CD3 delta and CD3 epsilon on heterodimer-specific antibodies

Abstract

This invention provides novel human CD3 antigen-binding polypeptides, their preparation, and their use in the treatment and / or diagnosis of various diseases. [Solution] The isolated monoclonal antigen-binding protein that binds to CD3, and the isolated monoclonal antibody binds to an epitope on CD3 that includes at least one residue selected from CD3 epsilon:K73 and S83, which include a specific sequence, and CD3 delta K82 and C93, which include another specific sequence.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 610,764, filed on December 27, 2017, the disclosure of which is hereby incorporated by reference in its entirety. Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The name of the aforementioned ASCII copy, created on March 5, 2019, is TNO-0010-WO4_SL.txt, and its size is 39,719 bytes.

[0002] The present invention relates to novel human CD3 antigen - binding polypeptides, their preparation, and their use in the treatment and / or diagnosis of various diseases, to bispecific antibody molecules capable of activating immune effector cells, and to their use in the diagnosis and / or treatment of various diseases.

Background Art

[0003] Background The body's immune system plays a role in defense against infection, injury, and cancer. The humoral immune system and the cellular immune system, two separate but interrelated systems, work together to protect the body. The humoral system is mediated by soluble factors called antibodies and neutralizes products recognized as foreign in the body. In contrast, the cellular system includes cells such as T cells and macrophages that remove and neutralize foreign invaders.

[0004] Activation of T cells is important for stimulating the immune response. T cells exhibit immunological specificity and drive most cellular immune responses. T cells do not secrete antibodies but are required for antibody secretion by B lymphocytes. T cell activation requires the involvement of a number of 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 membrane glycoprotein with disulfide - bonded heterodimers, chains, alpha and beta (α and β), or gamma and delta (γ and δ). The TcR is non - covalently associated with a complex of invariant proteins called CD3.

[0005] T cells are known to exert a potent anti-tumor effect in a number of experimental settings. Antibodies that can effectively mobilize T cells against tumor cells were available, for example, as bispecific antibodies directed against tumor-associated antigens (TAAs), as well as agonistic 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 specific lysis of cells bearing each TAA.

[0006] However, anti-CD3 bispecific antibodies can direct T cell-mediated lysis towards malignant cells, but clinical trials with CD3-based bSAbs have shown high toxicity in patients. Non-specific T cell activation from bSAbs can occur antigen-independently due to Fc / Fc receptor (FcR) interactions or antigen-dependently when the antigen is expressed on both normal and tumor cells. Both mechanisms may have been responsible for the toxicity observed in previous clinical trials. See, for example, Link et al. (1998) Int. J. Cancer 77(2):251-6; Durben et al. Molecular Therapy (2015); 23 4, 648-655. As a result of the resulting cytokine release syndrome, it has been an important block in the development of these antibodies for therapeutic purposes.

[0007] The interaction between the T cell receptor (TCR) and its peptide-MHC ligand determines T cell activity. The binding properties of this interaction have been studied in great detail and have been shown to regulate T cell function. The strength and nature of the TCR-peptide / MHC interaction determine whether T cells exert effector function or are inactivated and deleted. Antibodies against CD3 activate T cells by altering the conformation of the CD3ε chain, and depending on the epitope, which may have either agonist or antagonist effects on T cells (Yoon et al., 1994 Immunity 1:563-569). Given the significant side effects of many T cell agonists, it may be preferable to maintain a potent antitumor effect while reducing the release of pro-inflammatory cytokines. However, partial agonist anti-CD3 antibodies alter the CD3ε chain to a suboptimal state, resulting in ineffective signaling, while most anti-CD3 antibodies are full agonists for both pathways. It remains unclear whether these effector functions can be isolated. Many existing anti-CD3 antibodies (e.g., SP-34, UCHT1, OKT3) have affinity in the range of 1–50 nM KD, which may not be optimal for therapeutic use.

[0008] CD3-specific antibodies and bispecific antibodies derived therefrom are provided by the present invention.

[0009] public CD3 antibodies are disclosed, for example, in U.S. Patents 5,585,097, 5,929,212, 5,968,509, 6,706,265, 6,750,325, 7,381,803, and 7,728,114. Bispecific antibodies having CD3 binding specificity are disclosed, for example, in U.S. Patents 7,262,276, 7,635,472, 7,862,813, and 8,236,308, each specifically incorporated herein by reference. CD3 binding antibody sequences are provided in concurrently pending application PCT US2017 / 038377, which is specifically incorporated herein by reference. [Overview of the Initiative]

[0010] overview The composition and its method of use bind to CD3 and activate signal transduction via CD3, for example, CD3 + This invention provides antibodies for activating T cells. The antibodies are characterized by their binding to a CD3 epitope to which the F2B antibody binds, which may be referred to herein as the F2B epitope. The F2B antibody comprises the set of CDR sequences of SEQ ID NO: 1 and the fixed light chain sequence of SEQ ID NO: 19. In some embodiments, the antibody that binds to the F2B epitope comprises heavy chain variable region sequences other than those shown in SEQ ID NOs: 1-18.

[0011] Antibodies that bind to the F2B epitope offer significant advantages in terms of biological activity. The antibodies maintain effective tumor cell lysis while minimizing the release of toxic cytokines. In some embodiments, binding of anti-CD3 antibodies to the F2B epitope is characterized by a reduced tendency to induce cytokine release, such as IL-2 and IFNγ, upon binding to competent T cells. Without being constrained by theory, binding to specific epitopes recognized by F2B is considered to provide the unique and beneficial properties of the antibodies described herein.

[0012] Antibodies that bind to the F2B epitope are approximately 10 -6 ~about 10 -11 The selection may be based on the binding affinity to CD3 within the following ranges. Anti-CD3 antibodies with affinity (KD) of 50 nM or greater, 100 nM or greater, 500 nM or greater, or 1 μM or greater may be 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 that is approximately 200% or less of the maximum cytokine release observed with F2B antibodies, and may be approximately 150% or less, 125% or less, or 100% or less, and may be smaller than the maximum value observed for F2B in comparative assays can be selected.

[0013] In comparative in vitro assays, antibodies that induce maximal IL-2 and IL-10 release of less than 20%, 30%, or 50% of the maximal release of a control anti-CD3 antibody, such as OKT-3 or TNB-383B, may be selected.

[0014] The F2B epitope is characterized by binding to at least one residue selected from CD3 epsilon (SEQ ID NO: 23): K73 and S83, and CD3 delta (SEQ ID NO: 24): K82 and C93. In some embodiments, the epitope includes a region of CD3 epsilon defined by K73, N74, I75, G76, S77, D78, E79, D80, H81, L82, and S83. In some embodiments, the epitope includes one or both of K73 and S83. In some embodiments, the epitope includes a region of CD3 delta defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, and C93. In some embodiments, the epitope includes one or both of K82 and C93. In some embodiments, the F2B epitope comprises a conformational epitope containing both CD3 delta and CD3 epsilon residues. In some embodiments, the conformational epitope contains residues CD3 K73 and S83; and CD3 K82 and C93, respectively. In some embodiments, antibodies that bind to the F2B epitope do not cross-react with cynomolgus monkey CD3 protein.

[0015] In some embodiments, the antibody that binds to the F2B epitope is determined by a competitive assay between the antibody disclosed herein and other antibodies. In some embodiments, the antibody binds to a specific residue of CD3 that reduces cytokine release.

[0016] In some embodiments, bispecific or multispecific antibodies are provided that include at least a heavy chain variable region derived from an antibody that binds to an F2B epitope. The bispecific antibody includes a heavy chain variable region of an antibody that is specific to at least a protein other than CD3, and may include heavy chain and light chain variable regions. In some such embodiments, the second antibody specifically binds to tumor-associated antigens, such as target antigens like integrins, pathogen antigens, checkpoint proteins, etc. Various forms of bispecific antibodies, including but not limited to single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and multiples thereof, are within the scope of the present invention.

[0017] In some embodiments, the F2B epitope-binding antibody of the present invention includes a CD3-binding variable region paired with a light chain. In some embodiments, the light chain includes a variable region containing the variable region sequence described in SEQ ID NO: 19, or a set of CDR sequences and framework sequences of SEQ ID NO: 19. Various Fc sequences are used, including but not limited to human IgG1, IgG2a, IgG2b, IgG3, IgG4, etc. In some embodiments, the second arm of the bispecific antibody includes a variable region that specifically binds to tumor-associated antigens. In some embodiments, the second arm of the bispecific antibody includes a variable region that specifically binds to BCMA. In some embodiments, the anti-BCMA arm is a single-stranded variable region, for example, as shown in Figure 2B.

[0018] In other embodiments, pharmaceutical compositions are provided comprising at least the CD3-binding VH domain of the present invention, an antibody or antibody-like protein having monospecificity, bispecificity, or the like containing at least the CD3-binding VH domain of the present invention, and a pharmaceutically acceptable excipient. The composition may be lyophilized, suspended in a solution, or provided in unit dose formulations.

[0019] In several embodiments, methods for treating cancer are provided, the methods comprising administering an effective amount of the antibody of the present invention, such as monospecificity or bispecificity, to an individual in need thereof. When the antibody is bispecific, a second antigen-binding site may specifically bind to a tumor antigen, checkpoint protein, etc. In various embodiments, cancer is selected from the group consisting of ovarian cancer, breast cancer, gastrointestinal cancer, brain tumor, head and neck cancer, prostate cancer, colon cancer, lung cancer, leukemia, lymphoma, sarcoma, carcinoma, neuronal tumor, squamous cell carcinoma, germ cell tumor, metastasis, undifferentiated tumor, seminoma, melanoma, myeloma, neuroblastoma, mixed cell tumor, and neoplasm formation due to infectious pathogens.

[0020] In some embodiments, methods for treating infectious diseases are provided, the methods comprising administering an effective amount of the antibody of the present invention, such as monospecific or bispecific, to an individual in need. If the antibody is bispecific, a second antigen-binding site may specifically bind to a pathogenic antigen, such as bacteria, viruses, or parasites.

