CD3 binding antibodies

Abstract

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

Description

[0001] This application is a divisional application of the invention patent application of the same name 201780062822.4 filed on June 20, 2017.

[0002] 1. Cross-reference

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 394,360, filed September 14, 2016, and U.S. Provisional Patent Application No. 62 / 491,908, filed April 28, 2017, which are incorporated herein by reference in their entireties. 2. Background technology

[0005] The body's immune system functions as a defense against infection, injury, and cancer. Two separate but interrelated systems, the humoral immune system and the cellular immune system, work together to protect the body. The humoral system is mediated by soluble factors (called antibodies) that neutralize products that the body recognizes as foreign. In contrast, the cellular system involves cells, such as T cells and macrophages, that remove and neutralize foreign invaders.

[0006] The activation of T cells is essential for stimulating immune responses. T cells exhibit immunospecificity and direct most cellular immune responses. Although T cells do not secrete antibodies, they are necessary for B lymphocytes to secrete antibodies. T cell activation requires the participation of many cell surface molecules, such as the T cell receptor complex and CD4 or CD8 molecules. Antigen-specific T cell receptors (TcRs) consist of heterodimeric membrane glycoproteins with disulfide bonds of chains α and β (alpha and beta) or γ and δ (gamma and delta). TcR is non-covalently linked to a complex of an invariant protein called CD3.

[0007] It is known that T cells exert potent anti-tumor effects in many experimental settings. Antibodies that can effectively recruit T cells against tumor cells are already available as, for example, bispecific antibodies against tumor-associated antigens (TAAs) and agonistic T cell membrane proteins such as the TCR / CD3 complex and CD28. These bispecific antibodies are able to activate T cells, regardless of their TCR specificity, thereby leading to specific lysis of cells carrying the corresponding TAA.

[0008] However, although anti-CD3 bispecific antibodies can redirect T cell-mediated lysis toward malignant cells, clinical trials using CD3-based bsAbs have shown high toxicity in patients. Nonspecific T cell activation from bsAbs can occur in an antigen-independent manner due to Fc / Fc receptor (FcR) interactions, or in an antigen-dependent manner when the antigen is expressed on both normal cells and tumor cells. These two mechanisms may be the cause of the toxicity observed in previous clinical studies. (See, for example, Link et al. (1998) Int. J. Cancer 77 (2): 251-6; Durben et al. Molecular Therapy (2015); 23 4, 648–655). Due to the resulting cytokine release syndrome, there has always been a huge obstacle in developing these antibodies for therapeutic purposes.

[0009] The interaction between the T cell receptor (TCR) and its peptide-MHC ligand determines the activity of T cells. The binding characteristics of this interaction have been studied in great detail and shown to control T cell function. The intensity and nature of the TCR-peptide / MHC interaction determine whether T cells exert effector functions or are inactivated and missing. Antibodies against CD3 activate T cells by changing the conformation of the CD3ε chain, and can have an exciting or antagonistic effect on T cells depending on the epitope (Yoon et al., 1994Immunity1:563-569). In view of the significant side effects of many T cell agonists, it may be preferred to maintain an effective anti-tumor effect while reducing the release of proinflammatory cytokines. However, some agonistic anti-CD3 antibodies may suboptimally change the CD3ε chain, resulting in ineffective signaling, and most anti-CD3 antibodies are full agonists of two pathways. It is not clear whether these effector functions can be separated. Many existing anti-CD3 antibodies (e.g., SP-34, UCHT1, OKT3) have an affinity in the range of 1-50nM KD, but this may not be optimal for therapeutic use.

[0010] The present invention provides CD3-specific antibodies and bispecific antibodies derived from the CD3-specific antibodies.

[0011] publication

[0012] CD3 antibodies are disclosed, for example, in U.S. Pat. Nos. 5,585,097; 5,929,212; 5,968,509; 6,706,265; 6,750,325; 7,381,803; 7,728,114. Bispecific antibodies having CD3 binding specificity are disclosed, for example, in U.S. Pat. Nos. 7,262,276; 7,635,472; 7,862,813; and 8,236,308, each of which is expressly incorporated herein by reference. 3. Summary of the invention

[0014] Compositions and methods of using a family of closely related antibodies that bind to CD3 and activate signaling through CD3, such as activating CD3 + T cells. The antibody family comprises a set of CDR sequences as defined herein. The antibody family provides a number of benefits that are useful for use as one or more clinical therapeutic agents. The antibodies within the family include members with a range of binding affinities, allowing the selection of specific sequences with desired affinity. The ability to fine-tune affinity is particularly important for managing the level of CD3 activation in treated individuals and thereby reducing toxicity.

[0015] In some embodiments, the anti-CD3 antibody has a CD3 affinity of about 10 -6 About 10 -11Affinity (KD) in the range of about 50nM or more, 100nM or more, 500nM or more or 1μM or more affinity (KD) anti-CD3 antibodies can be expected to more closely simulate TCR / MHC interactions and minimize toxic cytokine release while maintaining effective tumor cell lysis. In some embodiments, anti-CD3 antibodies are characterized or selected for the tendency to reduce cytokine release (e.g., for the release of IL-2 and IFNγ) when combined with competent T cells. Antibodies that optimize tumor cell killing and reduce cytokine release can be selected for therapeutic use, such as antibodies within the antibody sequence family described herein, which induce cytokine release less than about half of the maximum value observed for family members in comparative determinations, and may be less than, for example, about 25% of the maximum value observed for family members in comparative determinations. In some embodiments, bispecific or multispecific antibodies are provided, the antibodies at least comprising a heavy chain variable region from the antibody family and may comprise a heavy chain and light chain variable region provided herein. The bispecific antibody comprises at least a heavy chain variable region of an antibody specific for a protein other than CD3, and may comprise a heavy chain variable region and a light chain variable region. In some such embodiments, the second antibody specifically binds to a tumor-associated antigen, a targeted antigen (e.g., integrin, etc.), a pathogen antigen, a checkpoint protein, etc. Various forms of bispecific antibodies fall within the scope of the present invention, including but not limited to single-chain polypeptides, double-chain polypeptides, three-chain polypeptides, four-chain polypeptides, and multiples thereof.

[0016] The CD3-specific antibodies each comprise a VH domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. As an example, for CDR1, CDR2 and CDR3, the Family 2 CDR sequences may be located in the region of about amino acid residues 26-33; 51-58; and 97-112 of the provided exemplary variable region sequences listed in SEQ ID NOs: 1-18, respectively. Those skilled in the art will appreciate that if different framework sequences are selected, the CDR sequences may be in different positions, although the order of the sequences will generally remain the same.

[0017] The CDR sequence of the family 2 antibody may have the following sequence formula: X represents a variable amino acid, which may be a specific amino acid as shown below.

[0018] CDR1

[0019] G1 F2 T3 F4 X5 X6 Y7 A8

[0020] in:

[0021] X5 can be any amino acid; in some embodiments, X5 is D, A, or H; in some embodiments, X5 is D.

[0022] X6 can be any amino acid; in some embodiments, X6 is D or N; in some embodiments, D6 is D.

[0023] In some embodiments, the CDR1 sequence of the Family 2 anti-CD3 antibody comprises residues 26-33 of the sequence set forth in any one of SEQ ID NOs: 1-18.

[0024] CDR2

[0025] I 1’ S 2’ W 3’ N 4’ S 5’ G 6’ S 7’ I 8’

[0026] In some embodiments, the CDR2 sequence of the Family 2 anti-CD3 antibody comprises residues 51-58 of the sequence set forth in any one of SEQ ID NOs: 1-18.

[0027] CDR3

[0028] A 1” K 2” D 3” S 4” R 5” G 6” Y 7” G 8” X 9” Y 10” X 11” X 12” G 13” G 12” A 15” Y 16”

[0029] in:

[0030] X 9” Can be any amino acid, in some embodiments, X 9” is D or S; in some embodiments, X 9” It is D;

[0031] X 11” Can be any amino acid, in some embodiments, X 11” is R or S;

[0032] X12” Can be any amino acid, in some embodiments, X 12” It is L or R.

[0033] In some embodiments, the CD3 sequence of the Family 2 anti-CD3 antibody has the formula AKDSRGYGDYX 11” X 12” GGAY, where X 11” and X 12” is as defined above. In some embodiments, the CDR3 sequence of the Family 2 anti-CD3 antibody comprises residues 97-112 of the sequence set forth in any one of SEQ ID NOs: 1-18. In some embodiments, the CD3 binding VH domain of the Family 2 antibody is paired with a light chain variable region domain. In some such embodiments, the light chain is a fixed light chain.

[0034] In some embodiments, the light chain comprises a VL domain having CDR1, CDR2 and CDR3 sequences in a human VL framework. The CDR sequences may be those of SEQ ID NO: 19. In some embodiments, for CDR1, CDR2, CDR3, the CDR1 sequence comprises amino acid residues 27-32; 50-52; 89-97, respectively.

[0035] In some embodiments, the CDR sequence of the antibody of the present invention is a sequence with at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity relative to a CDR sequence or a group of CDR sequences in SEQ ID NO: 1-18. In some embodiments, the CDR sequence of the present invention comprises one, two, three or more amino acid substitutions relative to a CDR sequence or a group of CDR sequences in any one of SEQ ID NO: 1-18. In some embodiments, the one or more amino acid substitutions are relative to the family 2 formula provided above, one or more of positions 5 or 10 of CDR1, positions 2, 6 or 7 of CDR2, and positions 1, 8, 9 or 10 of CDR3.

[0036] In some embodiments, the bispecific antibodies of the present invention comprise a CD3 binding variable region described herein paired with a light chain. In some embodiments, the light chain comprises a variable region sequence listed in SEQ ID NO: 19, or a variable region comprising a set of CDR sequences and a framework sequence in SEQ ID NO: 19. Various Fc sequences can be used, including but not limited to human IgG1, IgG2a, IgG2b, IgG3, IgG4, etc. In some embodiments, the second arm of the bispecific antibody comprises a variable region that specifically binds to a tumor-associated antigen. In some embodiments, the second arm of the bispecific antibody comprises a variable region that specifically binds to BCMA. In some embodiments, the anti-BCMA arm is a single chain variable region, such as Figure 2B In some embodiments, the anti-BCMA arm comprises the variable region sequence listed in SEQ ID NO: 20; or the tandem variable region sequence listed in SEQ ID NO: 21. The Fc sequence of the anti-BCMA arm can be, but is not limited to, human IgG1, IgG2a, IgG2b, IgG3, IgG4, etc. The CDR sequences can be those contained in SEQ ID NO: 20. In some embodiments, for CDR1, CDR2, and CDR3, the CDR sequences comprise amino acid residues 26-33; 51-58; 97-108, respectively.

[0037] In other embodiments, a pharmaceutical composition is provided, comprising at least a CD3 binding VH domain of the present invention, such as a monospecific, bispecific, etc. antibody or antibody-like protein comprising at least a CD3 binding VH domain of the present invention; and a pharmaceutically acceptable excipient. The composition can be lyophilized, suspended in a solution, etc., and can be provided in a unit dose formulation.

