CD3-delta / epsilon heterodimer specific antibodies

Abstract

The present invention provides novel human CD3 antigen binding polypeptides as well as their preparation and use in the treatment and / or diagnosis of various diseases, as well as bispecific antibody molecules capable of activating immune effector cells as well as their use in the diagnosis and / or treatment of various diseases.

Description

[0001] This application is a divisional application of CN201880084359.8.

[0002] Cross - reference to related applications

[0003] This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 610,764, filed on December 27, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Sequence listing

[0005] This application contains a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on September 25, 2023, named TNO - 0010 - WO4 ST26.xml, and is 64,689 bytes in size. Field of the invention

[0006] The present invention relates to novel human CD3 antigen - binding polypeptides and their preparation and use in the treatment and / or diagnosis of various diseases, as well as bispecific antibody molecules capable of activating immune effector cells and their use in the diagnosis and / or treatment of various diseases. Background art

[0007] The body's immune system serves to defend against infection, injury, and cancer. Two independent but interrelated systems, the humoral immune system and the cellular immune system, work together to protect the body. The humoral system is mediated by soluble factors (called antibodies) that neutralize products recognized by the body as foreign. In contrast, the cellular system involves cells such as T cells and macrophages that remove and neutralize foreign invaders.

[0008] The activation of T cells is crucial for stimulating an immune response. T cells exhibit immune specificity and direct most of the cellular immune response. Although T cells do not secrete antibodies, they are required for B lymphocytes to secrete antibodies. T - cell activation requires the participation of many cell - surface molecules such as the T - cell receptor complex and CD4 or CD8 molecules. The antigen - specific T - cell receptor (TcR) consists of a disulfide - linked heterodimeric membrane glycoprotein having chains alpha and beta (α and β) or gamma and delta (γ and δ). The TcR is non - covalently linked to a complex with an invariant protein called CD3.

[0009] It is known that T cells play an effective anti-tumor role in many experimental settings. Antibodies capable of effectively recruiting T cells against tumor cells are already available as, for example, bispecific antibodies directed against tumor-associated antigens (TAAs) and activating T cell membrane proteins such as the TCR / CD3 complex and CD28. These bispecific antibodies can activate T cells regardless of their TCR specificity, resulting in the specific lysis of cells carrying the corresponding TAA.

[0010] However, while anti-CD3 bispecific antibodies can redirect T cell-mediated lysis towards malignant cells, clinical trials using CD3-based bsAbs have shown high toxicity in patients. Non-specific T cell activation from bsAbs can occur in an antigen-independent manner due to Fc / Fc receptor (FcR) interactions or in an antigen-dependent manner when the antigen is expressed on both normal and tumor cells. These two mechanisms may be responsible for the toxicity observed in previous clinical studies. (See, for example, Link et al. (1998) Int. J. Cancer 77(2):251-6; Durben et al. Molecular Therapy (2015); 23 4,648–655). There has been a significant hurdle in developing these antibodies for therapeutic purposes due to the cytokine release syndrome that is produced.

[0011] The interaction of the T cell receptor (TCR) with its peptide-MHC ligand determines the activity of T cells. The binding characteristics of this interaction have been studied in great detail and shown to control T cell function. The strength and nature of the TCR-peptide / MHC interaction determine whether T cells exert effector functions or become inactivated and anergic. Antibodies against CD3 activate T cells by altering the conformation of the CD3ε chain and can be agonistic or antagonistic to T cells depending on the epitope (Yoon et al., 1994 Immunity 1:563-569). Given the significant side effects of many T cell agonists, it may be preferable to maintain an effective anti-tumor effect while reducing the release of pro-inflammatory cytokines. However, partially agonistic anti-CD3 antibodies may sub-optimally alter the CD3ε chain, resulting in ineffective signaling, and most anti-CD3 antibodies are full agonists of both pathways. It is not clear whether these effector functions can be separated. Many existing anti-CD3 antibodies (e.g., SP-34, UCHT1, OKT3) have affinities in the 1-50 nM KD range, which may not be optimal for therapeutic use.

[0012] The present invention provides CD3-specific antibodies and bispecific antibodies derived from said CD3-specific antibodies.

[0013] Publication

[0014] CD3 antibodies are disclosed, for example, in U.S. Patent Nos. 5,585,097; 5,929,212; 5,968,509; 6,706,265; 6,750,325; 7,381,803; 7,728,114. Bispecific antibodies having CD3 binding specificity are disclosed, for example, in U.S. Patent Nos. 7,262,276; 7,635,472; 7,862,813; and 8,236,308, each of which is hereby expressly incorporated by reference. CD3 binding antibody sequences are provided in co-pending application PCT US2017 / 038377 (which is hereby expressly incorporated by reference). SUMMARY OF THE INVENTION

[0015] Compositions of antibodies and methods of use thereof are provided, which antibodies bind to CD3 and activate signal transduction through CD3, such as activation of CD3 + T cells. The antibodies are characterized by binding to a CD3 epitope bound by the F2B antibody, which CD3 epitope may be referred to herein as the F2B epitope. The F2B antibody comprises a set of CDR sequences of SEQ ID NO:1 and a constant light chain sequence of SEQ ID NO:19. In some embodiments, the antibody that binds to the F2B epitope comprises a heavy chain variable region sequence other than the sequences shown in SEQ ID NOs:1-18.

[0016] Antibodies that bind to the F2B epitope provide significant benefits in terms of biological activity. The antibody minimizes the release of toxic cytokines while maintaining effective tumor cell lysis. In some embodiments, the anti-CD3 antibody that binds to the F2B epitope is characterized by a reduced tendency to induce cytokine release (e.g., release of IL-2 and IFNγ) upon binding to naïve T cells. Without being bound by theory, it is believed that binding to the specific epitope recognized by F2B provides the unique and beneficial properties of the antibodies described herein.

[0017] Antibodies that bind to the F2B epitope can be selected for a binding affinity (KD) for CD3 in the range of about 10 -6 to about 10 -11 . Anti-CD3 antibodies with an affinity (KD) of 50 nM or greater, 100 nM or greater, 500 nM or greater or 1 μM or greater are desirable to more closely mimic TCR / MHC interactions and minimize the release of toxic cytokines while maintaining effective tumor cell lysis. Antibodies that induce cytokine release can be selected that do not exceed about 200% of the maximum cytokine release observed with the F2B antibody, and can be no more than about 150%, no more than 125%, no more than 100%, and can be less than the maximum value observed for F2B in a comparative assay.

[0018] Antibodies that can induce no more than 20%, no more than 30%, and no more than 50% of the maximum IL-2 and IL-10 release of a control anti-CD3 antibody (such as OKT-3 or TNB-383B) in a comparative in vitro assay can be selected.

[0019] The F2B epitope is characterized by binding to at least one residue selected from CD3ε (SEQ ID NO:23): K73 and S83; and CD3δ (SEQ ID NO:24) K82 and C93. In some embodiments, the epitope comprises the region of CD3ε defined by K73, N74, I75, G76, S77, D78, E79, D80, H81, L82, S83. In some embodiments, the epitope comprises one or both of K73 and S83. In some embodiments, the epitope comprises the region of CD3δ defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, C93. In some embodiments, the epitope comprises one or both of K82 and C93. In some embodiments, the F2B epitope comprises a conformational epitope involving residues of both CD3δ and CD3ε. In some embodiments, the conformational epitope comprises each of the residues CD3□K73 and S83, CD3□K82 and CD93. In some embodiments, the antibody that binds to the F2B epitope does not cross-react with cynomolgus monkey CD3 protein.

[0020] In some embodiments, the antibody that binds to the F2B epitope is determined by a competition assay between the antibody disclosed herein and other antibodies. In some embodiments, the antibody binds to specific residues of CD3 that reduce cytokine release.

[0021] In some embodiments, bispecific or multispecific antibodies are provided, which at least comprise the heavy chain variable region from an antibody that binds to the F2B epitope. The bispecific antibody at least comprises the heavy chain variable region of an antibody that is specific for a protein other than CD3, and may comprise a heavy chain variable region and a light chain variable region. In some such embodiments, the second antibody specifically binds to a tumor-associated antigen, a target antigen (such as integrin, etc.), a pathogen antigen, a checkpoint protein, etc. Various forms of bispecific antibodies are within the scope of the present invention, including but not limited to single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and their multiple-chain polypeptides.

[0022] In some embodiments, the F2B epitope-binding antibody of the invention comprises a CD3-binding variable region paired with a light chain. In some embodiments, the light chain comprises the variable region sequence listed in SEQ ID NO:19, or a variable region comprising a set of CDR sequences and framework sequences in SEQ ID NO:19. A variety of Fc sequences can be used, including but not limited to human IgG1, IgG2a, IgG2b, IgG3, IgG4, etc. In some embodiments, the second arm of the bispecific antibody comprises a variable region that specifically binds to a tumor-associated antigen. In some embodiments, the second arm of the bispecific antibody comprises a variable region that specifically binds to BCMA.

[0023] In other embodiments, there is provided a pharmaceutical composition comprising at least the CD3-binding VH domain of the invention, such as a monospecific, bispecific, etc. antibody or antibody-like protein comprising at least the CD3-binding VH domain of the invention; and a pharmaceutically acceptable excipient. The composition can be lyophilized, suspended in solution, etc., and can be provided in unit dosage form.

[0024] In some embodiments, there is provided a method for treating cancer, the method comprising administering to an individual in need an effective dose of a monospecific, bispecific, etc. antibody of the invention. In the case where the antibody is bispecific, the second antigen-binding site can specifically bind to a tumor antigen, a checkpoint protein, etc. In various embodiments, the cancer is selected from the group consisting of: ovarian cancer, breast cancer, gastrointestinal cancer, brain cancer, head and neck cancer, prostate cancer, colon cancer, lung cancer, leukemia, lymphoma, sarcoma, carcinoma, neurocytic tumor, squamous cell carcinoma, germ cell tumor, metastasis, undifferentiated tumor, seminoma, melanoma, myeloma, neuroblastoma, mixed cell tumor, and neoplasia caused by infectious agents.

[0025] In some embodiments, there is provided a method for treating an infectious disease, the method comprising administering to an individual in need an effective dose of a monospecific, bispecific, etc. antibody of the invention. In the case where the antibody is bispecific, the second antigen-binding site can specifically bind to a pathogen antigen, such as a bacterium, a virus, or a parasite.