[0021] In other embodiments, a method for producing a bispecific antibody of the present invention is provided, comprising expressing an antibody sequence, for example, one or more light chain coding sequences and one or more heavy chain coding sequences, in a single host cell. In various embodiments, the host cell may be a prokaryotic cell or a eukaryotic cell such as a mammalian cell.

[0022] Aspects of the present invention include a method for producing an antigen-binding protein as described herein, comprising growing host cells under conditions that allow for protein expression, and isolating the protein from the cells and / or cell culture medium.

[0023] Aspects of the present invention include therapeutic methods comprising administering an effective dose of an antigen-binding protein or a pharmaceutical composition described herein to an individual.

[0024] Aspects of the present invention relate to the use of antigen-binding proteins described herein in the preparation of agents for the treatment of diseases.

[0025] Aspects of the present invention include antigen-binding proteins described herein for use in the treatment of diseases.

[0026] In some embodiments, the method or use includes a human subject (e.g., an individual that is human). [Brief explanation of the drawing]

[0027] This invention is best understood from the following detailed description in conjunction with the accompanying drawings. The patent or application file includes at least one drawing made in color. A copy of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fees. As is common practice, it is emphasized that the various features in the drawings are not at the same scale. Conversely, the dimensions of the various features are enlarged or reduced as appropriate for clarity. The drawings include the following figures:

[0028] [Figure 1]1A-1C. Figure 1A shows the alignment of the CDR1, 2, and 3 regions of antibody family 2 members with sequence numbers 1-18, which specifically bind to human CD3, corresponding to residues 26-33, 51-58, and 97-112. Figure 1A discloses the CDR1 sequences as sequence numbers 31, 31, 31, 31, 31-35, 35, 31, 31, 36, 31, 35, 31, 31, and 31, all in order of appearance; the CDR2 sequence "ISWNSGSI" as sequence number 28; and the CDR3 sequences as sequence numbers 37, 37-39, 39, 38, 38, 38, 38, 38, 38, 40, 39, 38-39, 37, 37, and 37. Figure 1B shows the fixed light chain CDR1, 2, and 3 regions (SEQ ID NO: 19), as well as exemplary anti-BCMA sequences (SEQ ID NO: 20 and 21). Figure 1B discloses the CDR1 sequence as SEQ ID NOs. 41-42 and 42, the CDR2 sequence as SEQ ID NOs. 43-44 and 44, and the CDR3 sequence as SEQ ID NOs. 45-46 and 46, all in order of appearance. Figure 1C provides the CDR sequence of the reference anti-CD3 antibody (SEQ ID NO: 22), ID 304704. Figure 1C discloses SEQ ID NOs. 47-49, all in order of appearance. [Figure 2] A schematic diagram of the molecule TNB-383B, which has an anti-CD3 arm (CD3_F2B or ID:312557) and a high-affinity anti-BCMA arm. [Figure 3] Dose-response curves of cytokine release by PBMCs treated with TNB-383B and positive control. Pre-cultured PBMCs were stimulated with increasing concentrations of positive control (black cross) or TNB-383B (black square). The positive control in this experiment was a bispecific anti-CD3 / anti-BCMA antibody (TNB-384B). The anti-CD3 arm of this bispecific antibody recognizes different epitopes of human CD3 with high affinity (kD=30nM, also known as), while the anti-BCMA arm is the same as the anti-BCMA arm of TNB-383B. This positive control showed a cytokine secretion profile similar to OKT3 (data not shown). [Figure 4]Activation profiles of T cell subsets after overnight stimulation with TNB-383B and a positive control (TNB-384B). Shows plateau concentrations for the positive control (132 ng / ml, black) and TNB-383B (1320 ng / ml, pattern). Cells were analyzed after a 24-hour incubation step as described in Example 1. CD69 expression was analyzed by gating the T cell subsets (percentage of positive cells and mean fluorescence intensity (MFI) of positive cells). The graph shows median and range values ​​obtained from three donors. [Figure 5] Sequence coverage of DEPC-labeled CD3 delta (SEQ ID NO: 51) and CD3 epsilon (SEQ ID NO: 50) after proteolytic digestion and analysis by LC-MS / MS. Shaded sequences indicate the presence of peptides with DEPC modifications. Uncovered sequences are embedded and inaccessible due to the lack of DEPC modification. [Figure 6] The number of Endo-GluC-derived peptides whose DEPC labeling was significantly reduced in the presence of mAb F2B. Peptides with at least a 15-fold difference in labeling are mapped to the CD3 delta (D) (SEQ ID NO: 51) and CD3 epsilon (E) (SEQ ID NO: 50)ECD positions, respectively (shaded). [Figure 7] The effects of CD3 epsilon-derived proteolytic peptides on their DEPC labeling. Residues that were found to be labeled are shown in bold and underlined. The greatest effects were observed at Lys73 and Lysine 85. Figure 7 discloses, in order of appearance, chymotrypsin sequences as SEQ ID NOs. 52-53, 53-54, 54-55, 55, 55-56, 56-57, and 57, Glu-C sequences as SEQ ID NOs. 58, 58-59, 59, 59-60, and 60, and trypsin sequences as SEQ ID NOs. 61-64. [Figure 8] The epitope of the mAb F2B of the CD3 delta subunit identified by DEPC labeling. Figure 8 discloses sequence numbers 51, 51, and 51. [Figure 9]The CD3 delta / epsilon complex was elucidated by ribbon plotting of the X-ray structure. Residues important for interaction with mAB F2B are highlighted by space emphasis. [Figure 10] Mapping of CD3 epsilon epitopes obtained by DEPC labeling. Figure 10 discloses sequence numbers 50 and 65, in order of appearance. [Modes for carrying out the invention]

[0029] Detailed explanation To facilitate understanding of the present invention, several terms are defined below.

[0030] Before describing the activators and methods, it should be understood that the present invention is not limited to the specific methodologies, products, apparatus, and factors described, and that such methods, apparatus, and formulations may naturally change. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and do not limit the scope of the present invention, which is limited only by the appended claims.

[0031] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless otherwise explicitly indicated by the context. For example, a reference to “candidate drug” refers to one or a mixture of such candidates, and a reference to “the method” includes references to equivalent processes and methods known to those skilled in the art.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, formulations, and methodologies described herein and that may be used in connection with the inventions described herein.

[0033] Where a range of values ​​is provided, each intervening value is understood to be one-tenth of the lower limit unit between the upper and lower limits of that range and any other stated or intervening value, unless otherwise clearly indicated in the context. These smaller upper and lower limits may independently be included in smaller ranges and are included within the stated range, subject to any specifically excluded limitations. Where a stated range includes one or both limits, the range excluding either of those limits is also included in the present invention.

[0034] In the following description, numerous specific details are given to provide a more complete understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out in the absence of one or more of these specific details. In other examples, features and procedures well known to those skilled in the art are not described in order to avoid obscuring the invention.

[0035] Generally, conventional methods of protein synthesis, recombinant cell culture, protein isolation, and recombinant DNA technology, which are within the scope of the art of those skilled in the art, are employed in the present invention. Such techniques are well described in the literature; see, for example, Maniatis, Fritsch & 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).

[0036] definition "Includes" means that the enumerated elements are required in the composition / method / kit, but other elements may be included to form the composition / method / kit, etc., within the scope of the claims.

[0037] "Essentially consisting of" means a limitation on the scope of a composition or method described in a particular material or process that does not substantially affect the basic and novel features(plural) of the present invention.

[0038] "Consists of" means the exclusion of any element, process or component not specified in the claim from the composition, method or kit.

[0039] The terms “treatment,” “to treat,” etc., are used herein in general to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or eliminates adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in a mammal and includes, hereafter, (a) preventing the onset of the disease in a subject that may be predisposed to the disease but has not yet been diagnosed with it, (b) inhibiting the disease, i.e., preventing its development, or (c) alleviating or regressing the disease. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient, is of particular note. Such treatment is preferably performed prior to the complete loss of function in the affected tissue. Therapies of the subject may be administered during, and possibly after, the symptomatic stages of the disease.

[0040] The "therapeutic dose" refers to the amount of activator necessary to provide a therapeutic benefit to the subject. For example, the "therapeutic dose" is the amount that induces, improves, or otherwise causes improvement of disease-related pathological symptoms, disease progression, or physiological condition, or improves resistance to the disorder.

[0041] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being evaluated for treatment and / or treated. In one embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. The subject may be a human, but may also include other mammals, in particular mammals useful as experimental models of human diseases, such as mice and rats.

[0042] The terms “cancer,” “neoplasm,” and “tumor” are used interchangeably herein and refer to cells exhibiting autonomous, unregulated growth, such as an abnormal proliferation phenotype characterized by a marked loss of control over cell proliferation. Cells targeted for detection, analysis, or treatment in this application include precancerous (e.g., benign), malignant, premetastatic, metastatic, and nonmetastatic cells. Cancers of virtually any tissue are known. The term “cancer load” refers to a quantum of cancer cells or tumor volume of interest. Therefore, reducing the cancer load means reducing the number of cancer cells or tumor volume of interest. As used herein, the term “cancer cell” refers to any cell that is a cancer cell or derived from a cancer cell, e.g., a clone of a cancer cell. Many types of cancer are known to those skilled in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, and circulating cancers such as leukemias, including B-cell leukemias and T-cell leukemias. Examples of cancer include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, and brain tumors.

[0043] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors, such as natural killer cells, neutrophils, and macrophages, recognize bound antibodies on target cells, causing the target cells to lyse. 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.

[0044] "Effector cells" are white blood cells that express one or more constant-region receptors and perform effector functions.

[0045] "Cytokines" are proteins released by one cell that act as intercellular mediators on other cells. While not limited to these, target cytokines include those released by activated T cells, such as IL-2 and IFNγ.