[0038] In some embodiments, a method for treating cancer is provided, the method comprising administering an effective dose of a monospecific, bispecific, etc. antibody of the present invention to an individual in need thereof. In the case where the antibody is bispecific, the second antigen binding site can specifically bind to a tumor antigen, a checkpoint protein, etc. In various embodiments, the cancer is selected from the group consisting of: ovarian cancer, breast cancer, gastrointestinal cancer, brain cancer, head and neck cancer, prostate cancer, colon cancer, lung cancer, leukemia, lymphoma, sarcoma, carcinoma, neural cell tumor, squamous cell carcinoma, germ cell tumor, metastasis, undifferentiated tumor, seminoma, melanoma, myeloma, neuroblastoma, mixed cell tumor, and neoplasia caused by infectious agents.

[0039] In some embodiments, a method for treating an infectious disease is provided, the method comprising administering an effective dose of a monospecific, bispecific, etc. antibody of the present invention to an individual in need thereof. In the case where the antibody is bispecific, the second antigen binding site can specifically bind to a pathogen antigen, such as a bacterium, virus, or parasite.

[0040] In other embodiments, a method for producing a bispecific antibody of the present invention is provided, the method comprising expressing an antibody sequence, such as one or more light chain coding sequences, one or more heavy chain coding sequences, in a single host cell. In various embodiments, the host cell can be a prokaryotic cell or a eukaryotic cell, such as a mammalian cell. 4. Description of the drawings

[0042] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawings will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, as is customary, the various features of the drawings are not to scale. Rather, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. Included in the drawings are the following figures:

[0043] Figures 1A-1C . Figure 1A Shown is an alignment of the CDR1, CDR2, and CDR3 regions of members of antibody Family 2 of SEQ ID NOs: 1-18 that specifically bind to human CD3, corresponding to residues 26-33; 51-58; and 97-112. Figure 1B Shown are the CDR1, CDR2, and CDR3 regions of a fixed light chain (SEQ ID NO: 19); and exemplary anti-BCMA sequences (SEQ ID NO: 20 and SEQ ID NO: 21). Figure 1C The CDR sequences of reference anti-CD3 antibody (SEQ ID NO:22), ID 304704 are provided.

[0044] Figure 2A-2E . Schematic model of a bispecific human antibody. Figure 2A Anti-CD3:anti-tumor antigen bispecific antibodies with common light chain (3 unique chains total). Figure 2B Anti-CD3:anti-tumor antigen bispecific antibody with 2 unique light chains (4 unique chains total). Figure 2C Anti-CD3: anti-tumor antigen bispecific antibody with heavy chain tumor antigen binding domain chain only (3 unique chains). Figure 2DAnti-CD3:anti-tumor antigen bispecific antibody with scFv tumor antigen binding domains (3 unique chains total). Figure 2E Anti-CD3:anti-tumor antigen bispecific antibody with scFv anti-CD3 binding domain (3 unique chains total).

[0045] Figure 3 .The anti-CD3 Family 2 data table summarizes the behavior of anti-CD3 antibodies in monospecific and bispecific formats. Column 1 shows the sequence ID of the anti-CD3 VH sequence. Column 2 shows the MFI value for Jurkat cell binding of the parental monospecific anti-CD3. Column 3 shows the MFI value for cyno T cell binding of the parental monospecific anti-CD3. Column 4 shows the name of the aCD3:aBCMA bispecific antibody. Column 5 shows the picograms of IL-2 released by pan T cells stimulated by the indicated doses of the bispecific antibody binding to BCMA protein coated on plastic. Column 6 shows the picograms of IL-6 released by pan T cells stimulated by the indicated doses of the bispecific antibody binding to BCMA protein coated on plastic. Column 7 shows the picograms of IL-10 released by pan T cells stimulated by the indicated doses of the bispecific antibody binding to BCMA protein coated on plastic. Column 8 shows the picograms of IFN-γ released by pan T cells stimulated by the indicated doses of the bispecific antibody binding to BCMA protein coated on plastic. The 9th column shows the picograms of TNFα released by pan T cells stimulated by the indicated doses of bispecific antibodies that bind to BCMA protein coated on plastic. The 10th column shows the EC50 of bispecific antibody-mediated lysis of U266 tumor cells in the presence of human pan T cells. The 11th column shows the percentage of lysis of U266 tumor cells in the presence of bispecific antibodies and human pan T cells at a dose of 333 ng / mL of bispecific antibodies. The 12th column shows the protein binding affinity of the anti-CD3 arm of the bispecific antibodies measured by Octet. The 13th column shows the MFI value of Jurkat cell binding of the bispecific antibodies.

[0046] Figure 4 . Bispecific antibody-mediated tumor cell lysis. Seven αCD3_fam2: aBCMA bispecific antibodies (each with a unique anti-CD3 arm and a common anti-BCMA arm) were tested for their ability to kill U266 BCMA+ tumor cells by redirecting activated primary T cells. In this experiment, U266 cells expressing BCMA were mixed with activated pan T cells at a 10:1E:T ratio, and bispecific antibodies were added. The x-axis shows the concentration of the antibody used, and the y-axis shows the lysis of tumor cells 6 hours after the addition of the antibody.

[0047] Figure 5 . Bispecific U266 killing activity is correlated with IL-2 release. The comparison of bispecific antibody-mediated tumor cell lytic activity and IL-2 cytokine release is shown in the scatter plot. The correlation between IL-2 production and U266 tumor cell lysis is R 2 =0.37.

[0048] Figure 6 . Bispecific U266 killing activity is correlated with IFN-γ release. The comparison of bispecific antibody-mediated tumor cell lytic activity and IFN-γ cytokine release is shown in the scatter plot. The correlation between IFN-γ production and U266 tumor cell lysis is R 2 =0.53.

[0049] Figure 7 . Bispecific U266 killing activity is correlated with anti-CD3 binding affinity. The comparison of U266 tumor cell lysis activity mediated by the bispecific antibody and anti-CD3 binding affinity is shown in the scatter plot. The correlation between U266 killing EC50 and protein binding affinity is R 2 =0.93.

[0050] Figures 8A-8D . Bispecific antibody-mediated tumor cell lysis. The ability of the αCD3_F1F:aBCMA bispecific antibody to kill three different BCMA+ tumor cells and one BCMA-negative cell line by redirecting activated primary T cells was determined. In this experiment, tumor cells were mixed with activated pan T cells at a 10:1 E:T ratio, and the bispecific antibody was added at the same time. Fig. 8A Showing the killing of RPMI-8226 cells, Figure 8B Showing the killing of NCI-H929 cells, Figure 8C shows killing of U-266 cells, and Fig.8D Killing of K562 cells (negative control) is shown. The x-axis shows the concentration of the antibody used and the y-axis shows the % lysis of tumor cells 6 hours after addition of the antibody.

[0051] Figures 9A-9D . Bispecific antibody-mediated IL-2 release. Levels of IL-2 cytokine release were measured after incubation of resting human T cells with various tumor cell lines and increasing doses of the αCD3_F1F:aBCMA bispecific antibody. Fig. 9A Shown is the release of IL-2 stimulated by RPMI-8226 cells. Fig. 9B Shown is the release of IL-2 stimulated by NCI-H929 cells, Fig. 9C shows IL-2 release stimulated by U-266 cells, and Fig.9D Shown is IL-2 release stimulated by K562 cells (negative control).

[0052] Figures 10A-10D Bispecific antibody-mediated IFN-γ release. Levels of IFN-γ cytokine release were measured after incubation of resting human T cells with various tumor cell lines and increasing doses of the αCD3_F1F:aBCMA bispecific antibody. Fig. 10A IFN-γ release by RPMI-8226 cell stimulation is shown. Fig. 10B IFN-γ release stimulated by NCI-H929 cells is shown. Fig. 10C shows IFN-g release stimulated by U-266 cells, and Fig. 10D Shown is IFN-γ release stimulated by K562 cells (negative control). 5. Specific implementation methods

[0054] To facilitate understanding of the present invention, a number of terms are defined below.

[0055] Before describing the present active agents and methods, it should be understood that the present invention is not limited to the specific methods, products, equipment and factors described, as such methods, equipment and formulations can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which will only be limited by the appended claims.

[0056] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "drug candidate" refers to one or a mixture of such candidates, and reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the devices, formulations, and methods described in the publications and that may be used in conjunction with the invention described herein.

[0058] When a range of values ​​is provided, it is understood that unless the context clearly dictates otherwise, each intervening value (to one-tenth of the unit of the lower limit) between the upper and lower limits of the range and any other stated or intervening value in the stated range is encompassed within the invention. The upper and lower limits of the smaller ranges that may be independently included in these smaller ranges are also encompassed within the invention, subject to any specifically excluded limits in the stated ranges. When the range includes one or two limits, ranges excluding one or both of those included limits are also included in the invention.

[0059] In the following description, a large number of specific details are set forth in order to provide a more detailed understanding of the present invention. However, it will be clear to those skilled in the art that the present invention can be implemented without one or more of these specific details. In other cases, well-known features and procedures well known to those skilled in the art are not described to avoid obfuscating the present invention.

[0060] In general, conventional methods of protein synthesis, recombinant cell culture and protein isolation within the skill of the art and recombinant DNA techniques are used in the present invention. Such techniques are fully explained in the literature, see, for example, Maniatis, Fritsch and Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0061] definition

[0062] "Comprising" means that the recited elements are required in the composition / method / kit, but other elements may be included to form a composition / method / kit etc. within the scope of the claim.

[0063] "Consisting essentially of" is intended to limit the scope of the described compositions or methods to specified materials or steps that do not materially affect the basic and novel characteristics of the invention.

[0064] "Consisting of" means excluding from the composition, method or kit any elements, steps or ingredients not specified in the claims.

[0065] The terms "treatment", "treating", etc. are used herein to generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms and / or may be therapeutic in terms of partially or completely curing a disease and / or an adverse effect attributable to the disease. "Treatment" as used herein encompasses the treatment of any disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting the development of the disease; or (c) alleviating the disease, i.e., causing the disease to regress. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of an ongoing disease, wherein the treatment stabilizes or alleviates the patient's adverse clinical symptoms. It is desirable to perform such treatment before the complete loss of function in the affected tissue. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0066] A "therapeutically effective amount" is intended to be the amount of an active agent required to confer a therapeutic benefit on a subject. For example, a "therapeutically effective amount" is an amount that induces, alleviates, or otherwise causes an improvement in pathological symptoms, disease progression, or a physiological state associated with a disease, or improves resistance to a condition.

[0067] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infection, etc. The subject can be a human, but also includes other mammals, particularly those mammals suitable for use as laboratory models of human diseases, such as mice, rats, etc.

[0068] The terms "cancer", "neoplasm" and "tumor" are used interchangeably herein to refer to cells that exhibit autonomous unregulated growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Target cells for detection, analysis or treatment in the present application include precancerous cells (e.g., benign cells), malignant cells, pre-metastatic cells, metastatic cells and non-metastatic cells. Cancers of almost every tissue are known. The phrase "cancer burden" refers to the amount of cancer cells or the volume of cancer in a subject. Therefore, reducing cancer burden refers to reducing the number of cancer cells or the volume of cancer in a subject. As used herein, the term "cancer cell" refers to any cell that is a cancer cell or is derived from a cancer cell, such as 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, etc.; and circulating cancers such as leukemias, specifically including B cell leukemias, T cell leukemias, etc. Examples of cancer include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.