[0026] In other embodiments, there is provided a method for producing the bispecific antibody of the invention, the method comprising expressing the antibody sequences in a single host cell, such as one or more light chain-encoding sequences, one or more heavy chain-encoding sequences. In various embodiments, the host cell can be a prokaryotic cell or a eukaryotic cell, such as a mammalian cell.

[0027] Aspects of the invention include methods of generating antigen-binding proteins as described herein, the methods comprising growing a host cell under conditions permitting expression of the protein, and isolating the protein from the cells and / or cell culture medium.

[0028] Aspects of the invention include methods of treatment, the methods comprising administering to an individual an effective dose of an antigen-binding protein as described herein or a pharmaceutical composition as described herein.

[0029] Aspects of the invention relate to the use of an antigen-binding protein as described herein in the preparation of a medicament for treating a disease.

[0030] Aspects of the invention include antigen-binding proteins for treating a disease as described herein.

[0031] In some embodiments, the method or use relates to a human subject (e.g., an individual as a human). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be most fully understood from the following detailed description when read in conjunction with the accompanying drawings. This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is to be emphasized that, in accordance with the common practice, the various features of the drawings are not to scale. On the contrary, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. Included in the drawings are the following figures:

[0033] Figures 1A - 1C . Figure 1A Alignment of the CDR1, CDR2, and CDR3 regions of members of antibody family 2 of SEQ ID NOs: 1-18 that specifically bind human CD3, the CDR1, CDR2, and CDR3 regions corresponding to residues 26-33; 51-58; and 97-112. Figure 1A Disclosed are CDR1 sequences of SEQ ID NOs: 31, 31, 31, 31, 31-35, 35, 31, 31, 36, 31, 35, 31, 31, and 31, a CDR2 sequence of "ISWNSGSI" as SEQ ID NO: 28, and CDR3 sequences of SEQ ID NOs: 37, 37-39, 39, 38, 38, 38, 38, 38, 38, 40, 39, 38-39, 37, 37, and 37, all sequences in the order of occurrence. Figure 1B Shows the CDR1, CDR2, and CDR3 regions of a fixed light chain (SEQ ID NO: 19); and exemplary anti-BCMA sequences (SEQ ID NO: 20 and SEQ ID NO: 21). Figure 1BThe CDR1 sequences are SEQ ID NO:41 - 42 and 42, the CDR2 sequences are SEQ ID NO:43 - 44 and 44, and the CDR3 sequences are SEQ ID NO:45 - 46 and 46, with all sequences arranged in the order of appearance respectively. Figure 1C Provide the CDR sequences of the reference anti - CD3 antibody (SEQ ID NO:22), ID 304704. Figure 1C SEQ ID NO:47 - 49 are disclosed, arranged in the order of appearance respectively.

[0034] Figure 2 . Schematic diagram of the molecule TNB - 383B with an anti - CD3 arm (CD3_F2B or ID:312557) and a high - affinity anti - BCMA arm.

[0035] Figure 3 . Dose - response curve of cytokine release from PBMCs treated with TNB - 383B and a positive control. Pre - cultured PBMCs were stimulated with a positive control (black crosses) or increasing concentrations of TNB - 383B (black squares). The positive control in this experiment is a bispecific anti - CD3 / anti - BCMA antibody (TNB - 384B). The anti - CD3 arm of this bispecific antibody recognizes different epitopes on human CD3 with high affinity (kD = 30 nM, also known as), but the anti - BCMA arm is the same as that of TNB - 383B. The positive control showed a cytokine secretion profile similar to OKT3 (data not shown).

[0036] Figure 4 . Activation profile of T - cell subsets after overnight stimulation with TNB - 383B and a positive control (TNB - 384B). Responses at a stable concentration of the positive control (132 ng / ml, black) and TNB - 383B (1320 ng / ml, patterned) are shown. As described in Example 1, cells were analyzed after a 24 - hour incubation step. After gating on T - cell subsets, the expression of CD69 (percentage of positive cells and mean fluorescence intensity (MFI) of positive cells) was analyzed. The graph shows the median and range obtained from three donors.

[0037] Figure 5 . Combined sequence coverage of DEPC - labeled CD3δ (SEQ ID NO:51) and CD3ε (SEQ ID NO:50) after proteolytic digestion and analysis by LC - MS / MS. The shaded sequences indicate the presence of peptides with DEPC modification. The uncovered sequences are buried and cannot be DEPC - modified.

[0038] Figure 6. In the presence of mAb F2B, the number of Endo-GluC-derived peptides with significantly reduced DEPC labeling. Peptides with at least a 15-fold difference in labeling degree were separately mapped to the positions (shaded) of the ECDs of CD3δ (D) (SEQ ID NO:51) and CD3ε (E) (SEQ ID NO:50).

[0039] Figure 7 . CD3ε-derived proteolytic peptides and their effect on DEPC labeling. Residues found to be labeled are shown in bold and underlined. The greatest effects were observed for Lys 73 and Lys 85. Figure 7 The chymotrypsin sequences are disclosed as SEQ ID NO:52-53, 53-54, 54-55, 55, 55-56, 56-57, and 57, the Glu-C sequences as SEQ ID NO : 58, 58-59, 59, 59-60, and 60, and the trypsin sequences as SEQ ID NO : 61-64, all sequences being presented in the order of appearance.

[0040] Figure 8 . Epitopes of mAb F2B of the CD3δ subunit identified by DEPC labeling. Figure 8 SEQ ID NO:51, 51, and 51 are disclosed.

[0041] Figure 9 . Ribbon diagram of the CD3δ / ε complex derived from the X-ray structure. Residues important for interaction with mAB F2B are highlighted by space filling.

[0042] Figure 10 . Mapping of the CD3ε epitope obtained by DEPC labeling. Figure 10 SEQ ID NO:50 and 65 are disclosed, presented in the order of appearance. Detailed Description

[0043] To facilitate understanding of the present invention, many terms are defined below.

[0044] Before describing the active agents and methods of the present invention, it should be understood that the present invention is not limited to the specific methods, products, devices, and factors described, as such methods, devices, and formulations can of course vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which will be defined only by the appended claims.

[0045] It must be noted that, unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents. Thus, for example, reference to "a drug candidate" refers to one or a mixture of such candidates, and reference to "a method" includes reference to equivalent steps and methods known to those skilled in the art, and the like.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For the purposes of describing and disclosing the devices, formulations, and methods described in the publications and which can be used in conjunction with the invention described herein, all publications mentioned herein are incorporated herein by reference.

[0047] When ranges of values are provided, it is to be understood that unless the context clearly dictates otherwise, each intervening value (to one-tenth of the unit of the lower limit) between the upper and lower limits of the stated range and any other stated value or intervening value in the stated range is included within the invention. The invention also encompasses the upper and lower limits of the smaller ranges that may independently be included within these smaller ranges, subject to any specific excluded limit in the stated range. Ranges excluding one or both of the included limits of such ranges are also included in the invention when the stated range includes one or both of the limits.

[0048] In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention. However, it will be clear to those skilled in the art that the invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures known to those skilled in the art have not been described so as not to obscure the invention.

[0049] Generally, conventional methods of protein synthesis, recombinant cell culture, and protein isolation within the scope of the art, as well as recombinant DNA techniques, are used in the present invention. Such techniques are fully explained in the literature, see, for example, Maniatis, Fritsch and Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0050] Definition

[0051] "Comprising" means that the recited elements are required in the composition / method / kits, but other elements may be included to form a composition / method / kits etc. within the scope of the claims.

[0052] "Consisting essentially of" means a limitation of the scope of a composition or method that describes the specified materials or steps that do not substantially affect the basic and novel features of the present invention.

[0053] "Consisting of" means excluding any element, step or ingredient not specified in the claims from the composition, method or kit.

[0054] As used herein, the terms "treatment", "treating", etc. generally mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms and / or can be therapeutic in terms of partially or completely curing a disease and / or an adverse effect attributable to the disease. "Treatment" as used herein encompasses the treatment of any disease in a mammal and includes: (a) preventing a disease from occurring in a subject who may be predisposed to the disease but has not been diagnosed as having the disease; (b) inhibiting a disease, i.e., arresting the development of the disease; or (c) alleviating a disease, i.e., causing regression of the disease. A therapeutic agent can be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of an ongoing disease, wherein the treatment stabilizes or alleviates the adverse clinical symptoms of the patient. Such treatment is desirably carried out before complete loss of function in the affected tissue. The subject therapy can be administered during the symptomatic stage of the disease and, in some cases, after the symptomatic stage of the disease.

[0055] A "therapeutically effective amount" is intended to be the amount of an active agent that confers a therapeutic benefit on a subject. For example, a "therapeutically effective amount" is an amount that induces, alleviates, or otherwise causes an improvement in a pathological symptom, disease progression, or physiological state associated with a disease or an improvement in resistance to a disorder.

[0056] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject", "individual", and "patient" include, but are not limited to, an individual with cancer, an individual with an autoimmune disease, an individual with a pathogen infection, etc. A subject can be a human and also includes other mammals, particularly those mammals that are suitable as laboratory models for human diseases, such as mice, rats, etc.

[0057] The terms "cancer", "neoplasm", and "tumor" are used interchangeably herein to refer to cells that exhibit autonomous, unregulated growth such that they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. Target cells for detection, analysis, or treatment in the present application include pre-cancerous cells (e.g., benign cells), malignant cells, pre-metastatic cells, metastatic cells, and non-metastatic cells. Cancers of almost every tissue are known. The phrase "cancer burden" refers to the amount or volume of cancer cells in a subject. Thus, reducing the cancer burden refers to reducing the number or volume of cancer cells in a subject. The term "cancer cell" as used herein refers to any cell that is a cancer cell or derived from a cancer cell, such as a clone of a cancer cell. Many types of cancers are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, etc.; and circulating cancers such as leukemias, specifically including B-cell leukemia, T-cell leukemia, etc. Examples of cancers include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.

[0058] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (such as natural killer cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and cause lysis of the target cell. ADCC activity can be evaluated using methods such as those described in U.S. Patent No. 5,821,337. ADCP refers to antibody-dependent cell-mediated phagocytosis.

[0059] "Effector cell" is a leukocyte that expresses one or more constant region receptors and performs effector functions.

[0060] "Cytokine" is a protein released by one cell to act as an intercellular mediator on another cell. Target cytokines include, but are not limited to, cytokines released from activated T cells, such as IL-2, IFNγ, etc.

[0061] "Non-immunogenic" refers to a substance that does not initiate, stimulate, or enhance an immune response, where the immune response includes an adaptive immune response and / or an innate immune response.