[0046] "Non-immunogenic" refers to a substance that does not initiate, induce, or enhance an immune response, including adaptive and / or innate immune responses.

[0047] The term "isolated" means that a substance is removed from its original environment (e.g., the natural environment if it exists naturally). 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 substances coexisting in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition can still be isolated in that it is not part of its natural environment.

[0048] "Pharmacologically acceptable excipients" generally mean excipients that are safe, non-toxic, and useful in the preparation of desirable pharmaceutical compositions, and include excipients acceptable for veterinary and human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous.

[0049] "Pharmacologically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and possess the desired pharmacological properties. Such salts include those that can be formed when acidic protons present in a compound can react with inorganic or organic bases. 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 amine bases, such as ethanolamine, diethanolamine, triethanolamine, tromethamine and N-methylglucamine. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) as well as organic acids (e.g., acetic acid, citric acid, maleic acid, and alkanes and arenesulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include those with carboxyl, sulfonyloxy and phosphonoxy groups present in a compound, such as C 1-6 This includes esters formed from alkyl esters. If two acidic groups are present, the pharmaceutically acceptable salt or ester may be a monoacid monosalt or ester or diacid or ester; similarly, if more than two acidic groups are present, some or all of such groups may be chlorinated or esterified. The compounds named in this invention may exist in unchlorinated or unesterified forms, or in chlorinated and / or esterified forms, and the naming of such compounds is intended to include both the original (unchlorinated and unesterified) compounds and their pharmaceutically acceptable salts and esters. Furthermore, certain compounds named in this invention may exist in two or more stereoisomers, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.

[0050] The terms "pharmaceutically acceptable," "physiologically tolerable," and their grammatical variations are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that a substance can be administered to or onto humans without producing undesirable physiological effects to the extent that they interfere with the administration of the composition.

[0051] The "homology" between two sequences is determined by sequence identity. When the two sequences being compared are of different lengths, sequence identity is preferably related to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues in the longer sequence. Sequence identity can conventionally be determined using a computer program such as the Bestfit program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711). Bestfit utilizes the local homology algorithm from Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to find the segment with the highest sequence identity between the two sequences. When using Bestfit or other sequence alignment programs to determine whether a particular sequence has, for example, 95% identity with the reference sequence of the present invention, the parameters are preferably adjusted so that the percentage of identity is calculated over the entire length of the reference sequence and so that a homology gap of up to 5% of the total number of nucleotides in the reference sequence is permitted. When using Bestfit, the so-called optional parameters are preferably left at their preset ("default") values. Deviations that appear in the comparison of a given sequence with the above-mentioned sequence of the present invention may be caused, for example, by additions, deletions, substitutions, insertions, or recombinations. Such sequence comparisons may also preferably be performed using the program "fasta20u66" (version 2.0u66, September 1998, William R. Pearson and the University of Virginia; WRPearson (1990), Methods in Enzymology 183, 63-98, attached example, and see also http: / / workbench.sdsc.edu / ). For this purpose, the "default" parameter settings may be used.

[0052] A "mutant" refers to a polypeptide that has an amino acid sequence that differs to some extent from the natural polypeptide sequence. Typically, amino acid sequence mutants will have at least about 80% sequence identity, and more preferably at least about 90% sequence homology. Amino acid sequence mutants may have substitutions, deletions, and / or insertions at specific positions within the reference amino acid sequence.

[0053] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which is a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated into a viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can be directed to the expression of a gene to which they are operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “recombinant vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this specification, “plasmid” and “vector” may be used interchangeably, as plasmids are the most commonly used form of vector.

[0054] As used herein, the term “host cell” (or “recombinant host cell”) is intended to mean a cell that has been genetically modified or can be genetically altered by the introduction of exogenous polynucleotides, such as recombinant plasmids or vectors. It should be understood that such a term is intended to refer not only to a specific target cell but also to the offspring of such a cell. Because certain alterations may occur in a given generation due to mutation or environmental influences, such offspring may not be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0055] "Binding affinity" generally refers to the sum of the 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 common methods known in the art, including those described herein. Low-affinity antibodies tend to bind weakly to antigens (or receptors) and dissociate easily, while high-affinity antibodies bind more strongly to antigens (or receptors) and remain bound for longer periods.

[0056] Unless otherwise specified, the term “conjugate” as used herein and claimed is defined as heteromolecules formed by the covalent bonding of one or more antibody fragments to one or more polymer molecules, wherein the heteromolecules are water-soluble, i.e., soluble in physiological fluids such as blood, and the heteromolecules do not include any structured aggregates. The conjugate of interest is PEG. In the context of the definitions herein, the term “structured aggregate” means (1) any aggregate of molecules in aqueous solution having a spheroid or spheroid shell structure such that the heteromolecules are not immobilized in a micelle or other emulsion structure, but rather in a lipid bilayer, vesicle or liposome, and (2) aggregates of molecules in a solid or insoluble form that do not release heteromolecules into solution upon contact with the aqueous phase, such as a chromatography bead matrix. Thus, the term “conjugate” as used herein includes the heteromolecules in precipitates, deposits, biodegradable matrices or other solids that can release heteromolecules into aqueous solution upon hydration of the solid.

[0057] As used herein, the term “label” means a detectable compound or composition that is directly or indirectly conjugated to an antibody. A label may be detectable on its own (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition.

[0058] "Solid phase" means a non-aqueous matrix to which the antibodies of the present invention can adhere. Examples of solid phases incorporated herein include glass (e.g., controlled-pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicones to which some or all of the solid phase is formed. In certain embodiments, depending on the context, the solid phase may constitute wells of an assay plate, and in others, it may be a purification column (e.g., an affinity chromatography column). The term also includes discontinuous solid phases of individual particles, such as those described in U.S. Patent No. 4,275,149.

[0059] Antibodies, also called immunoglobulins, typically contain at least one heavy chain and one light chain, with the amino-terminal domains of the heavy and light chains being sequence-variable. Therefore, they are generally referred to as variable region domains, or variable heavy (VH) domains or variable light (VH) domains. Conventionally, the two domains associate to form a specific binding region, but as discussed herein, specific binding can also be obtained with a variable sequence of the heavy chain alone, and various unnatural configurations of antibodies are known and used in the art.

[0060] A “functional” or “biologically active” antibody or antigen-binding molecule (including, in this specification, heavy-chain-only antibodies and bispecific triple-chain antibody-like molecules (TCAs)) is capable of exhibiting one or more of its intrinsic activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, e.g., a TCA, may have the ability to specifically bind to an antigen, and this binding may induce or modify cellular or molecular events such as signal transduction or enzymatic activity. Functional antibodies or other binding molecules, e.g., TCAs, may also block ligand activation of receptors or act as agonists or antagonists. The ability of an antibody or other binding molecule, e.g., a TCA, to exhibit one or more of its intrinsic activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0061] In this specification, the term "antibody" is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, polymers, multispecific antibodies (e.g., bispecific antibodies), antibodies consisting only of heavy chains, triple-chain antibodies, single-chain Fv, nanobodies, and antibody fragments insofar as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be derived from mouse, human, humanized, chimeric, or other species.

[0062] The term antibody may refer to a polypeptide comprising a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an antigen-binding site that immune-specifically binds to an antigen or part thereof of a target of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any kind (e.g., IgG, IgE, IgM, IgD, and IgA), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass of an immunoglobulin molecule, including an operational subclass having a modified Fc moiety that provides reduced or enhanced effector cell activity. The immunoglobulins may originate from any species. In one embodiment, the immunoglobulins are largely of human origin.

[0063] The term "variable" refers to the fact that specific portions of the variable domain differ significantly in sequence between antibodies, and these differences are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is not uniformly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The native heavy-chain and light-chain variable domains each contain four FRs that primarily employ a beta-sheet structure, linked by three hypervariable regions that form loops linking the beta-sheet structure, or in some cases, form parts of the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs, and together with the hypervariable regions from other chains, they contribute to the formation of the antibody's antigen-binding site (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as the antibody's involvement in antibody-dependent cytotoxicity (ADCC).

[0064] As used herein, the term “hypervariable region” refers to an amino acid residue of an antibody involved in antigen binding. The hypervariable region may include amino acid residues derived from the “complementarity-determining region” or “CDR,” and / or these residues derived from the “hypervariable loop.” “Framework region” or “FR” residues are variable domain residues other than the hypervariable region residues as defined herein.

[0065] While exemplary CDR designations are provided herein, those skilled in the art will understand that many definitions of CDR, including Kabat's, are commonly used (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. Chothia's definition is based on the location of structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877-883).While not limiting, other definitions of CDRs exist, including: 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. This includes the disclosures made in 1995;9:133-139, each of which is specifically incorporated herein by reference.

[0066] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within the population are identical except for any naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being able to be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method.

[0067] The antibodies described herein specifically include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the sequence of an antibody derived from another species or belonging to another antibody class or subclass, and a corresponding fragment of such an antibody, insofar as it exhibits the desired biological activity (U.S. Patent No. 4,816,567, and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). The chimeric antibodies of interest herein include “primatized” antibodies that include a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0068] As used herein, “intact antibody chain” includes a full-length variable region and a full-length constant region (Fc). An intact “conventional” antibody includes an intact light chain and an intact heavy chain, as well as the light chain constant domain (CL) and heavy chain constant domains CH1, hinge, CH2, and CH3 for secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domain may be the constant domain of the natural sequence (e.g., the human natural sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more “effector functions” that signify biological activity attributable to the antibody’s Fc constant region (natural 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 the effector profile, such as binding to Fc receptors.

[0069] Depending on the amino acid sequence of the Fc (constant domain) of their heavy chains, antibodies and various antigen-binding proteins can be offered as different "classes." There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The Ig form includes either a hinged or hingeless form (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US2005 / 0048572; 2004 / 0229310). The light chains of antibodies derived 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.