[0069] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (such as natural killer cells, neutrophils and macrophages) recognize bound antibody on a target cell and cause lysis of the target cell. ADCC activity can be assessed using methods such as those described in U.S. Pat. No. 5,821,337. ADCP refers to antibody-dependent cell-mediated phagocytosis.

[0070] "Effector cells" are leukocytes that express one or more constant region receptors and perform effector functions.

[0071] "Cytokine" is a protein released by one cell to act on another cell as an intercellular mediator. Target cytokines include, but are not limited to, cytokines released from activated T cells, such as IL-2, IFNγ, etc.

[0072] "Non-immunogenic" refers to a substance that does not elicit, stimulate or enhance an immune response, including an adaptive immune response and / or an innate immune response.

[0073] The term "isolated" refers to material that is removed from its original environment (e.g., its natural environment if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and / or such polynucleotides or polypeptides could be part of a composition and they would still be isolated because such a vector or composition is not part of its natural environment.

[0074] "Pharmaceutically acceptable excipients" refer to generally safe, non-toxic and desirable excipients suitable for use in preparing pharmaceutical compositions, and include excipients acceptable for veterinary use as well as human medical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, may be a gas.

[0075] "Pharmaceutically acceptable salts and esters" refer to salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when the acidic protons present in the compound are able to 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 organic bases, such as amine bases, such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed by carboxyl, sulfonyl and phosphonyl groups present in the compound, for example, C 1-6 Alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a monoacid monosalt or ester or a disalt or ester; and similarly when more than two acidic groups are present, some or all of such groups may be salified or acidified. The compounds named in the present invention may exist in unsalted or unesterified form, or in salified and / or esterified form, and the naming of such compounds is intended to include the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. In addition, certain compounds named in the present invention may exist in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (racemic or other) of such stereoisomers.

[0076] The terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof are used interchangeably in referring to compositions, carriers, diluents, and agents and mean that a material is capable of being administered to or administered to a human without producing undesirable physiological effects to an extent that would prohibit administration of the composition.

[0077] "Homology" between two sequences is determined by sequence identity. If the two sequences to be compared are of different lengths, sequence identity preferably relates to the percentage of nucleotide residues of the shorter sequence that are identical to the nucleotide residues of the longer sequence. Sequence identity can be determined routinely using computer programs such as Bestfit program (Wisconsin Sequence Analysis Package, Unix version 8, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711). Bestfit uses a local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489) to find the segment with the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has, for example, 95% identity with a reference sequence of the present invention, it is preferred to adjust the parameters so that the identity percentage is calculated over the full length of the reference sequence and a homology gap of up to 5% of the total number of nucleotides in the reference sequence is allowed. When using Bestfit, it is preferred to keep the so-called optional parameters at their pre-set ("default") values. Deviations that occur in the comparison between a given sequence and the above-mentioned sequence of the present invention may be caused, for example, by addition, deletion, substitution, insertion or recombination. Preferably, the program "fasta20u66" (version 2.0u66, September 1998, William R. Pearson and University of Virginia; see also WRPearson (1990), Methods in Enzymology 183, 63-98 with examples and http: / / workbench.sdsc.edu / ) can also be used to implement this sequence comparison. For this purpose, the "default" parameter setting can be used.

[0078] "Variant" refers to a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Typically, an amino acid sequence variant has at least about 80% sequence identity to the sequence, more preferably, at least about 90% homology. An amino acid sequence variant may have substitutions, deletions, and / or insertions at certain positions within the reference amino acid sequence.

[0079] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid that has been connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated with the host genome. In addition, some vectors can guide the expression of the gene to which they are operably connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, the expression vector used in recombinant DNA technology is often in the form of a plasmid. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used forms of vectors.

[0080] As used herein, the term "host cell" (or "recombinant host cell") is intended to refer to a cell that has been genetically altered or is capable of being genetically altered by the introduction of an exogenous polynucleotide (such as a recombinant plasmid or vector). It should be understood that such terms are intended to refer not only to the specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in the next generation due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0081] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or other binding molecule) and its binding partner (e.g., an antigen or receptor). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies bind to antigens (or receptors) weakly and dissociate easily, while high-affinity antibodies bind to antigens (or receptors) more tightly and remain bound longer.

[0082] Unless specifically indicated to the contrary, the term "conjugate" as described and claimed herein is defined as a heterogeneous molecule formed by covalently linking one or more antibody fragments to one or more polymer molecules, wherein the heterogeneous molecule is water-soluble, i.e., soluble in physiological fluids such as blood, and wherein the heterogeneous molecule does not contain any structured aggregates. The conjugate of interest is PEG. In the context of the foregoing definition, the term "structured aggregate" refers to (1) any molecular aggregate having a spheroid or spheroid shell structure in aqueous solution, such that the heterogeneous molecule is not in the form of a micelle or other emulsion structure and is not anchored to a lipid bilayer, vesicle or liposome; and (2) any molecular aggregate in a solid or insoluble form that does not release the heterogeneous molecule into solution when in contact with an aqueous phase, such as a chromatographic bead matrix. Thus, the term "conjugate" as defined herein encompasses the above-mentioned heterogeneous molecules in the form of a precipitate, sediment, bioerodible matrix, or other solid form capable of releasing the heterogeneous molecule into an aqueous solution upon solid hydration.

[0083] The term "label" as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze chemical alteration of a detectable substrate compound or composition.

[0084] "Solid phase" refers to a non-aqueous matrix to which the antibodies of the present invention can adhere. Examples of solid phases encompassed herein include those formed in part or in whole by glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicon tubes. In certain embodiments, depending on the context, the solid phase may include the wells of an assay plate; in other cases, it is a purification column (e.g., an affinity chromatography column). This term also includes discontinuous solid phases of discrete particles, such as those described in U.S. Pat. No. 4,275,149.

[0085] Antibodies, also known as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, wherein the sequences of the amino-terminal domains of the heavy and light chains are variable, and therefore are often referred to as variable region domains or variable heavy (VH) or variable light (VH) domains. The two domains conventionally associate to form a specific binding region, although as discussed herein, specific binding can also be obtained using only heavy chain variable sequences, and various non-natural configurations of antibodies are known and used in the art.

[0086] "Functionalized" or "biologically active" antibodies or antigen-binding molecules (including heavy chain-only antibodies and bispecific triabody-like molecules (TCAs) herein) are antibodies or antigen-binding molecules that are able to exert one or more of their natural activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule (e.g., TCA) may have the ability to specifically bind to an antigen and the binding may in turn cause or alter a cellular or molecular event, such as a signal transduction or an enzyme activity. A functional antibody or other binding molecule (e.g., TCA) may also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule (e.g., TCA) to exert one or more of its natural activities depends on several factors, including the correct folding and assembly of the polypeptide chains.

[0087] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies only, three-chain antibodies, single-chain Fv, nanobodies, etc., and also includes antibody fragments, as long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be mouse, human, humanized, chimeric or derived from other species.

[0088] The term antibody can refer to full-length heavy chains, full-length light chains, complete immunoglobulin molecules; or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen binding site that immunospecifically binds to a target antigen of interest or a portion thereof, these targets include but are not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can have any type (e.g., IgG, IgE, IgM, IgD and IgA), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc portions that provide reduced or enhanced effector cell activity. Immunoglobulins can be derived from any species. On the one hand, immunoglobulins are mostly of human origin.

[0089] The term "variable" refers to the fact that certain parts of the variable domains are widely different in sequence between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. In the light chain and heavy chain variable domains, the variability is concentrated in three segments called hypervariable regions. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FRs, mainly using a β-folded configuration connected by three hypervariable regions connected by loops, and in some cases, forming a part of the β-folded structure. The hypervariable regions of each chain are closely bound together by FRs, and together with the hypervariable regions from other chains, contribute to the formation of the antigen binding site of the antibody (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, Md.). Although the constant domains do not directly participate in the binding of antibodies to antigens, they exhibit various effector functions, such as antibody participation in antibody-dependent cellular toxicity (ADCC).

[0090] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region may comprise amino acid residues from a "complementarity determining region" or "CDR", and / or those residues from a "hypervariable loop". The "framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0091] The target variable region comprises at least one CDR sequence from the family 2 variable region provided herein, usually at least 2 CDR sequences and more usually 3 CDR sequences. Exemplary CDR names are shown in this article, but it will be appreciated by those skilled in the art that many definitions of CDR are commonly used, including the Kabat definition (see "Zhao et al. A germline knowledgebased computational approach for determining antibody complementaritydetermining regions." Mol Immunol.2010; 47: 694–700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the position of the structural loop region (Chothia et al. "Conformations ofimmunoglobulin hypervariable regions." Nature.1989; 342: 877–883).Alternative CDR definitions of interest include, but are not limited to, those disclosed by Honegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool." J Mol Biol. 2001; 309: 657-670; Ofran et al. "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes." J Immunol. 2008; 181: 6230-6235; Almagro "Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires." J Mol Recognit. 2004; 17: 132-143; and Padlan et al. "Identification of specificity-determining residues in antibodies.” Faseb J. 1995; 9: 133–139., each of which is expressly incorporated herein by reference.

[0092] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations that may be present in small amounts, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, unlike polyclonal antibody preparations including different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous because they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" indicates that the antibody is a characteristic obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the antibody to be produced by any ad hoc method.

[0093] The antibodies herein specifically include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in 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 a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of the antibodies, as long as they exhibit the desired biological activity (U.S. Pat. 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 comprising variable domain antigen binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0094] As used herein, a "complete antibody chain" is an antibody chain comprising a full-length variable region and a full-length constant region (Fc). A complete "conventional" antibody comprises a complete light chain and a complete heavy chain of a secreted IgG, as well as a light chain constant domain (CL) and a heavy chain constant domain CH1, hinge CH2 and CH3. Other isotypes such as IgM or IgA may have different CH domains. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. A complete antibody may have one or more "effector functions," which refer to those biological activities attributable to an antibody Fc constant region (a native sequence Fc region or an 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 change effector distribution, binding to Fc receptors, and the like.

[0095] Antibodies and various antigen-binding proteins can be provided as different classes depending on the amino acid sequence of the Fc (constant region) of their heavy chain. There are five main classes of heavy chain Fc regions: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into "subclasses" (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The Fc constant domains corresponding to different classes of antibodies can be referred to as α, δ, ε, γ and μ respectively. The subunit structure and the three-dimensional configuration of different classes of immunoglobulins are well known. Ig forms include hinge modifications or hingeless forms (Roux et al. (1998) J. Immunol. 161: 4083-4090; Lund et al. (2000) Eur. J. Biochem. 267: 7246-7256; US 2005 / 0048572; US 2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types (called kappa and lambda) based on the amino acid sequence of their constant domains.

[0096] A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary effector functions include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require the Fc region to interact with receptors, such as FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors, and low affinity FcRn receptors; and can be assessed using various assays disclosed, such as the definitions herein. A "dead" Fc is an Fc that has been mutagenized to retain activity with respect to, for example, extending serum half-life, but does not activate high affinity Fc receptors.

[0097] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, and naturally occurring variants thereof.

[0098] The amino acid sequence comprised by a "variant Fc region" differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or to the Fc region of a parent polypeptide, such as from about one to about ten amino acid substitutions in the native sequence Fc region or in the Fc region of a parent polypeptide, and preferably from about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology.