[0062] The term "isolated" means that a material has been removed from its original environment (e.g., if it is naturally occurring, its natural environment). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in a natural system is isolated. Such polynucleotides can be part of a vector and / or such polynucleotides or polypeptides can be part of a composition, and they are still isolated because such a vector or composition is not part of its natural environment.

[0063] "Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic and desirable for use in the preparation of pharmaceutical compositions, and includes excipients acceptable for veterinary as well as human pharmaceutical use. Such excipients can be solids, liquids, semi-solids, or in the case of aerosol compositions, gases.

[0064] "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include those that can be formed when an acidic proton present in a compound is capable of reacting with an inorganic or organic base. Suitable inorganic salts include those formed with alkali metals such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxyl, sulfonyl, and phosphonyl groups present in a compound, e.g., C 1-6 alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester can be a monoacid monosalt or ester or a disalt or ester; and similarly when more than two acidic groups are present, some or all of such groups can be salified or esterified. The compounds named in the present invention can exist in unsalted or unesterified form, or in salified and / or esterified form, and the naming of such compounds is intended to include the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. In addition, certain compounds named in the present invention can exist in more than one stereoisomeric form, and the naming of such compounds is intended to include all individual stereoisomers and all mixtures (racemic or otherwise) of such stereoisomers.

[0065] The terms "pharmaceutically acceptable", "physiologically tolerable" and their grammatical variants are used interchangeably when indicating a composition, vehicle, diluent and reagent and mean that the material can be administered to or applied to a human without producing an undesirable physiological effect to the extent that would prohibit the administration of the composition.

[0066] "Homology" between two sequences is determined by sequence identity. If the two sequences to be compared are of different lengths from each other, sequence identity preferably refers to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues of the longer sequence. Computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Unix version 8, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis 53711) can be routinely used to determine sequence identity. Bestfit uses the local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489) to find the segment with the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a specific sequence has, for example, 95% identity with a reference sequence of the present invention, it is preferred to adjust the parameters so that the percentage of identity is calculated over the full length of the reference sequence and allow for at most 5% homology gaps in the total number of nucleotides in the reference sequence. When using Bestfit, it is preferred to leave the so-called optional parameters at their preset ("default") values. Deviations that occur in the comparison between a given sequence and the sequences described above of the present invention may be caused, for example, by addition, deletion, substitution, insertion, or recombination. Preferably, the program "fasta20u66" (version 2.0u66, September 1998, William R. Pearson and University of Virginia; see also W. R. Pearson (1990), Methods in Enzymology 183, 63-98 and the accompanying examples and http: / / workbench.sdsc.edu / ) can also be used to perform such sequence comparisons. For this purpose, the "default" parameter settings can be used.

[0067] A "variant" refers to a polypeptide having an amino acid sequence that is somewhat different from that of a native sequence polypeptide. Generally, the amino acid sequence variant has at least about 80% sequence identity with the sequence, and more preferably, at least about 90% homology. The amino acid sequence variant may have substitutions, deletions, and / or insertions at certain positions within the reference amino acid sequence.

[0068] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell and, thereby, replicate with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors used in recombinant DNA techniques often take the form of plasmids. In the present specification, the terms "plasmid" and "vector" are used interchangeably because the plasmid is the most commonly used form of vector.

[0069] As used herein, the term "host cell" (or "recombinant host cell") is intended to refer to a cell that has been genetically altered or is capable of being genetically altered by the introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector. It should be understood that such terms are intended to refer not only to a particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein.

[0070] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or other binding molecule) and its binding partner (e.g., an antigen or receptor). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies bind antigens (or receptors) weakly and dissociate readily, while high-affinity antibodies bind antigens (or receptors) more tightly and remain bound for a longer time.

[0071] Unless specifically indicated to the contrary, the term "conjugate" as described and claimed herein is defined as a heterogeneous molecule formed by covalently linking one or more antibody fragments to one or more polymer molecules, wherein the heterogeneous molecule is water-soluble, i.e., soluble in physiological fluids such as blood, and wherein the heterogeneous molecule does not contain any structured aggregates. The target conjugate is PEG. In the context of the foregoing definition, the term "structured aggregate" means (1) any molecular aggregate having a globular or globular shell structure in aqueous solution such that the heterogeneous molecule does not assume the form of a micelle or other emulsion structure and is not anchored to a lipid bilayer, vesicle, or liposome; and (2) any molecular aggregate in solid or insoluble form that does not release the heterogeneous molecule into solution upon contact with an aqueous phase, such as a chromatographic bead matrix. Thus, the term "conjugate" as defined herein encompasses the foregoing heterogeneous molecules in the form of precipitates, sediments, bioerodible matrices, or other solids capable of releasing the heterogeneous molecule into an aqueous solution upon solid hydration.

[0072] The term "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze a chemical alteration of a detectable substrate compound or composition.

[0073] "Solid phase" refers to a non-aqueous matrix to which the antibodies of the present invention can adhere. Examples of solid phases encompassed herein include those formed in part or whole from glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicon. In certain embodiments, depending on the context, the solid phase can include the wells of an assay plate; in other instances, it is a purification column (e.g., an affinity chromatography column). This term also includes discontinuous solid phases of discrete particles, such as those described in U.S Patent No. 4,275,149.

[0074] Antibodies, also known as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, wherein the sequences of the amino-terminal domains of the heavy and light chains are variable and are thus commonly referred to as variable domain or variable heavy chain (VH) or variable light chain (VL) domains. The two domains conventionally associate to form a specific binding region, although, as discussed herein, specific binding can also be obtained using only heavy chain variable sequences, and various non-natural configurations of antibodies are known and used in the art.

[0075] "Functionalized" or "bioactive" antibodies or antigen-binding molecules (including the single-chain antibodies and bispecific triclonobody-like molecules (TCA) herein) are antibodies or antigen-binding molecules capable of exerting one or more of their native activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule (such as a TCA) may have the ability to specifically bind an antigen and such binding may in turn cause or alter cellular or molecular events such as signal transduction or enzyme activity. A functional antibody or other binding molecule (such as a TCA) may also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule (such as a TCA) to exert one or more of its native activities depends on several factors, including the correct folding and assembly of the polypeptide chains.

[0076] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, triclonobodies, single-chain Fvs, nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric or derived from other species.

[0077] The term antibody may refer to full-length heavy chains, full-length light chains, intact immunoglobulin molecules; or immunologically active portions of any of these polypeptides, i.e., polypeptides containing antigen-binding sites that immunospecifically bind a target antigen of interest or a portion thereof, these targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc portions that provide reduced or enhanced effector cell activity. Immunoglobulins can be derived from any species. In one aspect, the immunoglobulins are mostly of human origin.

[0078] The term "variable" refers to the fact that certain portions of the variable domains between antibodies vary extensively in sequence and are responsible for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the entire variable domain of the antibody. In the variable domains of the light and heavy chains, the variability is concentrated in three segments called hypervariable regions. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, predominantly adopting a β-sheet conformation connected by three hypervariable regions that form loops and, in some cases, form part of the β-sheet structure. The hypervariable regions of each chain are held tightly together by the FRs and, together with the hypervariable regions from the other chain, contribute to form the antigen-binding site of the antibody (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md.). Although the constant domains do not directly participate in antibody-antigen binding, they exhibit various effector functions, such as the antibody's participation in antibody-dependent cell cytotoxicity (ADCC).

[0079] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region may contain amino acid residues from "complementary determining regions" or "CDRs", and / or those residues from "hypervariable loops". "Framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0080] Exemplary CDR names are shown herein, however those skilled in the art will understand that many definitions of CDRs are commonly used, including the Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010;47:694–700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877–883).Target alternative CDR definitions include, but are not limited to, those disclosed below: Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001; 309:657–670; Ofran et al., “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008; 181:6230–6235; Almagro, “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004; 17:132–143; and Padlan et al., “Identification of specificity-determining residues in antibodies.” Faseb J. 1995; 9:133–139., each of which is hereby expressly incorporated by reference.

[0081] 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 except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody is highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Besides their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0082] The antibodies herein expressly include "chimeric" antibodies in which a portion of the heavy and / or light chain is the same or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is the same or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855). Target chimeric antibodies herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0083] As used herein, a "full antibody chain" is an antibody chain that comprises a full-length variable region and a full-length constant region (Fc). A full "conventional" antibody comprises a full light chain and a full heavy chain of a secreted IgG, as well as the light chain constant domain (CL) and the heavy chain constant domains CH1, hinge, CH2, and CH3. Other isotypes such as IgM or IgA may have different CH domains. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. A full antibody may have one or more "effector functions," which refers to those biological activities that can be attributed to the antibody Fc constant region (native sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include: C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors. Constant region variants include those that alter effector profile, binding to Fc receptors, etc.

[0084] Antibodies and various antigen-binding proteins can be provided in different classes depending on the amino acid sequence of the Fc (constant region) of their heavy chains. There are five main classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The Fc constant domains corresponding to antibodies of different classes can be referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modified or hinge-less forms (Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US2005 / 0048572; US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types (called κ and λ) based on the amino acid sequence of their constant domains.

[0085] A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary effector functions include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require interaction of the Fc region with receptors such as the FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors, and the low affinity FcRn receptor; and can be assessed using various assays as disclosed, for example, in the definitions herein. A "dead" Fc is an Fc that has been mutagenized to maintain activity with respect to, for example, extended serum half-life but does not activate high affinity Fc receptors.

[0086] A "native sequence Fc region" contains an amino acid sequence identical to that of a naturally occurring Fc region. Native sequence human Fc regions include, for example, the native sequence human IgG1 Fc region (non-A and A allotypes); the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, and naturally occurring variants thereof.

[0087] The amino acid sequence included in the "variant Fc region" differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, compared to the native sequence Fc region or the Fc region of the parental polypeptide, the variant Fc region has at least one amino acid substitution, such as from about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of the parental polypeptide, and preferably from about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% homology with the native sequence Fc region and / or with the Fc region of the parental polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology.

[0088] The variant Fc sequence may include three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution of IgG2 residues at 233 - 236 and IgG4 residues at positions 327, 330, and 331 into human IgG1 greatly reduces ADCC and CDC (see, for example, Armour KL. et al., 1999 Eur J Immunol. 29(8):2613 - 24; and Shields RL. et al., 2001. J Biol Chem. 276(9):6591 - 604). Other Fc variants are possible, including but not limited to variants in which regions capable of forming disulfide bonds are deleted or in which certain amino acid residues are eliminated at the N - terminus of the native Fc form or methionine residues are added thereto. Thus, in one embodiment of the present invention, one or more Fc portions of the scFc molecule may include one or more mutations in the hinge region to eliminate disulfide bonding. In another embodiment, the hinge region of Fc may be removed entirely. In still another embodiment, the molecule may include an Fc variant.