[0070] A “functional Fc region” possesses “effector function” of a naturally occurring Fc region. Exemplary effector functions include C1q binding, CDC, Fc receptor binding, ADCC, ADCP, and downregulation of cell surface receptors (e.g., B cell receptors). Such effector functions generally require the Fc region to interact with receptors, such as FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and low-affinity FcRn receptors, and can be evaluated using various assays, for example, as disclosed herein. A “dead” Fc is one that has been mutagenically altered to retain activity, for example with respect to an extended serum half-life, but does not activate high-affinity Fc receptors.

[0071] "Natural Fc regions" include amino acid sequences identical to those of naturally occurring Fc regions. Natural human Fc regions include, for example, natural human IgG1 Fc regions (non-A and A allotypes), natural human IgG2 Fc regions, natural human IgG3 Fc regions, and natural human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0072] A "mutant Fc region" includes an amino acid sequence different from the amino acid sequence of the Fc region of the natural sequence, due to at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the mutant Fc region has at least one amino acid substitution compared to the Fc region of the natural sequence or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the Fc region of the natural sequence or the Fc region of the parent polypeptide. The mutant Fc region described herein preferably has at least about 80% homology, most preferably at least about 90% homology, and more preferably at least about 95% homology.

[0073] The variant Fc sequence may contain 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 at 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 positions 233–236 and IgG4 residues at positions 327, 330, and 331 with human IgG1 significantly 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, but are not limited to those lacking a region capable of forming a disulfide bond, or those in which a specific amino acid residue is removed from the N-terminus of the native Fc form, or to which a methionine residue is added. Thus, in one embodiment of the present invention, one or more Fc portions of the ScFc molecule may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of Fc may be completely removed. In yet another embodiment, the molecule may include an Fc variant.

[0074] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting, deleting, or adding amino acid residues to enable complement binding or Fc receptor binding. For example, but not limited to, deletions may occur in complement binding sites such as C1q binding sites. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO97 / 34631 and WO96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

[0075] Fc may be in a form having the natural glycans, increased glycans compared to the natural form, or decreased glycans compared to the natural form, or it may be in a non-glycosylated or deglycosylated form. Increase, decrease, removal or other modification of glycans can be achieved by methods common in the art, such as chemical or enzymatic methods, or by expressing them in genetically engineered producing cell lines. Such cell lines may include microorganisms (e.g., Pichia Pastoris) and mammalian cell lines (e.g., CHO cells) that naturally express glycosylationases. Furthermore, microorganisms or cells can be engineered to express glycosylation enzymes or to be unable to express them (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 WO07 / 055916). As an example of cells engineered to alter sialylation activity, the alpha-2,6-sialyltransferase 1 gene has been engineered in Chinese hamster ovary cells and sf9 cells. Therefore, antibodies expressed by these manipulated cells are sialylated by exogenous gene products. Further methods for obtaining Fc molecules with modified amounts of sugar residues compared to multiple native molecules include, for example, separating the above molecules into glycosylated and non-glycosylated fractions using lectin affinity chromatography (see, for example, WO07 / 117505). The presence of specific glycosylated moieties has been shown to alter the function of immunoglobulins.For example, removal of glycans from Fc molecules leads to a sharp decrease in the binding affinity of the first complement component C1 to the C1q portion, and a decrease or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby preventing the induction of unnecessary immune responses in vivo. Further important modifications include sialylation and fucosylation; the presence of sialic acid in IgG correlates with anti-inflammatory activity (see, e.g., Kaneko, et al, Science 313:760 (2006)), while removal of fucose from IgG leads to an enhancement of ADCC activity (see, e.g., Shoj-Hosaka, et al, J. Biochem., 140:777 (2006)).

[0076] In alternative embodiments, the antibodies of the present invention may have Fc sequences with enhanced effector function, for example, by increasing binding affinity to FcγRIIIA and thereby enhancing ADCC activity. For example, fucose bound to an N-linked glycan at Asn-297 of Fc sterically hinders the interaction between Fc and FcγRIIIA, and removal of fucose by sugar manipulation can increase binding to FcγRIIIA, which is bridged to >50-fold higher ADCC activity compared to wild-type IgG1 control. Protein engineering has generated several mutants that increase the affinity of Fc binding to FcγRIIIA through amino acid mutations in the Fc portion of IgG1. In particular, the triple alanine mutant S298A / E333A / K334A shows a 2-fold increase in binding to FcγRIIIA and ADCC function. The S239D / I332E(2×) and S239D / I332E / A330L(3×) mutants exhibit a significant increase in binding affinity to FcγRIIIA and enhanced ADCC capacity in vitro and in vivo. Other Fc mutants identified by yeast display also showed improved binding to FcγRIIIA and enhanced tumor cell killing in mouse xenograft models. See, for example, Liu et al. (2014) JBC 289(6):3571-90, which is specifically incorporated herein by reference.

[0077] The term "antibody containing an Fc region" refers to an antibody that contains an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody purification or by recombination of the nucleic acid encoding the antibody. Therefore, antibodies having an Fc region according to the present invention may include antibodies that have or do not have K447.

[0078] "Fv" is a minimal antibody fragment containing a complete antigen recognition and antigen-binding site. The CD3-binding antibody of the present invention contains a tightly and non-covalently dimer of one heavy chain variable domain and one light chain variable domain, but may contain additional antibodies, e.g., VH in the absence of the VL sequence, for use in multispecific configurations. Although the affinity may be lower than that of the two-domain binding site, a single variable domain (or half of Fv containing only three antigen-specific hypervariable regions) also has the ability to recognize and bind to the antigen.

[0079] The Fab fragment also includes 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 in that it has a few residues added to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue(s) of the constant domain have at least one free thiol group. The F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments having a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0080] The "humanized" form of a non-human (e.g., rodent) antibody that includes a single-chain antibody is a chimeric antibody (including a single-chain antibody) that contains 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 further 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.

[0081] As used herein, the term "single-chain antibody" means a single polypeptide chain that includes one or more antigen-binding domains that bind to an epitope of an antigen, such domains being derived from or having sequence identity with the variable region of an antibody heavy or light chain. A portion of such variable region can be encoded by V H or V L gene segments, D and J H gene segments, or J L gene segments. The variable region can be a rearranged V H DJ H , V L DJ H , V H J L or V L J LThese can be encoded by gene segments. The V-, D-, and J- gene segments can originate from a variety of animals, including humans and birds, fish, sharks, mammals, rodents, non-human primates, camels, llamas, rabbits, and others.

[0082] The term "competing" when used in the context of antibodies competing for the same epitope means competition between antibodies such that the antibodies being tested (e.g., an antibody or an immunologically functional fragment thereof) prevent or inhibit (e.g., reduce) 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), as determined by the assay. Many types of competitive binding assays, e.g., solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competitive assays (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 labeling assays, solid-phase direct labeling sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIR (see, e.g., Cheung, et al., See Virology 176:546-552, 1990, and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82) can be used to determine whether one antibody competes with another.

[0083] Typically, such assays involve the use of purified antigen bound to a solid surface or cell carrying either an unlabeled test antibody or 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. Typically, the test antibody is present in excess. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to an adjacent epitope sufficiently proximal to the epitope to which the reference antibody binds to cause steric hindrance. Further details on methods for determining competitive binding are provided in the examples herein. Typically, when competitive antibodies are present in excess, the specific binding of the reference antibody to the common antigen is inhibited (e.g., reduced) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or more than 97%.

[0084] The term "epitope" includes any determinant groups to which an antibody, such as an F2B antibody, can bind. An epitope is a region of an antigen to which an antibody targeting that antigen binds, and when the antigen is a protein, it includes specific amino acids that directly contact the antibody. Epitope determinants can include a population of chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can have specific three-dimensional structural properties and / or specific charge properties. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on the target antigen in complex mixtures of proteins and / or macromolecules.

[0085] The CD3-binding antibody of the present invention is particularly useful in multispecific configurations, including, but is not limited to, bispecific antibodies and trifunctional antibodies. A wide variety of methods and protein configurations are known and are used in bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0086] First-generation BsMAbs consisted of two heavy chains and two light chains, each derived from two different antibodies. The two Fab regions were directed to two antigens. The Fc region consisted of two heavy chains and formed a third binding site with an Fc receptor on immune cells (see, e.g., Lindhofer et al., The Journal of Immunology, Vol 155, pp. 219-225, 1995). The antibodies may be derived from the same species or different species. For example, cell lines expressing rat and mouse antibodies secrete functionally bispecific Abs for preferentially species-restricted heavy and light chain pairing. In other embodiments, the Fc regions are designed solely to fit together in a specific manner.

[0087] Other types of bispecific antibodies contain chemically linked Fabs consisting only of Fab regions. Two chemically linked Fab or Fab2 fragments form an artificial antibody that binds to two different antigens, making it a type of bispecific antibody. Antigen-binding fragments (Fab or Fab2) of two different monoclonal antibodies are produced and linked by chemical means such as thioethers (see Glennie, MJ et al., Journal of Immunology 139, pp. 2367-75, 1987; Peter Borchmann et al., Blood, Vol.100, No.9, pp. 3101-3107, 2002).

[0088] Various other methods for producing multivalent artificial antibodies have been developed by recombinate fusion of the variable domains of two antibodies. A single-stranded variable fragment (scFv) is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, linked to 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. Bi-specific single-stranded variable fragments (di-scFv, bi-scFv) can be manipulated by linking two scFv with different specificities. A single peptide chain with two VH and two VL regions is produced, yielding a bivalent scFv.