[0099] The variant Fc sequence may comprise three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235 and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution of IgG2 residues at 233-236 and IgG4 residues at positions 327, 330, and 331 into human IgG1 greatly reduces ADCC and CDC (see, e.g., Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. et al., 2001. J Biol Chem. 276(9):6591-604). Other Fc variants are possible, including but not limited to variants in which regions capable of forming disulfide bonds are deleted or in which certain amino acid residues are eliminated or methionine residues are added to the N-terminus of the native Fc form. Thus, in one embodiment of the invention, one or more Fc portions of the scFc molecule may comprise one or more mutations in the hinge region to eliminate disulfide bonding. In another embodiment, the hinge region of the Fc may be removed as a whole. In still another embodiment, the molecule may comprise an Fc variant.

[0100] In addition, Fc variants can be constructed to remove or substantially reduce effector functions by substitution, deletion or addition of amino acid residues to achieve complement binding or Fc receptor binding. For example, but not limited to, deletions can occur in complement binding sites, such as C1q binding sites. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0101] Fc can be in the form of native sugar chains, sugar chains increased compared to the native form, or sugar chains reduced compared to the native form, or can be in the form of glycosylation (aglycosylated) or deglycosylation. The increase, reduction, removal or other modification of sugar chains can be achieved by common methods in the art, such as chemical methods, enzyme methods or by expressing the sugar chains in genetically engineered production cell lines. Such cell lines may include microorganisms that naturally express glycosylation enzymes, such as Pichia pastoris, and mammalian cell lines, such as CHO cells. In addition, the microorganism or cell can be engineered to express a glycosylation enzyme, or can be rendered incapable of expressing a glycosylation enzyme (see, e.g., Hamilton et al., Science, 313: 1441 (2006); Kanda et al., J. Biotechnology, 130: 300 (2007); Kitagawa et al., J. Biol. Chem., 269(27): 17872 (1994); Ujita-Lee et al., J. Biol. Chem., 264(23): 13848 (1989); Imai-Nishiya et al., BMC Biotechnology 7: 84 (2007); and WO 07 / 055916). As an example of a cell engineered to have altered sialylation activity, the α-2,6-sialyltransferase 1 gene has been engineered into Chinese hamster ovary cells and sf9 cells. The antibodies expressed by these engineered cells are therefore sialylated by the foreign gene product. Another method for obtaining Fc molecules with altered amounts of sugar residues compared to a variety of natural molecules includes, for example, separation of the various molecules into glycosylated and non-glycosylated fractions using lectin affinity chromatography (see, for example, WO 07 / 117505). The presence of specific glycosylated moieties has been shown to alter the function of immunoglobulins. For example, removal of sugar chains from Fc molecules results in a significant reduction in binding affinity to the C1q portion of the first complement component C1 and a reduction or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing an unwanted immune response in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been associated with anti-inflammatory activity (see, e.g., Kaneko et al., Science 313:760 (2006)), while removal of fucose from IgG results in enhanced ADCC activity (see, e.g., Shoj-Hosaka et al., J. Biochem., 140:777 (2006)).

[0102] In alternative embodiments, the antibodies of the invention may have an Fc sequence with enhanced effector function, for example by increasing its binding capacity to FcγRIIIA and increasing ADCC activity. For example, fucose attached to the N-linked glycan at Asn-297 of Fc sterically hinders the interaction of Fc with FcγRIIIA, and the removal of fucose by glycoengineering can increase binding to FcγRIIIA, which translates into ADCC activity >50 times higher than that of wild-type IgG1 controls. Protein engineering by amino acid mutations in the Fc portion of IgG1 has generated a variety of variants that increase the binding affinity of Fc to FcγRIIIA. Notably, the triple alanine mutant S298A / E333A / K334A exhibits a 2-fold increase in binding to FcγRIIIA and ADCC function. S239D / I332E (2X) and S239D / I332E / A330L (3X) variants have a significant increase in binding affinity to FcγRIIIA and enhanced ADCC ability in vitro and in vivo. Other Fc variants identified by yeast display also showed improved binding to FcγRIIIA and enhanced tumor cell killing in a mouse xenograft model. See, e.g., Liu et al. (2014) JBC 289 (6): 3571-90, which is specifically incorporated herein by reference.

[0103] The term "antibody containing an Fc region" refers to an antibody comprising an Fc region. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of nucleic acids encoding the antibody. Therefore, antibodies having an Fc region according to the present invention may include antibodies with or without K447.

[0104] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. The CD3 binding antibodies of the present invention comprise a dimer of one heavy and one light chain variable domain in tight, non-covalent association; however, other antibodies, e.g., for use in a multispecific configuration, may comprise a VH in the absence of a VL sequence. Even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although the affinity may be lower compared to the two-domain binding site.

[0105] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a small number of residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH herein denotes Fab' in which the cysteine ​​residues of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0106] "Humanized" forms of non-human (eg, rodent) antibodies (including single chain antibodies) are chimeric antibodies (including single chain antibodies) that contain minimal sequence derived from non-human immunoglobulin. See, e.g., 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 additional details, see US 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.

[0107] As used herein, the term "single-chain antibody" refers to a single polypeptide chain containing one or more antigen-binding domains that bind to an antigen epitope, wherein such domains are derived from or have sequence identity with the variable regions of an antibody heavy or light chain. Portions of such variable regions may consist of V H or V L Gene segments, D and J H Gene segment or J L The variable region can be encoded by rearranged V H DJ H 、V L DJ H 、V H J L or VL J L Gene segments encode V-, D- and J-gene segments can be derived from humans and various animals, including birds, fish, sharks, mammals, rodents, non-human primates, camels, llamas, rabbits, etc.

[0108] The CD3 binding antibodies of the present invention are particularly useful in multispecific configurations, including but not limited to bispecific antibodies, trifunctional antibodies, etc. Various methods and protein configurations are known and can be used for bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0109] The first generation of BsMAbs consists of two heavy chains and two light chains, each chain from two different antibodies. Two Fab regions are for two antigens. The Fc region consists of two heavy chains and forms a third binding site with the Fc receptor on immune cells (see, for example, Lindhofer et al., The Journal of Immunology, Vol. 155, pp. 219-225, 1995). The antibodies may be from the same or different species. For example, cell lines expressing rat and mouse antibodies secrete functional bispecific Abs due to preferential species-restricted heavy and light chain pairing. In other embodiments, the Fc region is designed to fit together only in a specific manner.

[0110] Other types of bispecific antibodies include chemically linked Fabs consisting only of Fab regions. Two chemically linked Fab or Fab2 fragments form an artificial antibody that is bound to two different antigens, thereby making it a type of bispecific antibody. The antigen-binding fragments (Fab or Fab2) of two different monoclonal antibodies are produced and chemically linked such as thioether (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).

[0111] Various other methods for producing multivalent artificial antibodies have been developed by recombinantly fusing the variable domains of two antibodies. Single-chain variable fragments (scFv) are fusion proteins of the variable regions of the heavy chain (VH) and light chain (VL) of immunoglobulins, connected to a short linker peptide of 10 to about 25 amino acids. The linker is usually rich in glycine to obtain flexibility, and serine or threonine to obtain solubility, and can connect the N-terminus of VH to the C-terminus of VL, and vice versa. Bispecific single-chain variable fragments (di-scFv, bi-scFv) can be engineered by connecting two scFvs with different specificities. A single peptide chain with two VH and two VL regions is produced, thereby producing a divalent scFv.

[0112] Bispecific tandem scFv is also referred to as bispecific T cell adapter (BiTE). Bispecific scFv can be produced with a linker peptide that is too short to fold together (about 5 amino acids) for two variable regions, thereby forcing scFv dimerization. This type is referred to as a double antibody (Adams et al., British journal of cancer 77, pp. 1405-12, 1998). Dual affinity retargeting (DART) platform technology (Macrogenics, Rockville, Md.). This fusion protein technology uses two single-chain variable fragments (scFv) 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-chain protein. Based on the immunoglobulin Fc region, one binding domain is located on the C-terminus, and the second binding domain is located on the N-terminus of the effector domain.

[0113] Tetravalent and bispecific antibody-like proteins also include DVD-Ig, which is engineered by two monoclonal antibodies (Wu, C. et al., Nature Biotechnology, 25, pp. 1290-1297, 2007). In order to construct DVD-Ig molecules, the V domains of two mAbs are fused in series by short joints (TVAAP), wherein the variable domains of the first antibody light chain (VL) are at the N-terminal, followed by other antibody VL and Ck, to form DVD-Ig protein light chains. Similarly, the variable regions of the heavy (VH) chains of two mAbs are fused in series by short joints (ASTKGP), wherein the first antibody is at the N-terminal, followed by another antibody and heavy chain constant domains, to form DVD-Ig protein heavy chains (VH1 / VL1). All light chains and heavy chain constant domains are retained in the DVD-Ig design because they are crucial for forming complete IgG-like molecules connected by disulfide bonds. Co-transfection of mammalian cells with expression vectors encoding DVD-Ig light and heavy chains results in the secretion of a single species of IgG-like molecule with a molecular weight of approximately 200 kDa. This molecule now has four binding sites, 2 for each mAb.

[0114] The term "bispecific three-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy chain / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody, the antibody comprising an Fc portion comprising a CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain and an antigen-binding domain that binds an epitope of a second antigen or a different epitope of the first antigen, wherein such binding domains are derived from or have sequence identity with the variable region of an antibody heavy chain or light chain. Portions of such variable regions may consist of V H and / or V L Gene segments, D and J H Gene segment or J L The variable region can be encoded by rearranged V H DJ H 、V L DJ H 、V H J L or V L J L Gene segment encoding.

[0115] TCA protein utilization "heavy chain antibody only" or "heavy chain antibody" or "heavy chain polypeptide" as used herein refers to a single chain antibody comprising the heavy chain constant regions CH2 and / or CH3 and / or CH4 but without a CH1 domain. In one embodiment, the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH2 and CH3 domain. In another embodiment, the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH2 domain. In another embodiment, the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In another embodiment, the heavy chain consists of an antigen binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but does not contain a hinge region. Heavy chain antibodies only may be in the form of dimers in which two heavy chains are disulfide bonded, covalently or non-covalently bonded to each other. The heavy chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses (such as IgM, IgA, IgD and IgE subclasses) are also included herein. In a specific embodiment, the heavy chain antibody has an IgG1, IgG2, IgG3 or IgG4 subtype, particularly an IgG1 subtype.

[0116] Heavy chain antibodies constitute about one quarter of the IgG antibodies produced by camelids (e.g., camels and llamas) (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains, but no light chains. Therefore, the variable antigen-binding portion is called a VHH domain and it represents the smallest naturally occurring complete antigen-binding site of only about 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity for a variety of antigens can be produced by immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be easily 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 have a single VH-like domain in their antibodies (which is called VNAR) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al. Molecular Immunology 40, 25-33 (2003)).