[0089] Alternatively, Fc variants can be constructed to remove or substantially reduce effector functions by substituting, deleting or adding amino acid residues to achieve complement binding or Fc receptor binding. For example but not limited to, deletions can occur in complement binding sites, such as in the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. Additionally, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0090] Fc can be in the form of having native glycans, increased glycans compared to the native form, or decreased glycans compared to the native form, or can be in an aglycosylated or deglycosylated form. The increase, decrease, removal, or other modification of glycans can be achieved by methods common in the art, such as chemical methods, enzymatic methods, or by expressing the glycans in genetically engineered production cell lines. Such cell lines can include microorganisms that naturally express glycosylation enzymes, such as Pichia pastoris, and mammalian cell lines, such as CHO cells. Additionally, the microorganisms or cells can be engineered to express glycosylation enzymes, or can be made unable to express glycosylation enzymes (see, for example, Hamilton et al., Science, 313:1441 (2006); Kanda et al., J. Biotechnology, 130:300 (2007); Kitagawa et al., J. Biol. Chem., 269(27):17872 (1994); Ujita-Lee et al., J. Biol. Chem., 264(23):13848 (1989); Imai-Nishiya et al., BMC Biotechnology 7:84 (2007); and WO 07 / 055916). As an example of cells engineered to have altered sialylation activity, the α-2,6-sialyltransferase 1 gene has been engineered into Chinese hamster ovary cells and sf9 cells. Antibodies expressed by these engineered cells are thus sialylated by the foreign gene product. Another method for obtaining Fc molecules having an altered amount of sugar residues compared to a variety of native molecules includes, for example, using lectin affinity chromatography to separate the variety of molecules into glycosylated and non-glycosylated fractions (see, for example, WO 07 / 117505). The presence of specific glycosylation moieties has been shown to alter the function of immunoglobulins. For example, removal of glycans from the Fc molecule results in a significant decrease in the binding affinity to the C1q portion of the first complement component C1 and a decrease or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thus not inducing unwanted immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been associated with anti-inflammatory activity (see, for example, Kaneko et al., Science 313:760 (2006)), while removal of fucose from IgG results in enhanced ADCC activity (see, for example, Shoj-Hosaka et al., J. Biochem., 140:777 (2006)).

[0091] In alternative embodiments, the antibodies of the invention may have an Fc sequence with enhanced effector function, e.g., by increasing its binding to FcγRIIIA and increasing ADCC activity. For example, fucose attached to the N-linked glycan at Asn-297 of the Fc sterically hinders the interaction of the Fc with FcγRIIIA, and removal of fucose by glycoengineering increases binding to FcγRIIIA, which translates into >50-fold higher ADCC activity compared to wild-type IgG1 controls. Protein engineering by amino acid mutations in the Fc portion of IgG1 has generated multiple variants that increase the binding affinity of the Fc for FcγRIIIA. Notably, the tri-alanine mutant S298A / E333A / K334A exhibits a 2-fold increase in binding to FcγRIIIA and ADCC function. The S239D / I332E (2X) and S239D / I332E / A330L (3X) variants have a significant increase in binding affinity for FcγRIIIA and enhanced ADCC capacity in vitro and in vivo. Other Fc variants identified by yeast display also show improved binding to FcγRIIIA and enhanced tumor cell killing in a mouse xenograft model. See, e.g., Liu et al. (2014) JBC 289(6):3571-90, which is specifically incorporated herein by reference.

[0092] The term “Fc region-containing antibody” refers to an antibody that comprises an Fc region. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, an antibody having an Fc region according to the invention may include an antibody having or not having K447.

[0093] “Fv” is the smallest antibody fragment that contains a complete antigen recognition and antigen-binding site. The CD3-binding antibodies of the invention comprise a dimer of one heavy-chain and one light-chain variable domain that are tightly, non-covalently associated; however, additional antibodies, e.g., for use in a multispecific configuration, may comprise a VH in the absence of a VL sequence. Even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind an antigen, although the affinity may be lower compared to a two-domain binding site.

[0094] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a small number of residues (including one or more cysteines from the antibody hinge region) at the carboxyl terminus of the heavy chain CH1 domain. Fab′-SH as used herein refers to a Fab' in which the cysteine residue of the constant domain carries at least one free thiol group. The F(ab')2 antibody fragment is initially produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical conjugations of antibody fragments are also known.

[0095] "Humanized" forms of non-human (e.g., rodent) antibodies (including single-chain antibodies) are chimeric antibodies (including single-chain antibodies) that contain a minimal sequence derived from a non-human immunoglobulin. See, for example, Jones et al., (1986) Nature 321:522-525; Chothia et al. (1989) Nature 342:877; Riechmann et al. (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al. (1993) J. Immunol. 151, 2623-2632; Werther et al. (1996) J. Immunol. Methods 157:4986-4995; and Presta et al. (2001) Thromb. Haemost. 85:379-389. For other details, see U.S. Patent Nos. 5,225,539; 6,548,640; 6,982,321; 5,585,089; 5,693,761; 6,407,213; Jones et al. (1986) Nature, 321:522-525; and Riechmann et al. (1988) Nature 332:323-329.

[0096] As used herein, the term "single-chain antibody" refers to a single polypeptide chain containing one or more antigen-binding domains that bind to epitopes of an antigen, wherein such domains are derived from the variable region of an antibody heavy or light chain or have sequence identity with the variable region. Portions of such variable regions may be encoded by V H or V L gene segments, D and J H gene segments or J L gene segments. The variable region may be formed by rearranged V H DJ H 、V L DJ H 、V H J L or V L JL Encoded by gene segments. The V-, D- and J-gene segments can be derived from humans and various animals, including birds, fish, sharks, mammals, rodents, non-human primates, camels, llamas, rabbits, etc.

[0097] The term "compete", when used in the context of antibodies competing for the same epitope, means competition between the antibodies as determined by an assay in which the test antibody (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antibody (e.g., a ligand or reference antibody) to a common antigen (e.g., CD3 or a fragment thereof). A variety of types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIA using I-125 (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).

[0098] Typically, such assays involve using a purified antigen bound to a solid surface or cell that carries an unlabeled test antibody and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of the test antibody. The test antibody is typically present in excess. Antibodies identified by competitive assays (competitor antibodies) include those that bind the same epitope as the reference antibody, as well as those that bind an adjacent epitope sufficiently close to the epitope bound by the reference antibody such that steric hindrance occurs. Additional details regarding methods for determining competitive binding are provided in the Examples herein. Generally, when the competitor antibody is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antibody to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0099] The term "epitope" includes any determinant capable of being bound by an antibody such as an F2B antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen and, when the antigen is a protein, includes specific amino acids that directly contact the antibody. Epitope determinants can include chemical reactive surface groupings of a molecule such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge characteristics. Generally, an antibody specific for a particular target antigen will preferentially recognize the epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0100] The CD3-binding antibodies of the present invention are particularly useful in multispecific configurations including, but not limited to, bispecific antibodies, trifunctional antibodies, and the like. A variety of methods and protein configurations are known and can be used for bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0101] First-generation BsMAbs consist of two heavy chains and two light chains, each from two different antibodies. The two Fab regions are directed against two antigens. The Fc region consists of two heavy chains and forms a third binding site with the Fc receptor on immune cells (see, e.g., Lindhofer et al., The Journal of Immunology, Vol. 155, pp. 219-225, 1995). The antibodies can be from the same or different species. For example, cell lines expressing rat and mouse antibodies secrete functional bispecific Abs due to preferential species-restricted heavy and light chain pairing. In other embodiments, the Fc regions are designed to pair together only in a specific manner.

[0102] Other types of bispecific antibodies include chemically linked Fabs consisting only of the Fab region. Two chemically linked Fabs or Fab2 fragments form an artificial antibody that binds to two different antigens, making it a type of bispecific antibody. Antigen-binding fragments (Fabs or Fab2) of two different monoclonal antibodies are produced and chemically linked, such as by thioether linkage (see Glennie, M J et al., Journal of immunology 139, pp. 2367-75, 1987; Peter Borchmann et al., Blood, Vol. 100, No. 9, pp. 3101-3107, 2002).

[0103] Various other methods for generating multivalent artificial antibodies have been developed by recombinant fusion of the variable domains of two antibodies. Single-chain variable fragments (scFvs) are fusion proteins of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, which are linked by a short linker peptide of 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. Bispecific single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs with different specificities. A single polypeptide chain with two VH and two VL regions is generated, resulting in a bivalent scFv.

[0104] Bispecific tandem scFvs are also known as bispecific T cell engagers (BiTEs). Bispecific scFvs can be generated with a linker peptide that is too short for the two variable regions to fold together (about 5 amino acids), thus forcing the scFv to dimerize. This type is called diabody (Adams et al., British journal of cancer 77, pp. 1405-12, 1998). The Dual Affinity ReTargeting (DART) platform technology (Macrogenics, Rockville, Md.). This fusion protein technology uses two single-chain variable fragments (scFvs) of different antibodies on a single polypeptide chain of about 55 kilodaltons. SCORPION Therapeutics (Emergent Biosolutions, Inc., Seattle, Wash.) combines two antigen-binding domains in a single-chain protein. Based on the immunoglobulin Fc region, one binding domain is located at the C-terminus and the second binding domain is located at the N-terminus of the effector domain.

[0105] The tetravalent and bispecific antibody-like proteins also include DVD-Ig, which was engineered from two monoclonal antibodies (Wu, C. et al., Nature Biotechnology, 25, pp. 1290-1297, 2007). To construct the DVD-Ig molecule, the V domains of the two mAbs were tandemly fused by a short linker (TVAAP) (SEQ ID NO:25), where the variable domain of the first antibody light chain (VL) is at the N-terminus, followed by the other antibody VL and Ck, to form the DVD-Ig protein light chain. Similarly, the variable regions of the heavy (VH) chains of the two mAbs were tandemly fused by a short linker (ASTKGP) (SEQ ID NO:26), where the first antibody is at the N-terminus, followed by the other antibody and the heavy chain constant domain, to form the DVD-Ig protein heavy chain (VH1 / VL1). All light and heavy chain constant domains are retained in the DVD-Ig design because they are essential for forming a disulfide-bonded intact IgG-like molecule. Co-transfection of mammalian cells with expression vectors encoding the DVD-Ig light and heavy chains results in the secretion of a single species of IgG-like molecule with a molecular weight of approximately 200 kDa. This molecule now has four binding sites, two for each mAb.