[0089] Bispecific tandem scFvs are also known as bispecific T cell engagers (BiTEs). Bispecific scFvs can be constructed using a linker peptide that forces dimerization of scFvs whose two variable regions are too short (about 5 amino acids) to fold together. This type is known as a diabody (Adams et al., British Journal of Cancer 77, p1405-12, 1998). Dual-Affinity Re-Targeting (DART) platform technology (Macrogenics, Rockville, Md). This fusion protein technology uses two single-stranded variable fragments (scFvs) of different antibodies on a single peptide chain of about 55 kilodaltons. SCORPION Therapeutics (Emergent Biosolutions, Inc., Seattle, Wash.) combines two antigen-binding domains in a single-stranded protein. Based on the immunoglobulin Fc region, one binding domain is located at the C-terminus, and a second binding domain is located at the N-terminus of the effector domain.

[0090] Tetravalent and bispecific antibody-like proteins also include DVD-Ig, which is engineered from two monoclonal antibodies (Wu, C. et al., Nature Biotechnology, 25, p1290-1297, 2007). To construct the DVD-Ig molecule, the V domains of two mAbs are combined with the variable domain of the N-terminal first antibody light (VL) chain and a short linker (TVAAP). (Sequence ID 25) These are fused in tandem, followed by other antibodies VL and Ck, to form the DVD-Ig protein light chain. Similarly, the variable regions of the heavy (VH) chains of the two mAbs are linked by a short linker (ASTKGP). (Sequence ID 26) This involves tandem fusion with the first antibody at the N-terminus, followed by fusion with other antibodies and the heavy chain constant domain to form the DVD-Ig protein heavy chain (VH1 / VL1). All light and heavy chain constant domains are conserved in the DVD-Ig design because they are crucial for the formation of a disulfide-bonded, fully IgG-like molecule. Simultaneous transfection of mammalian cells with an expression vector 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 has four binding sites, two from each mAb.

[0091] The term “bispecific triple-chain antibody-like molecule” or “TCA” is used herein to mean an antibody-like molecule comprising, essentially or consisting of, three polypeptide subunits, two of which are essentially or consist of one heavy chain and one light chain of a monoclonal antibody, comprising an antigen-binding region and at least one CH domain, or a functional antigen-binding fragment of such an antibody chain. This heavy / light chain pair has binding specificity to a first antigen. The third polypeptide subunit is essentially or consists of an antibody consisting only of a heavy chain comprising an Fc portion including 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, the such binding domain originating from or having sequence identity with a variable region of the antibody heavy or light chain. A portion of such a variable region is V H and / or V L Gene segments, D and J H gene segment, or J L It can be encoded by gene segments. The variable region is rearranged V H DJ H , V L DJ H , V H J L , or V L J L It can be encoded by a gene segment.

[0092] As used herein, “TCA protein utilizes heavy-chain-only antibodies,” “heavy-chain antibody,” or “heavy-chain polypeptide” means a single-chain antibody that includes the heavy-chain constant region CH2 domain and / or CH3 domain and / or CH4 domain but does not include the CH1 domain. In one embodiment, the heavy-chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH2 and CH3 domains. In another embodiment, the heavy-chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH2 domain. In a further embodiment, the heavy-chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH3 domain. Heavy-chain antibodies in which the CH2 and / or CH3 domains are cleaved are also included herein. In a further embodiment, the heavy chain consists of an antigen-binding domain and at least one CH(CH1, CH2, CH3, or CH4) domain but does not include the hinge region. The heavy-chain-only antibody may have two heavy chains disulfide-bonded, or otherwise may be in the form of a dimer covalently or noncovalently bonded to each other. Heavy chain antibodies may belong to the IgG subclass, but antibodies belonging to other subclasses such as the IgM, IgA, IgD, and IgE subclasses are also included herein. In certain embodiments, the heavy chain antibody is an IgG1, IgG2, IgG3, or IgG4 subtype, in particular an IgG1 subtype.

[0093] Heavy chain antibodies constitute about a 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 from two heavy chains but lack a light chain. As a result, the variable antigen-binding region is called the VHH domain, which represents the smallest naturally occurring intact antigen-binding site, consisting of only about 120 amino acids (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Highly specific and affinity heavy chain antibodies can be produced against various antigens by immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH moiety can be readily cloned and expressed in yeast (Frenken, LGJ, 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, MA et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to possess a single VH-like domain in the antibody 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)).

[0094] The antibodies or antigen-binding molecules described herein, including heavy-chain-only antibodies and bispecific triple-chain antibody-like molecules (TCAs), "bind" to the target antigen, but with sufficient affinity, and such antibodies or binding molecules are useful as diagnostic and / or therapeutic agents when targeting antigens and do not significantly cross-react with other proteins. In such embodiments, the degree of binding of the antibody or other binding molecule to the non-target antigen is 10% or less, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0095] protein The present invention binds to CD3 and activates signal transduction via CD3 (for example, CD3 + This invention provides a family of antibodies closely related to T cell activation. The antibodies within the family comprise a set of CDR sequences as defined herein, exemplified by the provided VH sequences, SEQ ID NOs. 1-18. The antibody family offers numerous benefits that contribute to its usefulness as a clinical therapeutic agent. The antibodies within the family comprise members with a range of binding affinities, allowing for the selection of specific sequences with desired affinity. The ability to fine-tune affinity is particularly important for controlling the level of CD3 activation in the treated individual, thereby reducing toxicity. For example, when targeting small, abundant tumor antigens (less than 10,000 molecules per cell), a high-affinity CD3 conjugate (<30 nM) is predicted to be preferable. When targeting very abundant tumor antigens (more than 50,000 molecules per cell), a CD3 conjugate with low affinity (>50 nM) is preferred. Apart from affinity, another consideration may be the tendency of the antibody to induce cytokine release, such as IL-2, IFNγ, etc., when a reduction in cytokine release is desired, upon binding to T cells.

[0096] In another embodiment, antibodies are provided that compete for specific binding to CD3 with one of the exemplary antibodies or functional fragments that bind to the epitopes described herein. Such antibodies may also bind to the same epitope as, or to, one of the antibodies described herein, or to an overlapping epitope. Antibodies and fragments that compete for or bind to the same epitope as the exemplary antibodies are expected to exhibit similar functional properties. The exemplary antibodies and fragments include those described above, including those having heavy and light chains, a variable region domain and a CDR as shown in Figure 1.

[0097] Such competing antibodies may bind to the F2B epitope but include a set of CDR sequences other than those shown in SEQ ID NOs: 1-18. The heavy chain CDR sequences may be substantially similar to, but not identical to, those shown in SEQ ID NOs: 1-18, and may include, for example, one amino acid substitution, one amino acid substitution, three amino acid substitutions, or more, where the changes may be found in one CDR sequence, two CDR sequences, or three CDR sequences. The light chain sequences may include the set of CDR sequences shown in SEQ ID NO: 19, or may include a different set of CDR sequences.

[0098] Residues directly involved in epitope binding, or those coated with antibodies, can be identified from the scan results. Therefore, these residues can provide indicators of the CD3 domain or region containing the binding region(s) to which the antibody binds.

[0099] The F2B epitope is characterized by binding to at least one residue selected from CD3 epsilon (SEQ ID NO: 23): K73 and S83, and CD3 delta (SEQ ID NO: 24): K82 and C93. In some embodiments, the epitope includes a region of CD3 epsilon defined by K73, N74, I75, G76, S77, D78, E79, D80, H81, L82, and S83. In some embodiments, the epitope includes one or both of K73 and S83. In some embodiments, the epitope includes a region of CD3 delta defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, and C93. In some embodiments, the epitope includes one or both of K82 and C93. In some embodiments, the F2B epitope comprises a conformational epitope containing both CD3 delta and CD3 epsilon residues. In some embodiments, the conformational epitope contains residues CD3ε K73 and S83; CD3δ K82 and C93, respectively. In some embodiments, antibodies that bind to the F2B epitope do not cross-react with cynomolgus monkey CD3 protein. Suitable antibodies, including but not limited to use as bispecific antibodies, may be selected from those provided herein for development and use. Affinity determination for candidate proteins may be carried out using methods known in the art, such as via-core assays. The antibody family consists of approximately 10 members. -6 ~about 10 -11 Kd may have affinity for CD3, and is not limited to, but approximately 10 -6 ~about 10 -10 , about 10 -6 ~about 10 -9 , about 10 -6 ~about 10 -8 , about 10 -8 ~about 10 -11 , about 10 -8 ~about 10 -10 , about 10 -8 ~about 10 -9 , about 10 -9 ~about 10 -11 , about 10 -9 ~about 10-10 , or any value within these ranges. Affinity selection can be confirmed, for example, by biological evaluations for T cell activation in in vitro or preclinical models and assessment of potential toxicity. The determination of cytokine release can be evaluated using any convenient method, including, but not limited to, the assays described in the examples.

[0100] Engagement of the T cell receptor (TCR) by binding to an MH peptide complex or an anti-TCR / CD3 antibody initiates T cell activation. Examples of anti-TCR / CD3 antibodies that activate T cells are OKT3 and UCHT1. These anti-CD3 antibodies cross-compete with each other for binding to CD3 on T cells and are routinely used in T cell activation assays. The anti-CD3 antibody of the present invention cross-competes with OKT3 for binding to human CD3. Depending on the binding affinity to CD3 and the epitope on CD3, the anti-CD3 antibody activated T cells with different functional outcomes. In vitro incubation of human T cells with low-affinity anti-CD3 antibodies resulted in incomplete T cell activation and low production of IL-2 and IL-10. In contrast, high-affinity CD3 conjugates activated T cells to produce significantly higher levels of IL-2 and other cytokines. Low-affinity anti-CD3 antibodies are considered partial agonists that selectively induce some effector functions, such as potent tumor killing and CD69 upregulation, while not inducing others, such as the production of IL-2 and IL-10. The strength of the interaction between CD3 and the recognized epitope resulted in qualitatively different activations of T cells. Maximum cytokine production of T cells activated by low-affinity anti-CD3 antibodies was lower than that of T cells activated by high-affinity anti-CD3 antibodies. In some embodiments, the antibodies of the present invention resulted in lower release of one or both of IL-2 and IL-10 when combined with T cells in the activation assay compared to a reference anti-CD3 antibody in the same assay, and the reference antibody may be ID304703 (SEQ ID NO: 22) or an antibody of equivalent affinity. The maximum release of IL-2 and / or IL-10 may be less than approximately 75% of the release by the reference antibody, less than approximately 50%, less than approximately 25%, and less than approximately 10% of the release by the reference antibody.