[0117] An antibody or antigen-binding molecule that "binds" an antigen of interest (including only heavy chain antibodies and bispecific triabody-like molecules (TCAs) herein) is an antibody or antigen-binding molecule that binds to an antigen with sufficient affinity to allow the antibody or binding molecule to be used as a diagnostic and / or therapeutic agent targeting the antigen and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody or other binding molecule to a non-targeted antigen will not exceed 10%, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0118] protein

[0119] The present invention provides a family of closely related antibodies that bind to CD3 and activate signaling through CD3, e.g., activate CD3 +T cells. The antibodies within the family include a set of CDR sequences as defined herein, and are exemplified by the VH sequences provided by SEQ ID NO: 1-18. The antibody family provides many benefits that are useful for use as one or more clinical therapeutic agents. The antibodies within the family include members with a range of binding affinities, thereby allowing the selection of specific sequences with desired affinity. The ability to fine-tune affinity is particularly important for managing the level of CD3 activation in the treated individual and thereby reducing toxicity. For example, if a low-abundance tumor antigen is targeted (less than 10,000 molecules per cell), a high-affinity CD3 binder (<30nM) is expected to be preferred. If a high-abundance tumor antigen is targeted (more than 50,000 molecules per cell), a CD3 binder with low affinity (>50nM) is preferred. From the affinity alone, it can be evaluated that the antibody is induced cytokine release (e.g., release IL-2, IFNγ, etc.) when combined with T cells, wherein a reduction in cytokine release may be desirable.

[0120] Suitable antibodies can be selected from those provided herein for development and use, including but not limited to use as bispecific antibodies. Determination of the affinity of the candidate protein can be performed using methods known in the art (e.g., Biacore measurement, etc.). Members of the antibody family may have an affinity for CD3 with a Kd of about 10 -6 to about 10 -11 About, including but not limited to: about 10 -6 to about 10 -10 About 10 -6 to about 10 -9 About 10 -6 to about 10 -8 About 10 -8 to about 10 -11 About 10 -8 to about 10 -10 About 10 -8 to about 10 -9 About 10 -9 to about 10 -11 About 10 -9 to about 10 -10 or any value within these ranges. Affinity selection can be confirmed by biological assessment of T cell activation and assessment of potential toxicity, for example, in vitro or in preclinical models. The determination of cytokine release can be evaluated using any convenient method, including but not limited to the assays described in the Examples.

[0121] T cell activation is initiated by binding to MH-peptide complexes or anti-TCR / CD3 antibodies to engage T cell receptors (TCR). Examples of anti-TCR / CD3 antibodies that activate T cells are OKT3 and UCHT1. These anti-CD3 antibodies cross-compete for binding to CD3 on T cells and are routinely used in T cell activation assays. The anti-CD3 antibodies of the present invention cross-compete with OKT3 for binding to human CD3. Depending on the binding affinity to CD3 and epitopes on CD3, anti-CD3 antibodies activate T cells with different functional results. In vitro incubation of human T cells with low-affinity anti-CD3 antibodies results in incomplete activation of T cells, low IL-2 and IL-10 production. In contrast, high-affinity CD3 binders activate T cells to produce significantly more IL-2 and other cytokines. Low-affinity anti-CD3 antibodies are considered partial agonists that selectively induce some effector functions, effective tumor killing, and CD69 upregulation without inducing others such as IL-2 and IL-10 production. The high-affinity binders of the present invention are full agonists of many immune effector functions of activated T cells. The intensity of the interaction with CD3 and the recognized epitopes qualitatively leads to different activations of T cells. The maximum cytokine production of T cells activated by low-affinity anti-CD3 antibodies is lower than the maximum activation of high-affinity anti-CD3 antibodies. In some embodiments, when compared with the reference anti-CD3 antibodies in the same assay, when combined with T cells in an activation assay, the antibodies of the present invention cause a lower release of one or both of IL-2 and IL-10, wherein the reference antibody can be ID 304703 (SEQ ID NO: 22) or an antibody with equivalent affinity. The maximum release of IL-2 and / or IL-10 may be less than about 75% of the release by the reference antibody, less than about 50% of the release by the reference antibody, less than about 25% of the release by the reference antibody, and may be less than about 10% of the release by the reference antibody.

[0122] In some embodiments of the present invention, bispecific or multispecific antibodies are provided, which may have any configuration discussed herein, including but not limited to three-chain bispecifics. The bispecific antibody comprises at least a heavy chain variable region of an antibody that is specific for a protein other than CD3, and may comprise a heavy chain variable region and a light chain variable region. In some such embodiments, the second antibody specifically binds to tumor-associated antigens, targeted antigens (e.g., integrins, etc.), pathogen antigens, checkpoint proteins, etc. Various forms of bispecific antibodies fall within the scope of the present invention, including but not limited to single-chain polypeptides, double-chain polypeptides, three-chain polypeptides, four-chain polypeptides, and multiples thereof.

[0123] The CD3-specific antibody family comprises a VH domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. As an example, for CDR1, CDR2 and CDR3, the CDR sequences may be located in the region of about amino acid residues 26-33; 51-58; and 97-112 of the provided exemplary variable region sequences listed in SEQ ID NOs: 1-18, respectively. Those skilled in the art will appreciate that if different framework sequences are selected, the CDR sequences may be in different positions, although generally the order of the sequences will remain unchanged.

[0124] The CDR sequence of the family 2 antibody may have the following sequence formula: X represents a variable amino acid, which may be a specific amino acid as shown below.

[0125] CDR1

[0126] G1 F2 T3 F4 X5 X6 Y7 A8

[0127] in:

[0128] X5 can be any amino acid; in some embodiments, X5 is D, A, or H; in some embodiments, X5 is D.

[0129] X6 can be any amino acid; in some embodiments, X6 is D or N; in some embodiments, D6 is D.

[0130] In some embodiments, the CDR1 sequence of the Family 2 anti-CD3 antibody comprises residues 26-33 of the sequence set forth in any one of SEQ ID NOs: 1-18.

[0131] CDR2

[0132] I 1’ S 2’ W 3’ N 4’ S 5’ G 6’ S 7’ I 8’

[0133] In some embodiments, the CDR2 sequence of the Family 2 anti-CD3 antibody comprises residues 51-58 of the sequence set forth in any one of SEQ ID NOs: 1-18.

[0134] CDR3

[0135] A 1” K 2” D 3” S 4” R 5”G 6” Y 7” G 8” X 9” Y 10” X 11” X 12” G 13” G 12” A 15” Y 16”

[0136] in:

[0137] X 9” Can be any amino acid, in some embodiments, X 9” is D or S; in some embodiments, X 9” It is D;

[0138] X 11” Can be any amino acid, in some embodiments, X 11” is R or S;

[0139] X 12” Can be any amino acid, in some embodiments, X 12” It is L or R.

[0140] In some embodiments, the CD3 sequence of the Family 2 anti-CD3 antibody has the formula AKDSRGYGDYX 11” X 12” GGAY, where X 11” and X 12” is as defined above. In some embodiments, the CDR3 sequence of the Family 2 anti-CD3 antibody comprises residues 97-112 of the sequence set forth in any one of SEQ ID NOs: 1-18.

[0141] 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 comprises a VL domain having CDR1, CDR2, and CDR3 sequences in a human VL framework. The CDR sequences may be those of SEQ ID NO: 19. In some embodiments, for CDR1, CDR2, CDR3, the CDR1 sequence comprises amino acid residues 27-32; 50-52; 89-97, respectively.

[0142] In some embodiments, the CDR sequences of the Family 2 antibodies have sequences that are at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to a CDR sequence or a set of CDR sequences in any one of SEQ ID NOs: 1-18. In some embodiments, the CDR sequences of the invention comprise one, two, three or more amino acid substitutions relative to a CDR sequence or a set of CDR sequences in any one of SEQ ID NOs: 1-18. In some embodiments, the one or more amino acid substitutions are one or more of positions 5 or 10 of CDR1, positions 2, 6 or 7 of CDR2, and positions 1, 8, 9 or 10 of CDR3 relative to the formula provided above.

[0143] When the protein of the invention is a bispecific antibody, one binding part (i.e., VH / VL combination or VH only) is specific for human CD3, while the other arm may be specific for target cells, including cancer cells such as ovarian cancer, breast cancer, gastrointestinal cancer, brain cancer, head and neck cancer, prostate cancer, colon cancer, and lung cancer, as well as blood tumors such as B cell tumors (including leukemias), lymphomas, sarcomas, carcinomas, neural cell tumors, squamous cell carcinomas, germ cell tumors, metastases, undifferentiated tumors, seminoma, melanoma, myeloma, neuroblastoma, mixed cell tumors, neoplasia caused by infectious agents, and cells of other malignancies; cells infected with pathogens, autoreactive cells causing inflammation and / or autoimmunity. The non-CD3 part may also be specific for immunomodulatory proteins, as will be described herein.

[0144] Tumor-associated antigens (TAAs) are relatively localized to tumor cells, whereas tumor-specific antigens (TSAs) are unique to tumor cells. TSAs and TAAs are usually part of intracellular molecules expressed on the cell surface as part of the major histocompatibility complex.

[0145] Tissue-specific differentiation antigens are molecules present on tumor cells and their normal cell counterparts. Tumor-associated antigens known to be recognized by therapeutic mAbs are divided into several different categories. Hematopoietic differentiation antigens are glycoproteins that are usually associated with the cluster of differentiation (CD) group and include CD20, CD30, CD33 and CD52. Cell surface differentiation antigens are a group of different glycoproteins and carbohydrates found on the surface of normal cells and tumor cells. Antigens involved in growth and differentiation signaling are usually growth factors and growth factor receptors. Growth factors that are targets for antibodies used in cancer patients include CEA, epidermal growth factor receptor (EGFR; also known as ERBB1), ERBB2 (also known as HER2), ERBB3, MET (also known as HGFR), insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EPHA3), tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B), and receptor activator of nuclear factor-κB ligand (RANKL; also known as TNFSF11). Antigens involved in angiogenesis are generally proteins or growth factors that support the formation of new microvasculature, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integrin αVβ3 and integrin α5β1. Tumor stroma and extracellular matrix are essential supporting structures for tumors. Stromal and extracellular matrix antigens that are therapeutic targets include fibroblast activation protein (FAP) and tenascin.

[0146] Examples of therapeutic antibodies that can be used in a bispecific configuration include, but are not limited to, rituximab; ibritumomab tiuxetan; tiuxetan; tositumomab; brentuximab; vedotin; gemtuzumab; ozogamicin; alemtuzumab; IGN101; adelimumab; labetuzumab; huA33; pertumumab; ogavuzumab; CC49 (mectinomab); cG250; J591; MOv18; MORAb-003 (farizumab); 3F8, ch14.18; KW-2871; hu3S193; IgN311; bevacizumab; IM-2C6; CDP791; edarizumab; volocizumab; cetuximab, panitumumab, nimotuzumab; 806; trastuzumab; pertuzumab; MM-121; AMG 102, METMAB; SCH 900105; AVE1642, IMC-A12, MK-0646, R1507; CP 751871; KB004; IIIA4; mapatumumab (HGS-ETR1); HGS-ETR2; CS-1008; denosumab; sirolimusumab; F19; and 81C6.

[0147] The most actively studied immune checkpoint receptors cytotoxic T lymphocyte-associated antigen 4 (CTLA4; also known as CD152) and programmed cell death protein 1 (PD1; also known as CD279) in the context of clinical cancer immunotherapy are both inhibitory receptors. The clinical activity of antibodies that block either of these receptors implies that anti-tumor immunity can be enhanced at multiple levels, and combination strategies can be intelligently designed, guided by mechanistic considerations and preclinical models.