[0106] The term "bispecific triabody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule that comprises, consists essentially of, or consists of three polypeptide subunits, two of which comprise a heavy chain and a light chain of a monoclonal antibody or a functional antigen-binding fragment of such an antibody chain that comprises an antigen-binding region and at least one CH domain, consists essentially of, or consists of. This heavy chain / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody, which antibody comprises an Fc portion that comprises CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen, where such binding domain is derived from the variable region of an antibody heavy or light chain or has sequence identity with the variable region of an antibody heavy or light chain. Portions of such variable regions may be encoded by V H and / or V L gene segments, D and J H gene segments or J L gene segments. The variable region may be encoded by a rearranged V H DJ H 、V L DJ H 、V H J L or V L J L gene segments.

[0107] As used herein, a "heavy chain only antibody" or "heavy chain antibody" or "heavy chain polypeptide" of TCA protein refers to a single-chain antibody that comprises the heavy chain constant regions CH2 and / or CH3 and / or CH4 but does not contain the CH1 domain. In one embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a part of the hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a part of the hinge region, and the CH2 domain. In another embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a part of the hinge region, and the CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In another embodiment, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but does not contain a hinge region. The heavy chain only antibody can be in a dimer form, in which two heavy chains are disulfide-bonded to each other, covalently or non-covalently linked to each other. The heavy chain antibody can belong to the IgG subclass, but antibodies belonging to other subclasses (such as IgM, IgA, IgD and IgE subclasses) are also included herein. In a specific embodiment, the heavy chain antibody has an IgG1, IgG2, IgG3 or IgG4 subtype, particularly the IgG1 subtype.

[0108] Heavy chain antibodies constitute approximately one quarter of the IgG antibodies produced by camelids (such as camels and llamas) (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but lack light chains. Thus, the variable antigen-binding portion is called the VHH domain and it represents the smallest naturally occurring complete antigen-binding site with a length of only about 120 amino acids (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity against various antigens can be generated by immunization (van der Linden, R.H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, L.G.J., et al. J. Biotechnol. 78, 11-21 (2000)). Their expression levels, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M.A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies (which is called VNAR) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0109] An antibody or antigen-binding molecule (including the heavy chain antibodies and bispecific tricbodibody-like molecules (TCA) herein) that "binds" to a target antigen is an antibody or antigen-binding molecule that binds to the antigen with sufficient affinity such that the antibody or binding molecule can be used as a diagnostic and / or therapeutic agent targeting the antigen and does not significantly cross-react with other proteins. In such embodiments, the degree of binding of the antibody or other binding molecule to a non-target antigen, as measured by fluorescence-activated cell sorting (FACS) analysis or radioimmuno-precipitation (RIA), will not exceed 10%.

[0110] Protein

[0111] The present invention provides a family of closely related antibodies that bind to CD3 and activate signal transduction through CD3, such as activating CD3 +T cells. Antibodies within the family comprise a set of CDR sequences as defined herein and are exemplified by the provided VH sequences of SEQ ID NO: 1-18. The antibody family provides a number of benefits useful as one or more clinical therapeutic agents. Antibodies within the family include members having a range of binding affinities, allowing selection of a particular sequence having a desired affinity. The ability to fine-tune the affinity is particularly important for managing the level of CD3 activation in the treated individual and thereby reducing toxicity. For example, if targeting a low-abundance tumor antigen (less than 10,000 molecules per cell), a high-affinity CD3 binder (<30 nM) is expected to be preferred. If targeting a high-abundance tumor antigen (more than 50,000 molecules per cell), a CD3 binder with low affinity (>50 nM) is preferred. Evaluation from affinity alone can be the tendency of the antibody to induce cytokine release (e.g., release of IL-2, IFNγ, etc.) upon binding to T cells, where reduced cytokine release may be desirable.

[0112] In another aspect, antibodies are provided that compete with one of the exemplified antibodies or functional fragments that bind to the epitopes described herein for specific binding to CD3. Such antibodies may also bind to the same epitope as one of the antibodies exemplified herein, or to an overlapping epitope. Antibodies and fragments that compete with the exemplified antibodies or bind to the same epitope as them are expected to exhibit similar functional properties. The exemplified antibodies and fragments include the above-mentioned antibodies and fragments, including those having the heavy and light chains, variable domain, and CDRs shown in FIG. 1.

[0113] Such competing antibodies may bind to the F2B epitope but comprise a set of CDR sequences other than those shown in SEQ ID NO: 1-18. The CDR sequences of the heavy chain may be substantially similar but not identical to the CDR sequences shown in SEQ ID NO: 1-18. For example, they may contain 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, or more substitutions in the CDR sequences, where the variations may be present in 1 CDR sequence, 2 CDR sequences, or 3 CDR sequences. The light chain sequences may comprise the set of CDR sequences shown in SEQ ID NO: 19; or they may comprise a different set of CDR sequences.

[0114] Residues directly involved in epitope binding or covered by the antibody can be identified from the scanning results. Thus, these residues can provide an indication of the domain or region of CD3 that contains one or more binding regions bound by the antibody.

[0115] The F2B epitope is characterized by binding to at least one residue selected from CD3ε (SEQ ID NO:23): K73 and S83, and CD3δ (SEQ ID NO:24) K82 and C93. In some embodiments, the epitope comprises the region of CD3ε defined by K73, N74, I75, G76, S77, D78, E79, D80, H81, L82, S83. In some embodiments, the epitope comprises one or both of K73 and S83. In some embodiments, the epitope comprises the region of CD3δ defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, C93. In some embodiments, the epitope comprises one or both of K82 and C93. In some embodiments, the F2B epitope comprises a conformational epitope comprising residues of both CD3δ and CD3ε. In some embodiments, the conformational epitope comprises each of CD3ε K73 and S83, CD3δ K82 and C93. In some embodiments, an antibody that binds to the F2B epitope does not cross-react with cynomolgus monkey CD3 protein. Suitable antibodies can be selected from those provided herein for development and use, including but not limited to use as bispecific antibodies. The affinity of candidate proteins can be determined using methods known in the art (e.g., Biacore measurements, etc.). Members of the antibody family can have an affinity for CD3, where the Kd is about 10 -6 to about 10 -11 or so, including but not limited to: about 10 -6 to about 10 -10 or so; about 10 -6 to about 10 -9 or so; about 10 -6 to about 10 -8 or so; about 10 -8 to about 10 -11 or so; about 10 -8 to about 10 -10 or so; about 10 -8 to about 10 -9 or so; about 10 -9 to about 10 -11 or so; about 10 -9 to about 10 -10 or so; or any value within these ranges. Affinity selection can be confirmed by biological evaluation of T cell activation and evaluation of potential toxicity in, for example, in vitro or preclinical models. The determination of cytokine release can be evaluated using any convenient method, including but not limited to the assays described in the Examples.

[0116] T cell activation is initiated by engaging the T cell receptor (TCR) by binding an MH-peptide complex or an anti-TCR / CD3 antibody. Examples of anti-TCR / CD3 antibodies that activate T cells are OKT3 and UCHT1. These anti-CD3 antibodies cross-compete for binding to CD3 on T cells and are routinely used in T cell activation assays. The anti-CD3 antibodies of the present invention cross-compete with OKT3 for binding to human CD3. Depending on the binding affinity for CD3 and the epitope on CD3, anti-CD3 antibodies activate T cells with different functional outcomes. Incubation of human T cells in vitro with low-affinity anti-CD3 antibodies results in incomplete activation of T cells, low production of IL-2 and IL-10. In contrast, high-affinity CD3 binders activate T cells to produce significantly more IL-2 and other cytokines. Low-affinity anti-CD3 antibodies are considered partial agonists that selectively induce some effector functions, effective tumor killing, and CD69 upregulation without inducing others such as IL-2 and IL-10 production. The strength of the interaction with CD3 and the recognized epitope qualitatively results in different activation of T cells. The maximum cytokine production of T cells activated by low-affinity anti-CD3 antibodies is lower than the maximum activation by high-affinity anti-CD3 antibodies. In some embodiments, when combined with T cells in an activation assay, the antibodies of the present invention result in lower release of one or both of IL-2 and IL-10 when compared to a reference anti-CD3 antibody in the same assay, wherein the reference antibody can be ID 304703 (SEQ ID NO:22) or an antibody with equivalent affinity. The maximum release of IL-2 and / or IL-10 can be less than about 75% of the release by the reference antibody, less than about 50% of the release by the reference antibody, less than about 25% of the release by the reference antibody, and can be less than about 10% of the release by the reference antibody.

[0117] In some embodiments of the present invention, bispecific or multispecific antibodies are provided, which may have any configuration discussed herein, including but not limited to trispecific. The bispecific antibody comprises at least the heavy chain variable region of an antibody specific for a protein other than CD3 and may comprise a heavy chain variable region and a light chain variable region. In some such embodiments, the second antibody specifically binds to a tumor-associated antigen, a target antigen (such as integrin, etc.), a pathogen antigen, a checkpoint protein, etc. Various forms of bispecific antibodies are within the scope of the present invention, including but not limited to single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and their multiple-chain polypeptides.

[0118] The CD3 - specific antibody family contains VH domains, and the VH domains contain CDR1, CDR2, and CDR3 sequences in the human VH framework. As an example, for CDR1, CDR2, and CDR3, the CDR sequences can be in the regions of approximately amino acid residues 26 - 33; 51 - 58; and 97 - 112 of the provided exemplary variable region sequences listed in SEQ ID NO: 1 - 18. Those skilled in the art will understand that if different framework sequences are selected, the CDR sequences can be in different positions, although generally the order of the sequences will remain unchanged.

[0119] The CDR sequences of the family 2 antibodies can have the following sequence patterns. X represents a variable amino acid, which can be a specific amino acid as shown below.

[0120] CDR1 (SEQ ID NO: 27)

[0121] G1 F2 T3 F4 X5 X6 Y7 A8

[0122] Where:

[0123] X5 can be any amino acid; in some embodiments, X5 is D, A, or H; in some embodiments, X5 is D.

[0124] X6 can be any amino acid; in some embodiments, X6 is D or N; in some embodiments, D6 is D.

[0125] In some embodiments, the CDR1 sequence of the family 2 anti - CD3 antibody contains residues 26 - 33 of the sequence listed in any of SEQ ID NO: 1 - 18.