[0101] In some embodiments of the present invention, bispecific antibodies or multispecific antibodies are provided, which may have any configuration discussed herein, but are not limited to triple-stranded bispecific antibodies. A bispecific antibody comprises a heavy-chain variable region of the antibody that is specific to at least a protein other than CD3, and may also comprise heavy-chain and light-chain variable regions. In some such embodiments, the second antibody specificity binds to tumor-associated antigens, such as target antigens like integrins, pathogen antigens, checkpoint proteins, etc. Various forms of bispecific antibodies, including but not limited to single-stranded polypeptides, double-stranded polypeptides, triple-stranded polypeptides, quadruple-stranded polypeptides, and multiples thereof, are within the scope of the present invention.

[0102] The family of CD3-specific antibodies includes a VH domain containing CDR1, CDR2, and CDR3 sequences within the human VH framework. For example, the CDR sequences in the exemplary variable region sequences provided in SEQ ID NOs. 1-18 may be located in the regions approximately 26-33, 51-58, and 97-112 amino acid residues, respectively. It is understood by those skilled in the art that while the CDR sequences may be in different positions when different framework sequences are selected, the sequence order generally remains the same.

[0103] The CDR sequences of Family 2 antibodies may have the following sequence formula, where X represents a variable amino acid, which may be a specific amino acid as shown below. JPEG2026090249000017.jpg29170 Here, X5 may be any amino acid, and in some embodiments X5 is D, A, or H, and in some embodiments X5 is D. X6 may be any amino acid, and in some embodiments X6 is D or N, and in some embodiments D6 is D. In some embodiments the CDR1 sequence of the family 2 anti-CD3 antibody includes the sequence described in any of SEQ ID NOs: 1-18, residues 26-33.

[0104] JPEG2026090249000018.jpg23170 In some embodiments, the CDR2 sequence of a Family 2 anti-CD3 antibody includes the sequence described in any of SEQ ID NOs: 1-18, residues 51-58.

[0105] JPEG2026090249000019.jpg10170JPEG2026090249000020.jpg8170Here, X 9’ X may be any amino acid, and in some embodiments, 9’ is D or S, and in some embodiments, X 9’ D is, X 11’ X may be any amino acid, and in some embodiments, 11’ is R or S, X12' may be any amino acid, and in some embodiments, X 12’ It is either L or R.

[0106] In some embodiments, the CDR3 sequence of Family 2 anti-CD3 antibodies is given by formula: JPEG2026090249000021.jpg11170 (Sequence ID 30) It has X 11’ and X 12’ This is defined above. In some embodiments, the CDR3 sequence of a Family 2 anti-CD3 antibody includes any of the sequences described in SEQ ID NOs. 1-18, residues 97-112.

[0107] In some embodiments, the CD3-binding VH domain is paired with a light chain variable region domain. In some such embodiments, the light chain is a fixed light chain. In some embodiments, the light chain includes a VL domain having CDR1, CDR2, and CDR3 sequences in the human VL framework. The CDR sequences may be those contained in SEQ ID NO: 19. In some embodiments, the CDR1 sequence includes amino acid residues 27-32, 50-52, and 89-97 for CDR1, CDR2, and CDR3, respectively.

[0108] In some embodiments, the CDR sequence of a Family 2 antibody has a sequence that has at least 85% identity, at least 90% identity, at least 95% identity, and at least 99% identity with respect to any one of the CDR sequences or sets of CDR sequences described in SEQ ID NOs. 1 to 18. In some embodiments, the CDR sequence of the present invention includes one, two, three or more amino acid substitutions with respect to any one of the CDR sequences or sets of CDR sequences described in SEQ ID NOs. 1 to 18. In some embodiments, the amino acid substitution(s) are one or more at positions 5 or 10 of CDR1, position 2, 6 or 7 of CDR2, and position 1, 8, 9 or 10 of CDR3 with respect to the above formula.

[0109] If the protein of the present invention is a bispecific antibody, one binding portion, i.e., the VH / VL combination or VH alone, may be specific to human CD3, while the other arm may be specific to target cells including cancer cells such as ovarian cancer, breast cancer, gastrointestinal tumors, brain tumors, head and neck cancers, prostate cancer, colon cancer, and lung cancer, as well as to hematological malignancies such as B-cell tumors, including leukemia, lymphoma, sarcoma, carcinoma, neuronal tumor, squamous cell carcinoma, germ cell tumor, metastasis, anaplastic tumor, seminoma, melanoma, myeloma, neuroblastoma, mixed cell tumor, and neoplasms caused by infectious agents, and other malignancies, pathogen-infected cells, autoreactive cells causing inflammation and / or autoimmunity. The non-CD3 portion may also be specific to immunomodulatory proteins, as described herein.

[0110] Tumor-associated antigens (TAAs) are relatively limited to tumor cells, while tumor-specific antigens (TSAs) are unique to tumor cells. TSAs and TAAs are typically intracellular molecular components expressed on the cell surface as part of the major histocompatibility complex.

[0111] 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 fall into several distinct categories. Hematopoietic differentiation antigens are typically glycoproteins associated with differentiation (CD) taxa, including CD20, CD30, CD33, and CD52. Cell surface differentiation antigens are a diverse group of glycoproteins and carbohydrates found on the surfaces of both normal and tumor cells. Antigens involved in proliferation and differentiation signaling are often growth factor and growth factor receptors. Growth factors targeted by 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 nuclear factor κB ligand receptor activator (RANKL, also known as TNFSF11). Antigens involved in angiogenesis are typically proteins or growth factors that support the formation of new microvascular systems, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integrin αVβ3, and integrin α5β1. The tumor stroma and extracellular matrix are essential supporting structures for tumors. Therapeutic targets, including stromal and extracellular matrix antigens, include fibroblast-activating proteins (FAPs) and tenascin.

[0112] Examples of therapeutic antibodies useful with bispecific configurations include, but are not limited to, rituximab, ibritumomab, tiuxetan, tositumomab, brentuximab, vedotin, gemtuzumab, ozogamicin, alemtuzumab, IGN101, adecatumumab, rabetuzumab, huA33, pemtumomab, olegovomab, CC49 (minretumomab), cG250, J591, MOv18, and MORAb-00. Examples include 3 (phaletuzumab), 3F8, ch14.18, KW-2871, hu3S193, IgN311, bevacizumab, IM-2C6, CDP791, etalacizumab, boroximab, cetuximab, panitumumab, nimotuzumab, 806, trastuzumab, pertuzumab, MM-121, AMG102, METMAB, SCH900105, AVE1642, IMC-A12, MK-0646, R1507, CP 751871, KB004, IIIA4, mapatumumab (HGS-ETR1), HGS-ETR2, CS-1008, denosumab, cibrotuzumab, F19, and 81C6.

[0113] In the context of clinical cancer immunotherapy, the most actively studied immune checkpoint receptors in the context of cytotoxic T lymphocyte-associated antigen 4 (CTLA4, also known as CD152) and programmed cell death protein 1 (PD1, also known as CD279) are both inhibitory receptors. The clinical activity of antibodies blocking either of these receptors means that antitumor immunity can be enhanced at multiple levels, and combinatorial strategies can be designed under the guidance of mechanistic considerations and preclinical models.

[0114] The two ligands for PD1 are PD1 ligand 1 (also known as PDL1, B7-H1, and CD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed on cancer cells and inhibits T cell activation / function by binding to its receptor PD1 on T cells.

[0115] Lymphocyte activation gene 3 (LAG3, also known as CD223), 2B4 (also known as CD244), B and T lymphocyte attenuators (BTLA, also known as CD272), T cell membrane protein 3 (TIM3, also known as HAVcr2), adenosine A2a receptor (A2aR), and a family of killer inhibitory receptors are each associated with the inhibition of lymphocyte activity and, in some cases, the induction of lymphocyte anergy. Antibody targeting of these receptors can be used in the methods of the present invention.

[0116] Agents that activate immunocostimulatory molecules are also useful in the methods of the present invention. Such agents include agonists or CD40 and OX40. CD40 is a costimulatory protein found on antigen-presenting cells (APCs) and is required for their activation. These APCs include phagocytic cells (macrophages and dendritic cells) as well as B cells. CD40 is part of the TNF receptor family. The main activation signaling molecules for CD40 are IFNγ and CD40 ligand (CD40L). Stimulation with CD40 activates macrophages.

[0117] The target anti-CCR4 (CD194) antibody contains a humanized monoclonal antibody against CC chemokine receptor 4 (CCR4), which has potential anti-inflammatory and antitumor 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 its ligand, CCL2, mediates the recruitment of regulatory T cells to tumors. Regulatory T cells suppress the response of antitumor T cells, and therefore their inhibition or deficiency is desirable.

[0118] Production of the protein of the present invention Antibodies can be prepared by chemical synthesis, but are typically produced by recombinant DNA techniques, such as co-expression of all chains constituting a protein in a single recombinant host cell, or co-expression of a heavy chain polypeptide and an antibody (e.g., a human antibody). Furthermore, the heavy and light chains of an antibody can also be expressed using a single polycistronic expression vector. Purification of individual polypeptides is achieved using standard protein purification techniques such as affinity (protein A) chromatography, size exclusion chromatography, and / or hydrophobic interaction chromatography. Bispecific substances differ sufficiently in size and hydrophobicity that purification can be carried out using standard procedures.

