table 3 provides the SMH identified for each of the heavy and light chain variable region sequences. In Table 3, each HC and VL variable region sequence is identifiable and mappable by the identity of their associated CD3 antibody.

[0230] Table 4 provides data pertaining to the described clusters of CD3 antibodies, i.e., 1-8. The table also shows the heavy and light chain gene usage and the percentage difference from the germline sequence. The corresponding and paired CDR3 sequences of the HC and VL are also provided. Within each cluster, there are related sequences, or "subclusters," e.g., 1.1 and 1.2. Surprisingly, several antibodies yielded identical heavy and light chain nucleotide sequences between antibodies.

[0231] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0232] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] Table 2-7 Table 2-8 Table 2-9 Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6

Claims

1. An anti-CD3 antibody or antibody fragment comprising a heavy chain (VH) CDR3 sequence comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59.

2. 2. The anti-CD3 antibody or antibody fragment of claim 1, wherein the antibody or fragment comprises a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117.

3. 2. The anti-CD3 antibody or antibody fragment of claim 1, wherein the antibody or fragment comprises a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175, and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175.

4. 2. The anti-CD3 antibody or antibody fragment of claim 1, wherein the antibody or fragment comprises a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233, and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

5. the antibody or fragment a) a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175; and b) a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

2. The anti-CD3 antibody or antibody fragment of claim 1, comprising:

6. Cluster: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; Clust 3.10; Clust 3.11; 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; Clust 4.2; 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 5.8; Clust An anti-CD3 antibody or antibody fragment comprising a heavy chain variable region and / or light chain variable region sequence comprising somatic hypermutation (SMH) of Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

7. The antibody or antibody fragment may be grouped into clusters: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; Clust 4.2; 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 5.8; Clust 6. The anti-CD3 antibody or antibody fragment of claim 6, comprising a heavy chain variable region comprising somatic hypermutation (SMH) of Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

8. The antibody or antibody fragment may be grouped into clusters: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; Clust 4.2; 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 5.8; Clust 6. The anti-CD3 antibody or antibody fragment of claim 6, comprising a light chain variable region comprising somatic hypermutation (SMH) of Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

9. The antibody or antibody fragment may be grouped into clusters: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; Clust 4.2; 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 5.8; Clust 7. The anti-CD3 antibody or antibody fragment of claim 6, comprising a heavy chain CDR3 sequence comprising somatic hypermutation (SMH) of Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

10. The antibody or antibody fragment may be grouped into clusters: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; Clust 4.2; 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 5.8; Clust 6. The anti-CD3 antibody or antibody fragment of claim 6, comprising a light chain CDR3 sequence comprising somatic hypermutation (SMH) of Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

11. A therapeutic composition comprising an anti-CD3 antibody or antibody fragment comprising a heavy chain (VH) CDR3 sequence comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59.

12. 12. The therapeutic composition of claim 11, wherein the antibody or fragment comprises a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117.

13. 12. The therapeutic composition of claim 11, wherein the antibody or fragment comprises a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175, and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175.

14. 12. The therapeutic composition of claim 11, wherein the antibody or fragment comprises a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233, and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

15. 12. The therapeutic composition of claim 11, wherein the therapeutic composition comprises a plurality of different CD3 antibodies, each of the different CD3 antibodies comprising a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175 and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175, and / or a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233 and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

16. Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; Clust 3.10; Clust 3.11; 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; Clust 4.2; 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 5.8; Clust 12. The therapeutic composition of claim 11, comprising a heavy chain variable region and / or light chain variable region sequence comprising somatic hypermutation (SMH) of Cluster 5.9; Cluster 6.1; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

17. 1. A method of treating a cancer patient with a therapeutic composition comprising at least one anti-CD3 antibody or fragment thereof, the anti-CD3 antibody or fragment comprising: a heavy chain (VH) CDR3 sequence comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59; a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117; a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175; and / or a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233. A method comprising:

18. The at least one anti-CD3 antibody or fragment thereof is selected from the group consisting of: Cluster 1.1; Cluster 1.2; Cluster 2.1; Cluster 2.2; Cluster 2.3; Cluster 2.3; Cluster 2.4; Cluster 3.1; Cluster 3.10; Cluster 3.11; Cluster 3.2; Cluster 3.3; Cluster 3.4; Cluster 3.5; Cluster 3.6; Cluster 3.6; Cluster 3.7; Cluster 3.8; Cluster 3.9; Cluster 4.1; Cluster 4.2; Cluster 5.1; Cluster 5.2; Cluster 5.3; Cluster 5.4; Cluster 5.5; Cluster 5.6; Cluster 5.7; Cluster 18. The method of claim 17, wherein the heavy chain variable region sequence comprises somatic hypermutation (SMH) of Cluster 5.8; Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

19. The at least one anti-CD3 antibody or fragment thereof is selected from the group consisting of: Cluster 1.1; Cluster 1.2; Cluster 2.1; Cluster 2.2; Cluster 2.3; Cluster 2.3; Cluster 2.4; Cluster 3.1; Cluster 3.10; Cluster 3.11; Cluster 3.2; Cluster 3.3; Cluster 3.4; Cluster 3.5; Cluster 3.6; Cluster 3.6; Cluster 3.7; Cluster 3.8; Cluster 3.9; Cluster 4.1; Cluster 4.2; Cluster 5.1; Cluster 5.2; Cluster 5.3; Cluster 5.4; Cluster 5.5; Cluster 5.6; Cluster 5.7; Cluster 18. The method of claim 17, wherein the light chain variable region sequence comprises somatic hypermutation (SMH) of Cluster 5.8; Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