[0148] The two ligands for PD1 are PD1 ligand 1 (PDL1; also known as B7-H1 and CD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed on cancer cells, and by binding to its receptor PD1 on T cells, it inhibits T cell activation / function.

[0149] Lymphocyte activation gene 3 (LAG3; also known as CD223), 2B4 (also known as CD244), B and T lymphocyte attenuator (BTLA; also known as CD272), T cell membrane protein 3 (TIM3; also known as HAVcr2), adenosine A2a receptor (A2aR) and the killer inhibitory receptor family are each associated with the inhibition of lymphocyte activity and, in some cases, with the induction of lymphocyte anergy. Antibody targeting of these receptors can be used in the methods of the invention.

[0150] Agents that excite immune co-stimulatory molecules can also be used in the methods of the present invention. Such agents include agonists or CD40 and OX40. CD40 is a co-stimulatory protein found on antigen presenting cells (APCs) and is necessary for their activation. These APCs include phagocytes (macrophages and dendritic cells) and B cells. CD40 is part of the TNF receptor family. The main activation signal transduction molecules of CD40 are IFNγ and CD40 ligand (CD40L). Macrophages are activated by CD40 stimulation.

[0151] Targeted anti-CCR4 (CD194) antibodies include humanized monoclonal antibodies against CC chemokine receptor 4 (CCR4) with potential anti-inflammatory and anti-tumor activity. CCR2 is expressed on inflammatory macrophages, which can be found in various inflammatory conditions, such as rheumatoid arthritis; and has also been identified as being expressed on tumor-promoting macrophages. CCR2 is also expressed on regulatory T cells, and the CCR2 ligand CCL2 mediates the recruitment of regulatory T cells into tumors. Regulatory T cells suppress the response to anti-tumor T cells and therefore require their inhibition or elimination.

[0152] Producing the Protein of the Invention

[0153] Although antibodies can be prepared by chemical synthesis, the antibodies are usually produced by methods of recombinant DNA technology, such as co-expression of all chains constituting proteins in a single recombinant host cell, or co-expression of heavy chain polypeptides and antibodies such as human antibodies. In addition, a single polycistronic expression vector can also be used to express antibody heavy and light chains. Standard protein purification techniques such as affinity (protein A) chromatography, size exclusion chromatography and / or hydrophobic interaction chromatography are used to achieve purification of individual polypeptides. The size and hydrophobicity of bispecific antibodies are sufficiently different so that standard methods can be used for purification.

[0154] The amount of antibody and heavy chain polypeptides produced in a single host cell can be minimized by engineering the constant regions of the antibody and heavy chains so that homodimerization is preferred over heterodimerization, for example, by introducing self-complementary interactions (see, for example, WO 98 / 50431 for possibilities such as the "protrusion into the cavity" strategy (see WO 96 / 27011)). Therefore, another aspect of the present invention is to provide a method for producing bispecific antibodies in a recombinant host, the method comprising the steps of: expressing in a recombinant host cell a nucleic acid sequence encoding at least two heavy chain polypeptides, wherein the differences in the heavy chain polypeptides in their constant regions are sufficient to reduce or prevent homodimer formation but increase bispecific formation.

[0155] When the proteins comprise three chains, such as FlicAb, they can be produced by co-expressing the three chains (two heavy chains and one light chain) constituting the molecule in a single recombinant host cell.

[0156] In order to recombinantly produce the protein herein, one or more nucleic acids encoding all chains (e.g., 2, 3, 4, etc.) are isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0157] In a preferred embodiment, the host cell according to the method of the present invention is capable of expressing human immunoglobulins at high levels, i.e. at least 1 pg / cell / day, preferably at least 10 pg / cell / day and even more preferably at least 20 pg / cell / day or more, without the need to amplify the single-chain nucleic acid molecule encoding the host cell.

[0158] Pharmaceutical composition

[0159] 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. As used herein, a pharmaceutically acceptable carrier is exemplary but not limited to an adjuvant, a solid carrier, water, a buffer or other carriers or combinations thereof used in the art to maintain therapeutic components.

[0160] Therapeutic formulations of proteins used according to the invention are prepared for storage, such as in the form of lyophilized formulations or aqueous solutions, by mixing the protein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamic acid, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; Sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0161] Anti-CD3 antibody formulations are disclosed, for example, in U.S. Patent Publication No. 20070065437, the entire disclosure of which is expressly incorporated herein by reference. Similar formulations can be used for the proteins of the present invention. The main components of such formulations are an effective pH buffer in the range of 3.0 to 6.2, a salt, a surfactant, and an effective amount of a bispecific antibody having anti-CD3 specificity.

[0162] How to use

[0163] Provided are methods for treating or alleviating a disease in a regimen, including but not limited to infections, autoimmune diseases, primary or metastatic cancers, etc., comprising contacting a target cell with an antigen-binding composition of the invention, particularly wherein the antigen-binding composition is a multispecific antibody suitable for the condition being treated, such as one of the binding moieties specifically binding to a tumor-associated antigen to treat the relevant cancer cells; a binding moiety specific for a target pathogen to treat the relevant infection, etc. Such methods comprise administering a therapeutically effective amount or effective dose of an agent of the invention to a subject in need of treatment, including but not limited to a combination of the agent and a chemotherapeutic drug, radiation therapy, or surgery.

[0164] The effective dose of the composition of the present invention for treating a disease varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is preventive or therapeutic. Typically, the patient is a human, but non-human mammals, such as companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., can also be treated. The therapeutic dose can be titrated to optimize safety and efficacy.

[0165] Dosage levels can be readily determined by an ordinary skilled clinician and can be modified as necessary, for example, as needed to alter the subject's response to treatment. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the particular mode of administration. Dosage unit forms typically contain between about 1 mg and about 500 mg of active ingredient.

[0166] In some embodiments, the therapeutic dose of the agent may be in the range of about 0.0001 to 100 mg / kg, and more typically 0.01 to 5 mg / kg of the host's body weight. For example, the dose may be 1 mg / kg body weight or 10 mg / kg body weight or in the range of 1-10 mg / kg. Exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. The therapeutic entity of the present invention is typically administered multiple times. The intervals between individual doses may be weekly, monthly, or annual. Intervals may also be irregular, as indicated by measuring blood levels of the therapeutic entity in the patient. Alternatively, the therapeutic entity of the present invention may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0167] In preventive applications, relatively low doses may be administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In other therapeutic applications, relatively high doses at relatively short intervals are sometimes required until disease progression is reduced or terminated, and preferably until the patient shows partial or complete improvement in disease symptoms. Thereafter, the patented product may be administered in a preventive manner.

[0168] In still other embodiments, the methods of the invention include treating, reducing or preventing tumor growth, tumor metastasis or tumor invasion of cancers, including carcinomas, blood cancers such as leukemias and lymphomas, melanomas, sarcomas, gliomas, etc. For prophylactic applications, the pharmaceutical composition or medicament is administered to a patient susceptible to or otherwise at risk of a disease in an amount sufficient to eliminate or reduce the risk, lessen the severity of the disease, or delay the onset of the disease, including the biochemical, histological and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during the development of the disease.

[0169] The composition for treating the disease can be administered parenterally, topically, intravenously, intratumorally, orally, subcutaneously, intraarterially, intracranially, intraperitoneally, intranasally or intramuscularly. Typical administration routes are intravenous or intratumoral, but other routes may be equally effective.

[0170] Typically, the composition is prepared as an injectable in the form of a liquid solution or suspension; solid forms suitable for dissolving or suspending in a liquid vehicle prior to injection can also be prepared. The preparation can also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymers to enhance the adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agent of the present invention can be administered in the form of a reservoir injection or implant formulation that can be formulated in a manner that allows for sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic and fully comply with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0171] The expression of LD can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example by determining the LD 50 (dose lethal to 50% of the population) and LD 100The toxicity of the proteins described herein is determined by the dose ratio between toxicity and therapeutic effect (the dose that is lethal to 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic for use in humans. The dosage of the proteins described herein is preferably within a range of circulating concentrations that includes an effective dose with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage may be selected by the individual physician in view of the patient's condition.

[0172] The pharmaceutical composition can be administered in a variety of unit dosage forms depending on the method of administration. For example, solid dosage forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules, and lozenges. It should be recognized that when administered orally, the composition of the present invention should be protected from digestion. This is typically achieved by complexing the molecule with the composition so that the molecule is resistant to acidic and enzymatic hydrolysis, or by packaging the molecule in an appropriate resistant carrier (such as a liposome or protective barrier). Methods for protecting proteins from digestion are well known in the art.

[0173] The composition for administration will generally include an antibody or other scavenger dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these preparations can vary widely, and will be selected mainly based on fluid volume, viscosity, patient weight, etc. according to the selected specific mode of administration and the needs of the patient (for example, Remington's Pharmaceutical Science (15th edition, 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., editor, 1996)).

[0174] Kits comprising the active agents of the invention and formulations thereof and instructions for use are also within the scope of the invention. The kits may also contain at least one additional agent, e.g., chemotherapeutic drugs, etc. The kits typically include a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit or otherwise accompanying the kit.

[0175] The composition can be used for therapeutic treatment. As described above, the composition is administered to the patient in an amount sufficient to substantially eliminate the targeted cells. An amount sufficient to achieve this purpose is defined as a "therapeutically effective dose", which can provide an improvement in overall survival. Single or multiple administrations of the composition can be administered according to the dose and frequency required and tolerated by the patient. The specific dose required for treatment will depend on the medical condition and medical history of the mammal and other factors, such as age, weight, sex, route of administration, efficiency, etc.

[0176] Now that the present invention is 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 and scope of the invention.

[0177] Example

[0178] Example 1 Genetically engineered rats expressing heavy chain-only antibodies

[0179] The human IgH locus was constructed and assembled in several parts, which involved modifying and ligating rat C region genes, and then ligating the locus to human V H 6-DJ H Then there will be a separate person V H The two BACs of the gene cluster are assembled with the coding (human V H 6-DJ H -BAC co-injection of rat C) fragments.

[0180] Transgenic rats carrying artificial heavy chain immunoglobulin loci in unrearranged configurations were produced. The constant region genes included encode IgM, IgD, IgG2b, IgE, IgA and 3' enhancers. RT-PCR and serum analysis (ELISA) of transgenic rats revealed the expression of only heavy chain antibodies of various isotypes in the productive rearrangement of transgenic immunoglobulin loci and serum. Transgenic rats were hybridized with rats of endogenous heavy chain and light chain loci with mutations previously described in U.S. Patent Publication 2009 / 0098134A1. Analysis of such animals demonstrated the inactivation of rat immunoglobulin heavy and light chain expression and the high-level expression of heavy chain antibodies with variable regions encoded by human V, D and J genes. The immunity of transgenic rats resulted in the high-titer serum response of antigen-specific heavy chain antibodies. These transgenic rats expressing heavy chain antibodies with human VDJ regions are referred to as UniRat.

[0181] Example 2 Genetically engineered rats expressing fixed light chain antibodies

[0182] From different (V H -DJ H )n The combination of rearranged H chains and unique L chains produces transgenic human antibody repertoires. To this end, rearranged L chains (human Vk-Jk1-Ck) were integrated into the rat germline by DNA microinjection, and the transgenic animals obtained were bred with previously described rat strains that naturally express human H chain repertoires (Osborn et al., 2013). This new rat strain was named OmniFlic.