[0119] The amounts of antibody and heavy chain polypeptide produced in a single host cell can be minimized, for example, by manipulating the constant regions of the antibody and heavy chain so that homodimerization is more favorable than heterodimerization, by introducing self-complementary interactions (see WO98 / 50431 for possibilities such as the “intraluminal extrusion” strategy (see WO96 / 27011)). Accordingly, another aspect of the present invention provides a method for producing a bispecific substance in recombinant host cells, the method comprising the step of expressing nucleic acid sequences encoding at least two heavy chain polypeptides in recombinant host cells, wherein the heavy chain polypeptides differ sufficiently in their constant regions to reduce or prevent homodimerization but increase bispecificity formation.

[0120] If a protein contains three chains (e.g., FlicAb), these can be produced in a single recombinant host cell by the co-expression of the three chains (two heavy chains and one light chain) that make up the molecule.

[0121] For the recombinant production of the proteins described herein, one or more nucleic acids encoding all the strands, e.g., 2, 3, 4, etc., are isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0122] In a preferred embodiment, host cells obtained by the method of the present invention can express high levels of human immunoglobulin, i.e., at least 1 pg / cell / day, preferably at least 10 pg / cell / day, more preferably at least 20 pg / cell / day, or more, without requiring amplification of single-stranded nucleic acid molecules in the host cells.

[0123] Pharmaceutical composition Another aspect of the present invention is to provide a pharmaceutical composition comprising one or more proteins of the present invention mixed with a suitable pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers used herein include, but are not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.

[0124] The therapeutic formulations of proteins used in accordance with the present invention are prepared for preservation by mixing a protein of the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized formulation or aqueous solution (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, e.g., serum albumin, gelatin. This includes proteins such as tin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or 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®, PLURONICS®, or polyethylene glycol (PEG).

[0125] Anti-CD3 antibody formulations are disclosed, for example, in U.S. Patent Publication No. 20070065437, the entire disclosure of which is incorporated herein by reference. Similar formulations may be used with the protein of the present invention. The main components of such formulations are a pH buffer, salt, surfactant, and an effective amount of a bispecific substance having anti-CD3 specificity, effective in the range of 3.0 to 6.2.

[0126] How to use In particular, when the antigen-binding composition is a multispecific antibody suitable for the condition being treated, for example, when one binding site (such as a specific binding site for a target pathogen for the treatment of an associated infection) specifically binds to a tumor-associated antigen for the treatment of associated cancer cells, methods for treating or mitigating diseases, including but not limited to infections, autoimmune diseases, and primary or metastatic cancers, are provided in regimens that involve contacting target cells with the antigen-binding composition of the present invention. Such methods include administering a therapeutically effective amount or an effective amount of the agent of the present invention to a subject in need of treatment, and include, but not limited to, combinations of the reagent with chemotherapeutic agents, radiotherapy, or surgery.

[0127] The effective dose of the composition of the present invention for the treatment of a disease varies depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, whether other drugs are being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but it may also be used to treat non-human mammals (e.g., companion animals such as dogs, cats, and horses, and experimental mammals such as rabbits, mice, and rats). The therapeutic dose may be gradually increased to optimize safety and efficacy.

[0128] The dosage level can be easily determined by a clinician with the usual skills and may be modified as needed, for example, to modify the subject's response to treatment. The amount of active ingredient that can be combined with the carrier material to manufacture a single dosage form varies depending on the host being treated and the specific method of administration. Dosage units generally contain approximately 1 mg to 500 mg of the active ingredient.

[0129] In some embodiments, the therapeutic dose of the drug may be in the range of approximately 0.0001 to 100 mg / kg of host body weight, more commonly 0.01 to 5 mg / kg. For example, the dosage may be 1 mg / kg body weight or 10 mg / kg body weight, or in the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration every two weeks, once a month, or once every three to six months. The therapeutic substance of the present invention is usually administered on multiple occasions. The interval between single doses may be weekly, monthly, or yearly. The interval may be irregular, as indicated by measuring the blood concentration of the therapeutic substance in the patient. Alternatively, the therapeutic substance of the present invention may be administered as a sustained-release formulation, in which case fewer doses are required. The dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0130] For preventative use, relatively low doses may be administered over a long period at relatively infrequent intervals. Some patients continue treatment for the remainder of their lives. For other therapeutic uses, relatively high doses may be required at relatively short intervals until the progression of the disease slows down or ceases, and preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered under a preventative management system.

[0131] In further embodiments, the methods of the present invention include the treatment, mitigation, or prevention of tumor growth, tumor metastasis, or tumor invasion of cancers such as carcinomas, hematological malignancies such as leukemia and lymphoma, melanoma, sarcoma, and glioma. For prophylactic use, a pharmaceutical composition or pharmacopoeia is administered to a patient at risk of disease or otherwise at risk of disease in an amount sufficient to eliminate or reduce the risk of disease, reduce the severity of disease, or delay the onset of disease, including the biochemical, histological, and / or behavioral symptoms of the disease (including its complications and intermediate pathological phenotypes that appear during the progression of the disease).

[0132] Compositions for the treatment of diseases may be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intra-arterial, intracranial, intraperitoneal, intranasal, or intramuscular means. Typical routes of administration are intravenous or intratumoral, but other routes may be equally effective.

[0133] Typically, the composition is prepared as an injectable preparation, either as a liquid solution or suspension, and a solid form suitable for a solution or suspension in a liquid vehicle prior to injection may also be prepared. The preparation may be emulsified or encapsulated in liposomes or microparticles such as polylactides, polyglycolides, or copolymers for an enhanced adjuvant effect, as described above (Langer, Science 249:1527, 1990, and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997). The agents of the present invention may be administered in the form of depot injections or implant preparations, which may be formulated in a manner that allows for sustained or pulsed release of the active ingredient. The pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all U.S. Food and Drug Administration Good Manufacturing Practice (GMP) regulations.

[0134] The toxicity of proteins described herein is, for example, LD50. 50 (A lethal dose for 50% of the population) or LD 100 The dose (a dose lethal to 100% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose-to-toxicity ratio is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dose range for use in humans. The protein doses described herein are preferably within the range of circulating concentrations that include effective doses with little to no toxicity. Dosages may vary within this range depending on the form of administration used and the route of administration utilized. The exact formulation, route of administration, and dosage may be selected by the individual physician, taking into account the patient's condition.

[0135] Pharmaceutical compositions can be administered in various unit dosage forms depending on the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules, and lozenges. It is recognized that the compositions of the present invention should be protected from digestion when administered orally. This is typically achieved by compounding molecules with the composition to make them resistant to acidic and enzymatic hydrolysis, or by packaging the molecules in a suitably resistant carrier such as a liposome or protective barrier. Means of protecting drugs from digestion are well known in the art.

[0136] The composition for administration typically comprises an antibody or other ablation agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the activator in these formulations can vary widely and will be selected according to the chosen specific mode of administration and the patient's needs, mainly based on body fluid volume, viscosity, body weight, etc. (e.g., Remington's Pharmaceutical Science (15th ed., 1980), and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0137] The scope of the present invention also includes a kit comprising the activator of the present invention and its formulation, as well as instructions for use. The kit may further include at least one additional reagent, such as a chemotherapeutic agent. The kit typically includes a label indicating the intended use of the kit's contents. The term "label" includes any written or recorded material on or supplied with the kit, or otherwise accompanying the kit.

[0138] The composition may be administered for therapeutic purposes. The composition is administered to the patient in an amount sufficient to substantially excise the target cells, as described above. An amount sufficient to achieve this is defined as a “therapeutably effective dose” that can result in an improvement in overall survival. Single or multiple doses of the composition may be administered depending on the required and tolerated dosage and frequency by the patient. The specific dose required for treatment depends on the mammal’s condition and medical history, as well as other factors such as age, weight, sex, route of administration, and efficiency.

[0139] Although the present invention has been fully described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. [Examples]

[0140] Example 1: Genetically modified rats expressing only heavy chain antibodies. The human IgH gene locus, human V H 6-DJ H It was constructed and assembled from several parts, including modifications and ligations of rat C region genes linked downstream of the region. Then, human V H Two BACs, each with a separate cluster of genes, were assembled (Human V H 6-DJ H - Rat C) Fragment was injected simultaneously with BAC encoding the fragment.

[0141] Transgenic rats were created that possessed an unrearranged artificial heavy chain immunoglobulin locus. The genes in the constant region included encode IgM, IgD, IgG2b, IgE, IgA, and the 3' enhancer. RT-PCR and serological analysis (ELISA) of the transgenic rats revealed productive rearrangement of the transgenic immunoglobulin locus and the expression of various isotypes of heavy chain-only antibodies in serum. The transgenic rats were crossed with rats possessing mutated endogenous heavy chain and light chain loci previously described in U.S. Patent Publication 2009 / 0098134 A1. Analysis of such animals showed inactivation of heavy chain and light chain expression of rat immunoglobulins, as well as high levels of expression of heavy chain antibodies with variable regions encoded by human V, D, and J genes. Immunization of transgenic rats resulted in the production of high titer serological responses of antigen-specific heavy chain antibodies. These transgenic rats expressing heavy-chain antibodies containing human VDJ regions were called UniRats.

[0142] Example 2: Genetically modified rats expressing immobilized light chain antibodies The transgenic human antibody repertoire is diverse (V) when combined with unique light chains. H -DJ H ) n It was generated from a rearranged heavy chain. For this purpose, the rearranged light chain human Vk-Jk1-Ck was incorporated into the rat germline by DNA microinjection, and the resulting transgenic animals were fed in a previously described rat strain that naturally expressed the human heavy chain repertoire (Osborn et al., 2013). This new rat strain was named OmniFlic.

[0143] Immunization of OmniFlic rats with many different antigens produced high levels of antigen-specific IgG, similar to other transgenic rats with the same IgH locus. Repertoire analysis by RT-PCR revealed highly variable V at high transcript and protein levels. HGene rearrangements were identified. Furthermore, only one light chain product was identified that was expressed even at high levels.