[0126] CDR2 (SEQ ID NO: 28)

[0127] I 1’ S 2’ W 3’ N 4’ S 5’ G 6’ S 7’ I 8’

[0128] In some embodiments, the CDR2 sequence of the family 2 anti - CD3 antibody contains residues 51 - 58 of the sequence listed in any of SEQ ID NO: 1 - 18.

[0129] CDR3 (SEQ ID NO: 29)

[0130] A 1” K [[ID=5~4]] 2” D 3” S4” R 5” G 6” Y 7” G 8” X 9” Y 10” X 11” X 12” G 13” G 12” A 15” Y 16”

[0131] Wherein:

[0132] X 9” can be any amino acid, and in some embodiments, X 9” is D or S; in some embodiments, X 9” is D;

[0133] X 11” can be any amino acid, and in some embodiments, X 11” is R or S;

[0134] X 12” can be any amino acid, and in some embodiments, X 12” is L or R.

[0135] In some embodiments, the CD3 sequence of the family 2 anti-CD3 antibody has the formula AKDSRGYGDYX 11” X 12” GGAY (SEQ ID NO:30), wherein X 11” and X 12” are as defined above. In some embodiments, the CDR3 sequence of the family 2 anti-CD3 antibody comprises the residues 97-112 of the sequence listed in any one of SEQ ID NOs: 1-18.

[0136] In some embodiments, the CD3-binding VH domain pairs with the light chain variable domain. In some such embodiments, the light chain is a fixed light chain. In some embodiments, the light chain comprises a VL domain having CDR1, CDR2, and CDR3 sequences in a human VL framework. The CDR sequences can be those of SEQ ID NO:19. In some embodiments, for CDR1, CDR2, CDR3, the CDR1 sequence comprises amino acid residues 27-32; 50-52; 89-97, respectively.

[0137] In some embodiments, the CDR sequences of the family 2 antibodies have sequences that have at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to one CDR sequence or a set of CDR sequences in any one of SEQ ID NOs: 1-18. In some embodiments, the CDR sequences of the present invention contain one, two, three, or more amino acid substitutions relative to one CDR sequence or a set of CDR sequences in any one of SEQ ID NOs: 1-18. In some embodiments, the one or more amino acid substitutions are in one or more of positions 5 or 10 of CDR1, positions 2, 6, or 7 of CDR2, or positions 1, 8, 9, or 10 of CDR3, relative to the formula provided above.

[0138] When the protein of the present invention is a bispecific antibody, one binding moiety (i.e., the VH / VL combination or just the VH) is specific for human CD3, while the other arm can be specific for target cells, which include cancer cells such as ovarian cancer, breast cancer, gastrointestinal cancer, brain cancer, head and neck cancer, prostate cancer, colon cancer, and lung cancer, etc., as well as hematological malignancies such as B cell tumors (including leukemia), lymphoma, sarcoma, carcinoma, neuronal cell tumors, squamous cell carcinoma, germ cell tumors, metastases, undifferentiated tumors, seminoma, melanoma, myeloma, neuroblastoma, mixed cell tumors, neoplasia caused by infectious agents, and cells of other malignancies; cells infected with a pathogen, autoreactive cells that cause inflammation and / or autoimmunity. The non-CD3 moiety can also be specific for an immunomodulatory protein, as described herein.

[0139] Tumor-associated antigens (TAAs) are relatively restricted to tumor cells, while tumor-specific antigens (TSAs) are unique to tumor cells. TSAs and TAAs are typically part of intracellular molecules that are expressed on the cell surface as part of the major histocompatibility complex.

[0140] Tissue-specific differentiation antigens are molecules present on tumor cells and their normal cell counterparts. Tumor-associated antigens known to be recognized by therapeutic mAbs are divided into several different categories. Hematopoietic differentiation antigens are glycoproteins usually associated with clusters of differentiation (CD) groups and include CD20, CD30, CD33, and CD52. Cell surface differentiation antigens are a diverse group of glycoproteins and carbohydrates found on the surfaces of normal and tumor cells. Antigens involved in growth and differentiation signaling are usually growth factors and growth factor receptors. Growth factors that are targets for antibodies in cancer patients include CEA, epidermal growth factor receptor (EGFR; also known as ERBB1), ERBB2 (also known as HER2), ERBB3, MET (also known as HGFR), insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EPHA3), tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B), and receptor activator of nuclear factor-κB ligand (RANKL; also known as TNFSF11). Antigens involved in angiogenesis are usually proteins or growth factors that support the formation of a new microvascular system and include vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integrin αVβ3, and integrin α5β1. Tumor stroma and extracellular matrix are essential supporting structures of tumors. Stromal and extracellular matrix antigens that are therapeutic targets include fibroblast activation protein (FAP) and tenascin.

[0141] Examples of therapeutic antibodies that can be used in bispecific configurations include, but are not limited to, rituximab; ibritumomab tiuxetan; tositumomab; brentuximab vedotin; gemtuzumab ozogamicin; alemtuzumab; IGN101; adalimumab; ranibizumab; huA33; pemtumomab; oregovomab; CC49 (muromonab); cG250; J591; MOv18; MORAb-003 (farletuzumab); 3F8, ch14.18; KW-2871; hu3S193; IgN311; bevacizumab; IM-2C6; CDP791; edrecolomab; volociximab; cetuximab, panitumumab, nimotuzumab; 806; trastuzumab; pertuzumab; MM-121; AMG 102, METMAB; SCH 900105; AVE1642, IMC-A12, MK-0646, R1507; CP 751871; KB004; IIIA4; mapatumumab (HGS-ETR1); HGS-ETR2; CS-1008; denosumab; ciltoxumab; F19; and 81C6.

[0142] The immunological checkpoint receptors cytotoxic T-lymphocyte associated antigen 4 (CTLA4; also known as CD152) and programmed cell death protein 1 (PD1; also known as CD279), which are most actively studied in the context of clinical cancer immunotherapy, are both inhibitory receptors. The clinical activity of antibodies that block either of these receptors implies that anti-tumor immunity can be enhanced at multiple levels, and combination strategies can be intelligently designed, guided by mechanistic considerations and preclinical models.

[0143] The two ligands of PD1 are PD1 ligand 1 (PDL1; also known as B7-H1 and CD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed on cancer cells, and by binding to its receptor PD1 on T cells, it inhibits T cell activation / function.

[0144] Lymphocyte activation gene 3 (LAG3; also known as CD223), 2B4 (also known as CD244), B and T lymphocyte attenuator (BTLA; also known as CD272), T cell membrane protein 3 (TIM3; also known as HAVcr2), adenosine A2a receptor (A2aR) and the killer inhibitory receptor family are each associated with the inhibition of lymphocyte activity and, in some cases, with the induction of lymphocyte anergy. Antibody targeting of these receptors can be used in the methods of the present invention.

[0145] Agents that agonize immunological co-stimulatory molecules can also be used in the methods of the present invention. Such agents include agonists of CD40 and OX40. CD40 is a co-stimulatory protein found on antigen-presenting cells (APCs) and is required for their activation. These APCs include phagocytes (macrophages and dendritic cells) and B cells. CD40 is part of the TNF receptor family. The main activating signaling molecule of CD40 is and CD40 ligand (CD40L). Macrophages are activated by CD40 stimulation.

[0146] The target anti-CCR4 (CD194) antibodies include humanized monoclonal antibodies against C-C chemokine receptor 4 (CCR4), which have potential anti-inflammatory and anti-tumor activity. CCR2 is expressed on inflammatory macrophages, which can be found in various inflammatory conditions such as rheumatoid arthritis; and has also been identified as being expressed on tumor-promoting macrophages. CCR2 is also expressed on regulatory T cells, and the CCR2 ligand CCL2 mediates the recruitment of regulatory T cells to tumors. Regulatory T cells inhibit the response against anti-tumor T cells and thus their inhibition or depletion is required.

[0147] Produce the proteins of the present invention

[0148] Although antibodies can be prepared by chemical synthesis, they are typically produced by methods of recombinant DNA technology, such as co-expressing all the chains that make up the protein in a single recombinant host cell, or co-expressing a heavy chain polypeptide and an antibody such as a human antibody. In addition, a single polycistronic expression vector can also be used to express the heavy and light chains of an antibody. Standard protein purification techniques, such as affinity (protein A) chromatography, size exclusion chromatography, and / or hydrophobic interaction chromatography, are used to purify the individual polypeptides. Bispecific antibodies are sufficiently different in size and hydrophobicity such that they can be purified using standard methods.

[0149] The amount of antibody and heavy chain polypeptide produced in a single host cell can be engineered by modifying the constant regions of the antibody and heavy chain such that homodimerization is favored over heterodimerization, for example, by introducing self-complementary interactions to minimize (see, for example, WO 98 / 50431 for access possibilities, such as the "protrude into the cavity" strategy (see WO 96 / 27011)). Accordingly, another aspect of the invention is to provide a method for producing a bispecific antibody in a recombinant host, the method comprising the steps of: expressing in a recombinant host cell a nucleic acid sequence encoding at least two heavy chain polypeptides, wherein the heavy chain polypeptides differ in their constant regions sufficiently to reduce or prevent homodimer formation but increase bispecific formation.

[0150] When the protein comprises three chains, such as FlicAb, they can be produced by co-expressing the three chains (2 heavy chains and 1 light chain) that make up the molecule in a single recombinant host cell.

[0151] To recombinantly produce the proteins herein, one or more nucleic acids encoding all the chains (e.g., 2, 3, 4, etc.) are isolated and inserted into a replicable vector for further cloning (amplifying the DNA) or for expression. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0152] In a preferred embodiment, the host cells of the method according to the invention are capable of expressing human immunoglobulins at a high level, i.e., at least 1 pg / cell / day, preferably at least 10 pg / cell / day, and even more preferably at least 20 pg / cell / day or more, without amplifying the nucleic acid molecules encoding the single chains in the host cells.

[0153] Pharmaceutical composition

[0154] Another aspect of the invention is to provide a pharmaceutical composition, which comprises one or more proteins of the invention mixed with a suitable pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier is exemplary but not limited to adjuvants, solid carriers, water, buffers or other carriers used in the art for maintaining therapeutic components or combinations thereof.

[0155] Therapeutic formulations of the proteins used according to the invention are prepared for storage by mixing the protein having the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (see, e.g., Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)), such as in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and include buffers, such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG).