20. The at least one anti-CD3 antibody or fragment thereof is selected from the group consisting of: Cluster 1.1; Cluster 1.2; Cluster 2.1; Cluster 2.2; Cluster 2.3; Cluster 2.3; Cluster 2.4; Cluster 3.1; Cluster 3.10; Cluster 3.11; Cluster 3.2; Cluster 3.3; Cluster 3.4; Cluster 3.5; Cluster 3.6; Cluster 3.6; Cluster 3.7; Cluster 3.8; Cluster 3.9; Cluster 4.1; Cluster 4.2; Cluster 5.1; Cluster 5.2; Cluster 5.3; Cluster 5.4; Cluster 5.5; Cluster 5.6; Cluster 5.7; Cluster 18. The method of claim 17, wherein the antibody comprises light chain variable region and heavy chain variable region sequences comprising somatic hypermutation (SMH) of Cluster 5.8; Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

21. 1. A bispecific or multispecific antibody that binds to CD3 and another antigen, comprising an anti-CD3 antibody or antibody fragment, wherein the targeting arm of the bispecific or multispecific antibody comprises a heavy chain (VH) CDR3 sequence comprising an amino acid sequence selected from one of SEQ ID NOs: 2-59, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-59.

22. 22. The bispecific or multispecific antibody of claim 21 , wherein the anti-CD3 antibody or fragment comprises a light chain CDR3 sequence (VL or λ) comprising an amino acid sequence selected from one of SEQ ID NOs: 60-117, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 60-117.

23. 22. The bispecific or multispecific antibody of claim 21 , wherein the anti-CD3 antibody or fragment comprises a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175 and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175.

24. 22. The bispecific or multispecific antibody of claim 21 , wherein the anti-CD3 antibody or fragment comprises a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233, and / or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

25. the anti-CD3 antibody or fragment thereof a) a heavy chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 118-175, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 118-175; and b) a light chain variable region comprising an amino acid sequence selected from one of SEQ ID NOs: 176-233, and / or a sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 176-233.

22. The bispecific or multispecific antibody of claim 21 , comprising:

26. 3.1; Cluster 3.2; Cluster 3.3; Cluster 3.4; Cluster 3.5; Cluster 3.6; Cluster 3.7; Cluster 3.8; Cluster 3.9; Cluster 4.1; Cluster 4.2; Cluster 5.1; Cluster 5.2; Cluster 5.3; Cluster 5.4; Cluster 5.5; Cluster 5.6; Cluster 5.7; Cluster 5.8; Cluster 5.9; Cluster 6.1; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6 ... 5.6; Clust 5.7; Clust 5.8; Clust 5.9; Clust 6.1; Clust 6.10; Clust 6.11; Clust 6.12; Clust 6.13; Clust 6.14; Clust 6.15; 6.16; Clust 6.17; Clust 6.2; Clust 6.3; Clust 6.4; Clust 6.5; Clust 6.6; Clust 6.7; Clust 6.8; Clust 6.9; Clust 7.1; 7.2; Clust 8.1l and / or Clust A bispecific or multispecific antibody comprising an anti-CD3 antibody or antibody fragment comprising a heavy chain variable region and / or a light chain variable region sequence comprising somatic hypermutation (SMH) of 8.

2.

27. The anti-CD3 antibody or antibody fragment is cluster: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; Clust 3.10; 3.11; Clust 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; 4.2; Clust 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 27. The bispecific or multispecific antibody of claim 26, comprising a heavy chain variable region comprising somatic hypermutation (SMH) of Cluster 5.8; Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

28. The anti-CD3 antibody or antibody fragment is cluster: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; Clust 3.10; 3.11; Clust 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; 4.2; Clust 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 27. The bispecific or multispecific antibody of claim 26, comprising a light chain variable region comprising somatic hypermutation (SMH) of Cluster 5.8; Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

29. The anti-CD3 antibody or antibody fragment is cluster: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; Clust 3.10; 3.11; Clust 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; 4.2; Clust 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 27. The bispecific or multispecific antibody of claim 26, comprising a heavy chain CDR3 sequence comprising somatic hypermutation (SMH) of Cluster 5.8; Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.

2.

30. The anti-CD3 antibody or antibody fragment is cluster: Clust 1.1; Clust 1.2; Clust 2.1; Clust 2.2; Clust 2.3; Clust 2.3; Clust 2.4; Clust 3.1; Clust 3.10; 3.11; Clust 3.2; Clust 3.3; Clust 3.4; Clust 3.5; Clust 3.6; Clust 3.6; Clust 3.7; Clust 3.8; Clust 3.9; Clust 4.1; 4.2; Clust 5.1; Clust 5.2; Clust 5.3; Clust 5.4; Clust 5.5; Clust 5.6; Clust 5.7; Clust 27. The bispecific or multispecific antibody of claim 26, comprising a light chain CDR3 sequence comprising somatic hypermutation (SMH) of Cluster 5.8; Cluster 5.9; Cluster 6.1; Cluster 6.10; Cluster 6.11; Cluster 6.12; Cluster 6.13; Cluster 6.14; Cluster 6.15; Cluster 6.16; Cluster 6.17; Cluster 6.2; Cluster 6.3; Cluster 6.4; Cluster 6.5; Cluster 6.6; Cluster 6.7; Cluster 6.8; Cluster 6.9; Cluster 7.1; Cluster 7.2; Cluster 8.1l and / or Cluster 8.2.

Images