[0144] Antigen-specific conjugates derived from OmniFlic were obtained by selection from NGS and cDNA libraries (yeast, E. coli, phage), and diverse heavy chain transcripts were identified during sequencing. For expression in mammalian cells, hypermutant heavy chain constructs were transfected in combination with the original transgenic Igk sequence. No mutational changes were observed in this rearranged Vk-Jk1-Ck, and the same light chain was consistently expressed along with various heavy chain products to generate monoclonal human IgG.

[0145] Example 3: Generation of antigen-specific antibodies in transgenic rats To produce antigen-specific heavy chain antibodies in rats, genetically modified rats were immunized using two methods.

[0146] Immunization using recombinant extracellular domains of PD-L1 and BCMA. Recombinant extracellular domains of PD-L1 and BCMA were purchased from R&D Systems, diluted in sterile saline, and combined with adjuvants. The immunogens were combined with either complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), or Titermax and Ribi adjuvants. The initial immunization (priming) with the immunogen in CFA or Titermax was administered to the left and right legs. Following the initial immunization with the immunogen in CFA, two or more immunizations (booster immunizations) with IFA, or four or more immunizations with Ribi, and one more immunization with Titermax were administered to each leg. This series of immunizations resulted in the development of B cells that produce high-affinity antibodies. The immunogen concentration was 10 micrograms per leg. Serum was collected from rats at the time of final blood collection to determine serum titer.

[0147] 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-epsilon / delta constructs at Aldevron, Inc. (Fargo, ND) using GENOVAC antibody technology. After final booster immunization and RNA isolation, influx region lymph nodes were collected. Following cDNA synthesis, the IgH heavy chain antibody repertoire was characterized by next-generation sequencing and proprietary software. Candidate antigen-specific VH sequences demonstrating evidence of antigen-specific positive selection were selected. Hundreds of VH sequences encoding FlicAbS were selected for recombination and cloned into expression vectors. Subsequently, fully human FlicAb IgG1 antibodies were expressed in HEK cells for analysis by flow cytometry and ELISA. Human FlicAb was tested for binding to primary human T cells and Jurkat cells by flow cytometry. Furthermore, human FlicAbs were tested using recombinant CD3δε protein in ELISA. All FlicAbs exhibiting positive binding to human T cells are listed in Figure 1. Selected sequences were further characterized in T cell activation assays.

[0148] Example 4: Analysis of Treg cell activation CD69 is a cell surface marker of T cells that is upregulated by stimulation. In this experiment, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat using Biocol (1.077 g / ml density) and standard methods. The isolated PBMCs were incubated in complete medium for 48 hours (2 × 10⁶). 7 Pre-culture cells at 1 / ml, wash, and immerse in complete medium for 10 minutes. 6 The cells were resuspended in cells / ml. These cells were incubated for 24 hours in FACS tubes (BD Falcon Corning) coated with recombinant BCMA protein. The tubes were pre-coated overnight with recombinant BCMA protein at 10 micrograms / ml.

[0149] Cells were washed and stained with antibodies specific to 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 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). Cells were analyzed using a Cytoflex flow cytometer with appropriate templates. In contrast to currently available anti-CD3 bispecific molecules, TNB-383B preferentially activates CD4+ and CD8+ T cells over Treg cells. By preventing the activation of Treg cells, which are an immunosuppressive cell type, CD8+ T cell function can be enhanced, increasing immunodestruction of tumor targets. Data are shown in Figures 3 and 4.

[0150] Example 5: Expression and purification of CD3 delta / epsilon Fc fusion protein The recombinant antigens CD3 epsilon (SEQ ID NO: 23, extracellular domain (ECD) residues 22-105) and CD3 delta (SEQ ID NO: 24, ECD residues 23-126), shown in Figure 5, were cloned in frame with mouse IgG1 Fc, transiently co-expressed, and purified from CHO cell culture medium. A C-terminal His tag added to the CD3 epsilon subunit was used for affinity capture of the CD3 delta / epsilon complex by IMAC using a standard protocol and for elution with imidazole. Sequence EPEA (Sequence ID 66) A second purified affinity tag, including a (C tag), was added to the C-terminus of the CD3 delta subunit.

[0151] Example 6: Epitope mapping of mAb CD3 F2B Labeling of surface residues Epitope mapping was achieved by surface residues labeled with diethyl pyrocarbonate DEPC (reference), which covalently reacts with the amino acids histidine, lysine, tyrosine, cysteine, serine, threonine, and the accessible side chain of the free N-terminal amino group. DEPC labeling was performed at an antibody:antigen molar ratio of 30:1 to promote complex formation. DEPC-labeled antibody-antigen complexes were captured using Capture Select C-tag affinity matrix (Thermo Fisher Scientific), and excess antibody was thoroughly washed away. Affinity resin-bound CD3 delta / epsilon were subjected to standard reduction and alkylation methods before digestion with trypsin, chymotrypsin, and endopeptidase Glu-C. The released peptides were LC- M Analysis was performed by / MS. The same DEPC labeling experiment was carried out in the absence of CD3 antibody F2B, followed by affinity catcher, reduction / alkylation, and protease digestion. Each digestion was performed in triple replication. The sequence coverage derived from mass spectrometry in the absence of mAb CD3 F2B was high, as shown in Figure 6.

[0152] DEPC labeling of CD3 delta / epsilon is affected by the binding of mAb CD3 F2B (heavy chain of SEQ ID NO: 1 and light chain of SEQ ID NO: 19). We analyzed peptides obtained from three proteolytic digests for the DEPC-modified residues that are significantly reduced due to the presence of mAb CD3F2B during labeling. Figure 7 shows a representative example of the affected peptide obtained from digestion with endopeptidase Glu-C.

[0153] The effect of amino acid residues on labeling is considered significant if a) DEPC uptake decreases by more than 15 times and b) it is detected in at least two of the three digests. The affected CD3 epsilon peptide is shown in Figure 7. Consistent and significant labeling effects were observed at Lys73 in both chymotrypsin and Glu-C peptides. Furthermore, Glu-C peptides I57-E86 and D80-E86 provide further evidence that the epitope of mAb F2B extends to at least lysine 85 within the epsilon chain.

[0154] Affected CD3 delta peptides were also identified. The sequence stretch affected by one label was common to each of the three digestion sets. These data suggest an epitope of mAb F2B from lysine 82 to cysteine ​​93. Figure 8 shows the epitopes identified in the CD3 delta protein (shaded). Figure 9 shows the crystal structure of CD3 delta / epsilon (PDB ID code 1XIW, Arnett K. et.al, Proc Natl Acad Sci US A. 2004;101(46):16268-16273) with the affected residues of each CD3 subunit shown in space-filling mode.

[0155] Example 7: CD3 Family 2 antibody recognizes epitopes different from OKT3 and SP34. Using the same epitope mapping approach, we confirmed a known interaction between the therapeutic antibody OKT3 and CD3 delta / epsilon (Salmeron, A., Sanchez-Madrid, F., Ursa, MA, Fresno, M. & Alarcon, B. (1991) J. Immunol. 147, 3047-3052). The cross-reactive antibody SP-34 was also characterized (US Patent No. 8,236,308, 2012). This antibody recognizes the epitope within the extended EF loop of CD3 epsilon and does not require CD3 delta for binding.

[0156] The applicants' dataset confirms that the CD3 antibody F2B binds to a different epitope compared to OKT3 and SP-34. Figure 10 shows the alignment of human and cynomolgus monkey ECDs. The CD3 mAbs provided herein bind to loops not present in the cynomolgus monkey homologs. This further explains why these antibodies do not exhibit monkey cross-reactivity to the CD3 complex. Furthermore, neither OKT3 nor SP-34 directly binds to the CD3 delta.

[0157] The examples are provided to those skilled in the art to provide a complete disclosure and explanation of the manufacturing method and use of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or the only experiments to be performed. Efforts have been made to ensure accuracy with respect to the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should be taken into consideration. Unless otherwise noted, parts are by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0158] Although the present invention has been described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications and substitutions of equivalents can be made without departing from the true spirit and scope of the invention. Furthermore, many modifications may be made to adapt specific circumstances, materials, composition of substances, processes, process steps, or a set of steps to the object, spirit, and scope of the invention. All such modifications are intended to fall 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 comprising at least one residue selected from CD3 epsilon (SEQ ID NO: 23): K73 and S83, and CD3 delta (SEQ ID NO: 24): K82 and C93.

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

3. The isolated monoclonal antibody according to claim 1, wherein the epitope on CD3 includes a region of CD3 epsilon 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 to 3, wherein the epitope comprises a conformational epitope containing both CD3 delta and CD3 epsilon residues.

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

6. The antigen-binding protein according to any one of claims 1 to 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 to 6, wherein when the antigen-binding protein binds to a T cell, it induces cytokine release that is approximately 200% or less of the maximum cytokine release observed with an F2B antibody.

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

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

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

11. The antigen-binding protein according to any one of claims 1 to 10, wherein the variable region of the light chain includes a set of CDR sequences in sequence 19.

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

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

15. The antigen-binding protein according to claim 14, wherein the Fc region is manipulated to reduce its effector function.

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

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

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

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

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

21. The aforementioned protein further comprises a variable heavy chain domain specific to proteins other than CD3, When the antigen-binding protein is brought into contact with T cells in an activation assay, it induces the release of one or both IL-2 and IL-6 at reduced levels compared to the reference anti-CD3 antibody. An antigen-binding protein according to any one of claims 1 to 20, which induces more than 30% tumor 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 to proteins other than CD3 is a heavy chain-only domain.

23. The antigen-binding protein according to claim 21, wherein the variable heavy chain domain specific to proteins 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 to 24, wherein the protein other than CD3 is a tumor-associated antigen.

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

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

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

29. A pharmaceutical composition according to claim 28, in a unit dose formulation.

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

31. A vector comprising the polynucleotide described in claim 30.

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

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

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

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

36. An antigen-binding protein according to any one of claims 1 to 27, for use in the treatment of a disease.

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

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