[0156] Anti-CD3 antibody formulations are disclosed, for example, in U.S. Patent Publication No. 20070065437, the entire disclosure of which is expressly incorporated herein by reference. Similar formulations can be used for the proteins of the invention. The main components of such formulations are a pH buffer, salts, surfactants and an effective amount of a bispecific antibody specific for anti-CD3 that is effective in the range of 3.0 to 6.2.

[0157] Method of Use

[0158] Methods for treating or alleviating a disease in a subject are provided, the disease including but not limited to infections, autoimmune diseases, primary or metastatic cancers, etc., the method comprising contacting a target cell with an antigen-binding composition of the present invention, particularly wherein the antigen-binding composition is a multispecific antibody suitable for the condition being treated, such as wherein one binding moiety specifically binds a tumor-associated antigen to treat associated cancer cells; a binding moiety specific for a target pathogen for treating an associated infection, etc. Such methods include administering to a subject in need of treatment a therapeutically effective amount or effective dose of an agent of the present invention, including but not limited to combinations of the agent with chemotherapeutic drugs, radiation therapy, or surgery.

[0159] The effective dose of a composition of the present invention for treating a disease varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Generally, the patient is human, but non-human mammals can also be treated, such as companion animals like dogs, cats, horses, etc., and laboratory mammals like rabbits, mice, rats, etc. The therapeutic dose can be titrated to optimize safety and efficacy.

[0160] The dose level can be readily determined by a ordinarily skilled clinician and modified as needed, for example to alter the subject's response to the treatment. The amount of active ingredient that can be combined with a carrier substance to produce a single dosage form varies depending on the host being treated and the particular mode of administration. Dosage unit forms generally contain between about 1 mg and about 500 mg of active ingredient.

[0161] In some embodiments, the therapeutic dose of the agent can be in the range of about 0.0001 to 100 mg / kg of the host body weight, and more typically 0.01 to 5 mg / kg. For example, the dose can be 1 mg / kg body weight or 10 mg / kg body weight or in the range of 1 - 10 mg / kg. Exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. Usually, the therapeutic entity of the present invention is administered multiple times. The interval between individual doses can be weekly, monthly, or annually. The interval can also be irregular, as indicated by measuring the blood level of the therapeutic entity in the patient. Alternatively, the therapeutic entity of the present invention can be administered as a sustained-release formulation, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the polypeptide in the patient.

[0162] In prophylactic applications, relatively low doses may be administered at relatively infrequent intervals over a long period of time. Some patients continue treatment for the remainder of their lives. In other therapeutic applications, relatively high doses at relatively short intervals may be required until disease progression is reduced or terminated, and preferably until the patient shows partial or complete improvement of the disease symptoms. Thereafter, the pharmaceutical of the present patent may be administered prophylactically.

[0163] In still other embodiments, the methods of the invention include treating, reducing, or preventing tumor growth, tumor metastasis, or tumor invasion of cancer, including carcinoma, hematological cancers such as leukemia and lymphoma, melanoma, sarcoma, glioma, and the like. For prophylactic applications, the pharmaceutical composition or agent is administered to a patient susceptible to or otherwise at risk of the disease in an amount sufficient to eliminate or reduce the risk, alleviate the severity of the disease, or delay the onset of the disease (including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during the development of the disease).

[0164] Compositions for treating a disease may be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular routes. The typical route of administration is intravenous or intratumoral, but other routes may be equally effective.

[0165] Generally, the compositions are prepared as injectables in the form of liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection may also be prepared. The formulations may also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide, or copolymers to enhance adjuvant effects, as discussed above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the invention may be administered in the form of depot injections or implant formulations formulated in a manner that permits sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the United States Food and Drug Administration.

[0166] It can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., by determining LD 50 (the dose lethal to 50% of the population) and LD 100(The dose lethal to 100% of the population) is used to determine the toxicity of the proteins described herein. The dose ratio between toxicity and therapeutic effect is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic for use in the human population. The doses of the proteins described herein are preferably within a range that includes the circulating concentrations of effective doses with minimal or no toxicity. The doses may vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0167] The pharmaceutical compositions can be administered in a variety of unit dosage forms depending on the method of administration. For example, solid dosage forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules, and lozenges. It should be recognized that when administered orally, the compositions of the present invention should be protected from digestion. This is generally accomplished by complexing the molecule with the composition to render the molecule resistant to acidic and enzymatic hydrolysis, or by packaging the molecule in a suitable resistant carrier, such as a liposome or protective barrier. Methods for protecting proteins from digestion are well known in the art.

[0168] Compositions for administration will generally comprise an antibody or other scavenger dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline, etc. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity modifying agents, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and will be selected primarily based on the fluid volume, viscosity, patient weight, etc., according to the particular mode of administration selected and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th Edition, 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0169] Kits containing the active agents of the present invention, their formulations, and instructions for use are also within the scope of the present invention. The kits may also contain at least one additional reagent, for example, a chemotherapeutic agent, etc. The kits generally include a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material provided on, with, or otherwise accompanying the kit.

[0170] The composition can be administered for therapeutic treatment. As described above, the composition is administered to a patient in an amount sufficient to substantially eliminate the targeted cells. The amount sufficient to achieve this is defined as a "therapeutically effective dose", and such amount can provide an improvement in overall survival rate. The composition can be administered in a single or multiple doses according to the dosage and frequency that the patient requires and can tolerate. The specific dose required for treatment will depend on the medical condition and medical history of the mammal, as well as other factors such as age, weight, sex, route of administration, efficacy, etc.

[0171] The present invention has been described in sufficient detail. It will be clear to those of ordinary skill in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

[0172] Examples

[0173] Example 1: Genetically engineered rats expressing only heavy chain antibodies

[0174] The human IgH locus was constructed and assembled in several parts, which involved modifying and ligating the rat C region genes, and then ligating the locus downstream of the human V H 6-D-J H region. Then two BACs with separate human V H gene clusters were co-injected with a BAC encoding the assembled (human V H 6-D-J H -rat C) fragment.

[0175] Transgenic rats carrying an artificial heavy chain immunoglobulin locus in an unrearranged configuration were generated. The constant region genes included encode IgM, IgD, IgG2b, IgE, IgA, and 3' enhancer. RT-PCR and serum analysis (ELISA) of the transgenic rats revealed productive rearrangement of the transgenic immunoglobulin locus and expression of only heavy chain antibodies of various isotypes in the serum. The transgenic rats were crossed with rats having mutated endogenous heavy chain and light chain loci as previously described in U.S. Patent Publication 2009 / 0098134A1. Analysis of such animals demonstrated inactivation of rat immunoglobulin heavy chain and light chain expression and high-level expression of heavy chain antibodies with variable regions encoded by human V, D, and J genes. Immunization of the transgenic rats resulted in a high-titer serum response producing antigen-specific heavy chain antibodies. These transgenic rats expressing heavy chain antibodies with human VDJ regions are called UniRat.

[0176] Example 2: Genetically engineered rats expressing fixed light chain antibodies

[0177] from those having different (V H -D-J H ) nThe combination of rearranged H chains with unique L chains generates a transgenic human antibody repertoire. To this end, rearranged L chains (human Vk-Jk1-Ck) were integrated in the rat germline by DNA microinjection, and the obtained transgenic animals were mated with a rat strain that naturally expresses the human H chain repertoire described previously (Osborn et al., 2013). This new rat strain was named OmniFlic.

[0178] Immunization of OmniFlic rats with a number of different antigens generates high levels of antigen-specific IgG, similar to other transgenic rats carrying the same IgH locus. Lineage analysis by RT-PCR identified highly variable V H -gene rearrangements at high transcript and protein levels. In addition, only one L-chain product that was also expressed at high levels was identified.

[0179] Antigen-specific binders from OmniFlic were obtained by NGS and selected from cDNA libraries (yeast, E. coli, phage), which identified different H-chain transcripts after sequencing. For expression in mammalian cells, hypermutated H-chain constructs were transfected in combination with the original transgenic Igk sequence. In this rearranged Vk-Jk1-Ck, mutational changes were not allowed, and the same L-chain was always expressed with various H-chain products to generate monoclonal human IgG.

[0180] Example 3: Antigen-specific antibodies are generated in transgenic rats,

[0181] To generate antigen-specific heavy chain antibodies in rats, genetically engineered rats were immunized in two ways.

[0182] Immunize with the recombinant extracellular domains of PD-L1 and BCMA. The recombinant extracellular domains of PD-L1 and BCMA were purchased from R&D Systems and diluted with sterile saline and combined with adjuvants. The immunogens were combined with complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) or Titermax and Ribi adjuvants. The first immunization (prime) with the immunogen in CFA or Titermax was administered in the left and right legs. After the first immunization with the immunogen in CFA, two additional immunizations (boosts) in IFA or four additional immunizations in Ribi and one additional immunization in Titermax were administered in each leg. This immunization schedule led to the development of B cells that produce high-affinity antibodies. The immunogen concentration was 10 micrograms per leg. Serum was collected from the rats at the final bleeding to determine the serum titer.

[0183] To generate anti-human CD3δε antibodies, genetically engineered rats were immunized using a DNA-based immunization protocol. OmniFlic rats were immunized with human and cynomolgus monkey CD3-ε / δ constructs using GENOVAC antibody technology at Aldevron, Inc. (Fargo, ND). Draining lymph nodes were harvested and RNA isolated after the final boost. After cDNA synthesis, IgH heavy chain antibody repertoires were characterized by next-generation sequencing and proprietary software. Candidate antigen-specific VH sequences showing evidence of positive selection for antigen specificity were selected. Hundreds of VH sequences encoding FlicAb were selected for gene assembly and cloned into expression vectors. Subsequently, fully human FlicAb IgG1 antibodies were expressed in HEK cells for analysis by flow cytometry and ELISA. The binding of human FlicAb to primary human T cells and Jurkat cells was tested by flow cytometry. In addition, human FlicAb was tested using recombinant CD3δε protein in ELISA. All FlicAb with positive binding to human T cells are listed in Figure 1. The selected sequences were further characterized in T cell activation assays.

[0184] Example 4: Analysis of activation of Treg cells

[0185] CD69 is a cell surface marker on T cells that is upregulated upon stimulation. In this experiment, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Biocol (density 1.077 g / ml) and standard methods. The isolated PBMCs were pre-cultured in complete medium at 2 x 10 7 cells / ml for 48 hours, washed and resuspended in complete medium at 10 6 cells / ml. These cells were incubated in FACS tubes (BD Falcon Corning) coated with recombinant BCMA protein for 24 hours. The tubes were pre-coated overnight with 10 μg / ml of recombinant BCMA protein.