[0183] Immunization of OmniFlic rats with many different antigens produced high levels of antigen-specific IgG, similar to other transgenic rats carrying the same IgH locus. Repertoire analysis by RT-PCR identified highly variable V H -Gene rearrangement. In addition, only one L-chain product was identified that was also expressed at high levels.

[0184] Antigen-specific binders from OmniFlic were obtained by NGS and selected from cDNA libraries (yeast, E. coli, phage), which identified different H-chain transcripts after sequencing. For expression in mammalian cells, the hypermutated H-chain constructs were transfected in combination with the original transgenic Igk sequence. In this rearranged Vk-Jk1-Ck, mutational changes were not allowed and the same L-chain was always expressed with various H-chain products to produce monoclonal human IgG.

[0185] Example 3 Production of antigen-specific antibodies in transgenic rats,

[0186] To generate antigen-specific heavy chain antibodies in rats, genetically engineered rats expressing HER2 were immunized in two ways.

[0187] Immunization with recombinant extracellular domains of PD-L1 and BCMA. Recombinant extracellular domains of PD-L1 and BCMA were purchased from R&D Systems and diluted with sterile saline and combined with adjuvants. The immunogen was combined with complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) or Titermax and Ribi adjuvants. The first immunization (priming) of the immunogen in CFA or Titermax was administered in the left and right legs. After the first immunization with the immunogen in CFA, another two immunizations (boosting) in IFA or another 4 immunizations in Ribi and another immunization in Titermax were administered in each leg. This immune sequence leads to the development of B cells that produce high-affinity antibodies. The immunogen concentration is 10 micrograms per leg. Serum was collected from rats at the time of final bleeding to determine serum titers.

[0188] To generate anti-human CD3δε antibodies, genetically engineered rats were immunized using a DNA-based immunization protocol.

[0189] OmniFlic rats were immunized with human and cynomolgus macaque CD3-ε / δ constructs at Aldevron, Inc. (Fargo, ND) using GENOVAC antibody technology. Draining lymph nodes were harvested after the final boost and RNA was isolated. After cDNA synthesis, the IgH heavy chain antibody repertoire was characterized by next-generation sequencing and our proprietary in-house software. Candidate antigen-specific VH sequences showing evidence of antigen-specific positive selection were selected. Hundreds of VH sequences encoding FlicAb were selected for gene assembly and cloned into expression vectors. Subsequently, fully human FlicAb IgG1 antibodies were expressed in HEK cells for analysis by Flow and ELISA. Human FlicAb was tested for binding to primary human T cells and Jurkat cells by flow cytometry. In addition, human FlicAb was tested using recombinant CD3δε protein in ELISA. All FlicAbs with positive binding to human T cells are listed in Figures 1 and 2. The selected sequences were further characterized in T cell activation assays.

[0190] Example 4 Characterization of antibodies

[0191] Figure 3The data table summarizes the behavior of the anti-CD3 antibodies of Family 2 in monospecific and bispecific formats. Column 1 shows the sequence ID of the anti-CD3 VH sequence. Column 2 shows the MFI value for Jurkat cell binding of the parental monospecific anti-CD3. Column 3 shows the MFI value for cyno T cell binding of the parental monospecific anti-CD3. Column 4 shows the name of the aCD3:aBCMA bispecific antibody. Column 5 shows the picograms of IL-2 released by pan T cells stimulated by the indicated doses of the bispecific antibody binding to BCMA protein coated on plastic. Column 6 shows the picograms of IL-6 released by pan T cells stimulated by the indicated doses of the bispecific antibody binding to BCMA protein coated on plastic. Column 7 shows the picograms of IL-10 released by pan T cells stimulated by the indicated doses of the bispecific antibody binding to BCMA protein coated on plastic. Column 8 shows the picograms of IFN-γ released by pan T cells stimulated by the indicated doses of the bispecific antibody binding to BCMA protein coated on plastic. The 9th column shows the picograms of TNFα released by pan T cells stimulated by the indicated doses of bispecific antibodies that bind to BCMA protein coated on plastic. The 10th column shows the EC50 of bispecific antibody-mediated lysis of U266 tumor cells in the presence of human pan T cells. The 11th column shows the percentage of lysis of U266 tumor cells in the presence of bispecific antibodies and human pan T cells at a dose of 333 ng / mL of bispecific antibodies. The 12th column shows the protein binding affinity of the anti-CD3 arm of the bispecific antibodies measured by Octet. The 13th column shows the MFI value of Jurkat cell binding of the bispecific antibodies.

[0192] Example 5 Bispecific Antibody Characterization

[0193] Seven αCD3_fam2:aBCMA bispecific antibodies (each with a unique anti-CD3 arm and a common anti-BCMA arm) were tested for their ability to kill U266 BCMA+ tumor cells by redirecting activated primary T cells. In this experiment, BCMA-expressing U266 cells were mixed with activated pan T cells at a 10:1 E:T ratio, and the bispecific antibodies were added. Figure 4 Shown in Figure 5, the x-axis shows the concentration of the antibody used and the y-axis shows the % lysis of tumor cells 6 hours after addition of the antibody.

[0194] Comparison of bispecific antibody-mediated tumor cell lytic activity and IL-2 cytokine release in Figure 5 The correlation between IL-2 production and U266 tumor cell lysis is shown in the scatter plot. 2=0.37. Comparison of bispecific antibody-mediated tumor cell lytic activity and IFN-γ cytokine release Figure 6 The correlation between IFN-γ production and U266 tumor cell lysis is shown in the scatter plot. 2 =0.53. Comparison of U266 tumor cell lytic activity mediated by bispecific antibody and anti-CD3 binding affinity Figure 7 The correlation between U266 killing EC50 and protein binding affinity is shown in the scatter plot. 2 =0.93.

[0195] Example 6 Tumor cell lysis

[0196] The ability of the αCD3_F1F:αBCMA bispecific antibody to kill three different BCMA+ tumor cells and one BCMA-negative cell line by redirecting activated primary T cells was determined. In this experiment, tumor cells were mixed with activated panT cells at a 10:1 E:T ratio and the bispecific antibody was added. Figures 8A-8D Figure A shows the killing of RPMI-8226 cells, Figure B shows the killing of NCI-H929 cells, Figure C shows the killing of U-266 cells, and Figure D shows the killing of K562 cells (negative control). The x-axis shows the concentration of the antibody used, and the y-axis shows the lysis of tumor cells 6 hours after adding the antibody.

[0197] Levels of IL-2 cytokine release were measured after incubation of resting human T cells with various tumor cell lines and increasing doses of the αCD3_F1F:αBCMA bispecific antibody. Fig. 9A Shown is the release of IL-2 stimulated by RPMI-8226 cells. Fig. 9B Shown is the release of IL-2 stimulated by NCI-H929 cells, Fig. 9C shows IL-2 release stimulated by U-266 cells, and Fig.9D Shown is IL-2 release stimulated by K562 cells (negative control).

[0198] Levels of IFN-γ cytokine release were measured after incubation of resting human T cells with various tumor cell lines and increasing doses of the αCD3_F1F:aBCMA bispecific antibody. Fig. 10A IFN-γ release by RPMI-8226 cell stimulation is shown. Fig. 10B IFN-γ release stimulated by NCI-H929 cells is shown. Fig. 10C shows IFN-γ release stimulated by U-266 cells, and Fig. 10DShown is IFN-γ release stimulated by K562 cells (negative control).

[0199] The examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of the inventor's view of his invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Although every effort has been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental errors and deviations should still be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure.

[0200] Although the present invention has been described with reference to specific embodiments thereof, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made in order to adapt a particular situation, material, composition of matter, method, method step or steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

[0201] This manual also includes the following contents:

[0202] 1. An antigen binding protein specific for CD3, the antigen binding protein comprising:

[0203] A variable heavy chain domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR1 sequence has the formula:

[0204] G1 F2 T3 F4 X5 X6 Y7 A8

[0205] Wherein X5 and X6 can be any amino acid;

[0206] The CDR2 sequence has the following formula:

[0207] I 1’ S 2’ W 3’ N 4’ S 5’ G 6’ S 7’ I 8’

[0208] The CDR3 sequence has the formula

[0209] A 1” K 2” D 3” S 4” R 5” G6” Y 7” G 8” X 9” Y 10” X 11” X 12” G 13” G 12” A 15” Y 16”

[0210] Where X 9” , X 11” , X 12” It can be any amino acid;

[0211] and variable light chain domain.

[0212] 2. The antigen binding protein of embodiment 1, comprising a variable heavy chain domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR1, CDR2 or CDR3 is found in any one of SEQ ID NOs: 1-18;

[0213] and variable light chain domain.

[0214] 3. The antigen binding protein of embodiment 1, comprising a variable heavy chain domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequences are sequences that are at least 85% identical to a CDR sequence or a group of CDR sequences in any one of SEQ ID NOs: 1-18;

[0215] and variable light chain domain.

[0216] 4. The antigen binding protein of embodiment 1, comprising a variable heavy chain domain comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, wherein the CDR sequences are sequences having up to 3 amino acid substitutions relative to a CDR sequence or a group of CDR sequences in any one of SEQ ID NOs: 1-78 or SEQ ID NOs: 80-102;

[0217] and variable light chain domain.

[0218] 5. An antigen-binding protein as described in any one of embodiments 1-4, wherein the variable light chain domain comprises CDR1, CDR2 and CDR3 sequences in a human VL framework, wherein the CDR sequence is a sequence having up to 3 amino acid substitutions relative to a CDR sequence or a group of CDR sequences in SEQ ID NO:19; or wherein the CDR sequence is a sequence having at least 85% identity with a CDR sequence or a group of CDR sequences in SEQ ID NO:19.

[0219] 6. An antigen binding protein as described in any one of embodiments 1-5, wherein the variable heavy chain domain comprises the amino acid sequence listed in any one of SEQ ID NOs: 1-18.

[0220] 7. An antigen binding protein as described in any one of embodiments 1-6, wherein the variable light chain domain comprises the amino acid sequence of SEQ ID NO: 19.

[0221] 8. An antigen binding protein as described in any of embodiments 1-7, wherein when contacted with T cells in an activation assay, the antigen binding protein induces the release of reduced levels of one or both of IL-2 and IL6 relative to a reference anti-CD3 antibody.

[0222] 9. An antigen binding protein as described in any one of embodiments 1-8, which also comprises an Fc region.

[0223] 10. The antigen binding protein of embodiment 9, wherein the Fc region has been engineered to reduce effector function.

[0224] 11. An antigen binding protein as described in any one of embodiments 1-10, wherein the affinity (KD) of the protein for human CD3δε is about 10 -6 M to about 10 -11 About M.

[0225] 12. An antigen binding protein as described in any of embodiments 1-11, wherein the protein is a single chain.

[0226] 13. An antigen-binding protein as described in any one of embodiments 1-11, wherein the protein is a double-chain or multiple chains thereof

[0227] 14. An antigen binding protein as described in any of embodiments 1-11, wherein the protein is a three-chain.

[0228] 15. An antigen-binding protein as described in any one of embodiments 1-11, wherein the protein is a three-chain, and the two antigen-binding arms comprise an antibody heavy chain and a light chain

[0229] 16. An antigen binding protein as described in any of embodiments 1-11, wherein the protein further comprises a variable heavy chain domain that is specific for a protein other than CD3.