[0186] Wash the cells and stain with antibodies specific for different subsets of T cells, namely: (1) CD4-positive T cells (T4-anti-huCD4-ECD), (2) CD8-positive T cells (anti-huCD8-AF700), and (3) Treg cells are defined by positivity for CD4, CD25, and the intracellular marker Fox-p3 (anti-huCD25-PECy7, anti-Foxp3-AF647, anti-huCD25-PECy7, all antibodies obtained from Beckman). Analyze the cells on a Cytoflex flow cytometer using an appropriate template. Contrary to currently available anti-CD3 bispecific molecules, TNB-383B preferentially activates CD4+ and CD8+ T cells rather than Treg cells. Blocking the activation of Treg cells, an immunosuppressive cell type, can enhance CD8+ T cell function and increase the immune destruction of tumor targets. Data are as Figure 3 and Figure 4 shown.

[0187] Example 5: Expression and purification of CD3δ / ε Fc fusion protein

[0188] The Figure 5 recombinant antigens CD3ε (SEQ ID NO:23, extracellular domain (ECD) residues 22 - 105) and CD3δ (SEQ ID NO:24, ECD residues 23 - 126) shown were cloned in-frame with murine IgG1 Fc, transiently co-expressed, and purified from CHO cell culture medium. A C-terminal His-tag added to the CD3ε subunit was used for affinity capture of the CD3δ / ε complex by IMAC using a standard protocol and elution with imidazole. A second purification affinity tag with the sequence EPEA (SEQ ID NO:66) (C-tag) was added to the C-terminus of the CD3δ subunit.

[0189] Example 6: Epitope mapping of mAb CD3 F2B

[0190] Labeling of surface residues. Epitope mapping was achieved by surface residue labeling with diethyl pyrocarbonate (DEPC) (reference), which covalently reacts with the accessible side chains of the amino acids histidine, lysine, tyrosine, cysteine, serine, threonine, and the free N-terminal amino group. DEPC labeling was carried out at an antibody:antigen molar ratio of 30:1 to drive complex formation. The DEPC-labeled antibody-antigen complex was captured on a capture select C-tag affinity matrix (Thermo Fisher Scientific), and the excess antibody was removed by extensive washing. The affinity resin-bound CD3δ / ε was subjected to standard reduction and alkylation methods prior to digestion with trypsin, chymotrypsin, and endopeptidase Glu-C. The released peptides were analyzed by LC-MS / MS. The same DEPC labeling experiment was carried out in the absence of the CD3 antibody F2B, followed by affinity capture, reduction / alkylation, and protease digestion. Each digestion was performed in triplicate. In the absence of mAb CD3 F2B, the sequence coverage derived from mass spectrometry was high, as Figure 6 shown.

[0191] DEPC labeling of CD3δ / ε is affected by the binding of mAb CD3 F2B (heavy chain of SEQ ID NO:1 and light chain of SEQ ID NO:19). Peptides obtained from three proteolytic digests were analyzed, and there was a significant reduction in DEPC-modified residues due to the presence of mAb CD3F2B during the labeling process. Figure 7 Shown as a representative example is the affected peptide obtained from the digest using endopeptidase Glu-C.

[0192] An effect on amino acid residue labeling was considered significant if a) the reduction in DEPC incorporation was more than 15-fold and b) it was detected in at least two of the three digests. The affected CD3ε peptides are shown as Figure 7 shown. A consistent and significant labeling effect for Lyc73 was observed in both chymotrypsin peptides and Glu-C peptides. Additionally, Glu-C peptides I57-E86 and D80-E86 provided further evidence that the epitope of mAb F2B extends at least to lysine 85 within the ε chain.

[0193] Affected CD3δ peptides were also found. One sequence segment affected by the tag was common to each of the three digestion groups. These data suggest the epitope of mAb F2B from lysine 82 to cysteine 93. Figure 8 The identified epitope (shaded) in the CD3δ protein is described. Figure 9The crystal structure of CD3δ / ε is shown (PDB ID code 1XIW, Arnett K. et al., Proc Natl Acad Sci U S A. 2004;101(46):16268–16273), where the affected residues in each CD3 subunit are shown in space-filling mode.

[0194] Example 7: The CD3 family 2 antibody recognizes an epitope different from OKT3 and SP34.

[0195] The same epitope mapping method was used to confirm the known interaction between the therapeutic antibody OKT3 and CD3δ / ε (Salmeron, A., Sanchez-Madrid, F., Ursa, M.A., Fresno, M. & Alarcon, B. (1991) J. Immunol. 147, 3047-3052.). The cross-reactive antibody SP-34 has been characterized (U.S. Patent 8,236,308, 2012). This antibody recognizes an epitope within the extended E-F loop of CD3ε and does not require CD3δ for binding.

[0196] Our data set confirms that the CD3 antibody F2B binds a different epitope compared to OKT3 and SP-34. Figure 10 Alignment of human and cynomolgus macaque ECD is shown. The CD3 mAbs provided herein bind to loops that are not present in the cynomolgus macaque homologs, which further explains why these antibodies do not show monkey cross-reactivity to the CD3 complex. Additionally, neither OKT3 nor SP-34 binds directly to CD3δ.

[0197] Examples are presented to provide a complete disclosure and illustration to those of ordinary skill in the art of how to make and use the invention, and are not intended to limit the scope in which the inventors regard their invention, nor are they intended to represent that the following experiments are all or the only experiments conducted. Although efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius and pressure is at or near atmospheric pressure.

[0198] Although the present invention has been described with reference to its specific embodiments, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, method, method step or step to the purposes, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. An isolated monoclonal antigen-binding protein that binds to CD3, wherein the isolated monoclonal antibody binds to an epitope on CD3, and the epitope comprises at least one residue selected from CD3ε (SEQ ID NO: 23): K73 and S83; and CD3δ (SEQ ID NO: 23): K82 and C93.

2. The antigen-binding protein according to claim 1, wherein the epitope on CD3 comprises the region of CD3δ defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, C93.

3. The antigen-binding protein according to claim 1, wherein the epitope on CD3 comprises the region of CD3ε defined by K73, N74, I75, G76, S77, D78, E79, D80, H81, L82, S83.

4. The antigen-binding protein according to any one of claims 1-3, wherein the epitope comprises a conformational epitope having residues of both CD3ε and CD3δ.

5. The antigen-binding protein according to any one of claims 1-4, wherein the conformational epitope comprises each of the residues CD3ε K73 and S83, CD3δ K82 and C93.

6. The antigen-binding protein according to any one of claims 1-5, wherein the antibody does not cross-react with cynomolgus monkey CD3 protein.

7. The antigen-binding protein according to any one of claims 1-6, wherein the antigen-binding protein induces cytokine release upon binding to T cells, and the cytokine release does not exceed about 200% of the maximum cytokine release observed for the F2B antibody.

8. The antigen-binding protein according to any one of claims 1-7, wherein the binding affinity for CD3 is 50 nM or greater.

9. The isolated monoclonal antigen-binding protein according to any one of claims 1-8, wherein the isolated monoclonal antigen-binding protein is a human antibody.

10. The isolated monoclonal antigen-binding protein according to any one of claims 1-8, wherein the isolated monoclonal antigen-binding protein is a humanized antibody.

11. The antigen-binding protein according to any one of claims 1-10, wherein the variable region of the light chain comprises a set of CDR sequences in SEQ ID NO:

19.

12. The antigen-binding protein according to any one of claims 1-11, wherein the variable light chain domain comprises the amino acid sequence of SEQ ID NO:

19.

13. The antigen-binding protein according to any one of claims 1-12, wherein the antibody comprises a set of CDR sequences other than those shown in SEQ ID NOs: 1-18.

14. The antigen-binding protein according to any one of claims 1-13, which further comprises an Fc region.

15. The antigen-binding protein according to claim 14, wherein the Fc region has been engineered to reduce effector function.

16. The antigen-binding protein according to any one of claims 1-15, wherein the protein is single-chain.

17. The antigen-binding protein according to any one of claims 1-15, wherein the protein is double-stranded or a multiple thereof.

18. The antigen-binding protein according to any one of claims 1-15, wherein the protein is triple-stranded.

19. The antigen-binding protein according to any one of claims 1-15, wherein the protein is triple-stranded, and the two antigen-binding arms comprise an antibody heavy chain and a light chain.

20. The antigen-binding protein according to any one of claims 1-15, wherein the protein further comprises a variable heavy chain domain specific for a protein other than CD3.

21. The antigen-binding protein according to any one of claims 1-20, wherein the protein further comprises a variable heavy chain domain specific for a protein other than CD3; wherein when contacting T cells in an activation assay, the antigen-binding protein induces a reduced level of release of one or both of IL-2 and IL6 relative to a reference anti-CD3 antibody; and induces more than 30% tumor cell cytotoxicity in a standard in vitro assay using tumor cells and human T cells.

22. The antigen-binding protein according to claim 21, wherein the variable heavy chain domain specific for a protein other than CD3 is a heavy chain domain only.

23. The antigen-binding protein according to claim 21, wherein the variable heavy chain domain specific for a protein other than CD3 further comprises a light chain variable region.

24. The antigen-binding protein according to claim 21, wherein the light chain variable region is the same as the light chain variable region of the CD3-binding region.

25. The antigen-binding protein according to any one of claims 21-24, wherein the protein other than CD3 is a tumor-associated antigen.

26. The antigen-binding protein according to any one of claims 21-24, wherein the protein other than CD3 is a pathogen antigen.

27. The antigen-binding protein according to any one of claims 21-24, wherein the protein other than CD3 is an immunomodulatory protein.

28. A pharmaceutical composition comprising the antigen-binding protein according to any one of claims 1-27.

29. The pharmaceutical composition according to claim 28, which is a unit dosage form.

30. A polynucleotide encoding the antigen-binding protein according to any one of claims 1-27.

31. A vector comprising the polynucleotide according to claim 30.

32. A cell comprising the vector according to claim 31.

33. A method for producing the antigen-binding protein according to any one of claims 1-27, the method comprising growing the cell according to claim 32 under conditions permitting the expression of the protein, and isolating the protein from the cell and / or the cell culture medium.

34. A method of treatment comprising administering to an individual an effective dose of the antigen-binding protein according to any one of claims 1-27, or the pharmaceutical composition according to claim 28.

35. Use of the antigen-binding protein according to any one of claims 1-27 in the preparation of a medicament for the treatment of a disease.

36. The antigen-binding protein according to any one of claims 1-27, which is used for treating a disease.

37. The method or use according to any one of claims 34-36, wherein the individual is a human.

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