[0230] 17. The antigen binding protein of any one of embodiments 1-16, wherein the protein further comprises a variable heavy chain domain specific for a protein other than CD3;

[0231] wherein when contacted with T cells in an activation assay, the antigen binding protein induces the release of reduced levels of one or both of IL-2 and IL6 relative to a reference anti-CD3 antibody; and

[0232] Induced more than 30% tumor cytotoxicity in standard in vitro assays using tumor cells and human T cells.

[0233] 18. An antigen binding protein as described in embodiment 16, wherein the variable heavy chain domain specific for a protein other than CD3 is only a heavy chain domain.

[0234] 19. An antigen binding protein as described in embodiment 16, wherein the variable heavy chain domain specific for a protein other than CD3 also comprises a light chain variable region.

[0235] 20. An antigen binding protein as described in embodiment 16, wherein the light chain variable region is the same as the light chain variable region of the CD3 binding region.

[0236] 21. An antigen binding protein as described in any of embodiments 16-20, wherein the protein other than CD3 is a tumor-associated antigen.

[0237] 22. An antigen binding protein as described in any of embodiments 16-20, wherein the protein other than CD3 is a pathogen antigen.

[0238] 23. An antigen binding protein as described in any one of embodiments 16-20, wherein the protein other than CD3 is an immunomodulatory protein.

[0239] 24. A pharmaceutical composition comprising the antigen binding protein of any one of embodiments 1-23.

[0240] 25. The pharmaceutical formulation of embodiment 24, which is a unit dose formulation.

[0241] 26. A polynucleotide encoding an antigen binding protein as described in any one of embodiments 1-23.

[0242] 27. A vector comprising the polynucleotide as described in embodiment 26.

[0243] 28. A cell comprising the vector of embodiment 27.

[0244] 29. A method for producing an antigen binding protein as described in any one of embodiments 1-24, the method comprising growing the cell according to embodiment 29 under conditions that allow expression of the protein, and isolating the protein from the cell.

[0245] 31. A method of treatment comprising administering to an individual an effective dose of an antigen binding protein as described in any one of embodiments 1-23.

[0246] 32. The method of embodiment 30, wherein the individual is a human.

[0247] 33. A method for preparing an antigen binding protein that specifically binds to CD3, the method comprising:

[0248] Immunization of OmniFlic or UniRat animals with CD3; and

[0249] Identification of antigen-specific heavy chain sequences.

[0250] 34. A method for preparing the antigen binding protein of embodiment 16, the method comprising:

[0251] Immunizing an OmniFlic or UniRat animal with an antigen other than CD3; and identifying antigen-specific heavy chain sequences that can be combined in a molecule comprising an antigen binding protein as described in any of embodiments 1-15.

[0252] 35. A bispecific antigen binding protein, wherein the protein comprises a binding domain specific for CD3 and a binding domain specific for an antigen other than CD3;

[0253] wherein when contacted with T cells in an activation assay, the bispecific antigen binding protein induces release of reduced levels of one or both of IL-2 and IL-10 relative to a reference anti-CD3 antibody; and

[0254] Induced more than 30% tumor cytotoxicity in standard in vitro assays using tumor cells and human T cells.

[0255] 36. A bispecific antibody comprising:

[0256] (i) a CD3 binding variable region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1 in a human VH framework;

[0257] (ii) a variable light chain domain comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 19 in a human VL framework;

[0258] (iii) a single-chain anti-BCMA variable region comprising the CDR1, CDR2 and CDR3 sequences at positions 26-33, 51-58, 97-108 of SEQ ID NO: 20, respectively, in a single or tandem configuration.

[0259] 37. The bispecific antibody of embodiment 36, further comprising an Fc region.

[0260] 38. The bispecific antibody of embodiment 36, wherein the CD3 binding variable region comprises SEQ ID NO: 1, the light chain variable region comprises SEQ ID NO: 19, and the BCMA binding variable region comprises SEQ ID NO: 20 or SEQ ID NO: 21. Sequence Listing <110> Tenio Biotech Co., Ltd. Trinklein, Nathan vanSchooten, Wim Aldred, Shelley Force Harris, Katherine Pham, Duy <120> CD3 binding antibodies <130> TEBO-001WO2 <150> 62 / 394,360 <151> 2016-09-14 <150> 62 / 491,908 <151> 2017-04-28 <160> twenty two <170> FastSEQ for Windows version 4.0 <210> 1 <211> 123 <212> PRT <213> Homo sapiens <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 123 <212> PRT <213> Homo sapiens <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 123 <212> PRT <213> Homo sapiens <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 123 <212> PRT <213> Homo sapiens <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 123 <212> PRT <213> Homo sapiens <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 123 <212> PRT <213> Homo sapiens <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 7 <211> 123 <212> PRT <213> Homo sapiens <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 123 <212> PRT <213> Homo sapiens <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 123 <212> PRT <213> Homo sapiens <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 123 <212> PRT <213> Homo sapiens <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 123 <212> PRT <213> Homo sapiens <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 123 <212> PRT <213> Homo sapiens <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Ser Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 123 <212> PRT <213> Homo sapiens <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 123 <212> PRT <213> Homo sapiens <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Arg Gly Gly Ala Tyr 100 105 110 Cys Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 123 <212> PRT <213> Homo sapiens <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Ser Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 123 <212> PRT <213> Homo sapiens <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 123 <212> PRT <213> Homo sapiens <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 123 <212> PRT <213> Homo sapiens <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Gly Tyr Gly Asp Tyr Arg Leu Gly Gly Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 107 <212> PRT <213> Homo sapiens <400> 19 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 243 <212> PRT <213> Homo sapiens <400> 20 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gly Ile Arg Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gln Gly Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr Gly Met Ser Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser Gly Ile Arg 165 170 175 Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gln Gly 210 215 220 Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser <210> 21 <211> 119 <212> PRT <213> Homo sapiens <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gly Ile Arg Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gln Gly Glu Asn Asp Gly Pro Phe Asp His Arg Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 22 <211> 128 <212> PRT <213> Homo sapiens <400> 22 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile His Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Trp Arg His Asp Ile Phe Ala Ala Tyr Pro Tyr Tyr Tyr 100 105 110 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125

Claims

1. A bispecific antibody comprising: (a) a first binding moiety specific for human CD3 δε, wherein the first binding moiety comprises: (i) a first polypeptide subunit comprising a heavy chain variable (VH) domain comprising: CDR1, which consists of the amino acid sequence of GFTFDDYA, CDR2, which consists of the amino acid sequence of ISWNSGSI, and CDR3 consisting of the amino acid sequence of AKDSRGYGDYRLGGAY; and (ii) a second polypeptide subunit comprising a light chain variable (VL) domain comprising: CDR1, which consists of the amino acid sequence of QSVSSN, CDR2, which consists of the amino acid sequence of GAS, and CDR3, which consists of the amino acid sequence of QQYNNWPWT; and (b) a second binding moiety specific for human BCMA, wherein the second binding moiety is a heavy chain only binding moiety comprising: (i) a third polypeptide subunit, which comprises a VH domain, which comprises: CDR1, which consists of the amino acid sequence of GFTVSSYG, CDR2, which consists of the amino acid sequence of IRGSDGST, and CDR3, which consists of the amino acid sequence of AKQGENDGPFDH. 2 . The bispecific antibody according to claim 1 , wherein the CDR1, CDR2 and CDR3 sequences in the VH domain of the first polypeptide subunit are present in a human VH framework.

3. The bispecific antibody of claim 1, wherein the CDR1, CDR2 and CDR3 sequences in the VL domain of the second polypeptide subunit are present in a human VL framework.

4. The bispecific antibody according to claim 1, wherein The CDR1, CDR2 and CDR3 sequences in the VH domain of the first polypeptide subunit are present in a human VH framework; and The CDR1, CDR2 and CDR3 sequences in the VL domain of the second polypeptide subunit are present in a human VL framework. The bispecific antibody of claim 1 , wherein the first polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO:

1. The bispecific antibody of claim 1 , wherein the second polypeptide subunit comprises a VL domain having the amino acid sequence of SEQ ID NO:

19.

7. The bispecific antibody according to claim 1, wherein The first polypeptide subunit comprises a VH domain having an amino acid sequence of SEQ ID NO: 1, and The second polypeptide subunit comprises a VL domain having the amino acid sequence of SEQ ID NO:

19. The bispecific antibody of claim 1 , wherein the third polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO:

21. 9 . The bispecific antibody of claim 1 , wherein the third polypeptide subunit has an amino acid sequence of SEQ ID NO:

20. 10 . The bispecific antibody of claim 5 , wherein the third polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO:

21. The bispecific antibody according to claim 5 , wherein the third polypeptide subunit has an amino acid sequence of SEQ ID NO:

20. 12 . The bispecific antibody of claim 6 , wherein the third polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO:

21. The bispecific antibody according to claim 6 , wherein the third polypeptide subunit has an amino acid sequence of SEQ ID NO:

20.

14. A bispecific antibody comprising: (a) a first binding moiety specific for human CD3 δε, wherein the first binding moiety comprises: a first polypeptide subunit comprising a VH domain having an amino acid sequence of SEQ ID NO: 1, and A second polypeptide subunit comprising a VL domain having the amino acid sequence of SEQ ID NO: 19; and (b) a second binding moiety specific for human BCMA, wherein the second binding moiety is a heavy chain only binding moiety comprising: The third polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO:

21.

15. The bispecific antibody according to claim 14, wherein the third polypeptide subunit comprises: a first VH domain having the amino acid sequence of SEQ ID NO: 21, and a second VH domain having the amino acid sequence of SEQ ID NO: 21, wherein the first VH domain and the second VH domain are arranged in a tandem configuration.

16. The bispecific antibody of claim 15, wherein the first VH domain and the second VH domain of the third polypeptide subunit are arranged in a tandem configuration and connected by a linker.

17. A bispecific antibody comprising: (a) a first binding moiety specific for human CD3 δε, wherein the first binding moiety comprises: a first polypeptide subunit comprising a VH domain having an amino acid sequence of SEQ ID NO: 1, and A second polypeptide subunit comprising a VL domain having the amino acid sequence of SEQ ID NO: 19; and (b) a second binding moiety specific for human BCMA, wherein the second binding moiety is a heavy chain only binding moiety comprising: The third polypeptide subunit comprises a VH domain having the amino acid sequence of SEQ ID NO:

20. 18 . A pharmaceutical composition comprising the bispecific antibody according to claim 1 , and a pharmaceutically acceptable carrier.

19. A polynucleotide encoding the bispecific antibody of any one of claims 1 to 17.

20. A vector comprising the polynucleotide of claim 19.

21. A cell comprising the vector of claim 20.

22. A method for producing a bispecific antibody, the method comprising culturing the cell of claim 21 under conditions allowing expression of the bispecific antibody, and isolating the bispecific antibody from the cell.

23. A kit comprising the bispecific antibody according to any one of claims 1 to 17 and instructions for use.

24. Use of the bispecific antibody of any one of claims 1 to 17 in the preparation of a medicament for treating a BCMA-expressing cancer in a human subject in need thereof, wherein the BCMA-expressing cancer is a myeloma.

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